CONCEPT OF HEMOSTASIS. DISORDERS OF HEMOSTASIS.

 

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. Hemostasissystem 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, 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 activitybegin the first after the destroyed of vessels. Secondary hemostasis add after that in leucocytes, enzymes and nonenzymes components of plasma – clotting 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-specificpluripotent 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 μmand 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) Themegakaryocyte 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 – Villibrandtfactor, 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 activateprofibrinolisin, 11 – inhibitir of thromboplastin, 12 – antilighting factor).

 

 

 AMP, thromboxane A₂, Von-Willebrand factor, fibronectin, thrombospondin and several other platelet factors including a heparieutralizing factor-4.  

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 thephosphorylalion of myosin light chains. The latter interact with actin to facilitate granule movement and platele shape change, (g) Another product of membran phospholipid isdiacylglycerol which brings about secretion of granules. The contents of the granules which are poured into the plasma arc heparinase, Ca²⁺, adrenaline, kinins, fibrinogcn. factorVa

(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 speciallyeffective 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 hemostaticplugs, 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 endoperoxidenamely 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 whichthrombosthenin 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 andthromboxane 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 can not 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 a

ii)                dhesion 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 thromboplastinantecedent; 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 orthrombinasa 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-ibecomes 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 ofprothrombin 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 tissuethromboplastin 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. Russelviper 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.)

 

 

Hemostasis or haemostasis (from the Ancient Greek: αἱμόστασις haimóstasis “styptic (drug)”) is a process which causes bleeding to stop, meaning to keep blood within a damaged blood vessel (the opposite of hemostasis is hemorrhage). It is the first stage of wound healing. Most of the time this includes blood changing from a liquid to a solid state. All situations that may lead to hemostasis are portrayed by the Virchow’s triad. Intact blood vessels are central to moderating blood’s tendency to clot. The endothelial cells of intact vessels prevent blood clotting with a heparin-like molecule and thrombomodulin and prevent platelet aggregation with nitric oxide and prostacyclin. When endothelial injury occurs, the endothelial cells stop secretion of coagulation and aggregation inhibitors and instead secrete von Willebrand factor which initiate the maintenance of hemostasis after injury. Hemostasis has three major steps: 1) vasoconstriction, 2) temporary blockage of a break by a platelet plug, and 3) blood coagulation, or formation of a clot that seals the hole until tissues are repaired.

Aggregation of thrombocytes (platelets). Platelet rich human blood plasma (left vial) is a turbid liquid. Upon addition of ADP, platelets are activated and start to aggregate, forming white flakes (right vial)

Hemostasis occurs when blood is present outside of the body or blood vessels. It is the instinctive response for the body to stop bleeding and loss of blood. During hemostasis three steps occur in a rapid sequence. Vascular spasm is the first response as the blood vessels constrict to allow less blood to be lost. In the second step, platelet plug formation, platelets stick together to form a temporary seal to cover the break in the vessel wall. The third and last step is called coagulation or blood clotting. Coagulation reinforces the platelet plug with fibrin threads that act as a “molecular glue”.^[1] Platelets are a large factor in the hemostatic process. They allow for the creation of the “platelet plug” that forms almost directly after a blood vessel has been ruptured. Within seconds of a blood vessel’s epithelial wall being disrupted platelets begin to adhere to the sub-endothelium surface. It takes approximately sixty seconds until the first fibrin strands begin to intersperse among the wound. After several minutes the platelet plug is completely formed by fibrin.^[2] Hemostasis is maintained in the body via three mechanisms:

1. Vascular Spasm – Damaged blood vessels constrict. Vascular spasm is the blood vessels’ first response to injury. The damaged vessels will constrict (vasoconstrict) which reduces the amount of blood flow through the area and limits the amount of blood loss. This response is triggered by factors such as a direct injury to vascular smooth muscle, chemicals released by endothelial cells and platelets, and reflexes initiated by local pain receptors. The spasm response becomes more effective as the amount of damage is increased. Vascular spasm is much more effective in smaller blood vessels.^[1]

2. Platelet plug formation – Platelets adhere to damaged endothelium to form platelet plug (primary hemostasis) and then degranulate. This process is regulated through thromboregulation. Platelet Plug Formation: Platelets play one of the biggest factors in the hemostatic process. Being the second step in the sequence they stick together (aggregation) to form a plug that temporarily seals the break in the vessel wall. As platelets adhere to the collagen fibers of a wound they become spiked and much stickier. They then release chemical messengers such as adenosine diphosphate (ADP), serotonin and thromboxane A2. These chemicals are released to cause more platelets to stick to the area and release their contents and enhance vascular spasms. As more chemicals are released more platelets stick and release their chemicals; creating a platelet plug and continuing the process in a positive feedback loop. Platelets alone are responsible for stopping the bleeding of unnoticed wear and tear of our skin on a daily basis.^[3]

The second stage of Hemostasis involves platelets that move throughout the blood. When the platelets find an exposed area or an injury, they begin to form what is called a platelet plug. The platelet plug formation is activated by a glycoprotein called the Von Willebrand factor (vWF), which are found in the body’s blood plasma. When the platelets in the blood are activated, they then become very sticky so allowing them to stick to other platelets and adhere to the injured area.^[4][5]

There are a dozen proteins that travel along the blood plasma in an inactive state and are known as clotting factors. Once the platelet plug has been formed by the platelets, the clotting factorsbegin creating the platelet plug. When this occurs the clotting factors begin to form a collagen fiber called fibrin. Fibrin mesh is then produced all around the platelet plug, which helps hold the fibrin in place. Once this begins, red and white blood cells become caught up in the fibrin mesh which causes the clot to become even stronger.

3. Blood coagulation – Clots form upon the conversion of fibrinogen to fibrin, and its addition to the platelet plug (secondary hemostasis). Coagulation: The third and final step in this rapid response reinforces the platelet plug. Coagulation or blood clotting uses fibrin threads that act as a glue for the sticky platelets. As the fibrin mesh begins to form the blood is also transformed from a liquid to a gel like substance through involvement of clotting factors and pro-coagulants. The coagulation process is useful in closing up and maintaining the platelet plug on larger wounds. The release of Prothrombin also plays an essential part in the coagulation process because it allows for the formation of a thrombus, or clot, to form. This final step forces blood cells and platelets to stay trapped in the wounded area. Though this is often a good step for wound healing, it has the ability to cause severe health problems if the thrombus becomes detached from the vessel wall and travels through the circulatory system; If it reaches the heart or brain it could lead to stroke, heart attack, or pulmonary embolism. However, without this process the healing of a wound would not be possible.

 

Hemostasis can be achieved in various other ways if the body cannot do it naturally (or needs help) during surgery or medical treatment. When the body is under shock and stress, hemostasis is harder to achieve. Though natural hemostasis is most desired, having other means of achieving this is vital for survival in many emergency settings. Without the ability to stimulate Hemostasis the risk of hemorrhaging is great. During surgical procedures the types of hemostasis listed below can be used to control bleeding while avoiding and reducing the risk of tissue destruction. Hemostasis can be achieved by chemical agent as well as mechanical or physical agents. Which hemostasis type used is determined based on the situation.

·         Debates still continue to rise on the subject of hemostasis and how to handle situations with large injuries. If an individual did acquire a large injury resulting in extreme blood loss, then ahemostatic agent alone would not be very effective. Medical professionals continue to debate on what the best ways to assist a patient in a chronic state are; however, it is universally accepted that hemostatic agents are the primary tool for smaller bleeding injuries.

Some main types of hemostasis used in emergency medicine include:

·         Chemical/topical– This is a topical agent often used in surgery settings to stop bleeding. Microfibriller collagen is the most popular choice among surgeons because it attracts the patients natural platelets and starts the blood clotting process when it comes in contact with the platelets. This topical agent requires normal hemostatic pathway to be properly functional.^[

·         Direct pressure or pressure dressing– This type of hemostasis approach is most commonly used in situations where proper medical attention is not available. Putting pressure and/or dressing to a bleeding wound only slows the process of blood loss, allowing for more time to get to an emergency medical setting. Soldiers use this skill during combat when someone has been injured because this process allows for blood loss to be decreased, giving the system time to start coagulation^.

·         Sutures and ties– Sutures are often used to close an open wound, allowing for the injured area to stay free of pathogens and other unwanted debris to enter the site; however, it is also essential to the process of hemostasis. Sutures and ties allow for skin to be joined back together allowing for platelets to start the process of hemostasis at a quicker pace. Using sutures results in a quicker recovery period because the surface area of the wound has been decreased.

·         Physical agents ( gelatin sponge )– Gelatin sponges have been indicated as great hemostatic devices. Once applied to a bleeding area, a gelatin sponge quickly stops or reduces the amount of bleeding present. These physical agents are mostly used in surgical settings as well as after surgery treatments. These sponges absorb blood, allow for coagulation to occur faster, and give off chemical responses that decrease the time it takes for the hemostasis pathway to start.^[10]

The body’s hemostasis system requires careful regulation in order to work properly; if the blood does not clot sufficiently, bleeding disorders such as hemophilia can result. Over-active clotting can also cause problems; thrombosis, where blood clots form abnormally, can potentially cause embolisms, where blood clots break off and subsequently become lodged in a vein or artery.

Hemostasis disorders can develop for many different reasons. They may be congenital, due to a deficiency or defect in an individual’s platelets or clotting factors. A number of disorders can be acquired as well, such as in HELLP syndrome, which is due to pregnancy, or Hemolytic-uremic syndrome (HUS), which is due to E. coli toxins.

The process of preventing blood loss from a vessel or organ of the body is referred to as hemostasis. The term comes from the Ancient Greek roots “heme” meaning blood, and “stasis” meaning halting; Put together means the “halting of the blood”.^[1] The origin of hemostasis dates back as far as ancient Greece; first referenced to being used in the Battle of Troy. It started with the realization that excessive bleeding inevitably equaled death. Vegetable and mineral styptics were used on large wounds by the Greeks and Romans until the takeover of Egypt around 332BC by Greece. At this time many more advances in the general medical field were developed based off the study of Egyptian mummification practice, which led to greater knowledge of the hemostatic process. It was during this time that many of the veins and arteries running throughout the human body were found and the directions in which they traveled. Doctors of this time realized if these were plugged, blood could not continue to flow out of the body. Nevertheless it took until the invention of the printing press during the fifteenth century for medical notes and ideas to travel westward, allowing for the idea and practice of Hemostasis to be expanded

There is currently a lot of research being conducted on hemostasis. The most current research is based on genetic factors of hemostasis and how it can be altered to reduce the cause ofgenetic disorders that alter the natural process hemostasis.

Von Willebrand disease is associated with a defect in the ability of the body to create the platelet plug and the fibrin mesh that ultimately stops the bleeding. New research is concluding that the von Willebrand disease is much more common in adolescence. This disease negatively hinders the natural process of Hemostasis causing excessive bleeding to be a concern in patients with this disease. There are complex treatments that can be done including a combination of therapies, estrogen–progesterone preparations, desmopressin, and Von Willebrand factor concentrates. Current research is trying to find better ways to deal with this disease; however, much more research is needed in order to find out the effectiveness of the current treatments and if there are more operative ways to treat this disease.

 

Disorders of Blood Vessels and Vascular Tissues:

 

 

The following bleeding disorders result from pathology in the vessel area itself, with secondary leakage of blood. Most have as their hallmark a visible and usually palpable skin lesion. Testing performed on patients with these bleeding disorders reveals normal coagulation and occasionally increased bleeding times.

(i) Autoimmune or allergic purpura occurs most commonly in children and young adults. Its hallmark is a perivascular inflammatory lesion with serosanguineous leakage into the skin and the submucous and serosal areas.

(a) The lesions characteristically are symmetrical and on proximal extremities. These are palpable lesions.

 

(b) The syndrome is associated with streptococcal infections and drugs (e.g., penicillin).

(c) Lesions in the bowel may cause gastrointestinal symptoms; joint lesions cause arthritis.

(d) No specific therapy is uniformly helpful. Prognosis is good except in the 5%-10% of patients who develop glomerulonephritis.

(ii) Purpura associated with infections may be due to embolic occlusion of the microvasculature (e.g., endocarditis) or to endothelial injury by the infectious agent (e.g., Rickettsia). Biopsy and culture of the material may be helpful.

(iii) Structural malformations of vessels and vascular tissues are associated with the following disorders.

(a) Scurvy is a condition caused by vitamin C deficiency; collagen synthesis is impaired as a result of this deficiency. Vessel walls with poor collagen support are pliable and easily ruptured.

·         Physical findings associated with scurvy include perifollicular petechiae, gum bleeding, and subperiosteal hemorrhages. Also, bleeding time usually is prolonged.

·         Therapy with 1 g/day of vitamin C rapidly corrects all bleeding.

(b) Hereditary hemorrhagic telangiectasia is an autosomal dominant disorder associated with abnormally thin vessel walls and impaired vascular contractility. Such vessels are markedly friable, liable to burst with trauma, and unable to contract appropriately for primary hemostasis.

·         Physical findings include small, nodular violaceous lesions on the lips, face, ears, tongue, and gastrointestinal mucosa; these lesions blanch upon pressure. Bleeding is common, especially gastrointestinal bleeding with resultant iron deficiency anemia.

·         Diagnosis involves the association of three factors: recurrent hemorrhage, multiple telangiectases, and familial occurrence.

(c) Steroid therapy diminishes collagen synthesis, resulting in a syndrome of vascular fragility and skin bleeding.

(iv) Miscellaneous Vascular Conditions:

(a) Paraproteinemias, including cryoglobulinemias and amyloidosis, are associated with skin bleeding. Diagnosis requires demonstration of the paraprotein.

 

(b) Senile purpura occurs in elderly individuals as a result of degeneration and loss of dermal collagen, elastin, and subcutaneous fat. This disorder is characterized by benign purpura of the arms, and it is thought to be caused by shearing injury to blood vessels from the hypermobility of the skin on the thinned underlying tissue.

 

Disorders of Platelets:

 

Platelets play a role in the primary arrest of bleeding; abnormalities in these coagulation components result in prolonged bleeding times and lead to hemorrhagic diathesis. Platelet abnormalities are classified according to disorders of number and function.

 

Types:

 

(1) Thrombocytopenia and (2) Thrombocytopathia.

(1) Thrombocytopenia (i.e., decreased numbers of platelets) is the most common cause of abnormal bleeding.

 

General Considerations:

(i) With a platelet count of 100,000/mm³, bleeding time (and clinical bleeding, if the hemostatic system is stressed) begins to prolong. Most individuals experience petechiae or purpura with platelet counts between 50,000 and 20,000/mm³. More serious spontaneous bleeding may occur with platelet counts less than 20,000/mm³, and such bleeding is a high risk with counts less than 10,000/mm³.

 

(ii) Thrombocytopenia is an indication for a marrow examination, which will reveal either the presence or absence of the platelet precursors, megakaryocytes. The presence of megakaryocytes indicates either peripheral destruction or pooling of platelets. The absence of megakaryocytes indicates platelet production problems.

 

Mechanisms:

(i) Impaired platelet production.

(a) Etiology: Megakaryocytes may be selectively suppressed by certain agents (e.g., thiazide diuretics and ethanol). A special case of thrombocytopenia due to impaired platelet production is ineffective thrombopoiesis associated with the megaloblastic hematopoiesis seen in vitamin B₁₂ and folate deficiencies. Megakaryocytes are present in marrow but are abnormal (megaloblastic) in morphology and function. Their platelets are abnormal and destroyed in the marrow.

(b) Diagnosis is confirmed by a bone marrow smear that reveals marrow megakaryocytic hypoplasia.

(c) Therapy/treatment involves removal of the offending agent, if possible, or treatment of the underlying disease. Patients have essentially normal platelet half-lives and should be transfused with exogenous platelets if they are thrombocytopenic and bleeding, Thrombocytopenia associated with vitamin B₁₂ or folate deficiency is rapidly corrected by therapy with the deficient vitamin.

(d) Associated conditions. Impaired platelet production is associated with aplastic anemia, myelophthistic processes with replacement of marrow by tumor or fibrosis, and certain rare congenital syndromes (e.g., rubella infection with absent radii).

(ii) Abnormal platelet pooling results when platelets are sequestered from the circulation. Splenic platelet sequestration is the most common cause of abnormal platelet pooling.

(a) Pathophysiology: Normally, the spleen holds one-third of the circulating platelet pool. As splenomegaly occurs, higher numbers of platelets are sequestered and, thus, unavailable for hemostasis. In very large spleens, as much as 90% of the platelet pool may be sequestered; however, platelets in the peripheral circulation do have normal survival times.

(b) Diagnosis of hypersplenism is suggested by a moderate thrombocytopenia (platelet counts below 40,000 are unusual), a bone marrow smear that reveals adequate marrow megakaryocytes, and evidence of significant splenic enlargement.

(c) Clinical features in such cases are dominated by the underlying illness causing the splenomegaly (e.g., cirrhosis with portalhypertension).

(d) Therapy/treatment usually is not required, although splenectomy may correct the problem. Transfused platelets are sequestered in the same ratio and are less effective than in hypoactive marrow states.

(iii) Increased peripheral destruction of platelets is the most common form of thrombocytopenia. Conditions involving increased platelet destruction are characterized by shortened platelet survival and increased numbers of marrow megakaryocytes. These disorders are characterized as either immune or non-immune thrombocytopenic purpura.

(a) Immune thrombocytopenic purpura:

·         Idiopathic thrombocytopenic purpura (ITP) is the prototypical immune-mediated thrombocytopenia; no apparent exogenous causes for platelet destruction exist.
Clinical features. The acute variant of ITP occurs in children between the ages of 2 and 6 years and often occurs after a nonspecific viral illness. The chronic variant occurs in young adults, more commonly in young women. All ITP patients show varying degrees of thrombocytopenia, which in some acute cases is severe (i.e., associated with platelet counts under 1000/mm³), and all show increased marrow megakaryocytes. Other blood findings and cell lines are normal. Patients present with mucocutaneous bleeding with petechiae, purpura, mucosal bullae, and excessive bleeding after trauma.

Diagnosis requires exclusion of associated illnesses (e.g., SLE) and thrombocytopenia that is induced by drugs (e.g., quinine). Platelet antibody techniques are available to demonstrate the abnormal presence of such antibody on platelets and in the plasma of these patients.

Clinical course. The acute childhood variant often runs its course and resolves within 4-8 weeks; the adult form is more chronic and demonstrates relapses and remissions.

Therapy/treatment for the acute childhood form usually involves protection from trauma and, in some cases, a short course of steroids. Treatment of adult ITP is more complex and protracted, initially, high doses of steroids are given, with complete remission achieved in about 35% of cases. Splenectomy often is necessary and is associated with complete remission in about 65% of cases. Refractory patients may require immunosuppressive therapy. Although platelet transfusions should not be withheld in ITP patients who are bleeding, such exogenous transfusions may be less efficacious than in other thrombocytopenic states due to the same short survival of the platelets.

Prognosis: The overall prognosis is good; only 2%-3% of ITP patients die after 5 years.

·         Other immune-mediated thrombocytopenias with known eliciting agents for platelet-associated antibody include: post-transfusion thrombocytopenia due to isoantibodies; drug-induced thrombocytopenia (e.g., quinidine-induced); sepsis-associated thrombocytopenia (the incidence of thrombocytopenia with sepsis may be as high as 70%); and SLE.

Therapy involves appropriately addressing the underlying disease. Steroids are of questionable value except in SLE-related thrombocytopenia. Exogenous platelets suffer the same enhanced destruction.

 

(b) Non-immune thrombocytopenic purpura occurs in the following conditions:

·         With infections (e.g., virus and malaria).

·         Following massive blood transfusion with banked blood that is platelet-poor.

·         As part of disseminated intravascular coagulation syndrome.

·         With cardiac-valve prostheses.

·         As part of the syndrome of thrombotic thrombocytopenic purpura.

(2) Thrombocytopathia involves platelets that are adequate iumber but unable to function properly in hemostasis and in the primary arrest of bleeding.

(i) Description: Thrombocytopathia is characterized by:

(a) Platelet-type mucocutaneous bleeding.

(b) Normal platelet counts but prolonged bleeding times.

(c) Demonstrable abnormalities in platelet function testing (e.g., aggregometry).

(ii) Etiology/Cause:

(a) Drug-related platelet dysfunction is the most common cause of abnormal platelet function.

·         Aspirin permanently acetylates platelet membranes, impairing the platelet prostaglandin synthesis required for proper platelet function. Such impaired platelets may prolong bleeding times and cause bruising and increased hemorrhage with trauma.

·         Other anti-inflammatory drugs (e.g., indomethacin) cause similar dysfunction but differ from aspirin in that their effects disappear when the agent is withdrawn.

(b) Uremia-associated dysfunction.

·         The mechanism is unclear, although new data implicate uremic toxins in the dis-aggregation of the high molecular weight polymers of factor VIII that are required for proper platelet function.

·         Therapy: In bleeding uremic patients, this lesion may respond to dialysis. New data suggest that the administration of high molecular weight forms of factor VIII (e.g., cryoprecipitates) may temporarily correct this lesion.

(c) Congenital forms of platelet dysfunction include Glanzmann’s thrombasthenia (an intrinsic platelet disorder) and von Willebrand’s disease (a congenital absence of the high molecular weight forms of factor VIII required for platelet aggregation).

Abnormalities of platelet function are characterized by clinical bleeding of varying severity. In most cases, patients present with mucocutaneous bleeding or excessive hemorrhage following surgery or trauma. A platelet count and careful examination of the peripheral smear is essential in the initial evaluation of patients with mucocutaneous bleeding. When examining the peripheral smear, it is important to evaluate the relative size of platelets. Large platelets may be seen as a result of accelerated marrow production of platelets attributable to a hemorrhagic event or recovery from bone marrow suppression as a result of infections or drugs. Large platelets are also encountered in the setting of patients with accelerated platelet turnover (idiopathic thrombocytopenic purpura).

The bleeding time (BT) test has also been widely utilized as a means of accessing primary hemostatic response (platelet-injured vessel wall interaction). Unfortunately, the BT is relatively insensitive and, in many cases, nonspecific with respect to identifying abnormalities of primary hemostasis. The major variables are the inherent differences between individuals performing the BT and the various BT devices. The introduction of BT devices designed to decrease the variability of the depth of the induced wound was a major advance over the traditional Ivy BT test. Despite the introduction of the newer devices, there remains substantial variability between individuals performing BTs as well as the possible complication of scar formation at the test site (typically, the anterior-lateral aspect of the arm).

There are several variables in the BT in addition to the technical aspects of performing the test. BTs tend to be longer in females and decrease with aging. One cosmetic complication frequently seen in elderly patients who have experienced extensive sun exposure is the formation of a somewhat symmetrical subepidermal hemorrhage, which is attributable to blood dissecting into the subepidermis as opposed to exiting onto the surface of the skin at the site of the BT incision. The BT is also affected by the hematocrit and platelet mass. Patients with chronic renal disease and decreased hematocrit often have a prolonged BT. Increasing the hematocrit to >30% often will correct a prolonged BT in a patient with chronic renal disease. Abnormalities of connective tissue (e.g., Ehlers-Danlos syndrome) may produce abnormal BTs.

The BT together with the Rumpel-Leede test were the first attempts to evaluate platelet/vascular response to injury. The Rumpel-Leede test involved inflating a blood pressure cuff midway between systolic and diastolic pressure and leaving the cuff on for a period of time, which was variable depending on the patient’s tolerance for the procedure. The arm distal to the blood pressure cuff was evaluated for the presence of petechiae.

Platelet aggregation is an important component of laboratory testing in a patient with clinical findings suggestive of a primary hemostatic abnormality. The addition of an agonist (e.g., ADP, epinephrine, or collagen) to normal platelet-rich plasma produces an aggregation pattern characterized by a biphasic response when epinephrine is used as the agonist. The primary wave results from the addition of exogenous epinephrine, and the secondary wave reflects the “release reaction” of the dense bodies. With release, granular components are excreted through the open canalicular system. Abnormalities of the release reaction may be seen in patients with storage pool disease (characterized by loss of platelet nucleotides and serotonin from the dense granules; Table 1⇓ ). Dense body storage pool abnormalities have been described in Hermansky-Pudlak, Chédiak-Higashi, and Wiskott-Aldrich syndromes and thrombocytopenia with absent radii. Patients with afibrinogenemia or Glanzmann thrombasthenia (abnormalities of the GP IIb-IIIa receptor) lack both primary and secondary responses to various platelet agonists. Glanzmann thrombasthenia is an autosomal recessive defect that frequently is encountered in patient populations in which there is a high incidence of consanguinity.

Hereditary disorders of platelet function.¹

 

There are numerous reports of patients with selectively impaired aggregation response to various platelet agonists. Lack of response to epinephrine has been reported in patients with decreased α₂ adrenergic receptors. Isolated collagen receptor defects have been reported (decreased platelet GP Ia). It is important to appreciate the variability one may see in platelet aggregation studies. Often a lack of a secondary response is attributable to drugs (classically aspirin) that inhibit cyclooxygenase. The pharmaceutical industry is intensively developing various inhibitors of ADP receptors (ticlopidine and clopidogrel) and IIb-IIIa receptors.

Antagonists of platelet IIb/IIIa receptors.¹

 

 

Targets for platelet inhibitors.

The activation of platelets occurs when platelets adhere to subendothelial components of the vessel wall. After adhesion, release of platelet granular contents leads to platelet aggregation. Various drugs have been used to inhibit platelet activation. Ticlopidine and clopidogrel inhibit the ADP-induced activation pathway. Aspirin irreversibly blocks cyclooxygenase enzyme (Cox-1). This prevents the generation thromboxane A₂(TXA2), which is a potent activator of platelets. Various inhibitors of GP IIb/IIIa complex prevent platelet aggregation. Among the inhibitory drugs that have been developed recently are Abciximab (c7E3 Fab fragments), Eptifibatide (a cyclic peptide), and two peptidomimetics (lamifiban and tirofiban). Oral inhibitors include xemilofiban, DMP 802, and SR 121787. These three drugs are oral inhibitors of platelet function. 

 

 

Other tests that have been used in evaluating platelet function include the prothrombin consumption test (a test to evaluate the platelet contribution of activated phospholipids), flow cytometry to quantify surface GPs, receptor occupancy, and electron microscopy for evaluating ultrastructural anatomy.

Several recently developed instruments have been designed to assess the global platelet response. Examples include Xylum^®, PFA-100^® (Dade-Behring), and test systems marketed by Array and Medtronics. The Xylum and PFA-100 represent instruments that attempt to simulate the in vivo response of platelets to vascular injury. In the case of the PFA-100, two collagen-impregnated membranes, one with ADP and the other with epinephrine, are used to evaluate the platelet response. The patient’s citrated blood sample is aspirated under high shear rates (5000–6000 dyn/cm²) through a 150-μm diameter aperture in the center of a collagen-impregnated membrane. The endpoint is obtained when the flow of blood ceases. This test system is extremely sensitive to the presence of aspirin (epinephrine abnormal/ADP normal). The PFA-100 may be used to monitor antiplatelet drug therapy. Other new instruments under development offer promise in the monitoring of patients who are increasingly exposed to a greater variety of platelet antagonists. The PFA-100 has been used to screen patients for von Willebrand disease (VWD) and has been very effective in identifying these patients.

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Increasingly, desmopressin (DDAVP) is being used to manage patients with abnormalities of primary hemostasis, e.g., VWD, patients exposed to aspirin, and cirrhotic patients with bleeding complications. DDAVP triggers the release of VWF from Weibel-Palade bodies of vascular endothelium. DDAVP has also been used in the management of patients with mild to moderate hemophilia A (deficiencies of factor VIII).

Recombinant human erythropoietin has been used to manage bleeding in uremic patients. In cases of severe thrombocytopenia or iatrogenic inhibition of platelet function, the use of platelet concentrates is indicated.

In renal failure patients with hemorrhagic complications, correction of the hematocrit to >30% often will alleviate bleeding problems. The red cell mass is instrumental in “marginating” platelets to the endothelial-blood interface. The proximity of platelets to endothelium facilitates the primary hemostatic response after vascular injury.

The laboratory tests for evaluation of platelet function have been discussed above. Evaluation of the coagulation pathways relies on four relative simple tests: the activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), and fibrinogen assays. In addition, the availability of a test to monitor D-dimer is of considerable value. D-Dimer is increased in fibrinolysis; in addition, it is used as a negative predictor to rule out deep vein thrombosis. Laboratories must carefully select the correct test (sensitivity) and apply this test in an appropriate clinical situation.

The APTT is a test that evaluates the intrinsic pathway of coagulation. The APTT reagents comprise an activator (e.g., ellagic acid, celite, or kaolin) and phospholipids. The phospholipids may be either synthetic or derived from animal tissue (e.g., rabbit brain). With the exception of factors VII and XIII, the APTT evaluates all of the coagulation factors. The most common cause of a prolonged APTT is incorrect collection of the blood sample. Most frequently, this is attributable to obtaining blood through an indwelling line that has been flushed with heparin. In addition, a traumatic venipuncture may produce an abnormal APTT. A polycythemic blood sample may yield an abnormal APTT because of the excess amount of citrate available to chelate calcium in a polycythemic blood sample. Other causes of a prolonged APTT include factor deficiencies [VWD, hemophilia A (factor VIII deficiency), and hemophilia B (factor IX deficiency)] and the presence of circulating anticoagulants (also known as inhibitors). The most common circulating anticoagulant is the lupus anticoagulant (LA). Antibodies to factor VIII are also encountered in both hemophilia A patients and adults who occasionally have an acquired autoantibody against factor VIII. Hereditary and acquired factor deficiencies often produce an abnormal APTT. Most reagent manufacturers provide reagents that will yield an abnormal APTT when the concentration of factor VIII is <30–35% (0.30–0.35 kilounits/L). However, there is substantial variability between reagents. There may also be lot-to-lot variability of APTT reagents from the same manufacturer. Therefore, it is imperative when any aspect of the “system” (e.g., reagent-instrument combination or collection tubes) is changed to recalculate the reference interval and the relative sensitivity of the system to factor deficiencies and heparin.

The PT is the most commonly performed test of hemostasis. The PT evaluates the extrinsic pathway of coagulation (factors VII, X, V, II, and fibrinogen). The PT is used to monitor patients on oral anticoagulant therapy. With the recent introduction of sensitive PT reagents, the use of the international normalized ratio has become the standard reporting format for PT results. Patients receiving oral anticoagulant therapy in most cases have a targeted therapeutic range of an international normalized ratio of 2.0–3.0. There are exceptions, including mechanical valves, patients who re-thrombose when in the therapeutic range of 2.0–3.0, and patients with anti-phospholipid antibody syndrome. The PT may be prolonged in patients with disseminated intravascular coagulation, liver disease, or vitamin K deficiency.

The TT is a simple test. Thrombin (bovine or human) is added to citrated plasma. There are two variations of the TT: one uses calcium and the other does not. In individuals with fibrinogens <1000 mg/L, the TT will be prolonged. Other causes of a prolonged TT include the presence of heparin in a blood sample, dysfibrinogenemias, antibodies to thrombin, and gammopathies (e.g., multiple myeloma and Waldenström macroglobulinemia).

A functional assay for fibrinogen is part of the initial analysis of patients with bleeding disorders. Often, the TT is not prolonged in patients with hypofibrinogenemia until it is <1000 mg/L. A discrepancy between the functional assay and antigenic assay is encountered in patients with dysfibrinogenemia.

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VWD is the most common inherited disorder of hemostasis. The incidence of VWD in the population is ∼1%. It is found in all ethnic groups, and in many cases, patients remain undiagnosed. VWD is an autosomal dominant disorder affecting both males and females. Before puberty, easy bruising and epistaxis are the most frequently encountered clinical presentations. At the time of puberty, the frequency of epistaxis tends to decrease. In affected females, the chief complaint becomes one of menorrhagia (73). It is estimated that ∼10% of hysterectomies performed in the United States are the result of underlying occult VWD. With appropriate diagnosis and patient management, many unnecessary surgeries could be eliminated.

The diagnosis of VWD requires a careful patient/family history. Many patients with VWD are first diagnosed following an accident/trauma or surgery. Prolonged bleeding following surgery is often encountered in VWD patients. However, the laboratory diagnosis may be very difficult because of the “fluctuation” of VWF in the patient’s plasma. VWF responds to stress similar to other acute phase proteins, e.g., fibrinogen, fibronectin, and vitronectin. Therefore, it is not appropriate to test the patient for VWD in the setting of acute bleeding or stress.

VWF is synthesized in endothelial cells and megakaryocytes. In the endothelial cells, it is stored in the Weibel-Palade bodies with a range of molecular masses from 0.5 to >20 million Da. VWF is also found in the α granules of platelets. VWF will bind to collagen, particularly in situations of high shear stress. In addition, as discussed above, VWF will bind platelet receptors GP IIb/IIIa and GP Ib/IX/V. Many variants of VWD have been described. These include both qualitative and quantitative abnormalities as well as combinations of both defects. Table 8⇓ summarizes the current classification of VWD.

View this table:

Classification of VWD.

Subtype	Frequency	Genetic transmission	Clinical findings	FVIIIC2	VWF:Ag	VWF:Act	Multimers	DDAVP response
Type 1	70% of VWD	Autosomal dominant	Mild to moderate bleeding	Decreased	Decreased	Decreased	Normal distribution	+++3
Type 2A	10–15% of VWD	Autosomal dominant	Mild to moderate bleeding	Decreased	Decreased	Decreased	High and intermediate multimers absent	±
Type 2B	<5% of VWD	Autosomal dominant	Mild to moderate bleeding	Variable, decreased	Variable, decreased	Enhanced, RIPA	Absent large multimers	May be contraindicated
Type 2M	Rare	Autosomal dominant, missense mutation	Variable bleeding	Variable, decreased	Variable, decreased	Variable, decreased	Large and intermediate multimers present	−
Type 2N	Uncommon	Autosomal dominant, missense mutation	Variable bleeding	↓↓4 VIIIC	Variable	Variable	Normal	−
Type 3	Rare	Gene deletion, missense, nonsense mutation, autosomal recessive	Severe bleeding	Markedly decreased ↓↓↓	Markedly decreased ↓↓↓	Markedly decreased ↓↓↓	Absent	No response

 

 

Laboratory testing includes a BT or other means of analyzing platelet function. Recent reports utilizing the PFA-100 suggest that this system or similar new platelet analyzers are preferable to the classical BT. Not infrequently, one may encounter variability of the APTT in patients with VWD.

Tests to classify VWD include quantification of VWF. Initially, this was determined by Laurell rocket immunoelectrophoresis. More recently, ELISA assays as well as flow cytometry have been used with a greater degree of sensitivity. The ristocetin cofactor is a test to assess VWF activity. Ristocetin-induced platelet agglutination is the most widely used procedure. However, there are recent reports on the use of antibodies to the collagen binding site as a means of testing for VWF function. This assay system uses an ELISA format. A factor VIII:C (coagulant activity) assay is also a part of the evaluation for VWD. Multimeric analysis of VWF by agarose gel electrophoresis is very helpful in identifying variants of VWD. In many cases, this assay is not readily available. There are several reference centers nationwide that have substantial expertise in multimeric analysis of VWF.

Management of clinical bleeding in patients with VWD in many cases is relatively simple. DDAVP is used to manage epistaxis and provide prophylaxis for minor surgery. Blood product replacement therapy in the past relied primarily on cryoprecipitate. However, because of the risk of infection (e.g., hepatitis and HIV), the recommended replacement therapy of choice is Humate-P^® or other factor VIII concentrates with significant amounts of VWF. There is a VWF concentrate available in France. Other therapeutic modalities include ε-aminocaproic acid (Amicar^®) and tranexamic acid in the management of mucous membrane bleeding. Estrogens are also helpful in the management of VWD-related menorrhagia.

Acquired VWD may be seen in a variety of settings, including immunologic disorders, hypothyroidism, cardiac defects, and uremia.

 

HEMOPHILIA (FACTOR VIII, IX, XI DEFICIENCIES)

Hemophilia A is the oldest recognized hereditary bleeding disorder. It is sex-linked in transmission. The gene for hemophilia A is located on the long arm of the X chromosome. The gene spans 186 kb of DNA, and many mutations have been described. The inversion mutation accounts for 25% of mutations in hemophilia A patients. Fifty percent of patients with severe hemophilia A (<1% activity) carry the inversion mutation. There are several different variants of this mutation: type I distal (a3), type II proximal (a2), and type III. Hemophilia A is classified based on the amount of factor VIII activity. Patients with severe hemophilia A (<1% factor VIII activity) have joint bleeding with resulting hemarthroses as well as deep intramuscular bleeding. One of the major complications seen in the recent past was transmission of HIV in replacement blood products (factor VIII concentrates and cryoprecipitate). As a result, in the early 1980s, a large portion of the hemophilic population developed HIV positivity and AIDS. The recent introduction of recombinant factor VIII replacement therapy has immensely improved the management of hemophilia patients. One complication of replacement therapy, however, continues to present a challenge: the development of factor VIII inhibitors in a large percentage of severe hemophilia A patients. In these patients, replacement therapy or management of an acute bleed presents a challenge. Porcine factor VIII and activated factor VII are new products for this type of patient, and prothrombin complex concentrates (Autoplex^® and Feiba) are used.

 

Hereditary factor IX deficiency (hemophilia B) and hereditary factor XI deficiency (hemophilia C) are relatively common hereditary hemostatic disorders. Factor IX deficiency is very heterogeneous. Factor XI deficiency is primarily encountered in the Jewish population.

The most common acquired inhibitor of coagulation is the LA. LA is a member of the anti-phospholipid antibody (APA) family. When evaluating patients for potential APAs, it is necessary to do both coagulation testing to identify LA as well as ELISA assays to identify “anti-cardiolipin antibodies” and antibodies to β₂-glycoprotein I. APAs may be seen in many patient populations, e.g., after infection and in patients with autoimmune disease. Most APAs seen in the setting of infections have no clinical complications. However, a large percentage of APA patients with underlying autoimmune disease present with thrombotic complications involving both the arterial and venous circulation, as well as recurrent fetal loss/spontaneous abortion in women. APA syndrome is diagnosed based on the presence of clinical complications (e.g., thrombosis or recurrent spontaneous abortion) and positive laboratory testing for LA and/or anti-cardiolipin antibodies.

The laboratory diagnosis of LA requires a well-coordinated work-up using three screening procedures as recommended by the Scientific Subcommittee of the International Society of Thrombosis and Hemeostasis. The three most commonly used tests are a LA-sensitive APTT reagent, Staclot LA^®, and a dilute Russell viper venom time. More recently, the dilute PT has been used.