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Hemostasis /Coagulation
Thrombosis-Hemostasis-Hemorrhage
Topics
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Hemostasis
Coagulation
Regulation
Diseases
Pharmacology
Thrombin receptor
study
Hemostasis
Vessel Injury
Tissue
Factor
Neural
Blood Vessel
Platelet
Coagulation
Constriction
Activation
Activation
Primary hemostatic plug
Reduced
Blood flow
Platelet
aggregation
Stable Hemostatic Plug
Fibrin clot
Primary Hemostasis
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Primary hemostasis is defined as the formation of the primary platelet plug and
involves platelets, the blood vessel wall and von Willebrand factor.
As a general rule, abnormalities in primary hemostasis result in hemorrhage
from mucosal surfaces (epistaxis, melena, hematuria), petechial or ecchymotic
hemorrhages, and prolonged bleeding after venipuncture or wounds.
However, if the defect is severe, bleeding more typical of disorders of
secondary hemostasis, can result, e.g. intracavity hemorrhage.
A defect in primary hemostasis may have abnormal platelet number or function,
abnormal von Willebrand factor or defects in the blood vessel wall (very rare).
The normal endothelium prevents hemostasis by providing a physical barrier
and by secreting products, including NO and prostaglandin I2 (prostacyclin),
which inhibit platelet activation.
Following injury to the vessel wall, the initial event is vasoconstriction, which is
a transient, local.
Vasoconstriction not only retards extravascular blood loss, but also slows local
blood flow, enhancing the adherence of platelets to exposed subendothelial
surfaces and the activation of the coagulation process.
The formation of the primary platelet plug involves platelet adhesion followed
by platelet activation then aggregation to form a platelet plug.
VWF UNFOLDS UNDER SHEAR STRESS
The faster the blood flow, the stickier it gets
Platelet activation and Plug
Formation
Platelet adhesion:
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When endothelium is damaged, the normally isolated, underlying collagen is exposed to
circulating platelets, which bind collagen with collagen-specific glycoprotein Ia/IIa surface
receptors.
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This adhesion is strengthened further by von Willebrand factor(vWF), which is released from
the endothelium and from platelets; vWF forms additional links between the platelets'
glycoprotein Ib/IX/V and the collagen fibrils.
Platelet activation:
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The adhesions cause platelet activation and release of stored granules.
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The granules contains ADP, serotonin, platelet-activating factor (PAF), vWF, platelet factor 4,
and thromboxane A2 (TXA2), can activate additional platelets.
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The granules can activate a Gq-linked protein receptor cascade, resulting in increased
calcium in the platelets' cytosol, calcium activates protein kinase C (PKC) and PKC
activates phospholipase A2 (PLA2).
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PLA2 modifies the integrin membrane glycoprotein IIb/IIIa and increase its affinity
to fibrinogen.
Platelet aggregation:
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The activated platelets change shape from spherical to stellate, fibrinogen cross-links with
glycoprotein IIb/IIIa results aggregation of adjacent platelets.
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Thromboxane2, PAF, ADP and serotonin are platelet agonists, causing the activation and
recruitment of additional platelets to the adhered platelets. This activation is enhanced by
the generation of thrombin through the coagulation cascade; thrombin being an important
platelet agonist.
Primary platelet plug:
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The aggregation leads to the formation of the primary platelet plug and later stabilized by
the formation of fibrin.
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Platelets also contribute to secondary hemostasis (coagulation cascade) by providing a
phospholipid surface (this used to be called PF3) and receptors for the binding of
Aggregation of thrombocytes (platelets). Platelet rich human blood
plasma (left vial) is a turbid liquid. Upon addition of ADP, platelets are
activated and start to aggregate, forming white flakes (right vial)
Secondary Hemostasis_
_Coagulation Cascade
Components of coagulation cascade:
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Secondary hemostasis is defined as the formation of fibrin through the coagulation
cascade. This involves circulating coagulation factors, which act as enzymes (zymogens)
and cofactors (factors V and VIII), calcium and platelets (platelets provide a source of
phospholipid [PF3] and a binding surface upon which the coagulation cascade proceeds).
Deficiency:
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Defects in the coagulation cascade manifest more serious bleeding than primary
hemostasis, including bleeding into cavities (chest, joints) and subcutaneous
hematomas.
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Petechial hemorrhages are not seen in secondary hemostasis. These disorders do share
common bleeding symptoms with defects in primary hemostasis, including epistaxis and
bleeding after surgery or wounds.
Coagulation cascade pathways:
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The extrinsic pathway (Tissue factor pathway) involves the tissue factor and factor VII
complex, which activates factor X. It is the primary pathway for the initiation of blood
coagulation.
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The intrinsic pathway (contact activation pathway ) involves high-molecular weight
kininogen, prekallikrein, and factors XII, XI, IX and VIII. Factor VIII acts as a cofactor
(with calcium and platelet phospholipid) for the factor IX-mediated activation of factor X.
The common pathway:
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The extrinsic and intrinsic pathways converge at the activation of factor X.
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The common pathway involves the factor X-mediated generation of thrombin from
prothrombin (facilitated by factor V, calcium and platelet phospholipid), with the ultimate
production of fibrin from fibrinogen.
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Thrombin activate FXIII which crosslink fibrin to produce a firm clot.
Coagulation Cascade
Tissue factor pathway (extrinsic)
• The main role of the tissue factor pathway is to generate a "thrombin
burst,“ the most important constituent of the coagulation cascade in terms of
its feedback activation.
• FVII/FVIIa are always ready for any vessel break in circulation.
• Following damage to the blood vessel, FVII comes into contact with tissue
factor (TF) expressing cells (stromal fibroblasts and leukocytes) and forms
activated complex (TF-FVIIa).
• TF-FVIIa activates FIX and FX to FIXa and FXa.
• FXa and co-factor FVa form the prothrombinase complex and convert
prothrombin to thrombin.
• FVII can be activated by thrombin and FXa.
• Thrombin is quickly generated through the auto-regulatory cycle.
• The pathways contains a series of serine protease zymogens
and glycoprotein co-factors which are activated in the cascade, ultimately
resulting in amplification and cross-linked fibrin.
• All the reactions happen on cell surface and localized.
Contact activation pathway
(intrinsic)
• The contact activation pathway begins with formation of
the primary complex on collagen by high-molecular-weight
kininogen (HMWK), prekallikrein, and FXII (Hageman
factor).
• Prekallikrein is converted to kallikrein and FXII becomes
FXIIa.
• FXIIa converts FXI into FXIa.
• Factor XIa activates FIX, which with its co-factor FVIIIa
form the tenase complex and activates FX to FXa.
• The small amount of thrombin activates factor XI of the
intrinsic pathway and amplifies the coagulation cascade.
• Deficiencies of FXII, HMWK, and prekallikrein do not have
a bleeding disorder, an indication of minor role in
coagulation of intrinsic pathway.
• Contact activation system seems to be more involved in
inflammation and pathologic development.
The common pathway
Generation of thrombin and formation of clot
• The tissue factor and contact activation pathways both activate the "final
common pathway" through factor X, thrombin and fibrin.
• Activated Factor X (FXa), in the presence of factor V (FVa), calcium and
platelet phospholipid ("prothrombinase complex") convert prothrombin to
thrombin.
• Thombin, in turn, cleaves fibrinogen to form soluble fibrin monomers, which
then spontaneously polymerize to form the soluble fibrin polymer.
• Thrombin also activates factor XIII, which, together with calcium, crosslink the
soluble fibrin polymer and form stable crosslinked (insoluble) fibrin clot.
Thrombin has a large array of functions:
• The primary role is the conversion of fibrinogen to fibrin and fibrin clot.
• The first major role is to generate thrombin burst through combined actions of
the extrinsic and intrinsic pathway.
• Thrombin activates Factors VIII, V, XI to generate more Xa and thrombin.
• Factor XIII to crosslink the fibrin polymers.
• Thrombin activate platelet through its receptors on platelets, mobilize calcium
and promote aggregation.
• Excess amount of thrombin activate protein C and initiate fibrinolysis and
wound repair.
THROMBIN CONVERTS FIBRINOGEN TO FIBRIN
FIBRIN FORMS LARGE POLYMERS
Red blood cells trapped in a fibrin mesh
Anti-coagulant Pathway
Switch of coagulant to anti-coagulant pathways:
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The common pathway of coagulation cascade is maintained in a prothrombotic state by the
continued activation of FVII, FVIII and FIX to generate thrombin.
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Factor X, in the presence of factor V, calcium and platelet phospholipid ("prothrombinase complex")
together convert prothrombin to thrombin.
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When thrombin level reaches certain threshold, thrombin start to activate anticoagulatory pathway.
Tissue factor pathway inhibitor
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The extrinsic pathway is rapidly inhibited by a lipoprotein-associated molecule, called tissue factor
pathway inhibitor (TFPI).
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TFPI inhibits activation of FX (FXa) by TF-FVIIa and excessive TF-mediated activation of FVII and FX.
Protein C
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Thrombin activate the coagulation inhibitor protein C (in the presence of thrombomodulin). Protein
C is a major physiological anticoagulant. The activated form (APC), along with protein S and a
phospholipid, degrades FVa and FVIIIa. Deficiency of protein C and S may lead to thrombophilia (a
tendency to develop thrombosis).
Antithrombin
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Antithrombin is a serine protease inhibitor that degrades the serine proteases: thrombin, FIXa, FXa,
FXIa, and FXIIa. It is constantly active, but its adhesion to these factors is increased by the presence
of heparan sulfate or heparins.
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Deficiency of antithrombin (inborn or acquired) leads to thrombophilia.
Prostacyclin
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Prostacyclin (PGI2) is released by endothelium and activates platelet Gs protein-linked receptors,
activates adenylyl cyclase and increase of cAMP.
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cAMP inhibits platelet activation by decreasing cytosolic levels of calcium, inhibits the release of
granules and activation of additional platelets.
Inhibitors of Hemostasis
Primary hemostasis
Naturally occurring inhibitors of platelet function are prostacyclin and nitric oxide, which
are released by endothelial cells, and bradykinin from plasma. Acquired inhibitors of platelet
function are rare, whereas acquired inhibitors of von Willebrand factor occur in a variety of
diseases in human patients and result in acquired von Willebrand disease (avWD). More
commonly, platelet function is inhibited intentionally by the administration of therapeutic
agents for the prevention of thrombosis. These inhibitors include aspirin and antagonists of
GPIIb/IIIa.
Secondary hemostasis
The most important natural anticoagulant is antithrombin (AT) (also called antithrombin III
or ATIII). Antithrombin is an alpha2-globulin produced in the liver. It inhibits many activated
coagulation proteins (including factors II, IX, X, XI and XII), however thrombin (factor IIa) is
its main target. Antithrombin binding to thrombin is enhanced by heparin, which, in vivo, is
provided by degranulated mast cells or basophils and heparin-like glycosaminoglycans on
endothelial cells. This provides the basis for administration of heparin as an anticoagulant
for the treatment or prevention of thrombotic disorders. Antithrombin complexes with
thrombin, the complex is then removed by the monocyte-macrophage system.
Heparin cofactor II is a specific thrombin antagonist. Like AT, this also requires heparin for
activation, but in far greater concentrations.
Tissue factor pathway inhibitor is a lipoprotein-associated molecule that rapidly inhibits the
tissue factor pathway, thus allowing this pathway to only generate small amounts of
thrombin (which is sufficient to amplify the coagulation cascade, but not enough to produce
fibrin).
Regulatory Mechanisms
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Several inhibitory mechanisms prevent activated coagulation reactions from amplifying
uncontrollably, causing extensive local thrombosis or disseminated intravascular
coagulation. These mechanisms include
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Inactivation of coagulation factors:
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Inactivation of procoagulant enzymes
Fibrinolysis
Hepatic clearance of activated clotting factors
Plasma protease inhibitors (antithrombin, tissue factor pathway inhibitor, α2-macroglobulin,
heparin cofactor II) inactivate coagulation enzymes.
Antithrombin inhibits thrombin, factor Xa, factor XIa, and factor IXa.
Heparin enhances antithrombin activity.
Two vitamin K–dependent proteins, protein C and free protein S, form a complex that
inactivates factors VIIIa and Va by proteolysis.
Thrombin, when bound to a receptor on endothelial cells (thrombomodulin), activates
protein C. Activated protein C, in combination with free protein S and phospholipid
cofactors, proteolyzes and inactivates factors VIIIa and Va.
Fibrinolysis:
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Fibrin deposition and lysis must be balanced to maintain temporarily and subsequently remove
the hemostatic seal during repair of an injured vessel wall.
The fibrinolytic system dissolves fibrin by plasmin, a proteolytic enzyme.
Vascular endothelial cell released plasminogen and its activators, tissue plasminogen
activator (t-PA) secreted by endothelium, bind to fibrin, the activators cleave plasminogen into
plasmin and plasmin degrade fibrin clot.
Plasminogen Activators
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Tissue plasminogen activator (tPA), from endothelial cells, is a poor activator when
free in solution but an efficient activator when bound to fibrin in proximity to plasminogen.
Urokinase exists in single-chain and double-chain forms with different functional
properties. Single-chain urokinase cannot activate free plasminogen but, like tPA, can
readily activate plasminogen bound to fibrin. A trace concentration of plasmin cleaves
single-chain to double-chain urokinase, which activates plasminogen in solution as well
as plasminogen bound to fibrin. Epithelial cells that line excretory passages (eg, renal
tubules, mammary ducts) secrete urokinase, which is the physiologic activator of
fibrinolysis in these channels.
Streptokinase, a bacterial product not normally found in the body, is another potent
plasminogen activator.
Streptokinase , urokinase, and recombinant tPA (alteplase) have all been used
therapeutically to induce fibrinolysis in patients with acute thrombotic disorders.
Fibrinolysis is regulated by plasminogen activator inhibitors (PAIs) and plasmin inhibitors.
PAI-1, the most important PAI, inactivates tPA and urokinase and is released from
vascular endothelial cells and activated platelets.
The primary plasmin inhibitor is  2-antiplasmin, which quickly inactivates any free
plasmin escaping from clots. Some α2-antiplasmin is also cross-linked to fibrin polymers
by the action of factor XIIIa during clotting. This cross-linking may prevent excessive
plasmin activity within clots.
tPA and urokinase are rapidly cleared by the liver, which is another mechanism of
preventing excessive fibrinolysis.
Fibrinolytic pathway
Fibrin deposition and fibrinolysis must be balanced during repair of an injured blood
vessel wall. Injured vascular endothelial cells release plasminogen activators (tissue
plasminogen activator, urokinase), activating fibrinolysis. Plasminogen activators
cleave plasminogen into plasmin, which dissolves clots. Fibrinolysis is controlled by
plasminogen activator inhibitors (PAI-1) and plasmin inhibitors (α2-antiplasmin).
Tertiary Hemostasis
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Tertiary hemostasis is defined as the formation of plasmin, which is the main enzyme
responsible for fibrinolysis (breakdown of the clot).
At the same time as the coagulation cascade is activated, tissue plasminogen activator (tPA)
is released from endothelial cells. Release is stimulated by a variety of factors, including
hypoxia and bradykinin. Tissue plasminogen activator binds to plasminogen within the clot,
converting it into plasmin. Plasmin lyses both fibrinogen and fibrin (soluble and crosslinked)
in the clot, releasing fibrin(ogen) degradation products.
Abbreviations: tPA: tissue plasminogen activator; PAI: plasminogen activator
inhibitor; PLG: Plasminogen; AP: Antiplasmin; FDPs: Fibrin(ogen) degradation
products.
Regulation of Coagulation
Cascade
• Vitamin K is an essential factor for hepatic gamma-glutamyl
carboxylase that add carboxyl group to glutamic acid residues on
factors II, VII, IX and X, Protein S, Protein C and Protein Z.
• Vitamin K epoxide reductase, (VKORC) reduces vitamin K back to its
active form. VKORC is pharmacologically important target:
– warfarin and coumarins (acenocoumarol, phenprocoumon,
and dicumarol) create a deficiency of reduced vitamin K by
blocking VKORC, thereby inhibiting maturation of clotting factors.
– Vitamin K deficiency from other causes (malabsorption) or
impaired vitamin K metabolism in disease (hepatic failure) lead to
partially or totally non-gamma carboxylated coagulation factors.
• Calcium and phospholipid are required for the tenase and
prothrombinase complexes to function.
– Calcium mediates the binding of the terminal gamma-carboxy
residues on FXa and FIXa to the phospholipid surfaces expressed
by platelets
Antiplasmin
PAI-1
Tissue factor
Clotting Factors
Procoagulant
Protein C
Protein S
ATIII
TFPI
Fibrinolytic System
Anticoagulant
Clinical significance
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The best-known coagulation factor disorders are the hemophilias.
– hemophilia A, factor VIII deficiency, X-linked recessive disorders
– hemophilia B, factor IX deficiency, X-linked recessive disorders
– hemophilia C, factor XI deficiency, mild bleeding tendency, rare autosomal recessive
disorder
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Von Willebrand disease
– The most common bleeding disorder and autosomal recessive or dominant.
– Defect in von Willebrand factor (vWF) that mediates the binding of glycoprotein Ib
(GPIb) to collagen.
– Defect in activation of platelets and formation of primary hemostasis.
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Bernard-Soulier syndrome:
– Deficiency in GPIb. GPIb, the receptor for vWF, autosomal recessive disorder
– Defective in primary clot formation (primary hemostasis). Increased bleeding tendency.
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Thrombasthenia of Glanzmann and Naegeli (Glanzmann thrombasthenia):
– Defect in GPIIb/IIIa fibrinogen receptor complex, autosomal recessive
– Fibrinogen cannot cross-link platelets in primary hemostasis.
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Deficiency of Vitamin K:
– Clotting factor maturation depends on Vitamin K.
Anticoagulants
• Anti-platelet agents
– aspirin, dipyridamole, ticlopidine, clopidogrel and prasugrel;
glycoprotein IIb/IIIa inhibitors are used during angioplasty.
• Anticoagulants
– warfarin (and related coumarins) and heparin are the most
commonly used.
– Warfarin affects the vitamin K-dependent clotting factors (II, VII,
IX,X),
– heparin and related compounds increase the action of
antithrombin on thrombin and factor Xa.
– A newer class of drugs, the direct thrombin inhibitors, is under
development; some members are already in clinical use (such as
lepirudin).
– Also under development are small molecules that interfere with
enzymatic action of particular coagulation factors (rivaroxaban,
dabigatran, apixaban).
Role in immune system
• The coagulation system overlaps with the immune system.
– Coagulation can physically trap invading microbes in blood clots.
– Some products of the coagulation system can contribute to
the innate immune system by their ability to increase vascular
permeability and act as chemotactic agents for phagocytic cells.
– Some of the products of the coagulation system are
directly antimicrobial. beta-lysine, a protein produced by platelets
during coagulation, can lyse many Gram-positive bacteria by acting
as a cationic detergent.
• Many acute-phase proteins of inflammation are involved in
the coagulation system.
• In addition, pathogenic bacteria may secrete agents that
alter the coagulation system,
e.g. coagulase and streptokinase.
Disseminated intravascular coagulation (DIC)
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A pathologic process in which coagulation and fibrinolysis are inappropriately initiated in microvasculature,
resulting in systemic generation of thrombin.
Generation of thrombin produces widespread thrombosis which eventually leads to hemorrhage from
consumption of platelets and coagulation factors.
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Animal in DIC is also experiencing diffuse microvascular thrombosis which directly contributes to the high
morbidity and mortality.
DIC is always a secondary hemostatic disorder. Many conditions, including sepsis, heat stroke,
intravascular hemolysis, burns, shock, pancreatitis, neoplasia, or trauma can initiate DIC.
Most animals with acute or overt DIC are very ill and show a long-standing, serious disease.
In certain conditions, e.g. snake bites, pancreatitis (trypsin release) or certain neoplasms, the coagulation
cascade can be activated directly.
The main trigger for DIC in nearly all disease states is the pathologic exposure, expression, or release of
tissue factor.
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Inhibitors such as antithrombin and protein C are depleted when the body attempts to limit the over-activated hemostatic
system.
Plasmin and other proteases lyse the formed clots, liberating excessive amounts of FDPs and D-dimer.
Fibrinolysis contributes to hemorrhage by clot lysis, plasmin-mediated cleavage of coagulation factors, and the
anticoagulant effect of FDPs, inhibiting platelet function and fibrin polymerization.
The consumption of inhibitors allows unchecked activation of coagulation.
Tissue factor expression on monocytes/macrophages and endothelial cells is upregulated by cytokines (IL-6).
Tissue factor initiates coagulation through the extrinsic pathway of coagulation, amplified by excessive thrombin
At the same time, tissue plasminogen activator is released from endothelial cells and initiates systemic
fibrinolysis.
ADP, a platelet agonist, in intravascular hemolysis] or hyperfibrinogenemia facilitate DIC.
In some conditions, e.g. sepsis, the fibrinolytic pathway is actually downregulated by TNFa-mediated
release of plasminogen activator inhibitor. This aggravates widespread thrombosis.
Although DIC is not a primary event, if left unchecked it can cause death of the patient primarily due to
hypoxic injury of vital organs because of thrombosis.
Pharmacology_
Procoagulants
• Adsorbent chemicals (zeolites) and hemostatic agents are used in
sealing severe injuries quickly.
• Thrombin and fibrin glue are used surgically to treat bleeding
• Desmopressin is used to improve platelet function by
activating arginine vasopressin receptor 1A.
• Coagulation factor concentrates are used to treat hemophilia
• Prothrombin complex concentrate, cryoprecipitate and fresh frozen
plasma are commonly used coagulation factor products.
• Recombinant activated human factor VII is increasingly popular in the
treatment of major bleeding.
• Tranexamic acid and aminocaproic acid inhibit fibrinolysis, and lead to
a reduced bleeding rate.
• aprotinin was used in some forms of major surgery.
Role in disease
• Thrombosis
– Pathological development of blood clots.
– These clots may break free. An embolism occur when
the thrombus (blood clot) becomes a mobile embolus and migrates to
another part of the body
– This causes ischemia and often leads to ischemic necrosis of tissue.
– Most cases of venous thrombosis are due to acquired states (older age,
surgery, cancer, immobility) or inherited thrombophilias
• Disseminated intravascular coagulation (DIC)
• Tissue factor is a marker for tumor progression
• Thrombin receptors are expressed in both platelets and endothelial
cells. They participates in vascular development and are involved in
tumor growth. Thrombin receptors are classified as proteaseactivated receptors (PARs)
PAR signaling
Thrombin Receptor
Protease Activated Receptors
Vessel Damage
Protease Cascade
Thrombin
Fibrinogen
Fibrin
(blood clot)
Prothrombin
Cellular Effects Including:
platelet activation
endothelial cell secretion
v. smooth muscle proliferation
monocyte chemotaxis
Protease Activated Receptors
(PARs)
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Thrombin receptors are G-protein coupled receptors
There are 3 thrombin receptors, PAR1, PAR3 and PAR4
They are renamed Protease Activated Receptors (PARs) since PAR2 was
discovered
They are unique in that they carry their own tethered ligand and need
protease to unmask their N-terminus to activate itself
PAR1, 2 and 3 are in one genomic locus
Human PAR1 and mouse PAR3 have same platelet distribution and
thrombin sensitivity (3nM), but genetically not so close
Both human and mouse PAR4 have high thrombin sensitivity (10nM)
Mouse PAR3 has short C-terminus and do not have down stream signaling
PAR4 does not have hirudin domain for thrombin binding, so has low affinity
for thrombin
Mouse PAR3 act together with PAR4 to perform low thrombin sensitive
function
Why do we need low and high thrombin sensitivity?
What physiologic role do they each regulate?
Cellular effects of thrombin are mediated by
a family of G protein coupled receptors,
the PARs, or Protease Activated Receptors.
PAR1
PAR4
Thrombin-triggered events
in human platelets
PAR3
Widely expressed, Not well studied
PAR1
Endothelial, vessel develoment
PAR3Thrombin-triggered events
PAR4in mouse platelets
• Thrombin cleaves fibrinogen, activates platelets
and protein C, is an important turning point in
hemostasis
• Thrombin-mediated platelet activation directly
promote platelet aggregation, and hence PAR
signaling, is critical in control of hemostasis and
thrombosis.
• PARs are potential targets for drugs designed to
treat bleeding disorders, heart attacks, and strokes.
Biochemical Studies
PARs are activated by proteolytic
unmasking of a new amino terminus
Thrombin
N
C
g
C
a b
signal
N
Tethered Ligand
= SFLLRN for PAR1
C
g
ab
signal
Pars are irreversibly activated
PAR thrombin sensitivity = 3nM
PAR desensitization: No signaling before membrane repopulation
An analysis of PAR1 regulation using mutants:
Internalization
Intracellular pool generation
Termination of signaling
Comparing the regulation of PAR1 and PAR4
Activated PAR1 is subject to:
phosphorylation of S/T on C-tail
internalization
targeting to lysosomes and degradation
P
P
P
PAR1 is localized to both plasma membrane
and intracellular pools (in fibroblasts and In endothelial cells).
Receptors tonically cycle between these pools.
The cell surface is rapidly repopulated with uncleaved
receptors after transient thrombin treatment.
Lysosomal targeting and degradation of activated
receptors is probably an adaptation to the irreversible
mechanism by which PARs are activated.
The intracellular pool of PAR1 acts as a buffer, allowing
repopulation of the cell surface with uncleaved receptors
without new protein synthesis. This may allow quick recovery
of endothelial cells to maintain the ability to respond to
thrombin in a constant way despite previous exposures.
Measurement of PAR1 Internalization utilizing an
antibody to the amino terminal FLAG epitope
1.
2.
1. Label Cells with antibody at 4 °C.
2. Incubate at 37 °C with or without SFLLRN.
3. Measure amount of antibody remaining on cell surface
(enzyme-linked secondary antibody) / measure amount of antibody
accumulated inside cell (strip off surface antibody, lysis, ELISA).
In antibody uptake assays, PAR1 exhibits:
Agonist-independent internalization produced by tonic
cycling of antibody-labeled receptors.
Agonist-triggered internalization, defined simply as a
greater rate of internalization of antibody-labeled
receptors when agonist is present.
Thrombin
NRLLFS
FLAG
NH2
CC
L
K
I
E
S
S
COOH
Y
S
D
R YV
T
P
L
S
L
YS
KK
N
S S
Y I S N N L N S S C T DM K S AM L Q G
397
402
407
420
S
S
E
C
Q
S/T A in C-TAIL
numbers indicate truncation mutants
(stop “Z” replaces residue)
PAR1 mutants reveal that the two types of PAR1
internalization are promoted by separate signals in its
cytoplasmic tail:
agonist-independent
internalization
WT
C-Tail:S/T=>A
AG 397-406
Y397Z
+
+
-
agonist-dependent
internalizaton
+
+
-
These mutants are valuable tools to examine the role of PAR1
trafficking in cellular responses to thrombin.
Agonist-triggered internalization of PAR1
is dependent on phosphorylation of
its cytoplasmic tail.
Agonist-independent internalization of PAR1
can occur in the absence of phosphorylation
of its cytoplasmic tail.
Receptor cycling are promoted by residues
397 to 406.
No Agonist
With Agonist:
Model #1
With Agonist:
Model #2
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+
+
The AG 397-406 mutant exhibits entirely normal
agonist-triggered internalization and receptor
degradation in the near absence of
agonist-independent internalization.
This observation strongly suggests
that PAR1 activation increases the rate
and/or changes the mechanism
of its endocytosis (Model #2).
+
+
Termination of PAR1 signaling is promoted
by phosphorylation of its cytoplasmic tail
Mutation of all potential phosphorylation sites in the
cytoplasmic tail of PAR1 eliminated shut-off entirely.
Combined mutation of the five serines between residues
395 and 406 decreased the rate of receptor shut-off.
The internalization “machinery” is less discriminating than
the shut-off “machinery.”
Phosphorylation seems to promote shut-off of PAR1 through a
mechanism that is distinct from and probably faster than
internalization.
Hammes, Shapiro, and Coughlin (1999)
Thrombin
+
g
P
a b
P
g
P
P
P
a b
P
Thrombin
Thrombin
PAR1 PAR4
PAR3 PAR4
HUMAN
Platelet
Activation
MOUSE
Platelet
Activation
Why do platelets have two thrombin receptors?
Are the receptors simply redundant, or do they
have different signaling properties?
Calcium mobilization in human platelets
[Ca 2+] i
Thrombin
SFLLRN
(PAR1 agonist)
time (seconds)
AYPGKF
(PAR4 agonist)
Human PAR1 and PAR4 signal with different tempos
in both platelets and fibroblasts.
This difference may stem from diminished agonisttriggered phosphorylation of PAR4 relative to PAR1.
These findings provide the first hint that the two
thrombin receptors expressed in human platelets
may serve different roles.
Why do platelets have two thrombin receptors?
Knockout studies
PAR1 Knockout
• PAR1 knockout showed 50% embryonic
lethality
• Tie2-PAR1 transgenic expression can
rescue the phenotype
• Indication of PAR1 function in vascular
development
mPAR3 Knock-Out
• mPAR3 null mice survive to adulthood and
are fertile
• No gross bleeding phenotype
• Platelets lack responsiveness to low
thrombin concentrations (3nM)
• Platelets do respond to high thrombin
concentrations (10nM)
Thrombin response in PAR3 null
platelets
mPAR4 Knock-Out
• mPAR4 mice survive to adulthood and are
fertile
• No gross/spontaneous bleeding defect
• Platelets are normal by appearance,blood
counts, and expression of PAR3
• Tail bleeding time is extended from 2 min
to more than 20 min
• Thrombosis does not grow, does not form
firm plug
Mouse PAR4 Knock-Out
1.
2.
3.
4.
5.
mPAR4 mice survive to adulthood
and are fertile
No gross/spontaneous bleeding
defect
Platelets are normal by
appearance,blood counts, and
expression of PAR3
Tail bleeding time is extended from
2 min to more than 20 min
Thrombosis does not grow, does
not form firm plug
Conclusion
• Human PAR1 and mouse PAR3 are low
thrombin responser with clear roles in
hemostasis and angiogenesis
• Human PAR1 is a potential target for
thrombosis
• Drugs against hPAR1 has been developed
• Partial inhibitor of human PAR4 could be
better drug candidate for platelet related
thrombosis
Structure of hPAR1
and drug interaction