Vessel-platlet hemostasis.

June 3, 2024
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Vessel-platlet hemostasis.

Physiology of blood clotting.

Anticlotting mechanisms and fibrinolysis

 

1. Common characteristic of hemostasis system

Hemostasis is a complex process that prevents or terminates blood loss from a disrupted intravascular space. Four major physiologic events participate, both in sequence and interdependently, in the hemostatic process. Vascular constriction, platelet plug formation, fibrin formation, and fibrinolysis occur in that general order, but the products of each of these four processes are interrelated in such a way that there is a continuum and multiple reinforcements. The process is shown schematically.

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http://www.clevelandclinicmeded.com/medicalpubs/diseasemanagement/hematology-oncology/bleeding-disorders/images/bleeding-disorders-fig1_large.jpg

 In the normal blood vessel, endothelial cells function to prevent clotting. They interfere with platelet recruitment by inactivating adenosine diphosphate (ADP). Endothelial cells also release several products that inhibit the coagulation process. Heparan sulfate acts to catalyze the inhibition of thrombin by antithrombin. Thrombomodulin down-modulates the coagulation process through the activation of protein C. Prostaglandin I2 (prostacyclin) inhibits platelet aggregation, as does nitric oxide (in addition to its vasodilator effects).

Vascular Constriction

When a vessel is injured, several processes are initiated. Vasoconstriction is the initial vascular response to injury. It is more pronounced in vessels with medial smooth muscles, but occurs even at the capillary level. It is dependent upon local contraction of smooth muscle that has a reflex response to various stimuli. The initial vascular constriction occurs before any platelet adherence to the site of injury. Adherence of endothelial cells to adjacent endothelial cells may be sufficient to cause cessation of blood loss from the intravascular space. Vasoconstriction is subsequently linked to platelet plug formation. Thromboxane A2 (TXA2), which is derived from the release of arachidonic acid from platelet membranes during aggregation, is a powerful vasoconstrictor. Endothelin synthesized by injured endothelium is also a vasoconstrictor. Serotonin, 5-hydroxytryptamine (5-HT), released during platelet aggregation, is another vasoconstrictor, but it has been shown that when platelets have been depleted of serotonin in vivo, constriction is not inhibited. Bradykinin and fibrinopeptides in the coagulation schema also are capable of contracting vascular smooth muscle. Some patients with mild bleeding disorders and a prolonged bleeding time have, as their only abnormality, capillary loops that fail to constrict in response to injury.

A lateral incision in a small artery may remain open because of physical forces, whereas a similarly sized vessel that is completely transected may contract to the extent that bleeding ceases spontaneously. The vascular response to injury also is affected by the contribution of pressure provided by surrounding tissues. Bleeding from a small venule ruptured by trauma in the thigh of an athlete may be negligible because of the compressive effect of the surrounding muscle. In the same individual, bleeding from a similar vessel in the nasal mucosa may be significant. When there is low perivascular pressure, as seen in patients with muscle atrophy accompanying aging, in patients on prolonged steroid therapy, and in patients with Ehlers-Danlos syndrome, bleeding tends to be more persistent. Vascular abnormalities, such as hereditary hemorrhagic telangiectasia, may predispose the patient to bleeding from the involved region.

 

Vessel-platelets hemostasis

PLATELETS OR THROMBOCYTES

Platelet Function

Platelets are 2 to 4 um in diameter anucleate fragments of megakaryocytes with normal circulating numbers falling between 150,000 and 400,000/uL. Thrombopoietin is the predominant mediator of platelet production, although other inflammatory mediators, such as interleukin (IL)-6 and IL-11, may play a role. Up to 30 of circulating platelets may be sequestered in the spleen and can be released in response to catecholamines. If not consumed in a clotting reaction, platelets are normally removed by the spleen with an average life span of 7 to 10 days.

http://biomed.brown.edu/Courses/BI108/BI108_2005_Groups/10/pictures/web/platelets3.jpg

 

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.

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STAGES IN PLATELET DEVELOPMENT

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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.

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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:

                   I.                       . 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.

Stock Illustration - platelet. fotosearch  - search clipart,  illustration,  drawings and vector  eps graphics images

Stock Photograph - blood platelet.  fotosearch - search  stock photos,  pictures, images,  and photo clipart

 

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.

 

                II.                       . 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.

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

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

               V.                       . 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 vesselplatelets 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.)

Platelets play an integral role in hemostasis along two pathways: by forming a hemostatic plug and by contributing to thrombin formation. Platelets, which normally do not adhere to each other or to the vessel wall, form a plug that stops bleeding when vascular disruption occurs. Injury to the intimal layer in the vascular wall exposes subendothelial collagen to which platelets adhere within 15 seconds of the traumatic event. This requires von Willebrand factor (vWF), a protein in the subendothelium that is lacking in patients with von Willebrand’s disease. vWF binds to glycoprotein (GP) I/IX/V on the platelet membrane. Platelet adhesion also is mediated by an interaction between collagen in the subendothelium and GP IaIIa on the platelet surface. Following adhesion, the platelets expand and develop pseudopodal processes and also initiate a release reaction that recruits other platelets from the circulating blood to seal the disrupted vessel. Up to this point, this process is known as primary hemostasis, and the aggregation is reversible and is not associated with secretion. Heparin does not interfere with this reaction, which is why hemostasis can occur in the heparinized patient. ADP and serotonin are the principal mediators in this process of aggregation. Various prostaglandins have opposing activities. Arachidonic acid released from platelet membranes is converted by cyclooxygenase to prostaglandin G2 (PGG2) and then to prostaglandin H2 (PGH2), which, in turn, is converted to TXA2. TXA2 has potent vasoconstriction and platelet aggregation effects. The arachidonic acid may also be shuttled to adjacent endothelial cells and converted to prostacyclin (PGI2), which is a vasodilator and acts to inhibit platelet aggregation. Platelet cyclooxygenase is irreversibly inhibited by aspirin and reversibly blocked by nonsteroidal anti-inflammatory agents, but is not affected by COX-2 inhibitors. In the second wave of platelet aggregation, a release reaction occurs in which several substances including ADP, Ca2 +, serotonin, TXA2, and granule proteins are discharged. Fibrinogen is a required cofactor for this process, acting as a bridge for the GP IIbIIIa receptor on the activated platelets during formation of a platelet plug. The release reaction results in compaction of the platelets into an “amorphous” plug, in a process that is no longer reversible. Thrombospondin, another protein secreted by the granule, stabilizes fibrinogen binding to the activated platelet surface and strengthens the platelet-platelet interactions. Platelet factor 4 (PF4) and thromboglobulin are also secreted during the release reaction, as is an inhibitor of plasminogen activation. PF4 is a potent heparin antagonist and it blocks angiogenesis.

The second wave of platelet aggregation is inhibited by aspirin and nonsteroidal anti-inflammatory drugs (NSAIDs)(through the inhibition of cyclooxygenase), by cyclic adenosine monophosphate (cAMP), and nitric oxide. As a consequence of the release reaction, alterations occur in the phospholipids of the platelet membrane that allow calcium and clotting factors to bind to the platelet surface, forming enzymatically active complexes. The altered lipoprotein surface (sometimes referred to as platelet factor 3) catalyzes reactions that are involved in the conversion of prothrombin (factor II) to thrombin (factor Iia) by activated factor X (Xa) in the presence of factor V and calcium, and it is involved in the reaction by which activated factor IX (IXa), factor VIII, and calcium activate factor X. Platelets may also play a role in the initial activation of factors XI and XII.

If congenital abnormalities exist, they can result in abnormal aggregation, as a result of effects on either the “first wave” of aggregation (the intrinsic ability of platelets to aggregate) or the “second wave” of the process (granule release).

d) Investigation of vesselplatelets 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.)

 

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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.)

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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.

Coagulation

Factor VIIIa combines with factor IXa (generated by the extrinsic Xase as above or by factor XIa formed by thrombin or factor XIIa action on factor XI on the activated platelet membrane) to form the intrinsic factor Xase, which is responsible for the bulk of the conversion of factor X to Xa. Intrinsic Xase (the VIIIa-IXa complex) is about 50 times more effective at catalyzing factor X activation than is the extrinsic (TF-VIIa) Xase complex and five to six orders of magnitude more effective than is factor IXa alone.

Factor Xa combines with factor Va, also on the activated platelet membrane surface to form the prothrombinase complex, which is responsible for converting prothrombin to thrombin. As with the Xase complex, the prothrombinase is significantly more effective at catalyzing its substrate than is factor Xa alone. Once formed, thrombin leaves the membrane surface and converts fibrinogen by two cleavage steps into fibrin and two small peptides termed fibrinopeptides A and B. Removal of fibrinopeptide A permits end-to-end polymerization of the fibrin molecules, whereas cleavage of fibrinopeptide B allows side-to-side polymerization of the fibrin clot. This latter step is facilitated by thrombin-activatable fibrinolysis inhibitor (TAFI), which acts to stabilize the resultant clot.

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The coagulation system is exquisitely regulated. In addition to clot formation that must occur to prevent bleeding at the time of vascular injury, two related processes must exist to balance propagation of the clot before the entire vascular bed is thrombosed in response to a local insult. First, there is a feedback inhibition on the coagulation cascade, which deactivates the enzyme complexes leading to thrombin formation. Second, mechanisms of fibrinolysis allow for breakdown of the fibrin clot and subsequent repair of the injured vessel with deposition of connectivetissue.

Tissue factor pathway inhibitor (TFPI) blocks the extrinsic factor Xase complex (TF-VIIa), eliminating this catalyst’s production of factors Xa and IXa. TFPI is normally present in the circulation at low concentrations (~2.5 nmol/L), and additional inhibitor is present on the endothelium and can be released in response to heparin. Antithrombin III (AT-III) effectively neutralizes all of the procoagulant serine proteases and only weakly inhibits the TF-VIIa complex. The primary effect is to quench the production of thrombin. A third major mechanism of inhibition of thrombin formation is the protein C system. Upon its formation, thrombin binds constitutively to thrombomodulin and activates protein C to activated protein C (APC), which then forms a complex with its cofactor, protein S, on a phospholipid surface. The APC-protein S complex cleaves factors Va and VIIIa so they are no longer able to participate in the formation of Xase or prothrombinase complexes. Of interest is an inherited form of factor V that carries a genetic mutation, called factor V Leiden, that is resistant to cleavage by APC, thereby remaining active (procoagulant). Patients with factor V Leiden are predisposed to venous thromboembolic events. As a result of the three systems described above, feedback inhibition of thrombin formation exists at upstream, intermediate, and downstream portions of the coagulation cascade to “turn off” thrombin formation once the procoagulant sequence is initially activated.

The same thrombin-thrombomodulin complex that leads to formation of activated protein C also activates TAFI. In addition to clot stabilization, removal of the terminal lysine on the fibrin molecule by TAFI also renders the clot more susceptible to lysis by plasmin. Degradation of fibrin clot is accomplished by plasmin, a serine protease derived from the proenzyme plasminogen. Plasmin formation occurs as a result of one of several plasminogen activators. Tissue plasminogen activator (tPA) is made by the endothelium and other cells of the vascular wall and is the main circulating form of this family of enzymes. tPA is selective for fibrin-bound plasminogen so that endogenous fibrinolytic activity occurs predominately at the site of clot formation. The other major plasminogen activator, urokinase plasminogen activator (uPA), also produced by endothelial cells as well as by urothelium, is not selective for fibrin-bound plasminogen. Because of the complex nature of hemostasis, potential interference in the process can occur at many levels. Platelet number or function can be insufficient to adequately support coagulation. Abnormalities in platelet function may be caused by either endogenous (e.g., uremia) or exogenous (e.g., aspirin or other antiplatelet agents) factors. Alternatively, abnormalities in the clotting factors may underlie a problem of abnormal bleeding. As with platelet dysfunction, the cause may be an intrinsic defect in one of the factors (detailed later in this chapter) or may result from pharmacotherapy.

 

2. Evaluation 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.)

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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.)

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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

 

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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. In norm 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) ProstacydinIts 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.

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.

 

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 VITROIn 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

Evaluation of coagulation:

A)             Coagulogramme

Rate coagulation status can be based on coagulation, which includes the following indicators:

1. Time coagulation (by Lee-White).

2. Recalcification time of plasma.

3. Trombotest.

4. Prothrombin (thromboplastin) time.

5. Prothrombin (thromboplastin) code.

6. The concentration of fibrinogen.

7. Tolerance plasma heparin.

8. Heparyn’s time.

9. Fibrinolysis.

1. Time coagulation (by Lee-White) – this time from the taking of blood from a vein before the advent of clot in a test tube, which is located in a water bath at 37°C. In healthy people, clotting time ranges from 5 to 10 minutes. Clotting time less than 5 minutes. evidence of increased coagulation.

2. Plasma recalcification time – this time after plasma coagulation dekaltsynovanoyi adding to it a standard amount of calcium chloride. Normal is 60-120 seconds. Recalcification time more than 120 seconds indicates a reduced coagulation.

3. Trombotest – gives a subjective assessment of the fibrin clot formed during centering plasma oxalate in a solution of calcium chloride. It is possible to observe the seven degrees of coagulation:

I – Opalescence,

II – small grains of fibrin,

III – flakes of fibrin,

IV – threads of fibrin;

V – net of fibrin strands,

VI – loose fibrin clot;

VII – dense fibrin clot.

fibrinnetwork

The first three stages are observed in hypocoagulation, IV, V, VI degree attesting normal coagulation.

4. Prothrombin (thromboplastin) time – this time the subject of plasma coagulation atadding her tissue thromboplastin. Thus starts the conversion of prothrombin to thrombin. The normal prothrombin time is 12-15 seconds. Increased prothrombin time indicates a decrease in coagulation.

5. Prothrombin (thromboplastin) index – a ratio of prothrombin time donor and the subject person, expressed as a percentage. Normal ranges from 80 to 105%. The increase in prothrombin index indicates an increase in coagulation.

6. The concentration of fibrinogen iormal is 2-4 g / l.

7. Tolerance plasma to heparin – a clotting time of plasma by adding to it mixture heparin with Ca2+. Normally, it is 6-11 minutes.

8. Heparyn’s time – a time of coagulation of tissue thromboplastin-activated plasma by adding thereto heparin. Normal ranges from 50 to 60 seconds.

Figures 7 and 8 show the interaction of coagulation factors and anticoagulant – heparin. With their minimal sense is the need for anticoagulants.

9. Fibrinolysis. This indicator assesses the fibrinolytic system of blood flow from lysis of thrombus over time. By using M.A.Kotovschykovoyi, B.I.Kuznyka fibrinolysis iormal is 15-20%. Lower than 15% indicates a reduction in fibrinolysis.

B) Tromboelastography.

This is a graphic record of the process of blood coagulation and fibrinolysis.

 

 

R – time from the beginning of the fluctuations of the cell to the formation of the first fibrin strands. Characterizes the phase of coagulation.

K – the time since the beginning of clot formation to achieve a fixed level of its density. 3 Displays the phase of blood coagulation.

MA- maximum amplitude – displays the maximum density of the bunch. 80% of the number of MA and properties of platelets (the ability to aggregate), 20% – the number of formed fibrin.

Short review:

Thrombocytes (Platelets)

*               Platelets form in bone marrow by following steps, myeloid stem cells eventually become megakaryocytes whose cell fragments form platelets

*               Short life span – 5 to 9 days in bloodstream

*               Platelets release ADP and other chemicals needed for platelet plug formation

 

 

 

 

Hemostasis

*               Hemostasis – stoppage of bleeding in a quick & localized fashion when blood vessels are damaged

*                Prevents hemorrhage (loss of a large amount of blood)

*               Three major steps of Hemostasis:

1.                 Vascular spasm

2.                 Platelet Plug Formation

*                                        Aggregation and adhesion of platelets

3.                 Blood Clotting

*                                        Fibrin threads entangle platelets and RBCs to form blood clot

 

Vascular Spasm

*               Damage to blood vessel stimulates pain receptors

*               Reflex contraction of smooth muscle of small blood vessels

*               Can reduce blood loss for several hours, allowing other mechanisms to take over

*               Effective only for small blood vessels or arterioles, not major arteries

 

Platelet plug formation

*               Platelet Plug Formation steps:

1.                 Platelet Adhesion

*                                        Platelets stick to exposed collagen of vessel

2.                 Platelet Release Reaction

*                                        Platelets extend projections

*                                        Platelets release Thromboxane A2,  Serotonin & ADP activating other platelets

3.                 Platelet Aggregation

*                                        Platelets stick together forming a platelet plug

 

1) Platelet Adhesion

*               Platelets stick to exposed collagen underlying damaged endothelial cells in vessel wall

2) Platelet Release Reaction

*               Platelets activated by adhesion

*               Extend projections to make contact with each other

*               Release Thromboxane A2,  Serotonin  & ADP activating other platelets

*                Serotonin & Thromboxane A2 are vasoconstrictors decreasing blood flow through the injured vessel.

*                ADP causes stickiness

 

 

3) Platelet Aggregation

*               Activated platelets stick together and activate new platelets to form a mass called a platelet plug

*               Plug reinforced by fibrin threads formed during clotting process

 

Platelet plug formation

Blood Clotting

*               Blood drawn from the body thickens into a gel if not mixed with anticoagulant

*                Blood separates into liquid (serum) and a clot of insoluble fibers (fibrin) in which the blood cells are trapped

*               Substances required for clotting: 

*                Ca+2

*                Clotting Factors (enzymes made by liver)

*                Substances released by platelets or damaged tissues

*               Clotting is a cascade of reactions

*                Each clotting factor activates the next, in a specific sequence, resulting in the formation of fibrin threads

Coagulation

*               A set of reactions in which blood is transformed from a liquid to a gel

*               Coagulation follows intrinsic and extrinsic pathways

*               Common Pathway:   The final three steps

*                Prothrombinase (Prothrombin activator) is formed from Factor X

*                Prothrombinase converts Prothrombin into Thrombin

*                Thrombin catalyzes polymerization of Fibrinogen into a Fibrin mesh

*               

 

 

Two Pathways to Prothrombin Activator

*               May be initiated by either the intrinsic or extrinsic pathway

*                Triggered by tissue-damaging events

*                Involves a series of procoagulants

*                Each pathway cascades toward Factor X

*               Once Factor X has been activated, it complexes with Ca+2 ions, PF3, and Factor V to form Prothrombin activator (Prothrominase)

Coagulation Phase of Hemostasis

 

Coagulation Pathway

*               Prothrombinase & Ca+2 catalyze the conversion of Prothrombin to Thrombin

*               Thrombin & Ca+2 catalyze the polymerization of Fibrinogen into Fibrin

*                Insoluble fibrin strands form the structural basis of a clot

*                Fibrin causes RBCs and Platelets to become a gel-like plug

*               Thrombin & Ca+2 also activate Factor XIII (F13) that:

*                Cross-links fibrin mesh

*                Strengthens and stabilizes the clot

 

Coagulation Pathway

 

Clot Dissolution

*               Inactive plasminogen becomes plasmin, a fibrinolytic enzyme

*               Plasmin dissolves small clots at site of a completed repair  

*               Clot formation remains localized

*                blood flow disperses clotting factors

*               Basophils release heparin (anticoagulant), preventing inappropriate clots

 

Intravascular Clotting

*               Thrombosis

Clot (thrombus) formed in an unbroken blood vessel

*                                        Attached to rough inner lining of blood vessel

*                                        Blood flows too slowly (stasis) allowing clotting factors to build up locally & cause coagulation

*                May dissolve spontaneously or dislodge & travel

*               Embolus – free floating clot in the blood

*               Low dose aspirin blocks synthesis of thromboxane A2 & reduces inappropriate clot formation,

*                Helps to prevent strokes, myocardial infarctions.

 

 

 

References:

 

1. Review of Medical Physiology // W.F.Ganong. – 24th edition, 2012.

2. Textbook of Medical Physiology // A.C.Guyton, J.E.Hall. – Eleventh edition, 2005.

 

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