CONCEPT OF HEMOSTASIS

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 Уsystem of hemostasisФ (Hemostasis is the physiologic system, which supports the blood in the fluid condition and prevent bloodless. Hemostasis system vital necessary and functionally connect with the cardiovascular, breathing, endocrine and other systems.)

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

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

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

ќписание: fibrinmesh

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.

ќписание: trombocit

 

 

 

 

 

 

 

 

 

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 heparin neutralizing properties.

Platelet factor 5 - It acts as fibrinogen.

Platelet factor 6 -It acts as anti-fibrinolysin.

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

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

STAGES IN PLATELET DEVELOPMENT

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

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

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

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

 

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

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

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

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

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

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

4. Injury - This increases blood platelet count.

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

6. Hypoxia - This markedly increases platelet count.

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

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

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

a) Activation of platelets (To do their function platelets must to activate. In the case of activation the platelets form psevdopodias, change the form. There are 2 groups of activators Ц the first from platelets and second from another cells, plasma. The outside platelets factors, which are produce in plasma, other cell besides platelets Ц 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 Ц 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 Ц platelets produce substances, which are secures the hemostasis.

 

Function of platelets are:

1. hemostatic function Ц 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 Ц thrompostenin,

7 Ц antifibrinolizin,

8 Ц serotonin,

9 - fibrinstabilising factor,

10 Ц factor which activate profibrinolisin,

11 Ц inhibitir of thromboplastin,

12 Ц 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 heparin neutralizing 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 Ц 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 Ц transfer the enzymes, ADP, serotonin and other.

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

5. Regeneratory function Ц 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 Ц thrombostenin, 7 Ц antifibrinolizin, 8 Ц serotonin, 9 - fibrinstabilising factor, 10 Ц factor which activate profibrinolisin, 11 Ц inhibitir of thromboplastin, 12 Ц 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 heparin neutralizing 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 can not function properly. This results in a decreased adhesion and aggregation of platelets. On the other hand, thromboxane A2 decreases the activity of the enzyme adenylate cyclase thereby increasing thrombosthenin activity; this leads to more tendency of platelets for undergoing adhesion and aggregation.

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

c) Stages of vessel-platelets hemostasis (1. Shorting spasm of the vessels Ц 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 Ц activation of platelets and stick it to the place of defect in vessel wall. 3. Reverse aggregation of platelets Ц the thromb which are formed may make way for plasma. 4. Unreverse aggregation of platelets Ц the thromb which are formed can not may make way for plasma. 5. Retraction of platelets plug Ц 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 DukeТs method Ц to 3 minute in norm. 3. Sample of fragility of capillars Ц to 10 petechias in norm in a round with diameter 5 centimetres.)

 

 

ќписание: fibrinnetwork

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 Ц fibrinogen; II Ц prothrombine; III Ц thromboplastin of tissue; IV Ц ions of calcium; V Ц proaccelerin; VII Ц proconvertin; VIII Ц antihemophylic factor A; IX Ц Christmas factor or antihemofilic factor B; X Ц Stuart-Prower factor or prothrombinase; XI Ц plasma thromboplastin antecedent; XII Ц Hageman factor; XIII Ц fibrin stabilizing factor. Some of them are enzymes Ц II, VII, IX, X, XI, XII,XIII; otherare not Ц 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 Ц platelets thromboplastine Ц 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 Ц 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 Ц 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.)

 

ќписание: http://intranet.tdmu.edu.ua/data/teacher/video/fiz_pat/Video2/F210083_thrombosisandembolism1.jpg

ќписание: http://intranet.tdmu.edu.ua/data/teacher/video/fiz_pat/Video2/F210113_thrombosisandembolism2.jpg

 

ќписание: http://intranet.tdmu.edu.ua/data/teacher/video/fiz_pat/Video2/F210143_thrombosisandembolism3.jpg

 

ќписание: http://intranet.tdmu.edu.ua/data/teacher/video/fiz_pat/Video2/F210173_thrombosisandembolism4.jpg

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.

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

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

Properties of Various Factors Participating in Blood Coagulation

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

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

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

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

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

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

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

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

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

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

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

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

2. Valuation of clotting

a) Coagulogram (Time of clotting by Ly-Wait Ц 5-10 minutes; time of plasma recalcification Ц 60-120 seconds; thrombotest Ц IV, V, VI degree; thromboplastin time Ц 12-15 seconds; thromboplastin index Ц 80-105 %; concentration of fibrinogen Ц 2-4 g/L; tolerancy of plasma to heparin Ц 6-11 minutes; heparin time Ц 50-60 seconds; fibrinolysis Ц 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 bloodsТ beginning clot (from the taking the blood to the first waves of amplitude to 1 mm on thromboelastogramm) Ц 8-12 minutes; b) time of thromb producing (time of the first waves of amplitude of 1 mm to 20 mm on thromboelastogramm) Ц 5-8 minutes; c) maximum amplitude (this characteristic of thromb elasticity) Ц 45-60 mm.)