1. Blood grouping

2. Vessel-platelets (primary) hemostasis.

3. Coagulative (secondary) hemostasis.

 

Original blood transfusions were very risky , but we now know there are four different blood groups and successful transfusion depends on the antigens and antibodies present in the donor and recipient’s blood type. The four groups are known as A, B, AB and O. Each group has specific antigen markers on the red blood corpuscles and specific antibodies in the plasma. Each group is named for the antigen present on the red blood corpuscles.

If the wrong type of blood is transfused agglutination occurs ie clotting of red blood corpuscles. This incompatibility is caused by the recipient’s antibodies and the donor’s antigens

Eg if a blood group A recipient receives B type blood, antibody B in the recipient’s plasma would attach to antigen B on the donor red blood cells causing clumping of the red cells which can block blood vessels.

In this situation, the antibody A in the donor plasma is so dilute in comparison to the recipient’s antigen A on the red blood cells that it causes no adverse affect.

Blood group O has both antibodies in the plasma so can only receive O blood, but as O blood has no antigens it is the universal donor blood.

Group AB has no antibodies so can receive blood from any donor ie AB is the universal recipient.

Blood Group Systems

Blood is grouped on the basis of the type of aggluti­nogen present on its erythrocytes. Thus, blood group A has A agglutinogen on its erythrocytes while blood group M has agglutinogen-M on its erythrocytes. All blood groups obey, in whole or in part, Landsteiner’s law which states that: (1) when the blood contains a particular agglutinogen, its corresponding agglutinin is always absent in that blood, and (2) when a particular agglutinogen is absent in the blood, its correspond­ing agglutinin is always present in the blood. The first clause of the law is always true but the second clause is valid only for the ABO blood groups.

While innumerable agglutinogens have been de­ciphered in the blood, the important ones are those which are widely prevalent in the population and those which cause the worst transfusion reactions. These are called the major blood group systems, e.g., the ABO and the Rhesus (CDE) systems. Some blood groups are found only in a small proportion of the population and occasionally produce mild transfusion reactions. These are called the minor blood group systems, e.g., MN, P, etc. In addition to the major and minor blood groups, there are familial blood groups such as the Kell, Duffy, Diego, Lewis, Lutheran, Kidd, and many others that are named after individuals, mostly women, whose blood groups were detected during childbirth. These blood agglutinogens are prevalent only in a few families.

 

The ABO system comprises two agglutinogens A and B whose corresponding agglutinins are α and β. Ac­cordingly, there are 4 blood groups in the ABO system: Group A, which has A agglutinogen, group B which has B agglutinogen, group AB having both, and group O hav­ing neither. Group A has α agglutinin, group B has β agglutinin, group AB has neither, and group O has both  Both α and β agglutinins are immunoglob­ulin-M (IgM), which is very effective in causing aggluti­nation (clumping) of the red cells.

In India, about 22% of the population have A group, 33% have B group and 40% have O group blood. Only 5% have AB group blood. 85% of Caucasians are D+. Among Asians, over 99% are D+.

 

The Rhesus blood group agglutinogens were first dis­covered in the erythrocytes of rhesus monkeys, and hence the name. Rhesus blood group comprises a sys­tem of 3 agglutinogens: C, D, and E. However, for all practical purposes, the term Rhesus agglutinogen re­fers to the D agglutinogen which produces the worst transfusion reactions. Accordingly, the Rhesus system comprises only two blood groups: the Rhesus positive (Rh positive or D+) and the Rhesus negative (Rh nega­tive or D–) blood groups depending on the presence or absence of D agglutinogen

Unlike in the ABO system, there are no natural an­tibodies to rhesus agglutinogens. Anti-D antibodies develop only when a D– person is transfused with D+ blood. Once produced, these antibodies persist in blood for years and can produce serious reactions dur­ing a second transfusion.

Anti-D agglutinins are predominantly immuno­globulin G (IgG) and partly immunoglobulin M (IgM ). Unlike IgM which is very effective in agglutinating ag­glutinogen-bearing red cells, IgG does not agglutinate the red cells although they do react with the agglutinin. Such immunoglobulins which do not cause agglutina­tion are called incomplete antibodies. Although they do not agglutinate red cells, IgG-coated red cells still get lysed due to the activation of complement on their sur­face

Blood grouping

For ABO blood grouping, the test sample of blood or erythrocyte suspension is reacted with sera contain­ing α and β (called antiserum-A and antiserum-B). The sample is grouped according to the serum that aggluti­nates its red cells.

Rhesus blood grouping can be done in the same way as ABO grouping if the anti-D agglutinin used is of the IgM type. If the anti-D agglutinin used is IgG, the D+ red cells will get coated with anti-D agglutinin but there will be no agglutination of the cells. The coated red cells will agglutinate only on subsequent addition of Coombs’ (anti-immunoglobulin) serum .

Agglutination will also occur if the IgG anti-D is poten­tiated by adding albumin to it.

 

Genotypes and inheritance

The ABO phenotypes are controlled by a pair of codomi­nant alleles A and B. An individual who has inherited A-agglutinogen from one parent and B agglutinogen from the other parent will have the AB blood group. Similarly, an individual whose phenotypic blood group is B may have either the genotype BB (homozygous) or BO (heterozygous).

The Rh phenotypes are controlled by three sets (C, D, and E) of two alternative alleles (dominant and reces­sive). Each phenotype has a variable number of pos­sible genotypes. For example, cde has only one possible genotype, i.e., ccddee. CDE on the other hand can have eight possible genotypes, viz., CCDDEE, CCDDEe, CCD­dEE, CCDdEe, CcDDEE, CcDDEe, CcDdEE, and CcDdEe.

 

Agglutinogens and agglutinins

The ABO agglutinogens represent only a few of the ap­proximately one million agglutinogens present on an erythrocyte. The ABO agglutinogens are glycosphingo­lipids (oligosaccharide plus sphingolipid). The antigenicity of the agglutinogens resides in the oligosaccharide moiety. The ABO agglutinogens are present on the red cell membrane as peripheral proteins. O group cells contain a non-antigenic H substance from which both A and B agglutinogens are derived. The genes for A and B agglutinogens are located on chromosome 9. They code the synthesis of transferase-A and transferase-B, the two enzymes that are responsible for conversion of substance H into A and B agglutinogens.

ABO agglutinogens are not confined to erythrocytes alone; they are widely found in the secretory glands of gastrointestinal, respiratory, and genitourinary tracts. The secreted agglutinogens are however not glyco­sphingolipids but glycoproteins (oligosaccharide plus protein). Only about 80% of the population secretes ABO agglutinogens. They are called secretors. The rest are nonsecretors.

 

Rhesus agglutinogens Unlike the ABO agglutinogens, Rhesus agglutinogens are integral membrane proteins. They are not found anywhere other than on red cells.

 

ABO agglutinins                    The agglutinins α and β are absent at birth but develop over the first 3 to 6 months of life. They are produced as a result of exposure to ABO-like polysaccharides that are abundant in microbes, seeds, and plants. These natural antibodies are immunoglob­ulins of the IgM type. Subsequent exposures to ABO agglutinogens, as in the course of mismatched transfusion, also produce agglutinins. Such immune aggluti­nins are often of the IgG type.

 

Rhesus agglutinins       There are no natural antibodies to Rhesus agglutinogens. Agglutinins formed against them are of the IgG type.

RHESUS FACTOR

 

Around 85% of. population have an additional antigen termed D antigen on their erythrocyte membranes, they are designated rhesus* positive (RhD+). There are no innate antibodies to D antigen but the immune system in rhesus negative individuals (RhD-) can produce anti-D antibodies in response to a RhD+ transfusion. Anti-D antibodies can result in haemolysis and agglutination of RhD+ erythrocytes so it is important that Rhesus positive blood is not given to Rhesus negative recipients. 

 

*The name rhesus comes from the antigen’s first discovery in the Rhesus monkey.

 

Pregnant women who are RhD- may be carrying a RhD+ baby because the gene which codes for antigen D may be inherited from the father and it is possible that this could cause problems during future pregnancies if the maternal and foetal blood are mixed during the trauma of birth. In this case, the mother will produce antibodies to antigen D.  Thus, during subsequent pregnancies, the RhD- mother, previously sensitised to antigen D, could react to a RhD+ foetus by producing more anti-D antibodies and these could be passed on to the foetus via the placenta*, causing haemolytic disease of the newborn. This is a disease where breakdown of the baby’s blood cells releases haemoglobin that may result in jaundice and possible brain damage. It is therefore normal practice to inject RhD- mothers immediately after birth with anti-D antibodies and these destroy any RhD+ foetal cells before the mother produces any of her own anti-D antibodies.

 

*in the ABO system, it is quite possible for a mother to carry a baby with an incompatible blood group because there is no mixing of the blood. Anti-A and anti-B antibodies will not cross the placenta as they are large IgM type antibodies. The rhesus anti-D antibodies are smaller and therefore can gain access to foetal circulation via the placenta.

 

Importance of Blood Groups

Transfusion of blood    The main importance of blood groups lies in ensuring compatible blood transfusion. This is discussed in detail below.

Medicolegal importance        When the blood types of the parents are known, it can be stated with certainty which of the blood groups cannot be present in the offspring. This knowledge is useful in exclusion of pre­tenders in cases of disputed paternity. It is however not possible to state conclusively that a certain person is indeed the parent of a child. The predictive value is increased if several blood group systems are considered. With the use of DNA fingerprinting, the exclusion rate for paternity rises close to 100%.

Association with diseases      The incidence of certain diseases is related to the blood group. For example duodenal ulcers are twice as common in group O nonsecretors than in group A or B secretors, and tumors of the salivary glands, stomach, and pancreas are more common in group A than in group O individuals.

Blood Transfusion

Autologous blood transfusion

Autotransfusion is completely free from the risks of transfusion reactions and transfusion-transmitted dis­eases. In this, the patient’s own blood is withdrawn in advance of elective surgery and then transfused back if needed during the surgery. Up to 1.5 L of blood is with­drawn over a 3-week period while the person receives iron supplements.

Blood grouping and cross-matching

Autotransfusion is only occasionally possible. Mostly, the patient needs a blood donor. Preferably, the blood groups of the donor and the recipient should be the same. In emergency situations, there may be no time for finding out the blood group of the patient and even when known, the blood of the same group may not be available. In such situations, blood group O– may be transfused indiscriminately to all patients in dire need of transfusion. This is because O– group blood has no agglutinogens and the chances of fatal reactions oc­curring following a mismatched transfusion (O– group blood donated to persons with A+, B+ or AB+ blood groups) are the lowest. Persons with O– blood group are therefore called universal donors. In the same way, persons with AB blood group are universal recipients. In emergency situations, they can be transfused with any of the ABO blood groups. This is because AB group blood has neither α nor β agglutinins.

The idea of a universal donor is not always safe. Nor­mally, the α and β agglutinins present in the transfused O group blood are greatly diluted by the recipient’s plasma and therefore, are unable to lyse the recipient’s erythrocytes. However, the O group donor may have very high titers of α and β agglutinins, and these may cause hemolysis of the recipient’s erythrocytes. Such O group donors are called dangerous universal donors.

Transfusion of blood of the same group does not guarantee a reaction-free transfusion. The donor and recipient’s blood may be ABO and Rhesus compatible but the donor might have P agglutinogens and the re­cipient might have anti-P agglutinins. Since there are innumerable minor and familial blood groups that are never ascertained, the donor and recipient’s blood have to be directly tested (cross-matched) against each other. Cross matching may be major or minor. Major cross-matching involves testing the donor’s erythro­cytes against the recipient’s serum. Minor cross-match­ing involves testing the recipient’s erythrocytes against the donor’s serum.

Blood grouping, however, does help iarrowing down the search for compatible blood. For example, if the recipient is B+, the blood bank techniciaeeds to test only a few samples of B+ blood for the perfect compatibility. Without blood grouping, a much larger number of random blood samples would have to be cross-matched.

 

 

1. Common characteristic of hemostasis system (Hemostasis is very important for our life, because if we are live our hemostatic system is very strong. They are includes in a case of trauma, cutting the vessels etc.)

a) Determine the notion “system of hemostasis” (Hemostasis is the physiologic system, which supports the blood in the fluid condition and prevent bloodless. Hemostasis system vital necessary and functionally connect with the cardiovascular, breathing, endocrine and other systems.)

b) Functional-structure components of hemostasis system (The components of hemostasis are wall of the vessels, blood cells – platelets, erythrocytes, leucocytes, enzymes and nonenzymes components of plasma – clotting and anticlotting substances, fibrinolysis components of hemostasis.)

c) Mechanisms of hemostasis (There are 2 kinds of hemostasis. They are vessel–platelets (primary) and coagulative (secondary) hemostasis. Primary hemostasis activity begin the first after the destroyed of vessels. Secondary hemostasis add after that in case the primary hemostasis do not stopped the bloodless.)

2. Vessel–platelets hemostasis (or primary hemostasis include in clotting first of all after the destroyed the safe of vessel wall.)

PLATELETS OR THROMBOCYTES

In wet preparations of the blood the platelets appear as small (average diameter = 1.5 μm), colorless, moderately refractile bodies that are discoid or elliptical in shape. In stained smears they are round, oval or rod shaped. Platelets do not have nucleus. Their cytoplasm is hyaline, bright blue having azurophilic granules. Young platelets are larger than old ones.

Platelets have small mitochondria, glycogen granules, lipid inclusions and ferritin granules (siderosomes). On the basis of dry weight platelets have 60 % protein, 15 % lipids (phospholipids, arachidonic acid) and 8 % carbohydrate (mainly glycogen, heteropolysaccharides, complexes containing sialic acid). Their major energy source is derived from glucose by glycolysis. Their ATP content is 150 times more than that of RBCs. Their surface has glycoproteins in which there are receptors for thrombin and ADP.

Platelet proteins – About 20 proteins including thrombosthenin, albumin, pre-albumin, IgG, IgM, plasminogen and fibrinogen have been demonstrated in the platelets. Thrombosthenin is identical to actomyosin of muscle; it can be dissociated into two segments, A (actin) and M (myosin). Platelets also have ATP-ase activity including Mg²⁺-Ca²⁺ dependent type. The contraction of thrombosthenin underlines the phenomenon of clot retraction and may also be involved in platelet aggregation.

Platelet granules – At least 3 types of granules are present in the platelets. Their names along with their contents are given below:

i) Lysosomes; these have endoglycosidase and a heparin-cleaving enzyme.

ii)Dense granules; these have Ca²⁺; serotonin and ADP.

iii) Alpha granules; these have Von Willebrand factor, fibronectin, fibrospondin and a heparin-neutralizing factor (platelet factor 4).

The platelets have been shown to release seven factors that help in blood clotting.

Platelet factor 1 – It has been found to be the same as factor V.

Platelet factor 2 – It is the thromboplastic substance.

Platelet factor 3 – It is a phospholipoprotein, which behaves as thromboplastin.

Platelet factor 4 – It has heparieutralizing properties.

Platelet factor 5 – It acts as fibrinogen.

Platelet factor 6 –It acts as anti-fibrinolysin.

Platelet factor 7- It is the platelet co-thromboplaslin.

In addition, the platelets also release CPFA and CICA whose roles as activators of factor XII and XI respectively have been mentioned earlier. Platelets also provide surface for the activation of prothrombin to thrombin.

 

STAGES IN PLATELET DEVELOPMENT

1. Megakaryoblast – It is the first cell which can be morphologically characterized and identified to form platelets. It arises, as other blood cells, from the non-specific pluripotent stem cell (CPU). It is 15 to 50 μm in diameter and contains a large oval or kidney-shaped nucleus with several nucleoli. The cytoplasm is scanty and intensely basophilic and has no granules. Mitosis may be seen.

2. Pro-megakaryocyte – It is 20 to 80 μm in diameter. The nucleus is oval or irregular in shape; cytoplasm is more abundant and contains fine bluish granules.

3. Megakaryocyte – This cell is so called because it possesses up to 64 N chromosomes instead of the normal 2 N chromosomes (46) of ordinary somatic cell. This poly-ploidy is brought about by a sequence of events termed as endoreduplication in which nuclear material replicates without cytoplasmic division. It has a diameter of 35 to 160 μm and shows two distinct stages. In the first in which the cell is termed as megakaryocyte without granular platelets, the nucleus is either indented or has multiple lobulations. The cytoplasm is finely and diffusely granular. In the second stage, the cell cytoplasm becomes still more increased in amount and the cell is termed as megakaryocyte with granular platelets or meta-megakaryocyte. The platelets differentiate at the periphery of the cell and when the cell dies, these break off from its cytoplasm to enter the blood stream.

In a different nomenclature the megakaryoblast, promegakaryocyte and the mature granular megakaryocyte are called stage I, II and III megakaryocyte respectively.

 

The megakaryocyte occurs in the bone marrow very close to the sinusoidal membrane. It is changed to platelets by two methods: (i) it sends pseudopodia of cytoplasm into the lumen of sinuses through apertures in the sinus membrane. Later these separate from the parent cell and arc swept away by the blood stream as platelets, (ii) The megakaryocyte cytoplasm splits outside the lumen of sinuses, giving rise to 2,000 to 4,000 discrete units, the platelets, which enter the sinuses. The nucleus is left behind and degenerates.

The life span of the platelets is about 10 days in man. The spleen stores them as well as mainly sequestrates the damaged or effete (worn-out by age) platelets. Normally 80 % of the total platelets are in circulation and the remaining 20 % are in the spleen. If the spleen becomes enlarged, then it can store more platelets and this ratio may even be reversed. This may obviously result in a decreased blood platelet count, i.e. thrombocytopenia.

Factors affecting Blood Platelet Count – The average number of platelets in the blood is 250,000 (range being 180,000 to 320,000) per cu mm. Following factors affect the blood platelet count:

1. Age – The count tends to be lower in the newborn especially in prematurely born babies.

2. Menstrual cycle – There is a slight increase on the day of ovulation followed by a progressive fall during the 14 days prior to menstruation. A rapid rise occurs after the start of menses.

3. Pregnancy – There is a slight progressive fall during pregnancy which may fall further during the first stage of labor and on the first and second day after child-birth.

4. Injury – This increases blood platelet count.

5. Adrenaline – It increases platelet count by mobilizing platelets from the spleen, which normally stores about 20 % of the total platelets.

6. Hypoxia – This markedly increases platelet count.

7. Smoking – It tends to shorten platelet survival and produces hyper-aggregability of the platelets.

8. Nutritional deficiencies – Platelet count is low in deficiencies of vitamin B₁₂, folic acid and iron.

9. Thrombopoietin – This substance has been isolated from the blood of a thrombocytopcnic patient. The transfusion of this patient’s blood into normal persons resulted in an increase in blood platelet count, i.e. thrombocytosis. It has been shown that if large number of platelets is intravenously administered to a person, then there is a decrease in his own platelet production. On the other hand, removal of platelets from the blood stimulates platelet production. These studies show that some type of regulatory system docs control their production. Erythropoietin, which stimulates erythropoiesis is also believed to produce thrombocytosis.

a) Activation of platelets (To do their function platelets must to activate. In the case of activation the platelets form psevdopodias, change the form. There are 2 groups of activators – the first from platelets and second from another cells, plasma. The outside platelets factors, which are produce in plasma, other cell besides platelets – Villibrandt factor, ADP, epinephrine and norepinephrine. The platelets factors, which are produce by platelets serotonin, ADP, thromboxan A₂.)

b) Properties and function of platelets (Quantity of platelets is 180-320 G/L. Diameter of platelets is 1-4 micrometers, thickness – 0,5-0,75 micrometers. They are the little peace of megacariocytes cytoplasm (from one megacariocytes may develop few hundred of platelets). Platelets circulated in blood from 5 to 11 days and than destroyed in liver, lungs, spleen by the cells of macrophagal system. Functions of platelets are: 1. hemostatic function – platelets produce substances, which are secures the hemostasis.

 

Function of platelets are:

1. hemostatic function – platelets produce substances, which are secure the hemostasis. Its produce 12 platelets factors

1 – proaccelerin,

2- factor, which are increase the speed of development the fibronogen in fibrin,

3 – platelets thromboplastin,

4 – antiheparinic factor,

5 – factor which promote aggregation of platelets,

6 – thrompostenin,

7 – antifibrinolizin,

8 – serotonin,

9 – fibrinstabilising factor,

10 – factor which activate profibrinolisin,

11 – inhibitir of thromboplastin,

12 – antilighting factor.

 

Other classiffication of platelets factors. The platelets have been shown to release seven factors that help in blood clotting.

Platelet factor 1 – It has been found to be the same as factor V.

Platelet factor 2 – It is the thromboplastic substance.

Platelet factor 3 – It is a phospholipoprotein, which behaves as thromboplastin.

Platelet factor 4 – It has heparieutralizing properties.

Platelet factor 5 – It acts as fibrinogen.

Platelet factor 6 –It acts as anti-fibrinolysin.

Platelet factor 7- It is the platelet co-thromboplaslin.

2. Angiotrophic function – provide trophic of endotheliocytes of vessel wall, support structure and functions of microvessels. These function is realize by adgesion of platelets to endotheliocytes and injection the enzymes into the endotheliocytes. For one day near 35 G/L platelets do this function.

3. Transport function – transfer the enzymes, ADP, serotonin and other.

4. Phagocytosis function – the contain of platelets help to kill viruses and antigens bodies.

5. Regeneratory function – platelets have the growth factor, which help to grow the endothelial and muscles cells which are present in the vessel wall.

Its produce 12 platelets factors (1 – proaccelerin, 2- factor, which are increase the speed of development the fibronogen in fibrin, 3 – platelets thromboplastin, 4 – antiheparinic factor, 5 – factor which promote aggregation of platelets, 6 – thrombostenin, 7 – antifibrinolizin, 8 – serotonin, 9 – fibrinstabilising factor, 10 – factor which activate profibrinolisin, 11 – inhibitir of thromboplastin, 12 – antilighting factor).

 

Other auther determined such functions of Platelets

1. Role in Hemostasis -The platelets are responsible for the primary hemostasis which is brought about by the formation of the primary hemostatic plug which can effectively stop bleeding from capillaries; small arterioles and venules. Effective primary hemostasis requires three critical events, platelet adhesion, platelet activation and secretion and platelet aggregation.

(A) Platelet adhesion – This means attachment of platelets to non-platelet surfaces, e.g. to collagen and elastic fibers of blood vessels. This process is facilitated by Von-Willebrand factor. This factor becomes attached on one side to the collagen fibrils in the vessel wall, and on the other side to receptors over the platelet surface.

(B) Platelet activation and secretion – This occurs in many steps which are given below:

(a) Binding of platelet agonists, i.e. adrenaline, collagen and thrombin the platelet surface, (b) Activation of phospholipases A₂ and C. (c) Released arachidonic acid from the membrane phospholipid. (d) Conversion of arachidonic acid to thromboxane A₂, (c) Thromboxane-A₂ activates phospholipase-C which liberates still more arachidonic acid from the membrane phospholipid (f) Some inositol triphosphate is also liberated from phospholipids. This stimulate the movement of Ca²⁺ into the platelet cylosol and the phosphorylalion of myosin light chains. The latter interact with actin to facilitate granule movement and platele shape change, (g) Another product of membran phospholipid is diacylglycerol which brings about secretion of granules. The contents of the granules which are poured into the plasma arc heparinase, Ca²⁺, adrenaline, kinins, fibrinogcn. factor Va, AMP, thromboxane A₂, Von-Willebrand factor, fibronectin, thrombospondin and several other platelet factors including a heparieutralizing factor-4.

(C) Platelet aggregation or cohesion – The ADP released from the platelets modifies the platelet surface in such a manner that a fibrinogen molecule interacts with specific surface glycoprotein receptors on two adjacent platelets and links the two platelets by a glue-like effect. Aggregation of a large number of platelets results in the formation of small platelet plugs called primary hemostatic plugs or white thrombi; this lakes place within seconds alter injury and the process is called primary hemostasis. It is specially effective in preventing bleeding from small blood vessels such as capillaries, arterioles and venules. It should be noted that in addition to the formation of the primary hemostatic plugs, the platelets also contribute several factors which help blood clotting. However, the platelets required for clotting process are relatively much less and usually mild to moderate thrombocytopenia does not cause blood clotting disorders.

Aspirin and other non-steroid anti-inflammatory drugs inhibit the enzyme cyclo-oxygenase thus inhibiting platelet aggregation. These drugs are being used in the treatment and prevention of thrombolic disorders.

Three more factors have been found to be released during platelet release reaction. These are (i) contact product forming activity (CPFA) which contributes to activation of blood clotting factor XII; (ii) collagen induced coagulant activity (CICA) which helps in the activation of factor XI; (iii) Platelet derived growth factor; it stimulates the migration and growth of fibroblasts and smooth muscle cells within the vessel wall which is an important part of the repair process.

2. Other Functions – (i) Platelets are necessary for the maintenance of the vascular integrity. They seem to donate to the endothelial cells some material essential for their integrity. The platelets may themselves enter the endothelial cells to strengthen them. Platelets also seem to repair small or imperceptible vascular injuries by adhering to the basement membrane. Platelets have been shown to provide glycoprotein which helps in their adhesion to the sub-endothelial collagen.

(ii) Platelets transport all 5-hydroxytryptamine (serotonin) of blood and also carry K⁺.

(iii) They show slight phagocytic activity to carbon particles, immune complexes and virus particles.

(iv) Contraction of thrombosthenin causes retraction of the clot.

3. Role of Arachidonic Acid Derivatives in Platelet Functions – mammalian tissues the 20-C poly-unsaturated fatty acid, arachidonic acid, converted to cyclic endoperoxide namely PGG₂. This reaction is catalyzed t the enzyme cyclo-oxygcnase. PGG₂ is converted to PGH₂ by the enzyme endoperoxidase. Cyclo-oxygcnase and endoperoxidase are collectively called prostaglandin endoperoxide synthase. The fate of PGH₂ is given below.

(i) In the platelets the enzyme thromboxane synlhasc converts PGH₂ J thromboxane A₂ which is later converted to thromboxane B₂; the luuq however, is relatively inert.

(ii) In the arterial wall the enzyme prostacyclin synthase converts PGH₂ to PGI₂ which is also called prostacyclin.

These two compounds, i.e. thromboxane A₂ and prostacyclin possess opposite biological properties. Thromboxane A₂ is a powerful vasoconstrictor and promotes aggregation of platelets. As opposed to the actions of thromboxane A₂, prostacyclin is a vasodilator and prevents aggregation of platelets. In addition to preventing platelet aggregation, it also has disaggregatory action, i.e. it causes dispersion of any already present platelet aggregates c platelet thrombi. These two substances act through varying the activity of the enzyme adenylate cyclase. For example, prostacyclin activates this enzyme which catalyses the production of 3′, 5′, cyclic AMP (c-AMP); this in turn activates enzymatic process that leads to the binding of Ca²⁺ to a Ca-binding protein (calmodulin) in the platelets. This leads to a decreased availability of Ca²⁺ due to which thrombosthenin caot function properly. This results in a decreased adhesion and aggregation of platelets. On the other hand, thromboxane A₂ decreases the activity of the enzyme adenylate cyclase thereby increasing thrombosthenin activity; this leads to more tendency of platelets for undergoing adhesion and aggregation.

4. Role of platelets in atherosclerosis – The essence of atherosclerosis is the formation of atheromalic plaques. Platelets arc believed to contribute to this process. This may be brought about by the release of lysosomal enzymes and other toxic factors from the platelets which injure the vascular endothelium. Platelets also release a growth factor that stimulates proliferation of fibroblasts and migration of monocytes to the injured area. Thromboxane A₂ favors while prostacyclin inhibits the development of atherosclerosis. Prostacyclin which can be called a hormone is being used in the treatment of peripheral arteriosclerosis with good results. More recent work has shown that PGI₃ and thromboxane A₃, which possess one more unsaturated bond than PGI₂ and thromboxane A₂, are also produced in the body. PCI₃ is as potent anti-aggregator of platelets as PGI₂ but thromboxane A₃ is a weaker pro-aggregator than thromboxane A₂. Fish oil is rich in the precursor fatty acid (5, 8, 11, 14, 17-eicosa pentaenoic acid) and its consumption provides both prostacyclin A₃ and thromboxane A₃. As the latter has weak pro-aggregation effect on platelets while PGI₃ has a potent anti-aggregation effect on platelets, the simultaneous presence of both favors anti-aggregation activity of platelets. This has a preventive effect on thrombosis. Eskimos who cat a lot of fish oil have a relatively low incidence of coronary thrombosis.

c) Stages of vessel–platelets hemostasis (1. Shorting spasm of the vessels – vascular spasm duration to 1 minute is caused by catecholamins and other enzymes. Diameter of vessels decrease on ½-⅓. Mechanism of it development determine by secretion of serotonin and thromboxan A₂ from platelets and epinephrine from ending of sympathetic nerves. 2. Adgesion of platelets – activation of platelets and stick it to the place of defect in vessel wall. 3. Reverse aggregation of platelets – the thromb which are formed may make way for plasma. 4. Unreverse aggregation of platelets – the thromb which are formed caot may make way for plasma. 5. Retraction of platelets plug – decrease the size of plug, pack down the plug.)

d) Investigation of vessel–platelets hemostasis (1. Calculation of the platelets quantity 180-320 G/L. 2. Determination of duration of capillary bleeding after Duke’s method – to 3 minute iorm. 3. Sample of fragility of capillars – to 10 petechias iorm in a round with diameter 5 centimetres.)

COAGULATION OR CLOTTING OF THE BLOOD

Blood has two remarkable properties; it remains fluid while in blood vessels and clots when it is shed. Both these properties are essential for normal life. The blood contains substances or factors, which favor coagulation (pro-coagulants); it also has substances, which are anti-coagulants. An optimum balance of these two opposing factors is essential for a normal life. The clotting, in essence, is the formation of the insoluble protein fibrin from the soluble plasma protein fibrinogen.

A large number of substances take part in producing fibrin from fibrinogen in the coagulation of blood. The coagulation process actually is the property of plasma though it is commonly termed as clotting of blood. Although a complete understanding of the mode of action of the procoagulants is still not possible, but it can be said that clotting is produced by a complex series of reactions. Once initiated, the whole process proceeds like a chain reaction until clotting is complete. Three methods, which have been much employed for understanding the clotting mechanism are given below.

1. Appropriate techniques by which the clotting process can be stopped at any required stage followed by its re-start.

2. Studies on patients suffering from hemorrhagic diseases.

3. Experimental studies in animals; hemophilia occurs in dogs which have been used for research in this disease.

Blood Clotting Factors – The various factors, which are known to take part in the clotting process in various theories of blood coagulation are given below. These factors have been assigned numbers, which arc written in Roman pattern.

I. Fibrinogen

II. Prothrombin (Thrombin is factor II-a)

III. Thromboplaslin. This is the name given to a substance capable of converting prolhrombin to thrombin. It is present in tissues in an active form, the tissue thromboplastin, which is also called the tissue pro-coagulant material.

IV. Calcium ions.

V. Labile factor, Pro-accelerin, Accelerator or Ac globulin.

VI. It has been found to be the same as factor V; it is now obsolete.

VII. Stable factor, Pro-convertin, Auto-prothrombin-I.

VIII. Anti-hemophilic globulin (AHIG, Platelet cofactor-I. Anti-hemophilic factor A (AHF-A). This is the original compound called factor VIII. However, factor VIII has been found to have three subtypes. The original factor VIII (AHF-A) is now called factor VIII-C, C signifying coagulant action. The other two subtypes are factor VIII V.W. (also called Von-Willebrand protein) and factor VIII R.Ag (protein precipitated by specific rabbit anliserum).

IX. Christmas factor, Plasma thromboplastin component (PTC), Platelet co-factor-II, Auto-prolhrombin-II, Anti-hemophilic factor B.

X. Stuart-Prower factor.

XI. Plasma thromboplastin antecedent (PTA), Anti-hemophilic factor-C, Rosenthal factor.

XII. Hageman’s factor, Contact factor, Glass factor.

XIII. Fibrin stabilizing factor, Laki-Lorand factor, Transglutaminase, Pre-fibrinoligase.

 

In addition, the following factors are also associated with blood clotting process.

i) Von-Willebrand factor or the platelet adhesion factor. It is needed for platelet adhesion as well as for activity of factor VIII-C; it is called factor VIII V.W.

ii) Fitzgerald factor; it is the same as high mol. wt. kininogcn.

iii) Fletcher factor; it is pre-kallikrein.

1. Analysis of coagulative hemostasis mechanisms

a) Characteristics of clotting factors (There are 12 clotting factors: I – fibrinogen; II – prothrombine; III – thromboplastin of tissue; IV – ions of calcium; V – proaccelerin; VII – proconvertin; VIII – antihemophylic factor A; IX – Christmas factor or antihemofilic factor B; X – Stuart-Prower factor or prothrombinase; XI – plasma thromboplastin antecedent; XII – Hageman factor; XIII – fibrin stabilizing factor. Some of them are enzymes – II, VII, IX, X, XI, XII,XIII; other  are not – I, III, IV, V, VIII. The vitamin K is necessary for the functional activity of II, VII, IX, X factors.)

b) External mechanism of the first stage (3 factors from the injure tissues go to plasma and interactions with VII factor, the last is activated. VII active factor and IV factors form the complex 1a: III + VII active + IV, which is activated X factor.)

c) Inner mechanism of the first stage (Factor 3 of platelets – platelets thromboplastine – influence on XII factor. Active XII factor + XI is complex 1. Active XI factor activated IX factor. Active IX factor + VIII factor + IV factor is complex 2. Complex 1a and 2 are activate X factor. Factor X active + V + IV formed complex 3 or thrombinasa complex.)

d) Course of the second and third stages (The second stage – formation of thrombin from prothrombin. The third stage is formation of fibrin from fibrinogen. The last stage has 3 period; formation of fibrin-monomers; formation of fibrin S (solubilis); formation of fibrin I (insolubilis). Calcium is necessary for all stages.)

e) Regulation of the clotting mechanisms (Increase of clotting names hypercoagulation, decrease – hypocoagulation. Hypercoagulation may be in a stress cases. It depends on epinephrine, which concentration increased in the cases of stress. Epinephrine increase from the vessels walls factors from which produced prothrombinasa. In cases of big concentration epinephrine should activate XII factor in a bloodstream. It divides fats and fat acids, which have prothrombinase activity. After the hypercoagulation stage may be secondary hypocoagulation.)

 

 

 

Theories of Blood Coagulation

I. Classical theory of Morowitz (1905-1906) – Blood clotting was considered to take place in two stages.

(i) In the first stage prolhrombin is converted to thrombin by the enzyme prothrombinase, Ca²⁺ being necessary for this reaction.

(ii) In the second stage the thrombin acts as an enzyme on fibrinogen and converts it to fibrin.

II. Cascade or waterfall theory – For many decades, Morowitz’s theory was accepted. But great developments in this field resulted in several new theories, one of which is called cascade or waterfall theory because it involves a cascade of events; it is described below.

There are two systems of clotting, intrinsic and extrinsic, which converge upon what is called the final common pathway.

1. Intrinsic or the blood system – This system is called so, because all factors taking part in the process are derived from the blood itself and it can take place in pure blood (blood not contaminated with tissue juice) kept in a test tube. It is also called contact system because the process starts when blood comes in contact with a foreign surface, e.g. vascular sub-endothelial collagen or even glass. This process takes place in the following six stages. In the first five of these stages limited proteolysis converts an inactive factor to its active form. Each of these steps is regulated by plasma and cellular co-factors and Ca²⁺. The inactive and active blood clotting factors are distinguished by writing and a respectively after the factor.

Stage No. 1. Three plasma proteins, i.e. Hageman factor (XII), high mol. wt. kininogen and pre-kallikrein form a complex with vascular subendolhelial collagen. Factor XH-i becomes activated to Xll-a, which acceleates the conversion of pre-kallikrein to kallikrein which then accelerates the conversion of still more XII-i to XII-a.

Satge No. 2. Factor XII-a converts factor XI-i to XI-a.

Stage No. 3. Factor XI-a converts factor IX-i to IX-a.

Stage No. 4. Factor IX-a in the presence of factor VIII C, Ca²⁺ a platelet membrane lipoprotein (platelet factor 3) converts X-i to X-a.

Stage No. 5. Several factors take part in the conversion of prothrombin to thrombin. These include factor X-a, factor V-a, Ca²⁺ and phospholipids. Although the conversion of prothrombin to thrombin can take place on a phospholipid-rich surface, but it is accelerated several thousand-fold on the surface of activated platelets.

Stage No. 6. Conversion of fibrinogen to fibrin is brought about thrombin by the following mechanism. Fibrinogen is a symmetrical dimer; each half of its molecule has the following structure:

i) Alpha polypeptide joined to a short A-fibrinopeptide.

ii) Beta polypeptide joined to a short B-fibrinopeplide.

iii) Gamma polypeptide.

Fibrinogen can thus be represented by the structure, [Alpha(A), beta(B), gamma]₂. Thrombin catalyzes the breakdown of fibrinogen in such a way that a part of the molecule separates leaving behind a fibrin monomer.

[alpha(A), beta(B), gamma]₂ → [alpha, beta, gamma]₂ (Fibrin monomer) + 2[fibrinopeptide A + B]

However, the removal of fibrinopeptide B is not essential for coagulation. The fibrin monomers undergo polymerization giving rise to fibrin polymers; this process involves formation of hydrogen bonds between fibrin monomers. These fibrin polymers are unstable and the polymerization is readily reversed by inhibitors of H bond formation such as urea. The unstable fibrin polymers are then acted upon by factor XIII, which actually is an enzyme. Factor XIII is initially inactive but is activated by thrombin. It brings about the production of cross linkages between adjacent fibrin polymers. This process involves covalent bond formation between epsilon amino group of lysine and the gamma amide group of glutamine; NH₃ is evolved in this reaction. A clot which is much more stable and is insoluble in urea solution is thus produced. Even this fibrin clot is quite soft, but after some time it undergoes retraction during which serum oozes out of it. The platelets are of primary importance in this process of clot retraction. The result is a firm clot that can effectively seal a wounded vessel.

2. The extrinsic or the tissue system – This is called so because it needs the presence of tissue juice that contains tissue thromboplastin which is not present in blood. The tissue thromboplastin in the presence of factor VII and Ca²⁺ activates factor X-i to X-a. Subsequent reactions are the same as described under the intrinsic system and, being common to both the intrinsic and extrinsic systems, are designated as the final common pathway. Because the extrinsic system involves fewer steps than the intrinsic system, therefore it proceeds faster than the latter. For this reason, while the intrinsic system takes 2 to 6 minutes for clotting to take place, the extrinsic system takes as little as 15 seconds to do that.

III. Seeger’s hypothesis – This concept basically differs from the cascade theory in that prothrombin and factors VII, IX and X are considered to occur in a single molecular system and not separate from each other. This common molecule is believed to release all these clotting factors during clotting process. A common characteristic of all these clotting factors is that all of them require the presence of vitamin K for their biosynthesis. Factors VII, IX and X are designated by Seeger as autoprothrombin I, II and III respectively. The corresponding active forms of these factors arc called autoprothrombin A, B and C. There are serious objections to this hypothesis as various studies have shown that all these factors arc different and are quite distinct from each other.

Properties of Various Factors Participating in Blood Coagulation

Fibrinogen – It occurs in the plasma in a concentration of 0.35 gram per 100 ml, but is also present in lymph and many tissues. It has a mol. wt. of about 340,000. It is synthesized in the liver. It behaves as a globulin but is more easily precipitated, i.e. by precipitation with 25% ammonium sulfate. The normal plasma has about 15 times more fibrinogen than that required for blood clotting. Its solution is clotted by thrombin; Ca²⁺ are not needed in this reaction. Afibrinogenemia and dysfibrinogenemia are clinical conditions associated with bleeding; in the former the plasma fibrinogen is absent, while in the latter condition fibrinogen is present but is of abnormal type. Drugs namely Arvin and reptilase are used in therapeutics to control thrombosis; these convert the plasma fibrinogen to fibrin micro-clots that are removed from the circulation by fibrinolysis. In this way, hypofibrinogenemia is produced decreasing the blood clotting tendency. Arvin is obtained from the venom of the Malayan pit viper snake.

Prothrombin – It is the proenzyme, the precursor of thrombin. It contains 2 to 10 % carbohydrate in its molecule and has a mol. wt. of 69,000. Its plasma concentration is 10 to 15 mg per 100 ml.

Thromboplastin – It implies an activity which converts prothrombin to thrombin. All body tissues have this activity and therefore it is termed as tissue or intrinsic thromboplaslin. The brain, lung, placenta and testes are especially rich in it. It is a complex of phospholipids, lipoproteins and cholesterol. Tissue extracts, if injected intravenously, can cause widespread clotting of blood. However, tissue thromboplaslin is not active as such but it needs Ca²⁺ and factor VII for its activation which normally arc present in blood. Russel viper venom has a strong thromboplaslin activity and is used for slopping bleeding from superficial areas by its local application in diseases like hemophilia.

Calcium – Ca in ionic form, Ca²⁺, is essential for clotting of blood and it acts at many stages. Ca ions serve to form complexes with lipids, which take part in blood clotting. In health or disease blood has always sufficient Ca²⁺ for this purpose. In other words, a Ca²⁺ deficiency is never a cause of a prolonged clotting time in man.

Factor V – It is activated by small amount of thrombin which in turn leads to a greater formation of thrombin. But an excess of thrombin destroys it and causes its disappearance from serum. It is unstable in the citrated plasma. Its congenital deficiency is the cause of parahemophilia, a mild bleeding disorder.

Factor VII – It is stable on storage. It acts as co-thromboplastin in the working of extrinsic system of blood cloning. Its congenital deficiency has been seen very rarely. It has up to 50 % carbohydrate in its molecule.

Factor VIII-C – It is also called platelet cofactor-I and anti-hemophilic globulin. Its deficiency causes the classical hemophilia (now called hemophilia A). Hemophilia is discussed later in detail. This factor is readily inactivated in vitro.

Factor IX – It is also called Christmas factor because its deficiency was first demonstrated in a patient with the surname Christmas whose bleeding disease was named Christmas disease. This disease is also called hemophilia B.

Factor X – It is an alpha globulin present both in scrum and plasma. I deficiency is seen in both sexes equally as a congenital defect.

Factor XI – Its deficiency causes hemophilia C, which is a mild bleeding disease.

Factor XII – It is activated by surface contact and according to the cascade theory, this process initiates the series of reactions leading to blood clotting. Blood deficient in this factor docs not clot in lest tube, i.e. in vitro. If blood taken from a vein (without letting it being mixed with tissue juice) is placed in a lest tube lined with silicone, it does not clot; this is because the silicone layer is smooth and unwettable and does not permit the activation of factor XII for the same reason. Blood also clots much more slowly when placed in polythene tubes as compared to glass tubes. The deficiency of this factor is seen in persons with Hageman’s trait, but they do not generally show bleeding tendency. Its additional roles arc the activation of fibrinolytic system and the plasma kinin syslem. It is activated by contact with glass, negatively charged surfaces, collagen fibers, unbroken skin, sebum, long chain fatty acids, uric acid, fibrin, elastin and homocysteine.

Factor XIII – It is the enzyme transglutaminase, whose function has already been discussed. Persons with congenital deficiency of this factor have bleeding tendencies and poor wound healing. Their blood clots all right, but the clot, unlike the normal clot, is unstable and can be solubilized in 5 molar urea or 1 % monochloracetic acid solution.

2. Valuation of clotting

a) Coagulogram (Time of clotting by Ly-Wait – 5-10 minutes; time of plasma recalcification – 60-120 seconds; thrombotest – IV, V, VI degree; thromboplastin time – 12-15 seconds; thromboplastin index – 80-105 %; concentration of fibrinogen – 2-4 g/L; tolerancy of plasma to heparin – 6-11 minutes; heparin time – 50-60 seconds; fibrinolysis – 15-20 %.)

b) Thromboelastography (Thromboelastography is a method of regestration of plugs forming and characteristic of clot by thromboelastograph. The characteristic of clot in thromboelastogramm: a) time of bloods’ beginning clot (from the taking the blood to the first waves of amplitude to 1 mm on thromboelastogramm) – 8-12 minutes; b) time of thromb producing (time of the first waves of amplitude of 1 mm to 20 mm on thromboelastogramm) – 5-8 minutes; c) maximum amplitude (this characteristic of thromb elasticity) – 45-60 mm.)

Anticoagulative mechanisms. Fibrinolysis.

1. Common characteristic of physiological anticlotting substances (The tendency of blood to clot is balanced by a number of limiting reactions that tend to prevent clotting inside the blood vessels and to break down any clots that do form.)

a) Properties of antithrombin III (It is the most important anticoagulant in the blood. It inhibits thrombin, factors Xa, IXa, V, XIa, XII. It is basic plasma cofactor of heparin. Very faint inhibitor of plasmin and kallikrein.)

b) Importance of heparin (Blood does not ordinarily clot internally in the body. It is assumed that, unless there is access to injured surfaces, there are not enough thromboplastic substances liberated to convert prothrombin into thrombin and thus start the series of chemical reactions that results in clotting. Even so, additional safeguards are present in antiprothrombic substances such as heparin. This substance was originally found in the liver but is now thought to be produced by large basophilic cells (mast cells) in tissues of various organs. Heparin reduces the ability of the blood to clot by blocking the changeof prothrombin to thrombin. It can also be used to aid in reducing clots in cases in which internal clotting has already occurred. In either case it acts in conjunction with a plasma cofactor. Internal clotting is called thrombosis. The clot, or thrombus, can form in some blood vessel of the arm or leg and do comparatively little harm, but if it should block the blood supply to the brain or to the heart (coronary thrombosis), it can be very serious. Heparin form complex with antithrombin-III and transform it in anticoagulant with the negative action. Activate nonenzyme fibrinolysis.)

c) Role of alpha-2-macroglobulin, alpha-1-antitripsin, protein C (Alpha-2-macroglobulin is very large globulin molecule. It is a similar to antithrombin-heparin cofactor in that it combines with the proteolytic coagulation factors. Its activity is not accelerated by heparin. Its function is mainly to act as a binding agent for the coagulation factors and prevent their proteolytic action until they can be destroyed in various ways. It a faint inhibitor of thrombin, connect with plasmin. Alpha-1-antitripsin inhibits thrombin activity, IXa, XIa, XIIa factors, plasmin and kallilrein. Protein C inhibits VIIIa, Va factors. It activity depend of thrombin and vitamin K concentration.)

d) Functionation of secondary anticlotting substances (Primary anticoagulants are produce and present all time in plasma and secondary anticoagulants form in a case of blood clotting. They are antithrombin-I or fibrin and products of fibrinolysis or products of fibrinogen degradation. Fibrin is sorbs and inactivates thrombin and Xa factor. Products of fibrinolysis inactivate ending stage of clotting, IXa factor, platelets’ agregation.)

2. Fibrinolytic system (Fibrinolysis is begining with the retraction. The plasma proteins contain a globulin called plasminogen or profibrinolysin, which activated into plasmin or fibrinolysin.)

a) Stages of fibrinolysis

 

active                       prekallikrein

factor XII                activator                          Kinins

 

               Proactivator                 Activator

 

                              Plasminogen                  Plasmin

 

                                 Fibronogen                                 Fibrinogen

         Thrombin                                                           degradation

                                    Fibrin                                       products

 

b) Mechanisms of fibrinolysis activation (There are 2 mechanisms of fibrinolysis activation: external and internal. Main internal mechanism put in action by XIIa factor. External mechanism stimulated by protein activators of plasminogen, which are produce by vessel wall.)

c) Regulation of fibrinolysis (All processes are direct on increase the clotting mechanism, for example, epinephrine, which are increase in the case of stress. It promote the forming of prothrombinase, activating of XII factor, increasing of fats and fats acids. But after the clotting send up the anticlotting mechanism – hypocoagulation. Iorm it has independent regulation. The lysis of blood clots allows slow clearing of extraneous blood in the tissues and sometimes allows reopening of clotted vessels. Reopening of large vessels occurs only rarely. But an important function of the fibrinolysin system is to remove very minute clots from the millions of tiny peripheral vessels that eventually would all become occluded were there no way to cleanse them.)

d) Notion about nonenzymes fibrinolysis (It may be steroid hormons with anabolic function, which are increase producing of fibrinolysis activators by endothelium. Leucocytes ensure function of independent mechanism of fibrinolysis. It limited the size of thromb. Erythrocytes ensure function of independent mechanism of fibrinolysis too.)

3. Functionating of anticlotting mechanisms

a) Role of vessels endothelium in support blood in the fluid condition (1. Smooth surface of vessels endothelium. 2. Negative charge of endotheliocytes and blood cells and that’s why they are push away. 3. Present on the vessels wall thin layer of fibrin which adsorb clotting factors, especially thrombin. 4. Constant presence in blood anticlotting factors in a small doses. 5. Producing by endothelium prostaciclins, which are powerful inhibitors of platelets aggregation. 6. Ability of endothelium to produce and fix antithrombin-III.)

THE FIBRINOLYTIC SYSTEM

A proteolytic enzyme, fibrinolysin or plasmin, acts on fibrinogen and fibrin causing their breakdown or dissolution by converting them into smaller peptides which are soluble; these peptides are called fibrin/fibrinogen degradative products (FDP) as well as fibrin/fibrinogen split products (PSP). The FDP so produced have several important actions, which oppose hemostasis; in other words they promote bleeding.

1. They inhibit binding of fibrinogen to platelets and inhibit aggregation of platelets.

2. They inhibit thrombin formation and also destroy any thrombin formed.

3. They prevent fibrin polymerization.

Formation of plasmin – Normally plasmin occurs in blood plasma as its inactive precursor called pro-fibrinolysin or plasminogen. The conversion of plasminogen to plasmin involves cleavage of a single arginine-valinc bond and is catalyzed by an activator which itself occurs in an inactive form namely pro-activator. The pro-activator is changed to activator by enzymes called pre-kallikrein activators, which are derived from breakdown of active factor XII; in other words, the activators of pre-kallikrein are the fragments of active factor XII.

Factors increasing the formation of plasmin – These are discussed below.

1. Fibrinolysokinase – It is present in plasma, many tissues and in many secretions, e.g. saliva, milk and tears.

2. Bacterial enzymes – These include staphylokinase and streptokinase which are produced by staphylococci and streptococci respectively. Streptokinase is used by intravascular infusion for the clearance of thrombo-embolism.

3. Urokinase – It is present in urine and is being used in therapeutics to accelerate fibrinolysis. It is formed in the kidney; its commercial source is the fetal kidney tissue culture. The urinary plasmin produced under its influence is believed to have a role in keeping the renal tubules patent by preventing the deposition of fibrin within them. Fibrin may be derived from the fibrinogen present in trace amount in the renal tubular fluid. In the same way, plasmin activity present in milk, tears and semen serves to keep the corresponding duct systems patent.

4. Cytokinase – It is present in tissue cells, WBCs and platelets.

5. Hormones – Growth hormone and thyroid stimulating hormone (TSH).

6. Miscellaneous – Exercise, adrenaline, hypoxia, histamine, bacterial pyrogens, ischemia, shock, tissue damage and chloroform.

Factors inhibiting plasmin formation – The plasmin activation is inhibited by the following factors.

1. Epsilon aminocaproic acid (Trasylol and tranexamic acid) – It is used in therapeutics to inhibit plasmin system.

2. ACTHl – (Adrcnocorlicotropic hormone).

Control of plasmin activity – A trace of plasmin activity is present even iormal plasma. However, this plasmin activity is kept under check by an opposing factor called anti-plasmin which is 10 times more active than plasmin activity. The anti-plasmin activity is present in alpha-1 and alpha-2 globulins of plasma. When excessive amount of plasmin is formed, it brings about the following two types of effects:

(i) The plasmin gets bound to fibrin clots and breaks them to small fragments which arc cleared by macrophages. This is the physiological action.

(ii) A part of plasmin remains free in the plasma and enters circulation. However, its action is rapidly neutralised by the anti-plasmin. But if plasmin level of the plasma is too high, then it causes digestion of fibrinogen and also of factors V, VIII and XII. This is the pathological action. If this action is very marked, hypofibrinogenemia and deficiencies of factors V, VIII and XII result and lead to hemorrhage.

FACTORS PREVENTING BLOOD CLOTTING WITHIN BLOOD VESSELS

It has already been pointed out that blood must remain fluid within the blood vessels if life has to be normal. The factors, which help in preventing intravascular clotting (thrombosis), are given below.

1. Vascular endothelium – The normal endothelium of blood vessels, has been described to act as a non-wettable surface and docs not allow the activation of factor XII. Similarly the platelets also cannot adhere to normal endothelium. Both these factors prevent the initial steps in clotting. The endothelial cells arc also rich in fibrinolysin activators. In atherosclerosis the normal intima is replaced by a diseased one, and clotting is favored resulting in thrombosis. The endothelium can also be damaged by bacteria, injuries such i as fractures, pressure on veins, etc.

2. Chemical substances in blood – There arc certain substances in the blood, which normally antagonize any tendency towards clotting. These include the following:

(i) Heparin – It is produced by the mast cells of the connective tissue which occur especially in the lung and liver. Heparin is an acidic mucopolysaccharide composed of alternating sulfated D-glucosamine and D-glucuronic acid units. Up to 40 % of its molecular mass is H₂SO₄. This makes heparin the strongest organic acid in the body. Heparin is formed in small amounts continuously, but its release from the mast cells is greatly increased in conditions like peptone shock and anaphylactic shock. The mechanism of heparin action is manifold. It antagonizes all the stages of blood clotting, e.g. formation of active factor X, thrombin and fibrin. It also inhibits the agglutination of platelets thus decreasing the release of platelet factors. In combination with a proteiamely anti-thrombin, it inactivates thrombin. This is probably the most important action of heparin.

Heparin is water soluble. It has mol. wt. of about 17,000. It is much in therapeutics. Excessive doses of heparin lead to bleeding; this cc controlled by protaminc sulfatc intravenously. Heparin is negatively charged molecule and protamine sulfatc being positively charged neutralizes it readily.

(ii) Antithrombins – These arc substances present in plasma which can inactivate large amounts of thrombin very quickly. Thrombin formed during the clotting of only 10 ml of blood can clot the whole body blood. But normally the excess of thrombin is destroyed by antithrombins, and thus the extension of the clotting process to other body regions is prevented; the fibrin clot also adsorb a lot of thrombin and this helps in keeping the clotting process localized. Heparin binds to the lysine residue on the antithrombin molecule, which results in an enhanced activity of the latter. Antithrombins are of great physiological importance. Small amounts of thrombin arc believed to be formed even under normal circumstances, but antilhrombins immediately inactivate it and thus prevent thrombosis. The cervical glands of the medicinal leech (hirudo) contain a substance hirudin which has antithrombin activity. It therefore acts as an anticoagulant and enables the leech to suck blood for a long period from the area where it is applied.

(iii) Anlithromboplastin substances – Blood and body tissues contain substances, which neutralize any thromboplastin released by damage to tissues.

(iv) Protein C-protein-S system – Protein C is a vitamin-K dependent plasma protein. It first occurs in an inactive form. It is activated by thrombin. Its active form acts as a powerful protease; in the presence of a cofactor, thrombomodulin, present on the endothelial surface it digests factors V and VIII and thus clotting process is inhibited. Protein S has the role of accelerating the activation of protein C. These two proteins are believed to have a physiologic role in helping to keep the blood unclotted.

(v) Fibrinolysis – This process has an important role in keeping the blood in a fluid state under normal circumstances. It is believed that micro-thrombi are formed in the blood eveormally, but fibrinolysis liquefies these clots. Cellular phagocytosis of small particles of fibrin thrombi is also a protective mechanism.

(vi) Prostacydin – Its role has already been described under platelets.

3. Vigorous circulation of blood – Under normal circumstances this is an important factor in preventing intravascular clotting of blood. This is brought about by not allowing the reactants to accumulate at a certain site, which could initiate clotting. In conditions associated with stasis of blood (bed-ridden patients, congestive heart failure, polycylhemia, hyper-gammaglobulinemia), intravascular clotting is quite common. Use of oral contraceptive pills and pregnancy also produce venous stasis by causing venous dilatation. During vigorous circulation the formed elements of the blood including platelets arc kept away from the vessel wall. Moreover, it facilitates the mixing of the activated clotting factors with their inhibitors. These active clotting factors are also carried to the liver and the reticuloendothelial system, which take them up and destroy them.

ROLE OF LIVER AND VITAMIN K IN BLOOD COAGULATION

Several factors needed for blood coagulation arc formed in the liver. The formation of four of these factors, i.e. prothrombin and factors VII, IX and X is dependent upon the presence of vitamin K. A deficiency of vitamin K leads to a deficiency of these factors, which may produce a tendency to bleed easily. Substances, which antagonize vitamin K, such as dicumarol, also produce a deficiency of these factors producing hemorrhagic tendency. If the liver is severely diseased, it cannot make these factors at a normal rate and even the administration of vitamin K will fail to exert a beneficial effect on the hemorrhagic tendency. Other factors synthesized in the liver but independent of vitamin K are fibrinogen, factor V and factor XIII. The sites of the formation of factors VIII, XI and XII are .not known, although factor VIII is believed to be produced in the reticuloendothelial cells.

HEMOSTASIS

Three important mechanisms bring about hemostasis, which means the stoppage of bleeding from an injured area.

1. Local factors – These can be divided into two types.

(a) Vascular spasm – A localized vascular spasm is the first line of defence against bleeding and is brought about by the following changes:

(i) Local spasm of blood vessels which is of myogenic origin. It lasts for upto 20 minutes,

(ii) Vasoconstriction in large vessels which is of reflex (neurogenic) origin. It lasts for only a few minutes,

(iii) Liberation of serotonin, a vasoconstrictor, from the disintegrating platelets.

The early vascular response reduces blood flow and intravascular pressure and therefore facilitates consolidation of the hemostatic plug.

(b) Extra-vascular – These include subcutaneous tissue, muscle and skin, which help in stopping bleeding by covering and thus applying pressure over the wounded area. Wounds of the scalp area bleed more as there is paucity of connective tissue in this area; same is true about Little’s area in the nose where bleeding results in epistaxis. Old age, rheumatoid arthritis and excessive use of glucocorticoids also produce atrophy of the subcutaneous tissue with tendency to bleed easily.

2. Role of platelets (platelet plug) – The platelet plug is produced earlier than formation of fibrin and for this reason the platelet plug is said to produce primary hemostasis while fibrin is said to produce secondary hemostasis.

The formation of the platelet plug that stops bleeding from small blood vessels like capillaries is an important defence against bleeding; this process and the additional contribution of platelets to clotting process have already been described. Platelets also release serotonin, a vasoconstrictor.

3. Clotting of blood – This process is very important for maintaining the occlusive plug contributed by the platelets. As soon as blood comes in contact with extravascular tissues, the complex series of reactions leading to blood clotting arc initiated; thrombin formed in this process further activates release of ADP from platelets. The formation of fibrin clot reinforces the platelet plug and the combined fibrin-platelet plug (hemostatic plug) serves to seal the wound more effectively and prevents bleeding after the injured vessels re-open after some time. The clot then undergoes retraction, till after 24 hours its size is only 40% of the original. Platelets and ATP arc needed for the retraction of the clot. The retraction of the hemostatic plug results in a still more effective plugging of the wounded vessels.

Interaction Between Blood Clotting and Kallikrein Systems – It is quite complex. Both systems affect each other and greatly accelerate blood coagulation.

(i) Active factor XII or its breakdown product acts as an enzyme that increases the reaction, Prekallikrein → Kallikrein.

(ii) Kallikrein in turn greatly increases the activation of factor XII; active factor XII now activates factor XI and thus accelerates clotting process.

(iii) Kallikrein acts on kininogcn to release kinin which by producing vasodilatation brings more blood containing clotting factors to the site of injury thus intensifying the clotting process.

Fate of the blood clot – The initial hemostatic plug contains the platelets and fibrin. But as the platelets undergo autolysis, therefore after 24 to 48 hours the hemostatic plug consists almost entirely of fibrin. In the meantime fibrinolysis starts as the fibrinolytic system is activated and the destroyed leukocytes release proteolytic enzymes.

A small clot may later be completely liquefied by the process of fibrinolysis. In bigger clots a proliferation of blood vessels and connective tissue may take place and after two to three weeks it becomes a fibrous mass; this process is called organization of the clot. The defects in the vessel wall are covered with endothelium. If the defect in vessel wall is small, the endothelium grows from the ends. But when the defect is large, then the endothelial cells are produced by transformation of smooth muscle cells which migrate from the media of the vessel wall.

agents.

Functional element of microcirculation

Microcirculatory part of vascular system performs all blood functions. There are such types of vessels: arterioles, metarterioles, capillaries and venuls. Mean diameter of these vessels is less than 100 mcm. Arterioles, capillary bed venuls and lymphatic capillaries compose functional element of microcirculation. Main processes as blood-tissue exchange or lymph production are performed there. Mean diameter of capillaries is 3-6 mcm. The length of capillary vessel is near 750 mcm. Capillaries perform exchange in surface near 14000 mkm². Blood flow velocity in capillaries consists near 0.3 mm/s, which permits passing erythrocytes through capillary in 2-3 s.

 

Microcirculatory bed and functional types of capillaries

 

Depending on structure it distinguished three types of capillaries: somatic, visceral and sinusoidal. Capillaries walls are composed from one layer of endothelial cells and basal membrane. Endothelial cells are active elements of capillary bed. Endothelial cell may produce enzymes as antithrombin III endothelial relaxing factor, endothelial contracting factor, which may activate function of hormones and neurotransmitters on vessel’s wall or cause some physiological effects by it. It was determined that endotheliocites may contract and become voluminous. Endoteliocytes contain microfibrills, composed from actin, myosin and other contractive elements. Such structures are directed along cell basis and binds to cytoplasm in places of intracellular contacts. When microfibrills contracting two kinds of effects may be produced: both increasing intracellular split after contraction and increasing cell height and its’ prominence inside the vessel. Capillary wall has small splits and a lot of pores. In certain organs capillary walls have some specialties. In kidneys glomeruls, intestinal epithelium, capillaries are fenestrated. This specialty permits passing through endothelial cells water, ions and other even rather large molecules as aminoacids or fructose. In red bone marrow, liver and spleen capillaries have interrupted walls, which let passing even blood cells.

 Common characteristic of erythrocytes resistance

a) Determine the notion “resistance” and “hemolysis” (Resistancy is a property of erythrocytes to be hole in solution with different concentration. Hemolysis is a process of going out of hemoglobin from erythrocytes in plasma. It may be in cases of destroyed the erythrocytes membranes and without destroyed the erythrocytes. Level of osmotic resistance of erythrocytes is the concentration of NaCl in solution in which hemolyse erythrocytes. Minimal resistance of erythrocytes (0,50-0,45 % of NaCl) – in this concentration of NaCl destroyed erythrocytes with the smallest resistance. Maximal resistance of erythrocytes (0,34-0,32 % of NaCl) – in this concentration of NaCl destroyed all erythrocytes. Osmotic resistance of erythrocytes may decrease and hemolysis may be in a higher concentration of NaCl.)

b) Kinds of hemolysis (There are 3 kinds of hemolysis: biological, chemical, physical. Biological causes of hemolysis are poisons of snakes, for example; chemical – strong acids, ether, for example; mechanical – in patient with the pathology of membranes in a case of running, jumping, for example; temperature – higher temperature.)

2. Deformation properties of erythrocytes (Erythrocytes may pass through capillars, which have a smaller diameter than erythrocytes.)

a) Membrane mechanisms (Depends of structure of membrane, it flexible.)

b) Role of erythrocytes content (Depends of structure of hemoglobin.)

3. Physiological analysis of erythrocytes sedimentation speed (ESS) (In a blood which caot clotting erythrocytes sedimentation. Iorm erythrocytes sedimentation speed in male is 2-10 mm/hour, in female is 2-15 mm/hour. It necessary for diagnose the inflammatory processes in human organism.)

a) Notion about erythrocytes aggregation (In healthy person erythrocytes aggregation is absent. Erythrocytes aggregation is develop of connection between erythrocytes (small bridges) and forming chains.)

b) Influence of blood components on erythrocytes sedimentation speed (It may be 2 groups of factors – erythrocytes and plasma. Erythrocytes sedimentation speed is increase in a case of decrease the quantity of erythrocytes, decrease pH of blood, quantity of albumins. Erythrocytes sedimentation speed is increase in a case of increase the quantity of hemoglobin, volume of erythrocytes, protein – fibrinogen, cholesterol, gamma-globulins, increase pH of blood.)

c) Influence of physiological condition on erythrocytes sedimentation speed (Erythrocytes sedimentation speed increase iewborn, pregnancy women, in a case of different inflammation processes.)

 

Key words and phrases: platelet plug, agglutinate, clot, retraction, appears, fibrin threads, thromboxan A, growth factor of platelets, fragility, acselerator of thrombin, platelets thromboplastin, antiheparinic, clotting factor, antifibrinolitic factor, antithromboplastic factor, retractosim, platelets cofactor, fibrinstabilising factor, ADP, petechia, capillary bleeding, Duke time, active and inactive clotting factors, coagulogram, thromboelastography, clots creation, thromboplastinal suspension, opalescention, small grains of fibrin, floces of fibrin, threads of fibrin, net, formed by threads of fibrin, uncompact fibrin clot, compact fibrin clot, loose, tight, concentration of fibrinigen, thromboplastin index, recalcification, thromboplastin time, thromboelastogramm, thromb elasticity thromb elasticity, hypercoagulation, hypocoagulation, fibrinolysis, anticlotting factors, hypocoagulation, antithrombin-III, prostaciclins, plasminogen, fibronogen, fibrin, kallikrein, kinins, fibrinogen degradation products, plasminogen, plasmin, thrombin, antithrombin-I, protein C, vitamin K, antithrombin-III

1. How many and what components take place in vessel-platelets hemostasis?

a) 2; vessel’s wall and platelets; b) 3; vessel’s wall, platelets and plasma’s clotting components; c) 3; vessel’s wall, platelets and plasma’s anticlotting components; d) 3; vessel’s wall, platelets and blood clotting and anticlotting components; e) 4; vessel’s wall, erythrocytes, platelets and blood components

2. What is the normal quantity of platelets?

a) (4,0-9,0) x 10⁹/L; b) (16,0-32,0) x 10⁹/L; c) (180,0-320,0) x 10⁹/L; d) (3,7-4,7) x 10¹²/L; e) (4,0-5,1) x 10¹²/L

3. Patient S. in Konchalovsky test has 15 petechiars. What cells injures in this patient?

a) erythrocytes; b) leucocytes; c) basophyls; d) monocytes; e) platelets

4. Patient P. in Konchalovsky test has 1 petechiars. What cells injures in this patient?

a) erythrocytes; b) leucocytes; c) basophyls; d) platelets; e) nothing

5. In patient K. by Duke method time of bloodless is norm. What is the norm?

a) to 1 minute; b) to 3 minutes; c) to 5 minutes; d) to 10 minutes; e) to 15 minutes

6. In patient D. by Duke method time of bloodless is norm. What is the norm?

a) 0-3 minutes; b) 1-3 minutes; c) 0-5 minutes; d) 1-15 minutes; e) 1-10 minutes

7. What is the time of clotting by Ly-Wait quantity iorm?

a) not more than 3 minutes; b) 1-3 minutes; c) 5-10 minutes; d) to 5 minutes; e) to 10 minutes

8. What is the time of plasma recalcification in norm?

a) 1-3 second; b) to 3 second; c) to 5 second; d) 5-10 second; e) 60-120 second

9. What is the concentration of fibrinogen in norm?

a) not more than 3 g/L; b) 1-3 g/L; c) 2-4 G/L; d) 2-4 g/L; e) to 5 g/L

10. What substances is the secondary anticoagulant?

a) Antithrombin-III; b) Protein C; c) Heparin; d) Antithrombin-I; e) Alpha 2-antitripsin

11. What substances is the primary anticoagulant?

a) Antithrombin-I; b) Antithrombin-III; c) Kinin; d) Kallikrein; e) Globulins

12. From what factors beginning fibrinolysis?

a) III; b) V; c) VII; d) X; f) XII

13. What factor do not take place in fibrinolysis?

a) III; b) V; c) VIII; d) X; e) XII

Real-life situations to be solved:

1. The quantity of platelets in blood is 100,0 x 10⁹/L. Is it norm? What is the clotting mechanism in this patient?

2. The quantity of platelets in blood is 100,0 x 10⁹/L. What is the breach in this patient?

3. The quantity of platelets in blood is 340,0 x 10⁹/L. What is the breach in this patient?

4. Three-years old patient S. has Duke time 2 minutes. Tomorrow will be the operation on his finger. Do this patient need addition medicines, which correct hemostasis?

5. What is you opinion about the hemostasis of patient F. if you know that clotting time is 8 minutes, Dukes time is 2 minutes, quantity of platelets is 190 x 10⁹/L?

6. What are You opinion about the hemostasis of patient T. if You know that in thromboelastogramm time of bloods’ beginning clot – 10 minutes, time of thromb producing – 7 minutes, maximum amplitude – 52 mm?

7. You know that time of clotting by Ly-Wait is 6 minutes; time of plasma recalcification is 65 seconds; thrombotest of V degree; thromboplastin time is 12 seconds; thromboplastin index is 100 %; concentration of fibrinogen is 3,2 g/L; tolerancy of plasma to heparin is 8 minutes; heparin time is 57 seconds; fibrinolysis is 18 %. What is you opinion about the secondary hemostasis of patient L.?

8. You know that time of clotting by Ly-Wait is 3 minutes; time of plasma recalcification is 56 seconds; thrombotest of VII degree; thromboplastin time is 9 seconds; thromboplastin index is 133 %; concentration of fibrinogen 4 g/L; tolerancy of plasma to heparin is 6 minutes; heparin time is 50 seconds. What is You opinion about the secondary hemostasis of patient W.?

9. You know that time of clotting by Ly-Wait is 12 minutes; time of plasma recalcification is 132 seconds; thrombotest of III degree; thromboplastin time is 17 seconds; thromboplastin index is 88 %; concentration of fibrinogen is 2,1 g/L; tolerancy of plasma to heparin is 11 minutes; heparin time is 60 seconds. What is you opinion about the secondary hemostasis of patient J.?

10. What is the difference between primary and secondary anticoagulants?

 Answers for the Self-Control

1. d;  2. c; 3. e; 4. e; 5 b; 6. a;. 7. c;  8. e;  9. d;  10. d;  11. b; 12. e; 13. a

 

1 real-life situation – It isn’t norm. Norm is (180,0-320,0) x 10⁹/L. Clotting of blood is breaches (aggregation of platelets).

2 real-life situation – It is thrombocytopenia – decrease the quantity of platelets.

3 real-life situation – It is thrombocytosis – increase the quantity of platelets.

4 real-life situation – No, they do not, because the Duke’s time is to 3 minutes to all aged groups.

5 real-life situation – The hemostasis system is norm (clotting time is 5-10 minutes; Dukes time is to 3 minutes, quantity of platelets is 180-320 G/L.)

6 real-life situation – It is norm, because iorm time of bloods’ beginning clot is 8-12 minutes; time of thromb producing is 5-8 minutes; maximum amplitude is 45-60 mm.

7 real-life situation – The secondary hemostasis iorm, because in norm time of clotting by Ly-Wait – 5-10 minutes; time of plasma recalcification – 60-120 seconds; thrombotest – IV, V, VI degree; thromboplastin time – 12-15 seconds; thromboplastin index – 80-105 %; concentration of fibrinogen – 2-4 g/L; tolerancy of plasma to heparin – 6-11 minutes; heparinic time – 50-60 seconds; fibrinolysis – 15-20 %.

8 real-life situation – The secondary hemostasis in abnorm – it is hypercoagulation, because iorm time of clotting by Ly-Wait – 5-10 minutes; time of plasma recalcification – 60-120 seconds; thrombotest – IV, V, VI degree; thromboplastin time – 12-15 seconds; thromboplastin index – 80-105 %; concentration of fibrinogen – 2-4 g/L; tolerancy of plasma to heparin – 6-11 minutes; heparinic time – 50-60 seconds.

9 real-life situation – The secondary hemostasis in abnorm – it is hypocoagulation, because iorm time of clotting by Ly-Wait – 5-10 minutes; time of plasma recalcification – 60-120 seconds; thrombotest – IV, V, VI degree; thromboplastin time – 12-15 seconds; thromboplastin index – 80-105 %; concentration of fibrinogen – 2-4 g/L; tolerancy of plasma to heparin – 6-11 minutes; heparinic time – 50-60 seconds.

10 real-life situation – Primary anticoagulants are produce and present all time in plasma and secondary anticoagulants form in a case of blood clotting.

Determination of duration of capillary bleeding after Duke’s method

Make a puncture of the skin of the IV finger by sterilized scarification (3-4 mm depth).

In every 30 seconds attach to the place of puncture by filter paper without pressing. Disappearance of red color confirms us about stopping of bleeding.

In conclusion compare your result with physiological norm.

Calculation of platelets

Put a drop of immersion oil on smear of blood. Calculate 1000 of erythrocytes and platelets under 90x objective and 7x-glass of microscope. The contains of platelets in 1 L of blood may be determined by such formula:

X=(a x b) / 1000, where:

X – quantity of platelets in 1 L of blood; a – quantity of platelets during calculation of 1000 erythrocytes; b – quantity of erythrocytes in 1 L of blood.

To valuate the quantity of platelets in blood and draw in pencil the erythrocyte and platelets.

Determination of time of plasma’s recalcification

Put 0,3 ml of calcium solution into the test-tube. Place it into the “water bath” under 37ºC temperature. In 60 seconds add 0,1 ml of plasma and determine the time of clot’s creation.

In conclusion compare your results with physiological normal.

Tromboplastinal test of Quich

Put 0,1 ml of research blood and 0,1 ml of tromboplastinal suspension into the test-tube. Place the mixture into the “water bath” under 37ºC temperature for 60 seconds. Than add 0,1 ml of CaCl₂ solution. Determine the time of blood clot’s appearance. Using your result find the tromboplastinal index after this formula:

            tromboplastine time of healthy person

  T1= ————————————————— 100%

              tromboplastine time of your patient

In conclusion point out what is protrombine time and protrombine index. Compare your results with physiological norm.

Trombotest

Put 5 ml of CaCl₂ solution and 0,1 ml of oxalat plasma into the tube. Place into the “water bath” with 37 ºC temperature for 30 minutes.

You can observe 7 levels of coagulation: I – opalescention; II – small grains of fibrin; III – floces of fibrin; IV – threads of fibrin; V – net, formed by threads of fibrin; VI – uncompact fibrin clot; VII – compact fibrin clot.

The first 3 levels can be observed during hypocoagulation; IV, V, VI levels – normal coagulation; VII – hypercoagulation.

In conclusion point out the state of coagulative hemostasis.

Ethanol test

Put on the test-tube 0,1mL of ethanol solution and 0,4 mL of plasma. The content of test-tube mix and stay on the stage at the room temperature during 10 minutes. The gel is forming, when products of fibrinolisis are in the plasma. Test is negative iorm.

Protaminsulfate test

0,1 ml of protaminsulfate solution add to 0,4 ml of plasma. The contains of test-tube mix and stay on the stage. Through 10 minutes we determinate if gel clot is forming. The test is negative iorm.

What is conclusion about results?

Study of fibrinolysis

Water-bath with temperature 38 ⁰C is prepared. The test-tube, which contain 1 ml of blood from experimental animal (rat) stay there. Through every 10 minutes we bent and take the test-tube off water-bath. The criterions of the start of fibrinolisis are decrease of the clot and change the color of serum. It’s necessary to determinate this criterion throught 30 minutes and 1 hour after the start of the study.

To delineated of chenges.

What about do results witness?

SOME LABORATORY TESTS DONE FOR INVESTIGATING BLEEDING DISORDERS

(1) Clotting time – (Method of Lee and White). Without letting tissue juice to contaminate the blood, venous blood is withdrawn into a clean, dry, all glass syringe with a wide bore needle. A stopping watch is started the moment blood appears in the syringe and blood coagulation time is recorded from the moment. The needle is detached and 1 ml of blood is delivered into each of the four dry, chemically clean glass tubes with a size 10 by 1 cm. These tube should have been previously placed in a container of water (water bath maintained at 37 °C).

After 3 minutes have elapsed, each of these tubes is lifted from the water bath and is tilled very gently. The clotting time is taken when the tube can be inverted without its contents spilling. The clotting time for each tube is recorded separately and the clotting time is reported as the average of the four tubes. The normal clotting time by this method is 4 to 10 minutes. Clotting time is prolonged in hemophilia and other conditions associated with a deficiency of clotting factors. It remains normal in purpura because in this conditions there are no coagulation defects.

(2) Prothrombin time – (Quick’s one-stage method). Oxalated plasma of the patient is obtained and to it is added an excess of thromboplastin, which is usually an emulsion of rabbit’s brain. Calcium chloride solution is added. The time taken for clotting of the plasma to occur after the addition of calcium chloride is noted; it is termed as the prothrombin time and normally it is 12 to 14 seconds. The prothrombin time is increased in hypoprothrombinemia. However, the test is not specific for prothrombin deficiency and deficiencies of factors V and X and of fibrinogen also increase its duration.

(3) Bleeding time – (Duke’s method). When injury to a small vessel produces only a small defect, the platelet plug, i.e. the white thrombus alone can cause hemostasis. This is the basis for the bleeding time that is employed clinically to distinguish hemostatic deficiency caused by abnormalities of blood capillary wall from those caused by coagulation defects. In doing the test a standardized cut is made usually into the skin of the car lobule. The blood, which flows, is absorbed by a filter paper every 15 seconds. When the filter paper remains unstained on touching the wounded area, it shows that bleeding has stopped. The time taken by the bleeding to stop after the cut is made is the bleeding time. This test is simple but crude. In the majority of normal persons, bleeding lime ranges between 1 to 5 minutes. Bleeding time is prolonged in purpuras.

(4) Platelet count – This is normally 180,000 to 320,000 per cu mm of blood. Bleeding usually occurs when the count falls to 50,000 platelets per cu mm or below.

(5) Plasma fibrinogen level – This is found to be low in conditions associated with excessive fibrinolysis. In this condition the presence of fibrinogen degradative products (FDP) in the plasma and urine can be demonstrated.

(6) Assay of clotting factors – Factors V, VIII, IX, etc. can be quantitatively assessed in the blood.

(7) Capillary fragility test – A strong positive pressure (by inflating sphygmomanometercuff) or a negative pressure (by applying suction cups) is applied and the number of minute petechial hemorrhages (petechiac) is counted in the skin; their number is found to be much increased in conditions associated with an increased capillary fragility, e.g. purpuras.

METHODS OF KEEPING BLOOD FLUID IN VITRO – In order for the blood to be kept fluid, certain methods are employed.

These include the following:

(i) Addition of heparin. Its mechanism of anticoagulant action has been already described.

(ii) Addition of substances which take up Ca²⁺, e.g. citrate, oxalate, ethylene-diamine-tetra-acetic acid or EDTA. The last compound is an example of the chelating

Theories of Blood Coagulation

I. Classical theory of Morowitz (1905-1906) – Blood clotting was considered to take place in two stages.

(i) In the first stage prolhrombin is converted to thrombin by the enzyme prothrombinase, Ca²⁺ being necessary for this reaction.

(ii) In the second stage the thrombin acts as an enzyme on fibrinogen and converts it to fibrin.

II. Cascade or waterfall theory – For many decades, Morowitz’s theory was accepted. But great developments in this field resulted in several new theories, one of which is called cascade or waterfall theory because it involves a cascade of events; it is described below.

There are two systems of clotting, intrinsic and extrinsic, which converge upon what is called the final common pathway.

1. Intrinsic or the blood system – This system is called so, because all factors taking part in the process are derived from the blood itself and it can take place in pure blood (blood not contaminated with tissue juice) kept in a test tube. It is also called contact system because the process starts when blood comes in contact with a foreign surface, e.g. vascular sub-endothelial collagen or even glass. This process takes place in the following six stages. In the first five of these stages limited proteolysis converts an inactive factor to its active form. Each of these steps is regulated by plasma and cellular co-factors and Ca²⁺. The inactive and active blood clotting factors are distinguished by writing and a respectively after the factor.

Stage No. 1. Three plasma proteins, i.e. Hageman factor (XII), high mol. wt. kininogen and pre-kallikrein form a complex with vascular subendolhelial collagen. Factor XH-i becomes activated to Xll-a, which acceleates the conversion of pre-kallikrein to kallikrein which then accelerates the conversion of still more XII-i to XII-a.

Satge No. 2. Factor XII-a converts factor XI-i to XI-a.

Stage No. 3. Factor XI-a converts factor IX-i to IX-a.

Stage No. 4. Factor IX-a in the presence of factor VIII C, Ca²⁺ a platelet membrane lipoprotein (platelet factor 3) converts X-i to X-a.

Stage No. 5. Several factors take part in the conversion of prothrombin to thrombin. These include factor X-a, factor V-a, Ca²⁺ and phospholipids. Although the conversion of prothrombin to thrombin can take place on a phospholipid-rich surface, but it is accelerated several thousand-fold on the surface of activated platelets.

Stage No. 6. Conversion of fibrinogen to fibrin is brought about thrombin by the following mechanism. Fibrinogen is a symmetrical dimer; each half of its molecule has the following structure:

i) Alpha polypeptide joined to a short A-fibrinopeptide.

ii) Beta polypeptide joined to a short B-fibrinopeplide.

iii) Gamma polypeptide.

Fibrinogen can thus be represented by the structure, [Alpha(A), beta(B), gamma]₂. Thrombin catalyzes the breakdown of fibrinogen in such a way that a part of the molecule separates leaving behind a fibrin monomer.

[alpha(A), beta(B), gamma]₂ → [alpha, beta, gamma]₂ (Fibrin monomer) + 2[fibrinopeptide A + B]

However, the removal of fibrinopeptide B is not essential for coagulation. The fibrin monomers undergo polymerization giving rise to fibrin polymers; this process involves formation of hydrogen bonds between fibrin monomers. These fibrin polymers are unstable and the polymerization is readily reversed by inhibitors of H bond formation such as urea. The unstable fibrin polymers are then acted upon by factor XIII, which actually is an enzyme. Factor XIII is initially inactive but is activated by thrombin. It brings about the production of cross linkages between adjacent fibrin polymers. This process involves covalent bond formation between epsilon amino group of lysine and the gamma amide group of glutamine; NH₃ is evolved in this reaction. A clot which is much more stable and is insoluble in urea solution is thus produced. Even this fibrin clot is quite soft, but after some time it undergoes retraction during which serum oozes out of it. The platelets are of primary importance in this process of clot retraction. The result is a firm clot that can effectively seal a wounded vessel.

2. The extrinsic or the tissue system – This is called so because it needs the presence of tissue juice that contains tissue thromboplastin which is not present in blood. The tissue thromboplastin in the presence of factor VII and Ca²⁺ activates factor X-i to X-a. Subsequent reactions are the same as described under the intrinsic system and, being common to both the intrinsic and extrinsic systems, are designated as the final common pathway. Because the extrinsic system involves fewer steps than the intrinsic system, therefore it proceeds faster than the latter. For this reason, while the intrinsic system takes 2 to 6 minutes for clotting to take place, the extrinsic system takes as little as 15 seconds to do that.

III. Seeger’s hypothesis – This concept basically differs from the cascade theory in that prothrombin and factors VII, IX and X are considered to occur in a single molecular system and not separate from each other. This common molecule is believed to release all these clotting factors during clotting process. A common characteristic of all these clotting factors is that all of them require the presence of vitamin K for their biosynthesis. Factors VII, IX and X are designated by Seeger as autoprothrombin I, II and III respectively. The corresponding active forms of these factors arc called autoprothrombin A, B and C. There are serious objections to this hypothesis as various studies have shown that all these factors arc different and are quite distinct from each other.

Properties of Various Factors Participating in Blood Coagulation

Fibrinogen – It occurs in the plasma in a concentration of 0.35 gram per 100 ml, but is also present in lymph and many tissues. It has a mol. wt. of about 340,000. It is synthesized in the liver. It behaves as a globulin but is more easily precipitated, i.e. by precipitation with 25% ammonium sulfate. The normal plasma has about 15 times more fibrinogen than that required for blood clotting. Its solution is clotted by thrombin; Ca²⁺ are not needed in this reaction. Afibrinogenemia and dysfibrinogenemia are clinical conditions associated with bleeding; in the former the plasma fibrinogen is absent, while in the latter condition fibrinogen is present but is of abnormal type. Drugs namely Arvin and reptilase are used in therapeutics to control thrombosis; these convert the plasma fibrinogen to fibrin micro-clots that are removed from the circulation by fibrinolysis. In this way, hypofibrinogenemia is produced decreasing the blood clotting tendency. Arvin is obtained from the venom of the Malayan pit viper snake.

Prothrombin – It is the proenzyme, the precursor of thrombin. It contains 2 to 10 % carbohydrate in its molecule and has a mol. wt. of 69,000. Its plasma concentration is 10 to 15 mg per 100 ml.

Thromboplastin – It implies an activity which converts prothrombin to thrombin. All body tissues have this activity and therefore it is termed as tissue or intrinsic thromboplaslin. The brain, lung, placenta and testes are especially rich in it. It is a complex of phospholipids, lipoproteins and cholesterol. Tissue extracts, if injected intravenously, can cause widespread clotting of blood. However, tissue thromboplaslin is not active as such but it needs Ca²⁺ and factor VII for its activation which normally arc present in blood. Russel viper venom has a strong thromboplaslin activity and is used for slopping bleeding from superficial areas by its local application in diseases like hemophilia.

Calcium – Ca in ionic form, Ca²⁺, is essential for clotting of blood and it acts at many stages. Ca ions serve to form complexes with lipids, which take part in blood clotting. In health or disease blood has always sufficient Ca²⁺ for this purpose. In other words, a Ca²⁺ deficiency is never a cause of a prolonged clotting time in man.

Factor V – It is activated by small amount of thrombin which in turn leads to a greater formation of thrombin. But an excess of thrombin destroys it and causes its disappearance from serum. It is unstable in the citrated plasma. Its congenital deficiency is the cause of parahemophilia, a mild bleeding disorder.

Factor VII – It is stable on storage. It acts as co-thromboplastin in the working of extrinsic system of blood cloning. Its congenital deficiency has been seen very rarely. It has up to 50 % carbohydrate in its molecule.

Factor VIII-C – It is also called platelet cofactor-I and anti-hemophilic globulin. Its deficiency causes the classical hemophilia (now called hemophilia A). Hemophilia is discussed later in detail. This factor is readily inactivated in vitro.

Factor IX – It is also called Christmas factor because its deficiency was first demonstrated in a patient with the surname Christmas whose bleeding disease was named Christmas disease. This disease is also called hemophilia B.

Factor X – It is an alpha globulin present both in scrum and plasma. I deficiency is seen in both sexes equally as a congenital defect.

Factor XI – Its deficiency causes hemophilia C, which is a mild bleeding disease.

Factor XII – It is activated by surface contact and according to the cascade theory, this process initiates the series of reactions leading to blood clotting. Blood deficient in this factor docs not clot in lest tube, i.e. in vitro. If blood taken from a vein (without letting it being mixed with tissue juice) is placed in a lest tube lined with silicone, it does not clot; this is because the silicone layer is smooth and unwettable and does not permit the activation of factor XII for the same reason. Blood also clots much more slowly when placed in polythene tubes as compared to glass tubes. The deficiency of this factor is seen in persons with Hageman’s trait, but they do not generally show bleeding tendency. Its additional roles arc the activation of fibrinolytic system and the plasma kinin syslem. It is activated by contact with glass, negatively charged surfaces, collagen fibers, unbroken skin, sebum, long chain fatty acids, uric acid, fibrin, elastin and homocysteine.

Factor XIII – It is the enzyme transglutaminase, whose function has already been discussed. Persons with congenital deficiency of this factor have bleeding tendencies and poor wound healing. Their blood clots all right, but the clot, unlike the normal clot, is unstable and can be solubilized in 5 molar urea or 1 % monochloracetic acid solution.

The clot, or thrombus, can form in some blood vessel of the arm or leg and do comparatively little harm, but if it should block the blood supply to the brain or to the heart (coronary thrombosis), it can be very serious. Heparin form complex with antithrombin-III and transform it in anticoagulant with the negative action. Activate nonenzyme fibrinolysis.)

c) Role of alpha-2-macroglobulin, alpha-1-antitripsin, protein C (Alpha-2-macroglobulin is very large globulin molecule. It is a similar to antithrombin-heparin cofactor in that it combines with the proteolytic coagulation factors. Its activity is not accelerated by heparin. Its function is mainly to act as a binding agent for the coagulation factors and prevent their proteolytic action until they can be destroyed in various ways. It a faint inhibitor of thrombin, connect with plasmin. Alpha-1-antitripsin inhibits thrombin activity, IXa, XIa, XIIa factors, plasmin and kallilrein. Protein C inhibits VIIIa, Va factors. It activity depend of thrombin and vitamin K concentration.)

d) Functionation of secondary anticlotting substances (Primary anticoagulants are produce and present all time in plasma and secondary anticoagulants form in a case of blood clotting. They are antithrombin-I or fibrin and products of fibrinolysis or products of fibrinogen degradation. Fibrin is sorbs and inactivates thrombin and Xa factor. Products of fibrinolysis inactivate ending stage of clotting, IXa factor, platelets’ agregation.)

2. Fibrinolytic system (Fibrinolysis is begining with the retraction. The plasma proteins contain a globulin called plasminogen or profibrinolysin, which activated into plasmin or fibrinolysin.)

a) Stages of fibrinolysis

 

b) Mechanisms of fibrinolysis activation (There are 2 mechanisms of fibrinolysis activation: external and internal. Main internal mechanism put in action by XIIa factor. External mechanism stimulated by protein activators of plasminogen, which are produce by vessel wall.)

c) Regulation of fibrinolysis (All processes are direct on increase the clotting mechanism, for example, epinephrine, which are increase in the case of stress. It promote the forming of prothrombinase, activating of XII factor, increasing of fats and fats acids. But after the clotting send up the anticlotting mechanism – hypocoagulation. Iorm it has independent regulation. The lysis of blood clots allows slow clearing of extraneous blood in the tissues and sometimes allows reopening of clotted vessels. Reopening of large vessels occurs only rarely. But an important function of the fibrinolysin system is to remove very minute clots from the millions of tiny peripheral vessels that eventually would all become occluded were there no way to cleanse them.)

d) Notion about nonenzymes fibrinolysis (It may be steroid hormons with anabolic function, which are increase producing of fibrinolysis activators by endothelium. Leucocytes ensure function of independent mechanism of fibrinolysis. It limited the size of thromb. Erythrocytes ensure function of independent mechanism of fibrinolysis too.)

3. Functionating of anticlotting mechanisms

a) Role of vessels endothelium in support blood in the fluid condition (1. Smooth surface of vessels endothelium. 2. Negative charge of endotheliocytes and blood cells and that’s why they are push away. 3. Present on the vessels wall thin layer of fibrin which adsorb clotting factors, especially thrombin. 4. Constant presence in blood anticlotting factors in a small doses. 5. Producing by endothelium prostaciclins, which are powerful inhibitors of platelets aggregation. 6. Ability of endothelium to produce and fix antithrombin-III.)

THE FIBRINOLYTIC SYSTEM

A proteolytic enzyme, fibrinolysin or plasmin, acts on fibrinogen and fibrin causing their breakdown or dissolution by converting them into smaller peptides which are soluble; these peptides are called fibrin/fibrinogen degradative products (FDP) as well as fibrin/fibrinogen split products (PSP). The FDP so produced have several important actions, which oppose hemostasis; in other words they promote bleeding.

1. They inhibit binding of fibrinogen to platelets and inhibit aggregation of platelets.

2. They inhibit thrombin formation and also destroy any thrombin formed.

3. They prevent fibrin polymerization.

Formation of plasmin – Normally plasmin occurs in blood plasma as its inactive precursor called pro-fibrinolysin or plasminogen. The conversion of plasminogen to plasmin involves cleavage of a single arginine-valinc bond and is catalyzed by an activator which itself occurs in an inactive form namely pro-activator. The pro-activator is changed to activator by enzymes called pre-kallikrein activators, which are derived from breakdown of active factor XII; in other words, the activators of pre-kallikrein are the fragments of active factor XII.

Factors increasing the formation of plasmin – These are discussed below.

1. Fibrinolysokinase – It is present in plasma, many tissues and in many secretions, e.g. saliva, milk and tears.

2. Bacterial enzymes – These include staphylokinase and streptokinase which are produced by staphylococci and streptococci respectively. Streptokinase is used by intravascular infusion for the clearance of thrombo-embolism.

3. Urokinase – It is present in urine and is being used in therapeutics to accelerate fibrinolysis. It is formed in the kidney; its commercial source is the fetal kidney tissue culture. The urinary plasmin produced under its influence is believed to have a role in keeping the renal tubules patent by preventing the deposition of fibrin within them. Fibrin may be derived from the fibrinogen present in trace amount in the renal tubular fluid. In the same way, plasmin activity present in milk, tears and semen serves to keep the corresponding duct systems patent.

4. Cytokinase – It is present in tissue cells, WBCs and platelets.

5. Hormones – Growth hormone and thyroid stimulating hormone (TSH).

6. Miscellaneous – Exercise, adrenaline, hypoxia, histamine, bacterial pyrogens, ischemia, shock, tissue damage and chloroform.

Factors inhibiting plasmin formation – The plasmin activation is inhibited by the following factors.

1. Epsilon aminocaproic acid (Trasylol and tranexamic acid) – It is used in therapeutics to inhibit plasmin system.

2. ACTHl – (Adrcnocorlicotropic hormone).

Control of plasmin activity – A trace of plasmin activity is present even iormal plasma. However, this plasmin activity is kept under check by an opposing factor called anti-plasmin which is 10 times more active than plasmin activity. The anti-plasmin activity is present in alpha-1 and alpha-2 globulins of plasma. When excessive amount of plasmin is formed, it brings about the following two types of effects:

(i) The plasmin gets bound to fibrin clots and breaks them to small fragments which arc cleared by macrophages. This is the physiological action.

(ii) A part of plasmin remains free in the plasma and enters circulation. However, its action is rapidly neutralised by the anti-plasmin. But if plasmin level of the plasma is too high, then it causes digestion of fibrinogen and also of factors V, VIII and XII. This is the pathological action. If this action is very marked, hypofibrinogenemia and deficiencies of factors V, VIII and XII result and lead to hemorrhage.

FACTORS PREVENTING BLOOD CLOTTING WITHIN BLOOD VESSELS

It has already been pointed out that blood must remain fluid within the blood vessels if life has to be normal. The factors, which help in preventing intravascular clotting (thrombosis), are given below.

1. Vascular endothelium – The normal endothelium of blood vessels, has been described to act as a non-wettable surface and docs not allow the activation of factor XII. Similarly the platelets also cannot adhere to normal endothelium. Both these factors prevent the initial steps in clotting. The endothelial cells arc also rich in fibrinolysin activators. In atherosclerosis the normal intima is replaced by a diseased one, and clotting is favored resulting in thrombosis. The endothelium can also be damaged by bacteria, injuries such i as fractures, pressure on veins, etc.

2. Chemical substances in blood – There arc certain substances in the blood, which normally antagonize any tendency towards clotting. These include the following:

(i) Heparin – It is produced by the mast cells of the connective tissue which occur especially in the lung and liver. Heparin is an acidic mucopolysaccharide composed of alternating sulfated D-glucosamine and D-glucuronic acid units. Up to 40 % of its molecular mass is H₂SO₄. This makes heparin the strongest organic acid in the body. Heparin is formed in small amounts continuously, but its release from the mast cells is greatly increased in conditions like peptone shock and anaphylactic shock. The mechanism of heparin action is manifold. It antagonizes all the stages of blood clotting, e.g. formation of active factor X, thrombin and fibrin. It also inhibits the agglutination of platelets thus decreasing the release of platelet factors. In combination with a proteiamely anti-thrombin, it inactivates thrombin. This is probably the most important action of heparin.

Heparin is water soluble. It has mol. wt. of about 17,000. It is much in therapeutics. Excessive doses of heparin lead to bleeding; this cc controlled by protaminc sulfatc intravenously. Heparin is negatively charged molecule and protamine sulfatc being positively charged neutralizes it readily.

(ii) Antithrombins – These arc substances present in plasma which can inactivate large amounts of thrombin very quickly. Thrombin formed during the clotting of only 10 ml of blood can clot the whole body blood. But normally the excess of thrombin is destroyed by antithrombins, and thus the extension of the clotting process to other body regions is prevented; the fibrin clot also adsorb a lot of thrombin and this helps in keeping the clotting process localized. Heparin binds to the lysine residue on the antithrombin molecule, which results in an enhanced activity of the latter. Antithrombins are of great physiological importance. Small amounts of thrombin arc believed to be formed even under normal circumstances, but antilhrombins immediately inactivate it and thus prevent thrombosis. The cervical glands of the medicinal leech (hirudo) contain a substance hirudin which has antithrombin activity. It therefore acts as an anticoagulant and enables the leech to suck blood for a long period from the area where it is applied.

(iii) Anlithromboplastin substances – Blood and body tissues contain substances, which neutralize any thromboplastin released by damage to tissues.

(iv) Protein C-protein-S system – Protein C is a vitamin-K dependent plasma protein. It first occurs in an inactive form. It is activated by thrombin. Its active form acts as a powerful protease; in the presence of a cofactor, thrombomodulin, present on the endothelial surface it digests factors V and VIII and thus clotting process is inhibited. Protein S has the role of accelerating the activation of protein C. These two proteins are believed to have a physiologic role in helping to keep the blood unclotted.

(v) Fibrinolysis – This process has an important role in keeping the blood in a fluid state under normal circumstances. It is believed that micro-thrombi are formed in the blood eveormally, but fibrinolysis liquefies these clots. Cellular phagocytosis of small particles of fibrin thrombi is also a protective mechanism.

(vi) Prostacydin – Its role has already been described under platelets.

3. Vigorous circulation of blood – Under normal circumstances this is an important factor in preventing intravascular clotting of blood. This is brought about by not allowing the reactants to accumulate at a certain site, which could initiate clotting. In conditions associated with stasis of blood (bed-ridden patients, congestive heart failure, polycylhemia, hyper-gammaglobulinemia), intravascular clotting is quite common. Use of oral contraceptive pills and pregnancy also produce venous stasis by causing venous dilatation. During vigorous circulation the formed elements of the blood including platelets arc kept away from the vessel wall. Moreover, it facilitates the mixing of the activated clotting factors with their inhibitors. These active clotting factors are also carried to the liver and the reticuloendothelial system, which take them up and destroy them.

ROLE OF LIVER AND VITAMIN K IN BLOOD COAGULATION

Several factors needed for blood coagulation arc formed in the liver. The formation of four of these factors, i.e. prothrombin and factors VII, IX and X is dependent upon the presence of vitamin K. A deficiency of vitamin K leads to a deficiency of these factors, which may produce a tendency to bleed easily. Substances, which antagonize vitamin K, such as dicumarol, also produce a deficiency of these factors producing hemorrhagic tendency. If the liver is severely diseased, it cannot make these factors at a normal rate and even the administration of vitamin K will fail to exert a beneficial effect on the hemorrhagic tendency. Other factors synthesized in the liver but independent of vitamin K are fibrinogen, factor V and factor XIII. The sites of the formation of factors VIII, XI and XII are .not known, although factor VIII is believed to be produced in the reticuloendothelial cells.

HEMOSTASIS

Three important mechanisms bring about hemostasis, which means the stoppage of bleeding from an injured area.

1. Local factors – These can be divided into two types.

(a) Vascular spasm – A localized vascular spasm is the first line of defence against bleeding and is brought about by the following changes:

(i) Local spasm of blood vessels which is of myogenic origin. It lasts for upto 20 minutes,

(ii) Vasoconstriction in large vessels which is of reflex (neurogenic) origin. It lasts for only a few minutes,

(iii) Liberation of serotonin, a vasoconstrictor, from the disintegrating platelets.

The early vascular response reduces blood flow and intravascular pressure and therefore facilitates consolidation of the hemostatic plug.

(b) Extra-vascular – These include subcutaneous tissue, muscle and skin, which help in stopping bleeding by covering and thus applying pressure over the wounded area. Wounds of the scalp area bleed more as there is paucity of connective tissue in this area; same is true about Little’s area in the nose where bleeding results in epistaxis. Old age, rheumatoid arthritis and excessive use of glucocorticoids also produce atrophy of the subcutaneous tissue with tendency to bleed easily.

2. Role of platelets (platelet plug) – The platelet plug is produced earlier than formation of fibrin and for this reason the platelet plug is said to produce primary hemostasis while fibrin is said to produce secondary hemostasis.

The formation of the platelet plug that stops bleeding from small blood vessels like capillaries is an important defence against bleeding; this process and the additional contribution of platelets to clotting process have already been described. Platelets also release serotonin, a vasoconstrictor.

3. Clotting of blood – This process is very important for maintaining the occlusive plug contributed by the platelets. As soon as blood comes in contact with extravascular tissues, the complex series of reactions leading to blood clotting arc initiated; thrombin formed in this process further activates release of ADP from platelets. The formation of fibrin clot reinforces the platelet plug and the combined fibrin-platelet plug (hemostatic plug) serves to seal the wound more effectively and prevents bleeding after the injured vessels re-open after some time. The clot then undergoes retraction, till after 24 hours its size is only 40% of the original. Platelets and ATP arc needed for the retraction of the clot. The retraction of the hemostatic plug results in a still more effective plugging of the wounded vessels.

Interaction Between Blood Clotting and Kallikrein Systems – It is quite complex. Both systems affect each other and greatly accelerate blood coagulation.

(i) Active factor XII or its breakdown product acts as an enzyme that increases the reaction, Prekallikrein → Kallikrein.

(ii) Kallikrein in turn greatly increases the activation of factor XII; active factor XII now activates factor XI and thus accelerates clotting process.

(iii) Kallikrein acts on kininogcn to release kinin which by producing vasodilatation brings more blood containing clotting factors to the site of injury thus intensifying the clotting process.

Fate of the blood clot – The initial hemostatic plug contains the platelets and fibrin. But as the platelets undergo autolysis, therefore after 24 to 48 hours the hemostatic plug consists almost entirely of fibrin. In the meantime fibrinolysis starts as the fibrinolytic system is activated and the destroyed leukocytes release proteolytic enzymes.

A small clot may later be completely liquefied by the process of fibrinolysis. In bigger clots a proliferation of blood vessels and connective tissue may take place and after two to three weeks it becomes a fibrous mass; this process is called organization of the clot. The defects in the vessel wall are covered with endothelium. If the defect in vessel wall is small, the endothelium grows from the ends. But when the defect is large, then the endothelial cells are produced by transformation of smooth muscle cells which migrate from the media of the vessel wall.

agents.

Functional element of microcirculation

Microcirculatory part of vascular system performs all blood functions. There are such types of vessels: arterioles, metarterioles, capillaries and venuls. Mean diameter of these vessels is less than 100 mcm. Arterioles, capillary bed venuls and lymphatic capillaries compose functional element of microcirculation. Main processes as blood-tissue exchange or lymph production are performed there. Mean diameter of capillaries is 3-6 mcm. The length of capillary vessel is near 750 mcm. Capillaries perform exchange in surface near 14000 mkm². Blood flow velocity in capillaries consists near 0.3 mm/s, which permits passing erythrocytes through capillary in 2-3 s.

 

 

 

Key words and phrases: platelet plug, agglutinate, clot, retraction, appears, fibrin threads, thromboxan A, growth factor of platelets, fragility, acselerator of thrombin, platelets thromboplastin, antiheparinic, clotting factor, antifibrinolitic factor, antithromboplastic factor, retractosim, platelets cofactor, fibrinstabilising factor, ADP, petechia, capillary bleeding, Duke time, active and inactive clotting factors, coagulogram, thromboelastography, clots creation, thromboplastinal suspension, opalescention, small grains of fibrin, floces of fibrin, threads of fibrin, net, formed by threads of fibrin, uncompact fibrin clot, compact fibrin clot, loose, tight, concentration of fibrinigen, thromboplastin index, recalcification, thromboplastin time, thromboelastogramm, thromb elasticity thromb elasticity, hypercoagulation, hypocoagulation, fibrinolysis, anticlotting factors, hypocoagulation, antithrombin-III, prostaciclins, plasminogen, fibronogen, fibrin, kallikrein, kinins, fibrinogen degradation products, plasminogen, plasmin, thrombin, antithrombin-I, protein C, vitamin K, antithrombin-III

1. How many and what components take place in vessel-platelets hemostasis?

a) 2; vessel’s wall and platelets; b) 3; vessel’s wall, platelets and plasma’s clotting components; c) 3; vessel’s wall, platelets and plasma’s anticlotting components; d) 3; vessel’s wall, platelets and blood clotting and anticlotting components; e) 4; vessel’s wall, erythrocytes, platelets and blood components

2. What is the normal quantity of platelets?

a) (4,0-9,0) x 10⁹/L; b) (16,0-32,0) x 10⁹/L; c) (180,0-320,0) x 10⁹/L; d) (3,7-4,7) x 10¹²/L; e) (4,0-5,1) x 10¹²/L

3. Patient S. in Konchalovsky test has 15 petechiars. What cells injures in this patient?

a) erythrocytes; b) leucocytes; c) basophyls; d) monocytes; e) platelets

4. Patient P. in Konchalovsky test has 1 petechiars. What cells injures in this patient?

a) erythrocytes; b) leucocytes; c) basophyls; d) platelets; e) nothing

5. In patient K. by Duke method time of bloodless is norm. What is the norm?

a) to 1 minute; b) to 3 minutes; c) to 5 minutes; d) to 10 minutes; e) to 15 minutes

6. In patient D. by Duke method time of bloodless is norm. What is the norm?

a) 0-3 minutes; b) 1-3 minutes; c) 0-5 minutes; d) 1-15 minutes; e) 1-10 minutes

7. What is the time of clotting by Ly-Wait quantity iorm?

a) not more than 3 minutes; b) 1-3 minutes; c) 5-10 minutes; d) to 5 minutes; e) to 10 minutes

8. What is the time of plasma recalcification in norm?

a) 1-3 second; b) to 3 second; c) to 5 second; d) 5-10 second; e) 60-120 second

9. What is the concentration of fibrinogen in norm?

a) not more than 3 g/L; b) 1-3 g/L; c) 2-4 G/L; d) 2-4 g/L; e) to 5 g/L

10. What substances is the secondary anticoagulant?

a) Antithrombin-III; b) Protein C; c) Heparin; d) Antithrombin-I; e) Alpha 2-antitripsin

11. What substances is the primary anticoagulant?

a) Antithrombin-I; b) Antithrombin-III; c) Kinin; d) Kallikrein; e) Globulins

12. From what factors beginning fibrinolysis?

a) III; b) V; c) VII; d) X; f) XII

13. What factor do not take place in fibrinolysis?

a) III; b) V; c) VIII; d) X; e) XII

Real-life situations to be solved:

1. The quantity of platelets in blood is 100,0 x 10⁹/L. Is it norm? What is the clotting mechanism in this patient?

2. The quantity of platelets in blood is 100,0 x 10⁹/L. What is the breach in this patient?

3. The quantity of platelets in blood is 340,0 x 10⁹/L. What is the breach in this patient?

4. Three-years old patient S. has Duke time 2 minutes. Tomorrow will be the operation on his finger. Do this patient need addition medicines, which correct hemostasis?

5. What is you opinion about the hemostasis of patient F. if you know that clotting time is 8 minutes, Dukes time is 2 minutes, quantity of platelets is 190 x 10⁹/L?

6. What are You opinion about the hemostasis of patient T. if You know that in thromboelastogramm time of bloods’ beginning clot – 10 minutes, time of thromb producing – 7 minutes, maximum amplitude – 52 mm?

7. You know that time of clotting by Ly-Wait is 6 minutes; time of plasma recalcification is 65 seconds; thrombotest of V degree; thromboplastin time is 12 seconds; thromboplastin index is 100 %; concentration of fibrinogen is 3,2 g/L; tolerancy of plasma to heparin is 8 minutes; heparin time is 57 seconds; fibrinolysis is 18 %. What is you opinion about the secondary hemostasis of patient L.?

8. You know that time of clotting by Ly-Wait is 3 minutes; time of plasma recalcification is 56 seconds; thrombotest of VII degree; thromboplastin time is 9 seconds; thromboplastin index is 133 %; concentration of fibrinogen 4 g/L; tolerancy of plasma to heparin is 6 minutes; heparin time is 50 seconds. What is You opinion about the secondary hemostasis of patient W.?

9. You know that time of clotting by Ly-Wait is 12 minutes; time of plasma recalcification is 132 seconds; thrombotest of III degree; thromboplastin time is 17 seconds; thromboplastin index is 88 %; concentration of fibrinogen is 2,1 g/L; tolerancy of plasma to heparin is 11 minutes; heparin time is 60 seconds. What is you opinion about the secondary hemostasis of patient J.?

10. What is the difference between primary and secondary anticoagulants?

 Answers for the Self-Control

1. d;  2. c; 3. e; 4. e; 5 b; 6. a;. 7. c;  8. e;  9. d;  10. d;  11. b; 12. e; 13. a

 

1 real-life situation – It isn’t norm. Norm is (180,0-320,0) x 10⁹/L. Clotting of blood is breaches (aggregation of platelets).

2 real-life situation – It is thrombocytopenia – decrease the quantity of platelets.

3 real-life situation – It is thrombocytosis – increase the quantity of platelets.

4 real-life situation – No, they do not, because the Duke’s time is to 3 minutes to all aged groups.

5 real-life situation – The hemostasis system is norm (clotting time is 5-10 minutes; Dukes time is to 3 minutes, quantity of platelets is 180-320 G/L.)

6 real-life situation – It is norm, because iorm time of bloods’ beginning clot is 8-12 minutes; time of thromb producing is 5-8 minutes; maximum amplitude is 45-60 mm.

7 real-life situation – The secondary hemostasis iorm, because in norm time of clotting by Ly-Wait – 5-10 minutes; time of plasma recalcification – 60-120 seconds; thrombotest – IV, V, VI degree; thromboplastin time – 12-15 seconds; thromboplastin index – 80-105 %; concentration of fibrinogen – 2-4 g/L; tolerancy of plasma to heparin – 6-11 minutes; heparinic time – 50-60 seconds; fibrinolysis – 15-20 %.

8 real-life situation – The secondary hemostasis in abnorm – it is hypercoagulation, because iorm time of clotting by Ly-Wait – 5-10 minutes; time of plasma recalcification – 60-120 seconds; thrombotest – IV, V, VI degree; thromboplastin time – 12-15 seconds; thromboplastin index – 80-105 %; concentration of fibrinogen – 2-4 g/L; tolerancy of plasma to heparin – 6-11 minutes; heparinic time – 50-60 seconds.

9 real-life situation – The secondary hemostasis in abnorm – it is hypocoagulation, because iorm time of clotting by Ly-Wait – 5-10 minutes; time of plasma recalcification – 60-120 seconds; thrombotest – IV, V, VI degree; thromboplastin time – 12-15 seconds; thromboplastin index – 80-105 %; concentration of fibrinogen – 2-4 g/L; tolerancy of plasma to heparin – 6-11 minutes; heparinic time – 50-60 seconds.

10 real-life situation – Primary anticoagulants are produce and present all time in plasma and secondary anticoagulants form in a case of blood clotting.

SOME LABORATORY TESTS DONE FOR INVESTIGATING BLEEDING DISORDERS

(1) Clotting time – (Method of Lee and White). Without letting tissue juice to contaminate the blood, venous blood is withdrawn into a clean, dry, all glass syringe with a wide bore needle. A stopping watch is started the moment blood appears in the syringe and blood coagulation time is recorded from the moment. The needle is detached and 1 ml of blood is delivered into each of the four dry, chemically clean glass tubes with a size 10 by 1 cm. These tube should have been previously placed in a container of water (water bath maintained at 37 °C).

After 3 minutes have elapsed, each of these tubes is lifted from the water bath and is tilled very gently. The clotting time is taken when the tube can be inverted without its contents spilling. The clotting time for each tube is recorded separately and the clotting time is reported as the average of the four tubes. The normal clotting time by this method is 4 to 10 minutes. Clotting time is prolonged in hemophilia and other conditions associated with a deficiency of clotting factors. It remains normal in purpura because in this conditions there are no coagulation defects.

(2) Prothrombin time – (Quick’s one-stage method). Oxalated plasma of the patient is obtained and to it is added an excess of thromboplastin, which is usually an emulsion of rabbit’s brain. Calcium chloride solution is added. The time taken for clotting of the plasma to occur after the addition of calcium chloride is noted; it is termed as the prothrombin time and normally it is 12 to 14 seconds. The prothrombin time is increased in hypoprothrombinemia. However, the test is not specific for prothrombin deficiency and deficiencies of factors V and X and of fibrinogen also increase its duration.

(3) Bleeding time – (Duke’s method). When injury to a small vessel produces only a small defect, the platelet plug, i.e. the white thrombus alone can cause hemostasis. This is the basis for the bleeding time that is employed clinically to distinguish hemostatic deficiency caused by abnormalities of blood capillary wall from those caused by coagulation defects. In doing the test a standardized cut is made usually into the skin of the car lobule. The blood, which flows, is absorbed by a filter paper every 15 seconds. When the filter paper remains unstained on touching the wounded area, it shows that bleeding has stopped. The time taken by the bleeding to stop after the cut is made is the bleeding time. This test is simple but crude. In the majority of normal persons, bleeding lime ranges between 1 to 5 minutes. Bleeding time is prolonged in purpuras.

(4) Platelet count – This is normally 180,000 to 320,000 per cu mm of blood. Bleeding usually occurs when the count falls to 50,000 platelets per cu mm or below.

(5) Plasma fibrinogen level – This is found to be low in conditions associated with excessive fibrinolysis. In this condition the presence of fibrinogen degradative products (FDP) in the plasma and urine can be demonstrated.

(6) Assay of clotting factors – Factors V, VIII, IX, etc. can be quantitatively assessed in the blood.

(7) Capillary fragility test – A strong positive pressure (by inflating sphygmomanometercuff) or a negative pressure (by applying suction cups) is applied and the number of minute petechial hemorrhages (petechiac) is counted in the skin; their number is found to be much increased in conditions associated with an increased capillary fragility, e.g. purpuras.

METHODS OF KEEPING BLOOD FLUID IN VITRO – In order for the blood to be kept fluid, certain methods are employed.

These include the following:

(i) Addition of heparin. Its mechanism of anticoagulant action has been already described.

(ii) Addition of substances which take up Ca²⁺, e.g. citrate, oxalate, ethylene-diamine-tetra-acetic acid or EDTA. The last compound is an example of the chelating