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. Hemostasis system vital necessary and functionally connect with the
cardiovascular, breathing, endocrine and other systems.)
b) Functional-structure
components of hemostasis system (The
components of hemostasis are wall of the vessels, blood cells – platelets,
erythrocytes, leucocytes, enzymes and nonenzymes components of plasma –
clotting and anticlotting substances, fibrinolysis components of hemostasis.)
c) Mechanisms of hemostasis (There are 2 kinds of hemostasis. They are vessel-platelets (primary) and coagulative (secondary) hemostasis. Primary hemostasis activity begin the
first after the destroyed of vessels. Secondary hemostasis add after that in case the
primary hemostasis do not stopped the bloodless.)
2. Vessel-platelets hemostasis (or primary hemostasis include in clotting first of
all after the destroyed the safe of vessel wall.)
PLATELETS OR THROMBOCYTES
In wet preparations of the blood the platelets appear
as small (average diameter = 1.5 μm), colorless, moderately refractile
bodies that are discoid or elliptical in shape. In stained smears they are
round, oval or rod shaped. Platelets do not have nucleus. Their cytoplasm is
hyaline, bright blue having azurophilic granules. Young platelets are larger
than old ones.
Platelets have small mitochondria, glycogen granules, lipid
inclusions and ferritin granules (siderosomes). On the basis of dry weight
platelets have 60 % protein, 15 % lipids (phospholipids, arachidonic acid) and
8 % carbohydrate (mainly glycogen, heteropolysaccharides, complexes containing
sialic acid). Their major energy source is derived from glucose by glycolysis.
Their ATP content is 150 times more than that of RBCs. Their surface has
glycoproteins in which there are receptors for thrombin and ADP.
Platelet proteins - About 20 proteins including
thrombosthenin, albumin, pre-albumin, IgG, IgM, plasminogen and fibrinogen have
been demonstrated in the platelets. Thrombosthenin is identical to actomyosin
of muscle; it can be dissociated into two segments, A (actin) and M (myosin).
Platelets also have ATP-ase activity including Mg2+-Ca2+
dependent type. The contraction of thrombosthenin underlines the phenomenon of
clot retraction and may also be involved in platelet aggregation.
Platelet granules - At least 3 types of granules are
present in the platelets. Their names along with their contents are given
below:
i) Lysosomes; these have endoglycosidase and a heparin-cleaving
enzyme.
ii)Dense granules; these have Ca2+; serotonin and ADP.
iii) Alpha granules; these have Von Willebrand factor, fibronectin, fibrospondin
and a heparin-neutralizing factor (platelet factor 4).
The platelets have been shown to release seven factors that help in blood
clotting.
Platelet factor 1 - It has been found to be the same as factor V.
Platelet factor 2 - It is the thromboplastic substance.
Platelet factor 3 - It is a phospholipoprotein, which behaves as
thromboplastin.
Platelet factor 4 - It has 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 –
the first from platelets and second from another cells, plasma. The outside
platelets factors, which are produce in plasma, other cell besides platelets –
Villibrandt factor, ADP, epinephrine and norepinephrine. The platelets factors,
which are produce by platelets serotonin, ADP, thromboxan A2.)
b) Properties and function of platelets (Quantity
of platelets is 180-320 G/L. Diameter of platelets is 1-4 micrometers, thickness
– 0,5-0,75 micrometers. They are the little peace of megacariocytes cytoplasm
(from one megacariocytes may develop few hundred of platelets). Platelets
circulated in blood from 5 to 11 days and than destroyed in liver, lungs,
spleen by the cells of macrophagal system. Functions of platelets are: 1.
hemostatic function – platelets produce substances, which are secures the
hemostasis.
Function of platelets are:
1. hemostatic function – platelets produce substances, which are secure the
hemostasis. Its produce 12 platelets factors
1 - proaccelerin,
2- factor, which are increase the speed of development the fibronogen in
fibrin,
3 - platelets thromboplastin,
4 - antiheparinic factor,
5 - factor which promote aggregation of platelets,
6 – thrompostenin,
7 – antifibrinolizin,
8 – serotonin,
9 - fibrinstabilising factor,
10 – factor which activate profibrinolisin,
11 – inhibitir of thromboplastin,
12 – antilighting factor.
Other classiffication of platelets factors. The
platelets have been shown to release seven factors that help in blood clotting.
Platelet factor 1 - It has been found to be the same as factor V.
Platelet factor 2 - It is the thromboplastic substance.
Platelet factor 3 - It is a phospholipoprotein, which behaves as thromboplastin.
Platelet factor 4 - It has 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 – provide trophic of endotheliocytes of vessel
wall, support structure and functions of microvessels. These function is
realize by adgesion of platelets to endotheliocytes and injection the enzymes
into the endotheliocytes. For one day near 35 G/L platelets do this function.
3. Transport function – transfer the enzymes, ADP, serotonin and other.
4. Phagocytosis function – the contain of platelets help to kill viruses
and antigens bodies.
5. Regeneratory function – platelets have the growth factor, which help to
grow the endothelial and muscles cells which are present in the vessel wall.
Its produce 12 platelets factors (1 - proaccelerin, 2- factor, which are
increase the speed of development the fibronogen in fibrin, 3 - platelets
thromboplastin, 4 - antiheparinic factor, 5 - factor which promote aggregation
of platelets, 6 – thrombostenin, 7 – antifibrinolizin, 8 – serotonin, 9 -
fibrinstabilising factor, 10 – factor which activate profibrinolisin, 11 –
inhibitir of thromboplastin, 12 – antilighting factor).
Other auther determined such functions of
Platelets
1. Role in Hemostasis -The platelets are responsible
for the primary hemostasis which is brought about by the formation of the primary
hemostatic plug which can effectively stop bleeding from capillaries; small
arterioles and venules. Effective primary hemostasis requires three critical
events, platelet adhesion, platelet activation and secretion and platelet
aggregation.
(A) Platelet adhesion - This means attachment of platelets to non-platelet
surfaces, e.g. to collagen and elastic fibers of blood vessels. This
process is facilitated by Von-Willebrand factor. This factor becomes attached
on one side to the collagen fibrils in the vessel wall, and on the other side
to receptors over the platelet surface.
(B) Platelet activation and secretion - This occurs in many steps which are given below:
(a) Binding of platelet agonists, i.e. adrenaline, collagen
and thrombin the platelet surface, (b) Activation of phospholipases A2
and C. (c) Released arachidonic acid from the membrane phospholipid. (d)
Conversion of arachidonic acid to thromboxane A2, (c) Thromboxane-A2
activates phospholipase-C which liberates still more arachidonic acid from the
membrane phospholipid (f) Some inositol triphosphate is also liberated from
phospholipids. This stimulate the movement of Ca2+ into the platelet
cylosol and the phosphorylalion of myosin light chains. The latter interact
with actin to facilitate granule movement and platele shape change, (g) Another
product of membran phospholipid is diacylglycerol which brings about secretion
of granules. The contents of the granules which are poured into the plasma arc
heparinase, Ca2+, adrenaline, kinins, fibrinogcn. factor Va, AMP,
thromboxane A2, Von-Willebrand factor, fibronectin, thrombospondin
and several other platelet factors including a 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 – 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 in norm. 3. Sample of
fragility of capillars – to 10 petechias in norm in a round with diameter
COAGULATION OR CLOTTING OF THE
BLOOD
Blood has two remarkable properties; it remains fluid while in blood vessels
and clots when it is shed. Both these properties are essential for normal life.
The blood contains substances or factors, which favor coagulation
(pro-coagulants); it also has substances, which are anti-coagulants. An optimum
balance of these two opposing factors is essential for a normal life. The
clotting, in essence, is the formation of the insoluble protein fibrin from the
soluble plasma protein fibrinogen.
A large number of substances take part in producing fibrin from fibrinogen
in the coagulation of blood. The coagulation process actually is the property
of plasma though it is commonly termed as clotting of blood. Although a
complete understanding of the mode of action of the procoagulants is still not
possible, but it can be said that clotting is produced by a complex series of
reactions. Once initiated, the whole process proceeds like a chain reaction
until clotting is complete. Three methods, which have been much employed for
understanding the clotting mechanism are given below.
1. Appropriate techniques by which the clotting process can be stopped at
any required stage followed by its re-start.
2. Studies on patients suffering from hemorrhagic diseases.
3. Experimental studies in animals; hemophilia occurs in dogs which have
been used for research in this disease.
Blood Clotting Factors - The various factors, which are known to take part
in the clotting process in various theories of blood coagulation are given
below. These factors have been assigned numbers, which arc written in Roman
pattern.
I. Fibrinogen
II. Prothrombin (Thrombin is factor II-a)
III. Thromboplaslin. This is the name given to a substance capable of
converting prolhrombin to thrombin. It is present in tissues in an active form,
the tissue thromboplastin, which is also called the tissue pro-coagulant
material.
IV. Calcium ions.
V. Labile factor, Pro-accelerin, Accelerator or Ac globulin.
VI. It has been found to be the same as factor V; it is now obsolete.
VII. Stable factor, Pro-convertin, Auto-prothrombin-I.
VIII. Anti-hemophilic globulin (AHIG, Platelet cofactor-I. Anti-hemophilic
factor A (AHF-A). This is the original compound called factor VIII. However,
factor VIII has been found to have three subtypes. The original factor VIII
(AHF-A) is now called factor VIII-C, C signifying coagulant action. The other
two subtypes are factor VIII V.W. (also called Von-Willebrand protein) and
factor VIII R.Ag (protein precipitated by specific rabbit anliserum).
IX. Christmas factor, Plasma thromboplastin component (PTC), Platelet
co-factor-II, Auto-prolhrombin-II, Anti-hemophilic factor B.
X. Stuart-Prower factor.
XI. Plasma thromboplastin antecedent (PTA), Anti-hemophilic factor-C,
Rosenthal factor.
XII. Hageman's factor, Contact factor, Glass factor.
XIII. Fibrin stabilizing factor, Laki-Lorand factor, Transglutaminase,
Pre-fibrinoligase.
In addition, the following factors are also associated with blood clotting
process.
i) Von-Willebrand factor or the platelet adhesion factor. It is needed for
platelet adhesion as well as for activity of factor VIII-C; it is called factor
VIII V.W.
ii) Fitzgerald factor; it is the same as high mol. wt. kininogcn.
iii) Fletcher factor; it is pre-kallikrein.
1. Analysis
of coagulative hemostasis mechanisms
a) Characteristics of clotting factors (There are 12 clotting factors: I –
fibrinogen; II – prothrombine; III – thromboplastin of tissue; IV – ions of
calcium; V – proaccelerin; VII – proconvertin; VIII – antihemophylic factor A;
IX – Christmas factor or antihemofilic factor B; X – Stuart-Prower factor or
prothrombinase; XI – plasma thromboplastin antecedent; XII – Hageman factor;
XIII – fibrin stabilizing factor. Some of them are enzymes – II, VII, IX, X,
XI, XII,XIII; other are not –
I, III, IV, V, VIII. The vitamin K is necessary for the functional activity of
II, VII, IX, X factors.)
b) External mechanism of the first stage (3 factors from the injure tissues
go to plasma and interactions with VII factor, the last is activated. VII
active factor and IV factors form the complex 1a: III + VII active + IV, which
is activated X factor.)
c) Inner mechanism of the first stage (Factor 3 of platelets – platelets
thromboplastine – influence on XII factor. Active XII factor + XI is complex 1.
Active XI factor activated IX factor. Active IX factor + VIII factor + IV
factor is complex 2. Complex 1a and 2 are activate X factor. Factor X active +
V + IV formed complex 3 or thrombinasa complex.)
d) Course of the second and third stages (The second stage – formation of
thrombin from prothrombin. The third stage is formation of fibrin from
fibrinogen. The last stage has 3 period; formation of fibrin-monomers;
formation of fibrin S (solubilis); formation of fibrin I (insolubilis). Calcium
is necessary for all stages.)
e) Regulation of the clotting mechanisms (Increase
of clotting names hypercoagulation, decrease – hypocoagulation.
Hypercoagulation may be in a stress cases. It depends on epinephrine, which
concentration increased in the cases of stress. Epinephrine increase from the
vessels walls factors from which produced prothrombinasa. In cases of big
concentration epinephrine should activate XII factor in a bloodstream. It
divides fats and fat acids, which have prothrombinase activity. After the
hypercoagulation stage may be secondary hypocoagulation.)
Theories
of Blood Coagulation
I. Classical theory of Morowitz (1905-1906) - Blood clotting was considered
to take place in two stages.
(i) In the first stage prolhrombin is converted to thrombin by the enzyme
prothrombinase, Ca2+ being necessary for this reaction.
(ii) In the second stage the thrombin acts as an enzyme on fibrinogen and
converts it to fibrin.
II. Cascade or waterfall theory - For many decades, Morowitz's theory was
accepted. But great developments in this field resulted in several new
theories, one of which is called cascade or waterfall theory because it
involves a cascade of events; it is described below.
There are two systems of clotting, intrinsic and extrinsic, which converge
upon what is called the final common pathway.
1. Intrinsic or the blood system - This system is called so, because all factors taking
part in the process are derived from the blood itself and it can take place in
pure blood (blood not contaminated with tissue juice) kept in a test tube. It
is also called contact system because the process starts when blood comes in
contact with a foreign surface, e.g. vascular sub-endothelial collagen
or even glass. This process takes place in the following six stages. In the
first five of these stages limited proteolysis converts an inactive factor to
its active form. Each of these steps is regulated by plasma and cellular
co-factors and Ca2+. The inactive and active blood clotting factors
are distinguished by writing and a respectively after the factor.
Stage No. 1. Three plasma proteins, i.e. Hageman factor
(XII), high mol. wt. kininogen and pre-kallikrein form a complex with vascular
subendolhelial collagen. Factor XH-i becomes activated to Xll-a, which
acceleates the conversion of pre-kallikrein to kallikrein which then
accelerates the conversion of still more XII-i to XII-a.
Satge No. 2. Factor XII-a converts factor XI-i to XI-a.
Stage No. 3. Factor XI-a converts factor IX-i to IX-a.
Stage No. 4. Factor IX-a in the presence of factor VIII C, Ca2+
a platelet membrane lipoprotein (platelet factor 3) converts X-i to X-a.
Stage No. 5. Several factors take part in the conversion of
prothrombin to thrombin. These include factor X-a, factor V-a, Ca2+
and phospholipids. Although the conversion of prothrombin to thrombin can take
place on a phospholipid-rich surface, but it is accelerated several
thousand-fold on the surface of activated platelets.
Stage No. 6. Conversion of fibrinogen to fibrin is brought about
thrombin by the following mechanism. Fibrinogen is a symmetrical dimer; each
half of its molecule has the following structure:
i) Alpha polypeptide joined to a short A-fibrinopeptide.
ii) Beta polypeptide joined to a short B-fibrinopeplide.
iii) Gamma polypeptide.
Fibrinogen can thus be represented by the structure, [Alpha(A), beta(B),
gamma]2. Thrombin catalyzes the breakdown of fibrinogen in such a
way that a part of the molecule separates leaving behind a fibrin monomer.
[alpha(A), beta(B), gamma]2 → [alpha, beta, gamma]2
(Fibrin monomer) + 2[fibrinopeptide A + B]
However, the removal of fibrinopeptide B is not essential for coagulation.
The fibrin monomers undergo polymerization giving rise to fibrin polymers; this
process involves formation of hydrogen bonds between fibrin monomers. These
fibrin polymers are unstable and the polymerization is readily reversed by
inhibitors of H bond formation such as urea. The unstable fibrin polymers are then
acted upon by factor XIII, which actually is an enzyme. Factor XIII is
initially inactive but is activated by thrombin. It brings about the production
of cross linkages between adjacent fibrin polymers. This process involves
covalent bond formation between epsilon amino group of lysine and the gamma
amide group of glutamine; NH3 is evolved in this reaction. A clot
which is much more stable and is insoluble in urea solution is thus produced.
Even this fibrin clot is quite soft, but after some time it undergoes
retraction during which serum oozes out of it. The platelets are of primary
importance in this process of clot retraction. The result is a firm clot that
can effectively seal a wounded vessel.
2. The extrinsic or the tissue system - This is called so because it needs the presence of
tissue juice that contains tissue thromboplastin which is not present in blood.
The tissue thromboplastin in the presence of factor VII and Ca2+
activates factor X-i to X-a. Subsequent reactions are the same as described
under the intrinsic system and, being common to both the intrinsic and
extrinsic systems, are designated as the final common pathway. Because the
extrinsic system involves fewer steps than the intrinsic system, therefore it
proceeds faster than the latter. For this reason, while the intrinsic system
takes 2 to 6 minutes for clotting to take place, the extrinsic system takes as
little as 15 seconds to do that.
III. Seeger's hypothesis - This concept basically differs from the cascade
theory in that prothrombin and factors VII, IX and X are considered to occur in
a single molecular system and not separate from each other. This common
molecule is believed to release all these clotting factors during clotting
process. A common characteristic of all these clotting factors is that all of
them require the presence of vitamin K for their biosynthesis. Factors VII, IX
and X are designated by Seeger as autoprothrombin I, II and III respectively.
The corresponding active forms of these factors arc called autoprothrombin A, B
and C. There are serious objections to this hypothesis as various studies have
shown that all these factors arc different and are quite distinct from each
other.
Properties of Various Factors Participating in Blood Coagulation
Fibrinogen - It occurs in the plasma in a concentration of
Prothrombin - It is the proenzyme, the precursor of thrombin. It contains 2
to 10 % carbohydrate in its molecule and has a mol. wt. of 69,000. Its plasma
concentration is 10 to 15 mg per 100 ml.
Thromboplastin - It implies an activity which converts prothrombin to
thrombin. All body tissues have this activity and therefore it is termed as
tissue or intrinsic thromboplaslin. The brain, lung, placenta and testes are
especially rich in it. It is a complex of phospholipids, lipoproteins and
cholesterol. Tissue extracts, if injected intravenously, can cause widespread
clotting of blood. However, tissue thromboplaslin is not active as such but it
needs Ca2+ and factor VII for its activation which normally arc
present in blood. Russel viper venom has a strong thromboplaslin activity and
is used for slopping bleeding from superficial areas by its local application
in diseases like hemophilia.
Calcium - Ca in ionic form, Ca2+, is essential for clotting of
blood and it acts at many stages. Ca ions serve to form complexes with lipids,
which take part in blood clotting. In health or disease blood has always
sufficient Ca2+ for this purpose. In other words, a Ca2+
deficiency is never a cause of a prolonged clotting time in man.
Factor V - It is activated by small amount of thrombin which in turn leads
to a greater formation of thrombin. But an excess of thrombin destroys it and
causes its disappearance from serum. It is unstable in the citrated plasma. Its
congenital deficiency is the cause of parahemophilia, a mild bleeding disorder.
Factor VII - It is stable on storage. It acts as co-thromboplastin in the
working of extrinsic system of blood cloning. Its congenital deficiency has
been seen very rarely. It has up to 50 % carbohydrate in its molecule.
Factor VIII-C - It is also called platelet cofactor-I and anti-hemophilic
globulin. Its deficiency causes the classical hemophilia (now called hemophilia
A). Hemophilia is discussed later in detail. This factor is readily inactivated
in vitro.
Factor IX - It is also called Christmas factor because its deficiency was
first demonstrated in a patient with the surname Christmas whose bleeding
disease was named Christmas disease. This disease is also called hemophilia B.
Factor X - It is an alpha globulin present both in scrum and plasma. I
deficiency is seen in both sexes equally as a congenital defect.
Factor XI - Its deficiency causes hemophilia C, which is a mild bleeding
disease.
Factor XII - It is activated by surface contact and according to the
cascade theory, this process initiates the series of reactions leading to blood
clotting. Blood deficient in this factor docs not clot in lest tube, i.e. in
vitro. If blood taken from a vein (without letting it being mixed with
tissue juice) is placed in a lest tube lined with silicone, it does not clot;
this is because the silicone layer is smooth and unwettable and does not permit
the activation of factor XII for the same reason. Blood also clots much more
slowly when placed in polythene tubes as compared to glass tubes. The
deficiency of this factor is seen in persons with Hageman's trait, but they do
not generally show bleeding tendency. Its additional roles arc the activation
of fibrinolytic system and the plasma kinin syslem. It is activated by contact
with glass, negatively charged surfaces, collagen fibers, unbroken skin, sebum,
long chain fatty acids, uric acid, fibrin, elastin and homocysteine.
Factor XIII - It is the enzyme transglutaminase, whose
function has already been discussed. Persons with congenital deficiency of this
factor have bleeding tendencies and poor wound healing. Their blood clots all
right, but the clot, unlike the normal clot, is unstable and can be solubilized
in 5 molar urea or 1 % monochloracetic acid solution.
2. Valuation of clotting
a) Coagulogram (Time of clotting by Ly-Wait – 5-10 minutes; time of plasma
recalcification – 60-120 seconds; thrombotest – IV, V, VI degree;
thromboplastin time – 12-15 seconds; thromboplastin index – 80-105 %;
concentration of fibrinogen – 2-4 g/L; tolerancy of plasma to heparin – 6-11
minutes; heparin time – 50-60 seconds; fibrinolysis – 15-20 %.)
b)
Thromboelastography (Thromboelastography is a method of regestration of plugs
forming and characteristic of clot by thromboelastograph. The characteristic of
clot in thromboelastogramm: a) time of bloods’ beginning clot (from the taking
the blood to the first waves of amplitude to