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Hemostasis /Coagulation

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  1. Hemostasis /Coagulation Thrombosis-Hemostasis-Hemorrhage

  2. Topics • Hemostasis • Coagulation • Regulation • Diseases • Pharmacology • Thrombin receptor study

  3. Vessel Injury Tissue Factor Neural Coagulation Activation Blood Vessel Constriction Platelet Activation Primary hemostatic plug Reduced Blood flow Platelet aggregation Fibrin clot Stable Hemostatic Plug 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 byvon 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 containsADP,serotonin,platelet-activating factor (PAF), vWF, platelet factor 4, andthromboxane A2(TXA2), can activate additional platelets. • The granules can activate aGq-linked protein receptorcascade, resulting in increased calcium in the platelets' cytosol, calcium activatesprotein kinase C (PKC) and PKC activatesphospholipase A2(PLA2). • PLA2 modifies theintegrinmembrane glycoprotein IIb/IIIa and increase its affinity tofibrinogen. 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 withtissue factor(TF) expressing cells (stromalfibroblasts and leukocytes) and forms activated complex (TF-FVIIa). • TF-FVIIa activates FIX and FX to FIXa and FXa. • FXa and co-factor FVa form theprothrombinasecomplex 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 proteasezymogens andglycoprotein 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 oncollagenbyhigh-molecular-weight kininogen(HMWK), prekallikrein, andFXII (Hageman factor). • Prekallikreinis converted tokallikreinand FXII becomes FXIIa. • FXIIa converts FXI into FXIa. • Factor XIa activates FIX, which with its co-factor FVIIIa form thetenasecomplex 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, andprekallikreindo 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 • Thetissue factorandcontact activationpathways 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. Thrombinhas a large array of functions: • The primary role is the conversion offibrinogento 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 inhibitorprotein C(in the presence of thrombomodulin). Protein Cis 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 • Antithrombinis aserine 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 ofheparan sulfate orheparins. • Deficiency of antithrombin (inborn or acquired) leads to thrombophilia. Prostacyclin • Prostacyclin(PGI2) is released by endothelium and activates platelet Gsprotein-linked receptors, activatesadenylyl 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, 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: • 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 α2-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.

  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 (α2-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 Kis an essential factor for hepaticgamma-glutamyl carboxylasethat add carboxylgroup toglutamic acidresidues on factors II, VII, IX and X, Protein S, Protein C andProtein Z. • Vitamin K epoxide reductase, (VKORC) reduces vitamin K back to its active form. VKORC is pharmacologically important target: • warfarinandcoumarins (acenocoumarol,phenprocoumon, anddicumarol) 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. • Calciumandphospholipid 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 thehemophilias. • 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 duringangioplasty. • Anticoagulants • warfarin(and relatedcoumarins) andheparinare 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, thedirect 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 theimmune system. • Coagulation can physically trap invading microbes in blood clots. • Some products of the coagulation system can contribute to theinnate immune systemby their ability to increase vascular permeability and act aschemotactic agents for phagocytic cells. • Some of the products of the coagulation system are directlyantimicrobial. beta-lysine, a protein produced by platelets during coagulation, can lyse manyGram-positive bacteriaby acting as a cationic detergent. • Manyacute-phase proteinsofinflammationare involved in the coagulation system. • In addition, pathogenic bacteria may secrete agents that alter the coagulation system, e.g.coagulaseandstreptokinase.

  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 agentsare used in sealing severe injuries quickly. • Thrombin and fibringlueare used surgically to treat bleeding • Desmopressinis used to improve platelet function by activatingarginine vasopressin receptor 1A. • Coagulation factor concentrates are used to treathemophilia • Prothrombin complex concentrate,cryoprecipitateandfresh frozen plasmaare commonly used coagulation factor products. • Recombinant activated human factor VIIis increasingly popular in the treatment of major bleeding. • Tranexamic acidandaminocaproic acidinhibit fibrinolysis, and lead to a reduced bleeding rate. • aprotininwas used in some forms of major surgery.

  28. Role in disease • Thrombosis • Pathological development of blood clots. • These clots may break free. Anembolism occur when thethrombus(blood clot) becomes a mobile embolus and migrates to another part of the body • This causesischemiaand often leads to ischemic necrosisof tissue. • Most cases ofvenous thrombosisare due to acquired states (older age, surgery, cancer, immobility) or inheritedthrombophilias • 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 FibrinogenFibrin (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. 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 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. Thrombin N N C C C g g b b a a signal signal = SFLLRN for PAR1 PARs are activated by proteolytic unmasking of a new amino terminus Tethered Ligand

  36. 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

  37. Activated PAR1 is subject to: phosphorylation of S/T on C-tail internalization targeting to lysosomes and degradation P P P

  38. 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.

  39. 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.

  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. 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. In antibody uptake assays, PAR1 exhibits:

  42. Thrombin NRLLFS NH2 FLAG K C C L S S E S Y S S S Y S S P S E V I C Q D T 397 R L 402 407 L Y K 420 COOH N K Q A L G M C M K Y S D S N N L S N S T I numbers indicate truncation mutants (stop “Z” replaces residue) S/TA in C-TAIL

  43. PAR1 mutants reveal that the two types of PAR1 internalization are promoted by separate signals in its cytoplasmic tail: agonist-independent agonist-dependent internalization internalizaton WT C-Tail:S/T=>A AG 397-406 Y397Z + + - - + - + - These mutants are valuable tools to examine the role of PAR1 trafficking in cellular responses to thrombin.

  44. 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.

  45. + + With Agonist: Model #1 With Agonist: Model #2 No Agonist +

  46. 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). +

  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. Thrombin + g a b g a b P P P P P P

  49. 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?

  50. SFLLRN (PAR1 agonist) AYPGKF (PAR4 agonist) Thrombin [Ca 2+] i time (seconds) Calcium mobilization in human platelets