Title: Hemostasis /Coagulation
1Hemostasis /Coagulation
Thrombosis-Hemostasis-Hemorrhage
2Topics
- Hemostasis
- Coagulation
- Regulation
- Diseases
- Pharmacology
- Thrombin receptor study
3Hemostasis
4Primary Hemostasis
- 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.
5VWF UNFOLDS UNDER SHEAR STRESSThe faster the
blood flow, the stickier it gets
6Platelet activation and Plug Formation
- Platelet adhesion
- 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. - 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
- The adhesions cause platelet activation and
release of stored granules. - The granules contains ADP, serotonin, platelet-act
ivating factor (PAF), vWF, platelet factor 4,
and thromboxane A2 (TXA2), can activate
additional platelets. - 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). - PLA2 modifies the integrin membrane glycoprotein
IIb/IIIa and increase its affinity to fibrinogen.
- Platelet aggregation
- The activated platelets change shape from
spherical to stellate, fibrinogen cross-links
with glycoprotein IIb/IIIa results aggregation of
adjacent platelets. - 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
- The aggregation leads to the formation of the
primary platelet plug and later stabilized by the
formation of fibrin. - 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 coagulation factors.
7Aggregation 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)
8Secondary Hemostasis_ _Coagulation Cascade
- Components of coagulation cascade
- 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
- Defects in the coagulation cascade manifest more
serious bleeding than primary hemostasis,
including bleeding into cavities (chest, joints)
and subcutaneous hematomas. - 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
- 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. - 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
- The extrinsic and intrinsic pathways converge at
the activation of factor X. - 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. - Thrombin activate FXIII which crosslink fibrin to
produce a firm clot.
9Coagulation Cascade
10Tissue 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 comp
lex 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.
11Contact 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.
12The 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.
13THROMBIN CONVERTS FIBRINOGEN TO FIBRIN
FIBRIN FORMS LARGE POLYMERS
14Red blood cells trapped in a fibrin mesh
15Anti-coagulant Pathway
- Switch of coagulant to anti-coagulant pathways
- The common pathway of coagulation cascade is
maintained in a prothrombotic state by the
continued activation of FVII, FVIII and FIX to
generate thrombin. - Factor X, in the presence of factor V, calcium
and platelet phospholipid ("prothrombinase
complex") together convert prothrombin to
thrombin. - When thrombin level reaches certain threshold,
thrombin start to activate anticoagulatory
pathway. - Tissue factor pathway inhibitor
- The extrinsic pathway is rapidly inhibited by a
lipoprotein-associated molecule, called tissue
factor pathway inhibitor (TFPI). - TFPI inhibits activation of FX (FXa) by TF-FVIIa
and excessive TF-mediated activation of FVII and
FX. - Protein C
- 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
- 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. - Deficiency of antithrombin (inborn or acquired)
leads to thrombophilia. - Prostacyclin
- Prostacyclin (PGI2) is released by endothelium
and activates platelet Gs protein-linked
receptors, activates adenylyl cyclase and
increase of cAMP. - cAMP inhibits platelet activation by decreasing
cytosolic levels of calcium, inhibits the release
of granules and activation of additional
platelets.
16Inhibitors of Hemostasis
- Primary hemostasisNaturally 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 hemostasisThe 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).
17Regulatory Mechanisms
- Several inhibitory mechanisms prevent activated
coagulation reactions from amplifying
uncontrollably, causing extensive local
thrombosis or disseminated intravascular
coagulation. These mechanisms include - Inactivation of procoagulant enzymes
- Fibrinolysis
- Hepatic clearance of activated clotting factors
- Inactivation of coagulation factors
- Plasma protease inhibitors (antithrombin, tissue
factor pathway inhibitor, a2-macroglobulin,
heparin cofactor II) inactivate coagulation
enzymes. - Antithrombin inhibits thrombin, factor Xa, factor
XIa, and factor IXa. - Heparin enhances antithrombin activity.
- Two vitamin Kdependent 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
- 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.
18Plasminogen Activators
- 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 a2-antiplasmin,
which quickly inactivates any free plasmin
escaping from clots. Some a2-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.
19Fibrinolytic 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 (a2-antiplasmin).
20Tertiary Hemostasis
- 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.
21Regulation 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, phenprocoum
on, 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
22Antiplasmin
Protein C
PAI-1
Protein S
Tissue factor
TFPI
ATIII
Clotting Factors
Fibrinolytic System
Procoagulant
Anticoagulant
23Clinical significance
- 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 - 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. - Bernard-Soulier syndrome
- Deficiency in GPIb. GPIb, the receptor for vWF,
autosomal recessive disorder - Defective in primary clot formation (primary
hemostasis). Increased bleeding tendency. - Thrombasthenia of Glanzmann and Naegeli
(Glanzmann thrombasthenia) - Defect in GPIIb/IIIa fibrinogen receptor complex,
autosomal recessive - Fibrinogen cannot cross-link platelets in primary
hemostasis. - Deficiency of Vitamin K
- Clotting factor maturation depends on Vitamin K.
24Anticoagulants
- 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).
25Role 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.
26Disseminated intravascular coagulation (DIC)
- 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. - 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. - 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. - 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.
27Pharmacology_
- 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 a
nd 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.
28Role 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 protease-activated receptors (PARs)
29PAR signalingThrombin ReceptorProtease
Activated Receptors
30Vessel Damage
Protease Cascade
Thrombin
Prothrombin
Cellular Effects Including platelet
activation endothelial cell secretion v. smooth
muscle proliferation monocyte chemotaxis
Fibrinogen Fibrin
(blood clot)
31Protease Activated Receptors (PARs)
- 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?
32Widely expressed, Not well studied
PAR1 PAR3 Thrombin-triggered events PAR4 in
mouse platelets
Endothelial, vessel develoment
33- 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.
34Biochemical Studies
35PARs are activated by proteolytic unmasking of a
new amino terminus
Tethered Ligand
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40Measurement of PAR1 Internalization utilizing
an antibody to the amino terminal FLAG epitope
2.
1.
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).
41In antibody uptake assays, PAR1 exhibits
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45With Agonist Model 1
With Agonist Model 2
No Agonist
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47Termination 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)
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50Calcium mobilization in human platelets
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54Why do platelets have two thrombin receptors?
55Knockout studies
56PAR1 Knockout
- PAR1 knockout showed 50 embryonic lethality
- Tie2-PAR1 transgenic expression can rescue the
phenotype - Indication of PAR1 function in vascular
development
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63mPAR3 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)
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66Thrombin response in PAR3 null platelets
67mPAR4 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
68Mouse PAR4 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
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72Conclusion
- 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
73Structure of hPAR1 and drug interaction
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