Vessel-platlet hemostasis.
Physiology of blood clotting.
Anticlotting
mechanisms and fibrinolysis
1. Common characteristic of hemostasis system
Hemostasis is a complex process that prevents or terminates blood loss from
a disrupted intravascular space. Four major physiologic events participate,
both in sequence and interdependently, in the hemostatic process. Vascular
constriction, platelet plug formation, fibrin formation, and fibrinolysis occur
in that general order, but the products of each of these four processes are
interrelated in such a way that there is a continuum and multiple
reinforcements. The process is shown schematically.
In the normal blood vessel,
endothelial cells function to prevent clotting. They interfere with platelet
recruitment by inactivating adenosine diphosphate (ADP). Endothelial cells also
release several products that inhibit the coagulation process. Heparan sulfate
acts to catalyze the inhibition of thrombin by antithrombin. Thrombomodulin
down-modulates the coagulation process through the activation of protein C.
Prostaglandin I2 (prostacyclin) inhibits platelet aggregation, as
does nitric oxide (in addition to its vasodilator effects).
Vascular Constriction
When a vessel is injured, several
processes are initiated. Vasoconstriction is the initial vascular response to
injury. It is more pronounced in vessels with medial smooth muscles, but occurs
even at the capillary level. It is dependent upon local contraction of smooth
muscle that has a reflex response to various stimuli. The initial vascular
constriction occurs before any platelet adherence to the site of injury.
Adherence of endothelial cells to adjacent endothelial cells may be sufficient
to cause cessation of blood loss from the intravascular space. Vasoconstriction
is subsequently linked to platelet plug formation. Thromboxane A2
(TXA2), which is derived from the release of arachidonic acid from
platelet membranes during aggregation, is a powerful vasoconstrictor.
Endothelin synthesized by injured endothelium is also a vasoconstrictor.
Serotonin, 5-hydroxytryptamine (5-HT), released during platelet aggregation, is
another vasoconstrictor, but it has been shown that when platelets have been
depleted of serotonin in vivo, constriction is not inhibited. Bradykinin and
fibrinopeptides in the coagulation schema also are capable of contracting
vascular smooth muscle. Some patients with mild bleeding disorders and a
prolonged bleeding time have, as their only abnormality, capillary loops that
fail to constrict in response to injury.
A lateral incision in a small
artery may remain open because of physical forces, whereas a similarly sized
vessel that is completely transected may contract to the extent that bleeding
ceases spontaneously. The vascular response to injury also is affected by the
contribution of pressure provided by surrounding tissues. Bleeding from a small
venule ruptured by trauma in the thigh of an athlete may be negligible because
of the compressive effect of the surrounding muscle. In the same individual,
bleeding from a similar vessel in the nasal mucosa may be significant. When
there is low perivascular pressure, as seen in patients with muscle atrophy
accompanying aging, in patients on prolonged steroid therapy, and in patients
with Ehlers-Danlos syndrome, bleeding tends to be more persistent. Vascular
abnormalities, such as hereditary hemorrhagic telangiectasia, may predispose
the patient to bleeding from the involved region.
Vessel-platelets hemostasis
PLATELETS OR THROMBOCYTES
Platelet
Function
Platelets are 2 to 4 um in diameter anucleate fragments of megakaryocytes
with normal circulating numbers falling between 150,000 and 400,000/uL. Thrombopoietin
is the predominant mediator of platelet production, although other inflammatory
mediators, such as interleukin (IL)-6 and IL-11, may play a role. Up to 30
of circulating platelets may be sequestered in the spleen and can be released
in response to catecholamines. If not consumed in a clotting reaction,
platelets are normally removed by the spleen with an average life span of 7 to
10 days.
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:
I.
. Hemostatic
function – platelets produce substances, which are secure
the hemostasis. Its produce 12 platelets factors:
1 - proaccelerin,
2- factor, which are increase the speed of
development the fibronogen in fibrin,
3 - platelets thromboplastin,
4 - antiheparinic factor,
5 - factor which promote aggregation of platelets,
6 – thrompostenin,
7 – antifibrinolizin,
8 – serotonin,
9 - fibrinstabilising factor,
10 – factor which activate profibrinolisin,
11 – inhibitir of thromboplastin,
12 – antilighting factor.
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.
II.
.
Angiotrophic function – provide trophic of endotheliocytes of vessel wall,
support structure and functions of microvessels. These function is realize by
adgesion of platelets to endotheliocytes and injection the enzymes into the
endotheliocytes. For one day near 35 G/L platelets do this function.
III.
. Transport
function – transfer the enzymes, ADP, serotonin and other.
IV.
.
Phagocytosis function – the contain of platelets help
to kill viruses and antigens bodies.
V.
.
Regeneratory function – platelets have the growth factor, which help to grow
the endothelial and muscles cells which are present in the vessel wall.
Its produce 12 platelets factors (1 - proaccelerin, 2- factor, which are
increase the speed of development the fibronogen in fibrin, 3 - platelets
thromboplastin, 4 - antiheparinic factor, 5 - factor which promote aggregation
of platelets, 6 – thrombostenin, 7 – antifibrinolizin, 8 – serotonin, 9 -
fibrinstabilising factor, 10 – factor which activate profibrinolisin, 11 –
inhibitir of thromboplastin, 12 – antilighting factor).
Other auther determined such functions of
Platelets
1. Role in Hemostasis -The platelets are responsible
for the primary hemostasis which is brought about by the formation of the primary
hemostatic plug which can effectively stop bleeding from capillaries; small
arterioles and venules. Effective primary hemostasis requires three critical
events, platelet adhesion, platelet activation and secretion and platelet
aggregation.
(A) Platelet adhesion - This means attachment of platelets to non-platelet
surfaces, e.g. to collagen and elastic fibers of blood vessels. This
process is facilitated by Von-Willebrand factor. This factor becomes attached
on one side to the collagen fibrils in the vessel wall, and on the other side
to receptors over the platelet surface.
(B) Platelet activation and secretion - This occurs in many steps which are given below:
(a) Binding of platelet agonists, i.e. adrenaline,
collagen and thrombin the platelet surface,
(b) Activation of phospholipases A2 and C.
(c) Released arachidonic acid from the membrane
phospholipid.
(d) Conversion of arachidonic acid to thromboxane A2,
(c) Thromboxane-A2 activates
phospholipase-C which liberates still more arachidonic acid from the membrane
phospholipid
(f) Some inositol triphosphate is also liberated from
phospholipids. This stimulate the movement of Ca2+
into the platelet cylosol and the phosphorylalion of myosin light chains. The latter
interact with actin to facilitate granule movement and platele shape change,
(g) Another product of membran phospholipid is
diacylglycerol which brings about secretion of granules. The
contents of the granules which are poured into the plasma arc heparinase, Ca2+,
adrenaline, kinins, fibrinogcn. factor Va, AMP, thromboxane A2,
Von-Willebrand factor, fibronectin, thrombospondin and several other platelet
factors including a 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.)
Platelets play an integral role in hemostasis along two pathways: by
forming a hemostatic plug and by contributing to thrombin formation. Platelets, which normally do not adhere to each
other or to the vessel wall, form a plug that stops bleeding when vascular
disruption occurs. Injury to the intimal layer in the vascular wall exposes
subendothelial collagen to which platelets adhere within 15 seconds of the
traumatic event. This requires von Willebrand factor (vWF), a protein in the
subendothelium that is lacking in patients with von Willebrand's disease. vWF
binds to glycoprotein (GP) I/IX/V on the platelet membrane. Platelet adhesion
also is mediated by an interaction between collagen in the subendothelium and
GP IaIIa on the platelet surface. Following adhesion, the platelets expand and
develop pseudopodal processes and also initiate a release reaction that
recruits other platelets from the circulating blood to seal the disrupted
vessel. Up to this point, this process is known as primary hemostasis,
and the aggregation is reversible and is not associated with secretion. Heparin
does not interfere with this reaction, which is why hemostasis can occur in the
heparinized patient. ADP and serotonin are the principal mediators in this
process of aggregation. Various prostaglandins have opposing activities.
Arachidonic acid released from platelet membranes is converted by
cyclooxygenase to prostaglandin G2 (PGG2) and then to
prostaglandin H2 (PGH2), which, in turn, is converted to
TXA2. TXA2 has potent vasoconstriction and platelet
aggregation effects. The arachidonic acid may also be shuttled to adjacent
endothelial cells and converted to prostacyclin (PGI2), which is a
vasodilator and acts to inhibit platelet aggregation. Platelet cyclooxygenase
is irreversibly inhibited by aspirin and reversibly blocked by nonsteroidal
anti-inflammatory agents, but is not affected by COX-2 inhibitors. In the
second wave of platelet aggregation, a release reaction occurs in which several
substances including ADP, Ca2 +, serotonin, TXA2, and
granule proteins are discharged. Fibrinogen is a required cofactor for this
process, acting as a bridge for the GP IIbIIIa receptor on the activated
platelets during formation of a platelet plug. The release reaction results in
compaction of the platelets into an "amorphous" plug, in a process
that is no longer reversible. Thrombospondin, another protein secreted by the granule, stabilizes fibrinogen binding to the
activated platelet surface and strengthens the platelet-platelet interactions.
Platelet factor 4 (PF4) and thromboglobulin are also secreted during the
release reaction, as is an inhibitor of plasminogen activation. PF4 is a potent
heparin antagonist and it blocks angiogenesis.
The second wave of platelet aggregation is inhibited by aspirin and
nonsteroidal anti-inflammatory drugs (NSAIDs)(through the inhibition of
cyclooxygenase), by cyclic adenosine monophosphate (cAMP), and nitric oxide. As
a consequence of the release reaction, alterations occur in the phospholipids
of the platelet membrane that allow calcium and clotting factors to bind to the
platelet surface, forming enzymatically active complexes. The altered
lipoprotein surface (sometimes referred to as platelet factor 3) catalyzes
reactions that are involved in the conversion of prothrombin (factor II) to
thrombin (factor Iia) by activated factor X (Xa) in the presence of factor V and calcium, and it
is involved in the reaction by which activated factor IX (IXa), factor VIII,
and calcium activate factor X. Platelets may also play a role in the initial
activation of factors XI and XII.
If congenital abnormalities exist, they can result in abnormal aggregation,
as a result of effects on either the "first wave" of aggregation (the
intrinsic ability of platelets to aggregate) or the "second wave" of
the process (granule release).
d) Investigation of 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.
Coagulation
Factor VIIIa combines with
factor IXa (generated by the extrinsic Xase as above or by factor XIa formed by
thrombin or factor XIIa action on factor XI on the activated platelet membrane)
to form the intrinsic factor Xase, which is responsible for the bulk of the
conversion of factor X to Xa. Intrinsic Xase (the VIIIa-IXa complex) is about
50 times more effective at catalyzing factor X activation than is the extrinsic
(TF-VIIa) Xase complex and five to six orders of magnitude more effective than
is factor IXa alone.
Factor Xa combines with factor
Va, also on the activated platelet membrane surface to
form the prothrombinase complex, which is responsible for converting
prothrombin to thrombin. As with the Xase complex, the prothrombinase is
significantly more effective at catalyzing its substrate than is factor Xa
alone. Once formed, thrombin leaves the membrane surface and converts
fibrinogen by two cleavage steps into fibrin and two small peptides termed
fibrinopeptides A and B. Removal of fibrinopeptide A permits end-to-end
polymerization of the fibrin molecules, whereas cleavage of fibrinopeptide B
allows side-to-side polymerization of the fibrin clot. This latter step is
facilitated by thrombin-activatable fibrinolysis inhibitor (TAFI), which acts
to stabilize the resultant clot.
The coagulation system is
exquisitely regulated. In addition to clot formation that must occur to prevent
bleeding at the time of vascular injury, two related processes must exist to balance
propagation of the clot before the entire vascular bed is thrombosed in
response to a local insult. First, there is a feedback inhibition on the
coagulation cascade, which deactivates the enzyme complexes leading to thrombin
formation. Second, mechanisms of fibrinolysis allow for breakdown of the fibrin
clot and subsequent repair of the injured vessel with deposition of
connectivetissue.
Tissue factor pathway inhibitor (TFPI) blocks the extrinsic factor Xase
complex (TF-VIIa), eliminating this catalyst's production of factors Xa and
IXa. TFPI is normally present in the circulation at low concentrations (~2.5
nmol/L), and additional inhibitor is present on the endothelium and can be
released in response to heparin. Antithrombin III (AT-III) effectively
neutralizes all of the procoagulant serine proteases and only weakly inhibits
the TF-VIIa complex. The primary effect is to quench the production of
thrombin. A third major mechanism of inhibition of thrombin formation is the
protein C system. Upon its formation, thrombin binds constitutively to
thrombomodulin and activates protein C to activated protein C (APC), which then
forms a complex with its cofactor, protein S, on a phospholipid surface. The
APC-protein S complex cleaves factors Va and VIIIa so
they are no longer able to participate in the formation of Xase or
prothrombinase complexes. Of interest is an inherited form of factor V that
carries a genetic mutation, called factor V Leiden, that is resistant to
cleavage by APC, thereby remaining active (procoagulant). Patients with factor
V Leiden are predisposed to venous thromboembolic events. As a result of the
three systems described above, feedback inhibition of thrombin formation exists
at upstream, intermediate, and downstream portions of the coagulation cascade
to "turn off" thrombin formation once the procoagulant sequence is
initially activated.
The same thrombin-thrombomodulin complex that leads to formation of
activated protein C also activates TAFI. In addition to clot stabilization,
removal of the terminal lysine on the fibrin molecule by TAFI also renders the
clot more susceptible to lysis by plasmin. Degradation of fibrin clot is
accomplished by plasmin, a serine protease derived from the proenzyme
plasminogen. Plasmin formation occurs as a result of one of several plasminogen
activators. Tissue plasminogen activator (tPA) is made
by the endothelium and other cells of the vascular wall and is the main
circulating form of this family of enzymes. tPA is
selective for fibrin-bound plasminogen so that endogenous fibrinolytic activity
occurs predominately at the site of clot formation. The other major plasminogen
activator, urokinase plasminogen activator (uPA), also produced by endothelial
cells as well as by urothelium, is not selective for fibrin-bound plasminogen.
Because of the complex nature of hemostasis, potential interference in the
process can occur at many levels. Platelet number or function can be
insufficient to adequately support coagulation. Abnormalities in platelet
function may be caused by either endogenous (e.g., uremia) or exogenous (e.g.,
aspirin or other antiplatelet agents) factors. Alternatively, abnormalities in
the clotting factors may underlie a problem of abnormal bleeding. As with
platelet dysfunction, the cause may be an intrinsic defect in one of the
factors (detailed later in this chapter) or may result from pharmacotherapy.
2. Evaluation of clotting
a) Coagulogram (Time of clotting by Ly-Wait – 5-10 minutes; time of plasma
recalcification – 60-120 seconds; thrombotest – IV, V, VI degree;
thromboplastin time – 12-15 seconds; thromboplastin index – 80-105 %;
concentration of fibrinogen – 2-4 g/L; tolerancy of plasma to heparin – 6-11
minutes; heparin time – 50-60 seconds; fibrinolysis – 15-20 %.)
b) Thromboelastography (Thromboelastography is a method of regestration of
plugs forming and characteristic of clot by thromboelastograph. The
characteristic of clot in thromboelastogramm: a) time of bloods’ beginning clot
(from the taking the blood to the first waves of amplitude to
Anticoagulative mechanisms. Fibrinolysis.
1. Common characteristic of physiological anticlotting substances (The tendency
of blood to clot is balanced by a number of limiting reactions that tend to
prevent clotting inside the blood vessels and to break down any clots that do
form.)
a) Properties of antithrombin III (It is the most important anticoagulant
in the blood. It inhibits thrombin, factors Xa, IXa, V, XIa, XII.
It is basic plasma cofactor of heparin. Very faint inhibitor
of plasmin and kallikrein.)
b) Importance of heparin (Blood does not
ordinarily clot internally in the body. It is assumed that, unless there is
access to injured surfaces, there are not enough thromboplastic substances
liberated to convert prothrombin into thrombin and thus start the series of
chemical reactions that results in clotting. Even so, additional safeguards are
present in antiprothrombic substances such as heparin. This substance
was originally found in the liver but is now thought to be produced by large
basophilic cells (mast cells) in tissues of various organs. Heparin reduces the
ability of the blood to clot by blocking the changeof prothrombin to thrombin.
It can also be used to aid in reducing clots in cases in which internal
clotting has already occurred. In either case it acts in conjunction with a
plasma cofactor. Internal clotting is called thrombosis. The clot, or thrombus,
can form in some blood vessel of the arm or leg and do comparatively little
harm, but if it should block the blood supply to the brain or to the heart
(coronary thrombosis), it can be very serious. Heparin form complex with antithrombin-III and transform it in
anticoagulant with the negative action. Activate nonenzyme fibrinolysis.)
c) Role of alpha-2-macroglobulin, alpha-1-antitripsin, protein C (Alpha-2-macroglobulin
is very large globulin molecule. It is a similar to antithrombin-heparin
cofactor in that it combines with the proteolytic coagulation factors. Its
activity is not accelerated by heparin. Its function is mainly to act as a
binding agent for the coagulation factors and prevent their proteolytic action
until they can be destroyed in various ways. It a faint inhibitor of thrombin,
connect with plasmin. Alpha-1-antitripsin inhibits thrombin activity, IXa, XIa,
XIIa factors, plasmin and kallilrein. Protein C inhibits VIIIa, Va factors. It activity depend of thrombin
and vitamin K concentration.)
d) Functionation of secondary anticlotting substances (Primary
anticoagulants are produce and present all time in plasma and secondary
anticoagulants form in a case of blood clotting. They are antithrombin-I or
fibrin and products of fibrinolysis or products of fibrinogen degradation.
Fibrin is sorbs and inactivates thrombin and Xa factor. Products of
fibrinolysis inactivate ending stage of clotting, IXa factor, platelets' agregation.)
2. Fibrinolytic system (Fibrinolysis is begining with the retraction. The
plasma proteins contain a globulin called plasminogen or profibrinolysin, which
activated into plasmin or fibrinolysin.)
a) Stages of fibrinolysis
b) Mechanisms of fibrinolysis activation (There
are 2 mechanisms of fibrinolysis activation: external and internal. Main
internal mechanism put in action by XIIa factor. External mechanism stimulated
by protein activators of plasminogen, which are produce by vessel wall.)
c) Regulation of fibrinolysis (All processes are
direct on increase the clotting mechanism, for example, epinephrine, which are
increase in the case of stress. It promote the forming
of prothrombinase, activating of XII factor, increasing of fats and fats acids.
But after the clotting send up the anticlotting mechanism – hypocoagulation. In
norm it has independent regulation. The lysis of blood clots allows slow clearing
of extraneous blood in the tissues and sometimes allows reopening of clotted
vessels. Reopening of large vessels occurs only rarely. But an important
function of the fibrinolysin system is to remove very minute clots from the
millions of tiny peripheral vessels that eventually would all become occluded
were there no way to cleanse them.)
d) Notion about nonenzymes fibrinolysis (It may
be steroid hormons with anabolic function, which are increase producing of
fibrinolysis activators by endothelium. Leucocytes ensure function of
independent mechanism of fibrinolysis. It limited the size of thromb.
Erythrocytes ensure function of independent mechanism of fibrinolysis too.)
3. Functionating of anticlotting mechanisms
a) Role of vessels endothelium in support blood in the fluid condition (1.
Smooth surface of vessels endothelium. 2. Negative charge of endotheliocytes
and blood cells and that’s why they are push away. 3. Present on the vessels
wall thin layer of fibrin which adsorb clotting factors, especially thrombin.
4. Constant presence in blood anticlotting factors in a small
doses. 5. Producing by endothelium prostaciclins, which are powerful
inhibitors of platelets aggregation. 6. Ability of
endothelium to produce and fix antithrombin-III.)
THE FIBRINOLYTIC SYSTEM
A proteolytic enzyme, fibrinolysin or plasmin, acts on
fibrinogen and fibrin causing their breakdown or dissolution by converting them
into smaller peptides which are soluble; these peptides are called fibrin/fibrinogen
degradative products (FDP) as well as fibrin/fibrinogen split products (PSP).
The FDP so produced have several important actions, which oppose hemostasis; in
other words they promote bleeding.
1. They inhibit binding of fibrinogen to platelets and
inhibit aggregation of platelets.
2. They inhibit thrombin formation and also destroy
any thrombin formed.
3. They prevent fibrin polymerization.
Formation of plasmin - Normally plasmin occurs in
blood plasma as its inactive precursor called pro-fibrinolysin or plasminogen.
The conversion of plasminogen to plasmin involves cleavage of a single
arginine-valinc bond and is catalyzed by an activator which itself occurs in an
inactive form namely pro-activator. The pro-activator is changed to activator
by enzymes called pre-kallikrein activators, which are derived from breakdown
of active factor XII; in other words, the activators of pre-kallikrein are the
fragments of active factor XII.
Factors increasing the formation of plasmin - These
are discussed below.
1. Fibrinolysokinase - It is present in plasma, many tissues and in many
secretions, e.g. saliva, milk and tears.
2. Bacterial enzymes - These include staphylokinase and streptokinase which
are produced by staphylococci and streptococci respectively. Streptokinase is
used by intravascular infusion for the clearance of thrombo-embolism.
3. Urokinase - It is present in urine and is being used in
therapeutics to accelerate fibrinolysis. It is formed in the kidney; its
commercial source is the fetal kidney tissue culture. The urinary plasmin
produced under its influence is believed to have a role in keeping the renal
tubules patent by preventing the deposition of fibrin within them. Fibrin may
be derived from the fibrinogen present in trace amount in the renal tubular
fluid. In the same way, plasmin activity present in milk, tears and semen
serves to keep the corresponding duct systems patent.
4. Cytokinase - It is present in tissue cells, WBCs and platelets.
5. Hormones - Growth hormone and thyroid stimulating hormone (TSH).
6. Miscellaneous - Exercise, adrenaline, hypoxia, histamine, bacterial
pyrogens, ischemia, shock, tissue damage and chloroform.
Factors inhibiting plasmin formation - The plasmin
activation is inhibited by the following factors.
1. Epsilon aminocaproic acid (Trasylol and tranexamic
acid) - It is used in
therapeutics to inhibit plasmin system.
2. ACTHl - (Adrcnocorlicotropic hormone).
Control of plasmin activity - A trace of plasmin
activity is present even in normal plasma. However, this plasmin activity is
kept under check by an opposing factor called anti-plasmin which is 10 times
more active than plasmin activity. The anti-plasmin activity is present in
alpha-1 and alpha-2 globulins of plasma. When excessive amount of plasmin is
formed, it brings about the following two types of effects:
(i) The plasmin gets bound to fibrin clots and breaks
them to small fragments which arc cleared by
macrophages. This is the physiological action.
(ii) A part of plasmin remains free in the plasma and
enters circulation. However, its action is rapidly neutralised by the
anti-plasmin. But if plasmin level of the plasma is too high, then it causes
digestion of fibrinogen and also of factors V, VIII and XII. This is the
pathological action. If this action is very marked, hypofibrinogenemia and
deficiencies of factors V, VIII and XII result and lead to hemorrhage.
FACTORS PREVENTING BLOOD CLOTTING WITHIN BLOOD VESSELS
It has already been pointed out that blood must remain
fluid within the blood vessels if life has to be normal. The factors, which
help in preventing intravascular clotting (thrombosis), are given below.
1. Vascular endothelium - The normal endothelium of
blood vessels, has been described to act as a non-wettable surface and docs not
allow the activation of factor XII. Similarly the platelets also cannot adhere
to normal endothelium. Both these factors prevent the initial steps in
clotting. The endothelial cells arc also rich in fibrinolysin
activators. In atherosclerosis the normal intima is replaced by a
diseased one, and clotting is favored resulting in thrombosis. The endothelium
can also be damaged by bacteria, injuries such i as fractures, pressure on
veins, etc.
2. Chemical substances in blood - There arc certain
substances in the blood, which normally antagonize any tendency towards
clotting. These include the following:
(i) Heparin - It is
produced by the mast cells of the connective tissue which occur especially in
the lung and liver. Heparin is an acidic mucopolysaccharide composed of
alternating sulfated D-glucosamine and D-glucuronic acid units. Up to 40 % of
its molecular mass is H2SO4. This makes heparin the
strongest organic acid in the body. Heparin is formed in small amounts
continuously, but its release from the mast cells is greatly increased in
conditions like peptone shock and anaphylactic shock. The mechanism of heparin
action is manifold. It antagonizes all the stages of blood clotting, e.g. formation
of active factor X, thrombin and fibrin. It also inhibits the agglutination of
platelets thus decreasing the release of platelet factors. In combination with
a protein namely anti-thrombin, it inactivates thrombin. This is probably the
most important action of heparin.
Heparin is water soluble. It has mol. wt. of about
17,000. It is much in therapeutics. Excessive doses of heparin lead to
bleeding; this cc controlled by protaminc sulfatc intravenously. Heparin is
negatively charged molecule and protamine sulfatc being positively charged
neutralizes it readily.
(ii) Antithrombins - These arc substances present in plasma which can
inactivate large amounts of thrombin very quickly. Thrombin formed during the
clotting of only 10 ml of blood can clot the whole body blood. But normally the
excess of thrombin is destroyed by antithrombins, and thus the extension of the
clotting process to other body regions is prevented; the fibrin clot also
adsorb a lot of thrombin and this helps in keeping the clotting process localized.
Heparin binds to the lysine residue on the antithrombin molecule, which results
in an enhanced activity of the latter. Antithrombins are of great physiological
importance. Small amounts of thrombin arc believed to be formed even under
normal circumstances, but antilhrombins immediately inactivate it and thus
prevent thrombosis. The cervical glands of the medicinal leech (hirudo) contain
a substance hirudin which has antithrombin activity. It therefore acts as an
anticoagulant and enables the leech to suck blood for a long period from the
area where it is applied.
(iii) Anlithromboplastin substances - Blood and body tissues contain substances, which
neutralize any thromboplastin released by damage to tissues.
(iv) Protein C-protein-S system - Protein C is a vitamin-K dependent plasma protein. It
first occurs in an inactive form. It is activated by thrombin. Its active form
acts as a powerful protease; in the presence of a cofactor, thrombomodulin,
present on the endothelial surface it digests factors V and VIII and thus
clotting process is inhibited. Protein S has the role of accelerating the
activation of protein C. These two proteins are believed to have a physiologic
role in helping to keep the blood unclotted.
(v) Fibrinolysis - This process has an important role in keeping the
blood in a fluid state under normal circumstances. It is believed that
micro-thrombi are formed in the blood even normally, but fibrinolysis liquefies
these clots. Cellular phagocytosis of small particles of fibrin thrombi is also
a protective mechanism.
(vi) Prostacydin - Its role has already been described under platelets.
3. Vigorous circulation of blood - Under normal
circumstances this is an important factor in preventing intravascular clotting
of blood. This is brought about by not allowing the reactants to accumulate at
a certain site, which could initiate clotting. In conditions associated with
stasis of blood (bed-ridden patients, congestive heart failure, polycylhemia,
hyper-gammaglobulinemia), intravascular clotting is quite common. Use of oral
contraceptive pills and pregnancy also produce venous stasis by causing venous
dilatation. During vigorous circulation the formed elements of the blood
including platelets arc kept away from the vessel wall. Moreover, it
facilitates the mixing of the activated clotting factors with their inhibitors.
These active clotting factors are also carried to the liver and the
reticuloendothelial system, which take them up and destroy them.
ROLE OF LIVER AND VITAMIN K IN BLOOD COAGULATION
Several factors needed for blood coagulation arc
formed in the liver. The formation of four of these factors, i.e. prothrombin
and factors VII, IX and X is dependent upon the presence of vitamin K. A
deficiency of vitamin K leads to a deficiency of these factors, which may
produce a tendency to bleed easily. Substances, which antagonize vitamin K,
such as dicumarol, also produce a deficiency of these factors producing
hemorrhagic tendency. If the liver is severely diseased, it cannot make these
factors at a normal rate and even the administration of vitamin K will fail to
exert a beneficial effect on the hemorrhagic tendency. Other factors
synthesized in the liver but independent of vitamin K are fibrinogen, factor V
and factor XIII. The sites of the formation of factors VIII, XI and XII are
.not known, although factor VIII is believed to be produced in the
reticuloendothelial cells.
HEMOSTASIS
Three important mechanisms bring about hemostasis,
which means the stoppage of bleeding from an injured area.
1. Local factors - These can be divided into two
types.
(a) Vascular spasm - A localized vascular spasm is the first line of
defence against bleeding and is brought about by the following changes:
(i) Local spasm of blood vessels which is of myogenic
origin. It lasts for upto 20 minutes,
(ii) Vasoconstriction in large vessels which is of
reflex (neurogenic) origin. It lasts for only a few minutes,
(iii) Liberation of serotonin, a vasoconstrictor, from
the disintegrating platelets.
The early vascular response reduces blood flow and
intravascular pressure and therefore facilitates consolidation of the
hemostatic plug.
(b) Extra-vascular - These include subcutaneous tissue, muscle and skin,
which help in stopping bleeding by covering and thus applying pressure over the
wounded area. Wounds of the scalp area bleed more as there is paucity of
connective tissue in this area; same is true about Little's area in the nose
where bleeding results in epistaxis. Old age, rheumatoid arthritis and excessive
use of glucocorticoids also produce atrophy of the subcutaneous tissue with
tendency to bleed easily.
2. Role of platelets (platelet plug) - The platelet
plug is produced earlier than formation of fibrin and for this reason the
platelet plug is said to produce primary hemostasis while fibrin is said to
produce secondary hemostasis.
The formation of the platelet plug that stops bleeding
from small blood vessels like capillaries is an important defence against
bleeding; this process and the additional contribution of platelets to clotting
process have already been described. Platelets also release serotonin, a
vasoconstrictor.
3. Clotting of blood - This process is very important
for maintaining the occlusive plug contributed by the platelets. As soon as
blood comes in contact with extravascular tissues, the complex series of
reactions leading to blood clotting arc initiated; thrombin formed in this
process further activates release of ADP from platelets. The formation of
fibrin clot reinforces the platelet plug and the combined fibrin-platelet plug
(hemostatic plug) serves to seal the wound more effectively and prevents
bleeding after the injured vessels re-open after some time. The clot then
undergoes retraction, till after 24 hours its size is only 40% of the original.
Platelets and ATP arc needed for the retraction of the clot. The retraction of
the hemostatic plug results in a still more effective plugging of the wounded
vessels.
Interaction Between Blood
Clotting and Kallikrein Systems - It is quite complex. Both systems affect each
other and greatly accelerate blood coagulation.
(i) Active factor XII or its breakdown product acts as
an enzyme that increases the reaction, Prekallikrein → Kallikrein.
(ii) Kallikrein in turn greatly increases the activation
of factor XII; active factor XII now activates factor XI and thus accelerates
clotting process.
(iii) Kallikrein acts on kininogcn to release kinin
which by producing vasodilatation brings more blood containing clotting factors
to the site of injury thus intensifying the clotting process.
Fate of the blood clot - The initial hemostatic plug contains the platelets and
fibrin. But as the
platelets undergo autolysis, therefore after 24 to 48 hours the hemostatic plug
consists almost entirely of fibrin. In the meantime fibrinolysis starts as the
fibrinolytic system is activated and the destroyed leukocytes release
proteolytic enzymes.
A small clot may later be completely liquefied by the
process of fibrinolysis. In bigger clots a proliferation of blood vessels and
connective tissue may take place and after two to three weeks it becomes a
fibrous mass; this process is called organization of the clot. The defects in
the vessel wall are covered with endothelium. If the defect in vessel wall is
small, the endothelium grows from the ends. But when the defect is large, then
the endothelial cells are produced by transformation of smooth muscle cells
which migrate from the media of the vessel wall.
Functional element of
microcirculation
Microcirculatory part of vascular system performs all blood functions.
There are such types of vessels: arterioles, metarterioles, capillaries and
venuls. Mean diameter of these vessels is less than 100 mcm. Arterioles,
capillary bed venuls and lymphatic capillaries compose functional element of
microcirculation. Main processes as blood-tissue exchange or lymph production
are performed there. Mean diameter of capillaries is 3-6 mcm. The length of
capillary vessel is near 750 mcm. Capillaries perform exchange in surface near
14000 mkm2. Blood flow velocity in capillaries consists near 0.3
mm/s, which permits passing erythrocytes through capillary in 2-3 s.
SOME LABORATORY TESTS DONE FOR INVESTIGATING BLEEDING
DISORDERS
(1) Clotting time - (Method of Lee and White). Without
letting tissue juice to contaminate the blood, venous blood is withdrawn into a
clean, dry, all glass syringe with a wide bore needle.
A stopping watch is started the moment blood appears in the syringe and blood
coagulation time is recorded from the moment. The needle is detached and 1 ml
of blood is delivered into each of the four dry, chemically clean glass tubes
with a size 10 by
After 3 minutes have elapsed, each of these tubes is
lifted from the water bath and is tilled very gently. The clotting time is
taken when the tube can be inverted without its contents spilling. The clotting
time for each tube is recorded separately and the clotting time is reported as
the average of the four tubes. The normal clotting time by this method is 4 to
10 minutes. Clotting time is prolonged in hemophilia and other conditions
associated with a deficiency of clotting factors. It remains normal in purpura
because in this conditions there are no coagulation
defects.
(2) Prothrombin time - (Quick's one-stage method).
Oxalated plasma of the patient is obtained and to it is added an excess of
thromboplastin, which is usually an emulsion of rabbit's brain. Calcium
chloride solution is added. The time taken for clotting of the plasma to occur
after the addition of calcium chloride is noted; it is termed as the
prothrombin time and normally it is 12 to 14 seconds. The prothrombin time is
increased in hypoprothrombinemia. However, the test is not
specific for prothrombin deficiency and deficiencies of factors V and X and of
fibrinogen also increase its duration.
(3) Bleeding time - (Duke's method). When injury to a
small vessel produces only a small defect, the platelet plug, i.e. the
white thrombus alone can cause hemostasis. This is the basis for the bleeding
time that is employed clinically to distinguish hemostatic deficiency caused by
abnormalities of blood capillary wall from those caused by coagulation defects.
In doing the test a standardized cut is made usually into the skin of the car
lobule. The blood, which flows, is absorbed by a filter paper every 15 seconds.
When the filter paper remains unstained on touching the wounded area, it shows
that bleeding has stopped. The time taken by the bleeding to stop after the cut
is made is the bleeding time. This test is simple but crude. In the majority of
normal persons, bleeding lime ranges between 1 to 5 minutes. Bleeding time is
prolonged in purpuras.
(4) Platelet count - This is normally 180,000 to
320,000 per cu mm of blood. Bleeding usually occurs when the count falls to
50,000 platelets per cu mm or below.
(5) Plasma fibrinogen level - This is found to be low
in conditions associated with excessive fibrinolysis. In this condition the
presence of fibrinogen degradative products (FDP) in the plasma and urine can
be demonstrated.
(6) Assay of clotting factors - Factors V, VIII, IX,
etc. can be quantitatively assessed in the blood.
(7) Capillary fragility test - A strong positive
pressure (by inflating sphygmomanometercuff) or a negative pressure (by
applying suction cups) is applied and the number of minute petechial
hemorrhages (petechiac) is counted in the skin; their number is found to be
much increased in conditions associated with an increased capillary fragility, e.g.
purpuras.
METHODS OF KEEPING BLOOD FLUID IN VITRO - In order for the blood to be kept fluid, certain methods are employed.
These include the following:
(i) Addition of heparin. Its mechanism of
anticoagulant action has been already described.
(ii) Addition of substances which take up Ca2+,
e.g. citrate, oxalate, ethylene-diamine-tetra-acetic acid or EDTA. The
last compound is an example of the chelating
Evaluation of coagulation:
A)
Coagulogramme
Rate coagulation status can be based on coagulation,
which includes the following indicators:
1. Time coagulation (by Lee-White).
2. Recalcification time of plasma.
3. Trombotest.
4. Prothrombin (thromboplastin) time.
5. Prothrombin (thromboplastin) code.
6. The concentration of fibrinogen.
7. Tolerance plasma heparin.
8. Heparyn's time.
9. Fibrinolysis.
1. Time coagulation (by Lee-White) - this time from
the taking of blood from a vein before the advent of clot in a test tube, which
is located in a water bath at 37°C. In healthy people, clotting time ranges
from 5 to 10 minutes. Clotting time less than 5 minutes.
evidence of increased coagulation.
2. Plasma recalcification time - this time after
plasma coagulation dekaltsynovanoyi adding to it a standard amount of calcium
chloride. Normal is 60-120 seconds. Recalcification time more than 120 seconds
indicates a reduced coagulation.
3. Trombotest - gives a subjective assessment of the
fibrin clot formed during centering plasma oxalate in a solution of calcium
chloride. It is possible to observe the seven degrees of coagulation:
I - Opalescence,
II - small grains of fibrin,
III - flakes of fibrin,
IV - threads of fibrin;
V - net of fibrin strands,
VI - loose fibrin clot;
VII - dense fibrin clot.
The first three stages are observed in hypocoagulation,
IV, V, VI degree attesting normal coagulation.
4. Prothrombin (thromboplastin) time - this time the
subject of plasma coagulation atadding her tissue thromboplastin. Thus starts
the conversion of prothrombin to thrombin. The normal prothrombin time is 12-15
seconds. Increased prothrombin time indicates a decrease in coagulation.
5. Prothrombin (thromboplastin) index - a ratio of
prothrombin time donor and the subject person, expressed as a percentage.
Normal ranges from 80 to 105%. The increase in prothrombin index indicates an
increase in coagulation.
6. The concentration of fibrinogen in normal is 2-4 g
/ l.
7. Tolerance plasma to heparin - a clotting time of
plasma by adding to it mixture heparin with Ca2+. Normally, it is
6-11 minutes.
8. Heparyn's
time - a time of
coagulation of tissue thromboplastin-activated plasma by adding thereto
heparin. Normal ranges from 50 to 60 seconds.
Figures 7 and 8 show the interaction of coagulation
factors and anticoagulant - heparin. With their minimal sense is the need for
anticoagulants.
9. Fibrinolysis. This indicator assesses the
fibrinolytic system of blood flow from lysis of thrombus over time. By using
M.A.Kotovschykovoyi, B.I.Kuznyka fibrinolysis in normal is 15-20%. Lower than
15% indicates a reduction in fibrinolysis.
B) Tromboelastography.
This is a graphic record of the process of blood
coagulation and fibrinolysis.
R - time from the beginning
of the fluctuations of the cell to the formation of the first fibrin strands. Characterizes the phase of coagulation.
K - the time since the
beginning of clot formation to achieve a fixed level of its density. 3 Displays
the phase of blood coagulation.
MA- maximum amplitude - displays the maximum density
of the bunch. 80% of the number of MA and properties of platelets (the ability
to aggregate), 20% - the number of formed fibrin.
Short review:
Thrombocytes (Platelets)
Platelets form in bone marrow by following steps, myeloid stem cells
eventually become megakaryocytes whose cell fragments form platelets
Short life span - 5 to 9 days in bloodstream
Platelets release ADP and other chemicals needed for platelet plug
formation
Hemostasis
Hemostasis -
stoppage of bleeding in a quick & localized fashion when blood vessels are damaged
Prevents hemorrhage (loss of a large amount of blood)
Three major steps of Hemostasis:
1.
Vascular spasm
2.
Platelet Plug Formation
Aggregation and adhesion of platelets
3.
Blood Clotting
Fibrin threads entangle platelets and RBCs to form blood clot
Vascular Spasm
Damage to blood vessel stimulates pain receptors
Reflex contraction of smooth muscle of small blood vessels
Can reduce blood loss for several hours, allowing other mechanisms to
take over
Effective only for small blood vessels or arterioles, not major
arteries
Platelet plug formation
Platelet Plug Formation steps:
1.
Platelet Adhesion
Platelets stick to exposed collagen of vessel
2.
Platelet Release Reaction
Platelets extend projections
Platelets release Thromboxane A2, Serotonin & ADP activating
other platelets
3.
Platelet Aggregation
Platelets stick together forming a platelet plug
1) Platelet Adhesion
Platelets stick to exposed collagen underlying damaged endothelial
cells in vessel wall
2) Platelet Release
Reaction
Platelets activated by adhesion
Extend projections to make contact with each other
Release Thromboxane A2, Serotonin & ADP activating other platelets
Serotonin & Thromboxane A2 are vasoconstrictors decreasing
blood flow through the injured vessel.
ADP causes stickiness
3) Platelet Aggregation
Activated platelets stick together and activate new platelets to form a
mass called a platelet plug
Plug reinforced by fibrin threads formed during clotting process
Platelet plug formation
Blood Clotting
Blood drawn from the body thickens into a gel if not mixed with
anticoagulant
Blood separates into liquid (serum) and a clot of insoluble fibers
(fibrin) in which the blood cells are trapped
Substances required for clotting:
Ca+2
Clotting Factors (enzymes made by liver)
Substances released by platelets or damaged tissues
Clotting is a cascade of reactions
Each clotting factor activates the next, in a specific sequence,
resulting in the formation of fibrin threads
Coagulation
A set of reactions in which blood is transformed from a liquid to a gel
Coagulation follows intrinsic and extrinsic pathways
Common Pathway: The final three
steps
Prothrombinase (Prothrombin activator) is formed from Factor X
Prothrombinase converts Prothrombin into Thrombin
Thrombin catalyzes polymerization of Fibrinogen into a Fibrin
mesh
Two Pathways to
Prothrombin Activator
May be initiated by either the intrinsic or extrinsic pathway
Triggered by tissue-damaging events
Involves a series of procoagulants
Each pathway cascades toward Factor X
Once Factor X has been activated, it complexes with Ca+2
ions, PF3, and Factor V to form Prothrombin activator (Prothrominase)
Coagulation Phase of
Hemostasis
Coagulation Pathway
Prothrombinase & Ca+2 catalyze the conversion of Prothrombin
to Thrombin
Thrombin & Ca+2 catalyze the polymerization of Fibrinogen
into Fibrin
Insoluble fibrin strands form the structural basis of a clot
Fibrin causes RBCs and Platelets to become a gel-like plug
Thrombin & Ca+2 also activate Factor XIII (F13)
that:
Cross-links fibrin mesh
Strengthens and stabilizes the clot
Coagulation Pathway
Clot Dissolution
Inactive plasminogen becomes plasmin, a fibrinolytic enzyme
Plasmin dissolves small clots at site of a completed repair
Clot formation remains localized
blood flow disperses clotting factors
Basophils release heparin (anticoagulant), preventing
inappropriate clots
Intravascular Clotting
Thrombosis
Clot (thrombus) formed in an unbroken blood vessel
Attached to rough inner lining of blood vessel
Blood flows too slowly (stasis) allowing clotting factors to build up
locally & cause coagulation
May dissolve spontaneously or dislodge & travel
Embolus –
free floating clot in the blood
Low dose aspirin blocks synthesis of thromboxane A2 &
reduces inappropriate clot formation,
Helps to prevent strokes, myocardial infarctions.
References:
1. Review of Medical Physiology // W.F.Ganong. – 24th
edition, 2012.
2. Textbook of Medical Physiology //
A.C.Guyton, J.E.Hall. – Eleventh edition, 2005.