Hemostasis /Coagulation

1
Hemostasis /Coagulation
Thrombosis-Hemostasis-Hemorrhage
2
Topics

• Hemostasis
• Coagulation
• Regulation
• Diseases
• Pharmacology
• Thrombin receptor study

3
Hemostasis
4
Primary 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.

5
VWF UNFOLDS UNDER SHEAR STRESSThe faster the
blood flow, the stickier it gets
6
Platelet 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.

7
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)
8
Secondary 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.

9
Coagulation Cascade
10
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 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.

11
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.

12
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.

13
THROMBIN CONVERTS FIBRINOGEN TO FIBRIN
FIBRIN FORMS LARGE POLYMERS
14
Red blood cells trapped in a fibrin mesh
15
Anti-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.

16
Inhibitors 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).

17
Regulatory 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.

18
Plasminogen 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.

19
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 (a2-antiplasmin).
20
Tertiary 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.
21
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, 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

22
Antiplasmin
Protein C
PAI-1
Protein S
Tissue factor
TFPI
ATIII
Clotting Factors
Fibrinolytic System
Procoagulant
Anticoagulant
23
Clinical 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.

24
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).

25
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.

26
Disseminated 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.

27
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 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.

28
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 protease-activated receptors (PARs)

29
PAR signalingThrombin ReceptorProtease
Activated Receptors
30
Vessel Damage
Protease Cascade
Thrombin
Prothrombin
Cellular Effects Including platelet
activation endothelial cell secretion v. smooth
muscle proliferation monocyte chemotaxis
Fibrinogen Fibrin
(blood clot)
31
Protease 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?

32
Widely 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.

34
Biochemical Studies
35
PARs are activated by proteolytic unmasking of a
new amino terminus
Tethered Ligand
36
(No Transcript)
37
(No Transcript)
38
(No Transcript)
39
(No Transcript)
40
Measurement 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).
41
In antibody uptake assays, PAR1 exhibits
42
(No Transcript)
43
(No Transcript)
44
(No Transcript)
45
With Agonist Model 1
With Agonist Model 2
No Agonist

46
(No Transcript)
47
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)
48
(No Transcript)
49
(No Transcript)
50
Calcium mobilization in human platelets
51
(No Transcript)
52
(No Transcript)
53
(No Transcript)
54
Why do platelets have two thrombin receptors?
55
Knockout studies
56
PAR1 Knockout

• PAR1 knockout showed 50 embryonic lethality
• Tie2-PAR1 transgenic expression can rescue the
  phenotype
• Indication of PAR1 function in vascular
  development

57
(No Transcript)
58
(No Transcript)
59
(No Transcript)
60
(No Transcript)
61
(No Transcript)
62
(No Transcript)
63
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)

64
(No Transcript)
65
(No Transcript)
66
Thrombin response in PAR3 null platelets
67
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

68
Mouse PAR4 Knock-Out

1. mPAR4 mice survive to adulthood and are fertile
2. No gross/spontaneous bleeding defect
3. Platelets are normal by appearance,blood counts,
   and expression of PAR3
4. Tail bleeding time is extended from 2 min to more
   than 20 min
5. Thrombosis does not grow, does not form firm plug

69
(No Transcript)
70
(No Transcript)
71
(No Transcript)
72
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

73
Structure of hPAR1 and drug interaction
74
(No Transcript)
75
(No Transcript)
76
(No Transcript)
77
(No Transcript)
78
(No Transcript)
79
(No Transcript)
80
(No Transcript)