Unit IV

HEMOSTASIS, HEMORRHAGIC DISORDERS,

AND THROMBOSIS
 Hemostasis
• Hemostasis is a precisely orchestrated process involving platelets, clotting
factors, and endothelium that occurs at the site of vascular injury and
culminates in the formation of a blood clot, which serves to prevent or limit
the extent of bleeding.
Sequence of events
Arteriolar vasoconstriction occurs
immediately and markedly reduces blood
flow to the injured area. It
is mediated by reflex neurogenic
mechanisms and augmented by the local
secretion of factors such as endothelin,
a potent endothelium-derived
vasoconstrictor. This
effect is transient, however, and bleeding
would resume
if not for activation of platelets and
coagulation factors.
Disruption of the endothelium exposes
subendothelial von Willebrand factor (vWF) and
collagen, which promote platelet adherence and
activation. Activation of platelets result in a
dramatic shape change (from small rounded
discs to flat plates with spiky protrusions that
markedly increased surface area), as well as the
release of secretory granules. Within minutes the
secreted products recruit additional platelets,
which undergo aggregation to form a primary
hemostatic plug
Tissue factor is also exposed at the site of injury.
Tissue factor is a membrane-bound procoagulant
glycoprotein that is normally expressed by
subendothelial cells in the vessel wall, such as
smooth muscle cells and fibroblasts. Tissue
factor binds and activates factor VII, setting in
motion a cascade of reactions that culiminates in
thrombin generation. Thrombin cleaves
circulating fibrinogen into insoluble fibrin,
creating a fibrin meshwork, and also is a potent
activator of platelets, leading to additional
platelet aggregation at the site of injury. This
sequence, referred to as secondary hemostasis,
consolidates the initial platelet plug
 Polymerized fibrin and platelet aggregates
undergo contraction to form a solid,
permanent plug that prevents further
hemorrhage. At this stage, counterregulatory
mechanisms (e.g., tissue plasminogen activator,
t-PA) are set into motion that limit clotting to
the site of injury and eventually lead to clot
resorption and tissue repair.

https://www.youtube.com/watch?v=HFNWGCx_Eu4
 Role of platelets in hemostatsis
• Platelets play a critical role in hemostasis by forming the primary plug that
initially seals vascular defects and by providing a surface that binds and
concentrates activated coagulation factors.
• After a traumatic vascular injury, platelets encounter constituents of the
subendothelial connective tissue, such as vWF and collagen. On contact with
these proteins, platelets undergo a sequence of reactions that culminate in the
formation of a platelet plug
• Platelet adhesion is mediated largely via interactions with vWF, which acts as a bridge
between the platelet surface receptor glycoprotein Ib (GpIb) and exposed collagen.
• Platelets rapidly change shape following
adhesion, being converted from smooth discs
to spiky “sea urchins” with greatly increased
surface area. This change is accompanied by
alterations in glycoprotein IIb/IIIa that
increase its affinity for fibrinogen, and by the
translocation of negatively charged
phospholipids (particularly phosphatidylserine)
to the platelet surface. These phospholipids
bind calcium and serve as nucleation sites for
the assembly of coagulation factor complexes.
• Secretion (release reaction) of granule contents occurs along with changes in shape;
these two events are often referred to together as platelet activation.
• Platelet activation is triggered by several factors, including the coagulation factor thrombin
and ADP.
• Thrombin activates platelets through a special type of G-protein– coupled receptor
referred to as a protease-activated receptor (PAR), which is switched on by a proteolytic
cleavage carried out by thrombin.
• ADP is a component of dense body granules; thus, platelet activation and ADP release
begets additional rounds of platelet activation, a phenomenon referred to as recruitment.
• Activated platelets also produce the prostaglandin thromboxane A2 (TxA2), a potent
inducer of platelet aggregation.
• Aspirin inhibits platelet aggregation and produces a mild bleeding defect by inhibiting
cyclooxygenase, a platelet enzyme that is required for TxA2 synthesis.
• Platelet aggregation follows their activation. The conformational change in glycoprotein
IIb/IIIa that occurs with platelet activation allows binding of fibrinogen, a large bivalent
plasma polypeptide that forms bridges between adjacent platelets, leading to their
aggregation.
• Predictably, inherited deficiency of GpIIb-IIIa results in a bleeding disorder called
Glanzmann thrombasthenia).
• The initial wave of aggregation is reversible, but concurrent activation of thrombin stabilizes
the platelet plug by causing further platelet activation and aggregation, and by promoting
irreversible platelet contraction.
• Platelet contraction is dependent on the cytoskeleton and consolidates the aggregated
platelets.
• In parallel, thrombin also converts fibrinogen into insoluble fibrin, cementing the platelets
in place and creating the definitive secondary hemostatic plug.
• Entrapped red cells and leukocytes are also found in hemostatic plugs, in part due to
adherence of leukocytes to P-selectin expressed on activated platelets.
https://www.youtube.com/watch?v=R8JMfbYW2p4
 Coagulation Cascade
• A series of amplifying enzymatic reactions that leads to the deposition of an
insoluble fibrin clot.
• The cascade of reactions in the pathway can be likened to a “dance,” in
which coagulation factors are passed from one partner to the next. Each
reaction step involves an enzyme (an activated coagulation factor), a substrate
(an inactive proenzyme form of a coagulation factor), and a cofactor (a
reaction accelerator). These components are assembled on a negatively
charged phospholipid surface, which is provided by activated platelets.
https://www.youtube.com/watch?v=cy3a__OOa2M
• Factor VIIa/tissue factor complex is the most important activator of factor
IX
• Factor IXa/factor VIIIa complex is the most important activator of factor X
• Assembly of reaction complexes also depends on calcium, which binds to
γ-carboxylated glutamic acid residues that are present in factors II, VII, IX,
and X.
• The enzymatic reactions that produce γ-carboxylated glutamic acid use
vitamin K as a cofactor and are antagonized by drugs such as coumadin, a
widely used anticoagulant.
• The prothrombin time (PT) assay assesses the function of the proteins in the
extrinsic pathway (factors VII, X, V, II, and fibrinogen). In brief, tissue factor,
phospholipids, and calcium are added to plasma and the time for a fibrin clot to
form is recorded.
• The partial thromboplastin time (PTT) assay screens the function of the
proteins in the intrinsic pathway (factors XII, XI, IX, VIII, X, V, II, and
fibrinogen). In this assay, clotting of plasma is initiated by addition of negatively
charged particles (e.g., ground glass) that activate factor XII (Hageman factor)
together with phospholipids and calcium, and the time to fibrin clot formation is
recorded.
• Deficiencies of factors V, VII, VIII, IX, and X are associated with moderate to
severe bleeding disorders, and prothrombin deficiency is likely incompatible with
life.
• Factor XI deficiency is only associated with mild bleeding, and individuals with
factor XII deficiency do not bleed and in fact may be susceptible to thrombosis.
• The mild bleeding tendency due to factor XI deficiency is likely explained by the
ability of thrombin to activate factor XI (as well as factors V and VIII), a feedback
mechanism that amplifies the coagulation cascade.
• The paradoxical effect of factor XII deficiency may be explained by involvement of
factor XII in the fibrinolysis pathway
 Significance of thrombin
• Conversion of fibrinogen into crosslinked fibrin. Thrombin directly
converts soluble fibrinogen into fibrin monomers that polymerize into an
insoluble clot, and also amplifies the coagulation process, not only by
activating factor XI, but also be activating two critical co-factors, factors V
and VIII. It also stabilizes the secondary hemostatic plug by activating factor
XIII, which covalently cross-links fibrin.
• Platelet activation. Thrombin is a potent inducer of platelet activation and
aggregation through its ability to activate PARs, thereby linking platelet
function to coagulation.
 Significance of thrombin
• Pro-inflammatory effects. PARs are also expressed on inflammatory cells,
endothelium, and other cell types (Fig. 4-8), and activation of these receptors
by thrombinis believed to mediate proinflammatory effects that contribute to
tissue repair and angiogenesis.
• Anticoagulant effects. Remarkably, through mechanisms described later,
upon encountering normal endothelium thrombin changes from a
procoagulant to an anticoagulant. This reversal in function prevents clotting
from extending beyond the site of the vascular injury.
 Factors That Limit Coagulation.
• Once initiated, coagulation must be restricted to the site of vascular injury to
prevent deleterious consequences. One limiting factor is simple dilution;
blood flowing past the site of injury washes out activated coagulation factors,
which are rapidly removed by the liver. A second is the requirement for
negatively charged phospholipids, which, as mentioned, are mainly
provided by platelets that have been activated by contact with subendothelial
matrix at sites of vascular injury. However, the most important
counter-regulatory mechanisms involve factors that are expressed by intact
endothelium adjacent to the site of injury.
 Fibrinolytic cascade
• Activation of the coagulation cascade also sets into motion a fibrinolytic cascade that limits the size
of the clot and contributes to its later dissolution.
• Fibrinolysis is largely accomplished through the enzymatic activity of plasmin, which breaks down
fibrin and interferes with its polymerization.
• Plasmin is generated by enzymatic catabolism of the inactive circulating precursor plasminogen,
either by a factor XII–dependent pathway (possibly explaining the association of factor XII
deficiency and thrombosis) or by plasminogen activators.
• The most important plasminogen activator is t-PA; it is synthesized principally by endothelium and
is most active when bound to fibrin.
• Once activated, plasmin is in turn tightly controlled by counterregulatory factors such as α2-plasmin
inhibitor, a plasma protein that binds and rapidly inhibits free plasmin.
Significance of endothelium in hemostasis
• The balance between the anticoagulant and procoagulant activities of
endothelium often determines whether clot formation, propagation, or dissolution
occurs.
• Normal endothelial cells express a multitude of factors that inhibit the
procoagulant activities of platelets and coagulation factors and that augment
fibrinolysis. These factors act in concert to prevent thrombosis and to limit
clotting to sites of vascular damage.
• However, if injured or exposed to proinflammatory factors, endothelial cells lose
many of their antithrombotic properties.
• The antithrombotic properties of endothelium can be divided into activities
directed at platelets, coagulation factors, and fibrinolysis.
• Platelet inhibitory effects. An obvious effect of intact endothelium is to serve as a
barrier that shields platelets from subendothelial vWF and collagen. However,
normal endothelium also releases a number of factors that inhibit platelet activation
and aggregation. Among the most important are prostacyclin (PGI2), nitric oxide
(NO), and adenosine diphosphatase; the latter degrades ADP, already discussed as a
potent activator of platelet aggregation. Finally, endothelial cells bind and alter the
activity of thrombin, which is one of the most potent activators of platelets.
• Anticoagulant effects. Normal endothelium shields coagulation factors
from tissue factor in vessel walls and expresses multiple factors that
actively oppose coagulation, most notably thrombomodulin, endothelial
protein C receptor, heparin-like molecules, and tissue factor pathway
inhibitor.
• Thrombomodulin and endothelial protein C receptor bind thrombin and protein C, respectively, in
a complex on the endothelial cell surface.
• When bound in this complex, thrombin loses its ability to activate coagulation factors and platelets,
and instead cleaves and activates protein C, a vitamin K–dependent protease that requires a
cofactor, protein S.
• Activated protein C/protein S complex is a potent inhibitor of coagulation factors Va and VIIIa.
• Heparin-like molecules on the surface of endothelium bind and activate antithrombin III, which
then inhibits thrombin and factors IXa, Xa, XIa, and XIIa.
• Tissue factor pathway inhibitor (TFPI), like protein C, requires protein S as a cofactor and, as the
name implies, binds and inhibits tissue factor/factor VIIa complexes.
• Fibrinolytic effects. Normal endothelial cells synthesize t-PA, already
discussed, as a key component of the fibrinolytic pathway.
 Hemorrhagic disorders
Disorders associated with abnormal bleeding inevitably stem from primary or
secondary defects in vessel walls, platelets, or coagulation factors, all of
which must function properly to ensure hemostasis.
Defects of primary hemostasis (platelet defects or von Willebrand disease) often
present with small bleeds in skin or mucosal membranes. These bleeds typically
take the form of petechiae, minute 1- to 2-mm hemorrhages or purpura, which are
slightly larger(≥3 mm) than petechiae. It is believed that the capillaries of the mucosa
and skin are particularly prone to rupture following minor trauma and that under
normal circumstances platelets seal these defects virtually immediately. Mucosal
bleeding associated with defects in primary hemostasis may also take the form of
epistaxis (nosebleeds), gastrointestinal bleeding, or excessive menstruation
(menorrhagia). A feared complication of very low platelet counts
(thrombocytopenia) is intracerebral hemorrhage, which may be fatal.
Defects of secondary hemostasis (coagulation factor defects) often present
with bleeds into soft tissues (e.g., muscle) or joints. Bleeding into joints
(hemarthrosis) following minor trauma is particularly characteristic of
hemophilia.
Generalized defects involving small vessels often present with “palpable
purpura” and ecchymoses. Ecchymoses (sometimes simply called bruises)
are hemorrhages of 1 to 2 cm in size. In both purpura and ecchymoses, the
volume of extravasated blood is sufficient to create a palpable mass of blood
known as a hematoma. Purpura and ecchymoses are particularly characteristic
of systemic disorders that disrupt small blood vessels (e.g., vasculitis) or that
lead to blood vessel fragility (e.g., amyloidosis, scurvy).
 Clinical significance
• It depends on the volume of the bleed, the rate at which it occurs, and its location.
• Rapid loss of up to 20% of the blood volume may have little impact in healthy
adults; greater losses, however, can cause hemorrhagic (hypovolemic) shock
• Bleeding that is trivial in the subcutaneous tissues can cause death if located in the
brain
• Chronic or recurrent external blood loss (e.g., peptic ulcer or menstrual bleeding)
causes iron loss and can lead to an iron deficiency anemia. In contrast, when red
cells are retained (e.g., hemorrhage into body cavities or tissues), iron is recovered
and recycled for use in the synthesis of hemoglobin.
 Thrombosis
• Thrombosis is the process of formation of solid mass in circulation from the
constituents of flowing blood; the mass itself is called a thrombus.
• In contrast, a blood clot is the mass of coagulated blood formed in vitro e.g. in a
test tube. Haemostatic plugs are the blood clots formed in healthy individuals at the
site of bleeding e.g. in injury to the blood vessel.
• Haematoma is the extravascular accumulation of blood clot e.g. into the tissues.
• Haemostatic plugs are useful as they stop the escape of blood and plasma, whereas
thrombi developing in the unruptured cardiovascular system may be
life-threatening
1. Ischaemic injury. Thrombi may decrease or stop the blood supply to part of
an organ or tissue and cause ischaemia which may subsequently result in
infarction.
2. Thromboembolism. The thrombus or its part may get dislodged and be
carried along in the bloodstream as embolus to lodge in a distant vessel.
 Thrombogenesis
• Human beings possess inbuilt system by which the blood remains in fluid state
normally and guards against the hazards of thrombosis and haemorrhage. However,
injury to the blood vessel initiates haemostatic repair mechanism or
thrombogenesis.
• Virchow described three primary events which predispose to thrombus formation
(Virchow’s triad):
1. endothelial injury,
2. altered blood flow, and
3. hypercoagulability of blood.
 1. Endothelial injury
The integrity of blood vessel wall is important for maintaining normal blood flow. An intact
endothelium has the following functions:
i. It protects the flowing blood from the thrombogenic influence of subendothelium.
ii. It elaborates a few anti-thrombotic factors (thrombosis inhibitory factors) e.g.
a) Heparin-like substance which accelerates the action of antithrombin III and inactivates some other
clotting factors.
b) Thrombomodulin which converts thrombin into activator of protein C, an anticoagulant.
c) Inhibitors of platelet aggregation such as ADPase, PGI2 or prostacyclin.
d) Tissue plasminogen activator which accelerates the fibrinolytic activity.
iii. It can release a few prothrombotic factors which have procoagulant
properties (thrombosis favouring factors) e.g.
a) Thromboplastin or tissue factor released from endothelial cells.
b) von Willebrand factor that causes adherence of platelets to the subendothelium.
c) Platelet activating factor which is activator and aggregator of platelets.
d) Inhibitor of plasminogen activator that suppresses fibrinolysis.
• Vascular injury exposes the subendothelial connective tissue (e.g. collagen,
elastin, fibronectin, laminin and glycosaminoglycans) which are
thrombogenic and thus plays important role in initiating haemostasis as well
as thrombosis.
• Injury to vessel wall also causes vasoconstriction of small blood vessels
briefly so as to reduce the blood loss. Endothelial injury is of major
significance in the formation of arterial thrombi and thrombi of the heart,
especially of the left ventricle.
• A number of factors and conditions may cause vascular injury and predispose to
the formation of thrombi. These are as under:
i. Endocardial injury in myocardial infarction, myocarditis, cardiac surgery, prosthetic valves.
ii. Ulcerated plaques in advanced atherosclerosis.
iii. Haemodynamic stress in hypertension.
iv. Arterial diseases.
v. Diabetes mellitus.
vi. Endogenous chemical agents such as hypercholesterolaemia, endotoxins.
vii. Exogenous chemical agents such as cigarette smoke.
 2. Role of Platelets
• Following endothelial cell injury, platelets come to play a central role in normal
haemostasis as well as in thrombosis. The sequence of events is as under:
1. Platelet adhesion: The platelets in circulation recognise the site of endothelial injury and
adhere to exposed subendothelial collagen (primary aggregation); von Willebrand’s factor is
required for such adhesion between platelets and collagen. Normal non-activated platelets
have open canalicular system with cytoplasmic organelles (granules, mitochondria,
endoplasmic reticulum) dispersed throughout the cytoplasm. During the early adhesion
process, there is dilatation of canalicular system with formation of pseudopods and the
cytoplasmic organelles shift to the centre of the cell.
2. Platelet release reaction: The activated platelets then undergo release reaction
by which the platelet granules are released to the exterior. Two main types of
platelet granules are released:
i. Alpha granules containing fibrinogen, fibronectin, platelet derived growth factor, platelet
factor 4 (an antiheparin) and cationic proteins.
ii. Dense bodies containing ADP (adenosine diphosphate), ionic calcium, 5-HT (serotonin),
histamine and epinephrine.
As a sequel to platelet activation and release reaction, the phospholipid
complex-platelet factor 3 gets activated which plays important role in the intrinsic
pathway of coagulation.
3. Platelet aggregation: Following release of ADP, a potent platelet
aggregating agent, aggregation of additional platelets takes place (secondary
aggregation). This results in formation of temporary haemostatic plug.
However, stable haemostatic plug is formed by the action of fibrin,
thrombin and thromboxane A2.
 Role of coagulation system
• Coagulation mechanism is the conversion of the plasma fibrinogen into solid
mass of fibrin. The coagulation system is involved in both haemostatic
process and thrombus formation.
i. In the intrinsic pathway, contact with abnormal surface leads to activation of factor
XII and the sequential interactions of factors XI, IX, VIII and finally factor X,
alongwith calcium ions (factor IV) and platelet factor 3.
ii. In the extrinsic pathway, tissue damage results in the release of tissue factor or
thromboplastin. Tissue factor on interaction with factor VII activates factor X
iii. The common pathway begins where both intrinsic and extrinsic pathways
converge to activate factor X which forms a complex with factor Va and
platelet factor 3, in the presence of calcium ions. This complex activates
prothrombin (factor II) to thrombin (factor IIa) which, in turn, converts
fibrinogen to fibrin. Initial monomeric fibrin is polymerised to form
insoluble fibrin by activation of factor XIII.