Normal Haemostasis PDF 20/21

Normal Haemostasis Emma Moore Senior Lecturer Haematology & Transfusion Medicine MSc Haematology and Transfusion 20/21  Learning Outcomes Understand the haemostatic mechanisms involved in blood clotting in normal physiological conditions Understand the process of prim...

Normal Haemostasis Emma Moore Senior Lecturer Haematology & Transfusion Medicine MSc Haematology and Transfusion 20/21  Learning Outcomes Understand the haemostatic mechanisms involved in blood clotting in normal physiological conditions Understand the process of primary haemostasis and the cellular components involved Understand the role of the blood vessel itself in the clotting process Understand the process of secondary haemostasis and the cellular components involved Understand the process of fibrinolysis & it’s importance in haemostasis  Bleeding Clotting Normal Haemostasis Crucial functions of Haemostasis mechanisms: Maintain the fluidity of circulating blood in normal physiological conditions Haemostasis is the Upon vessel injury, limit & arrest bleeding by formation of a blood clot body’s normal Removal of blood clot upon completion of wound physiological response healing for the prevention of bleeding The Clotting Trilogy: Blood vessels Platelets Coagulation factors  Haemostatic mechanisms Haemostatic mechanisms; Primary haemostasis - involves interactions between blood vessels, platelets and von Willebrand factor (vWF) - Primary haemostatic plug Secondary haemostasis - Interaction of coagulation factors generate fibrin strands which strengthens the clot Fibrinolysis - degrades fibrin clot to prevent vascular occlusion  How do they interact? Vascular damage Haemorrhage Coagulation activation Vascular smooth Platelet adhesion muscle contraction Platelet activation Fibrin formation Vasoconstriction Platelet aggregation Platelet plug reinforcement Platelet plug formation Reduced blood loss  Blood vessels Blood vessel wall; Intima – inner layer, comprised of endothelial cells on a basement membrane of collagen and elastin fibres Media – smooth muscle layer, responsible for regulating vascular tone Intima Adventitia - outer layer comprised of collagen Media and fibroblasts to protect and anchor the vessel Adventitia  Healthy blood vessels PGI2 = prostacyclin; NO = nitric oxide; AT = antithrombin; HS = heparin sulphate; TM = thrombomodulin; APC = activated protein C; PS = protein S; TFPI = tissue factor pathway inhibitor; tPA = tissue plasminogen activator; EPCR = endothelial protein C receptor; A2 = thromboxane A2 receptor; uPAR = urokinase-type plasminogen activator receptor Anticoagulant properties: PGI2 & NO inhibit platelet function & are vasodilators Thrombomodulin binds free thrombin HS & EPCR activate Protein C – a natural anticoagulant Protein C inactivates FVa & FVIIa Heparin Sulphate enhances antithrombin’s inhibition of coagulation factors Protein S is a co-factor for Protein C & binds to phospholipids on surface of activated platelets Tissue Plasminogen Activator promotes clot lysis  Damaged blood vessels EC = endothelial cells; PAF = platelet activation factor; TXA2 = thromboxane A2; TF = tissue factor; VWF = von Willebrand factor; PAI-1 = plasminogen activator inhibitor-1; Procoagulant properties: Endothelial cells (EC) release endothelial-1 – potent vasoconstrictor Cells of adventitia express tissue factor (TF) – crucial for initialising coagulation cascade of secondary haemostasis via FVIIa binding Thromboxane A2 & PAF activates platelets by binding to vWF on EC wall Fibrinolysis is inhibited via PAI-1 Von Willebrand Factor (vWF) Produced by VWF gene on chromosome 12 Large multimeric glycoprotein (2050 αα long) Cleaved & regulated by ADAMTS13 Produced in: - Weible-palade bodies of endothelial cells FVIII vWF - α-granules of platelets - sub-endothelial connective tissue Carries ABO antigens (Note: Group O have lower mean levels of vWF than A, B or AB) Binds to FVIII protecting it from proteolytic degradation & increasing its plasma half life Mediates adhesion of platelets to sites of vascular injury via GP1b complex Primary haemostasis Vessel injury: Procoagulant material exposed to circulation Endothelial-1 released from epithelium causing vasoconstriction = ↓blood loss, & promotes platelet aggregation Exposed vWF activates and recruits circulating platelets by binding to GPIb receptor complex on platelet surface Platelets tend to flow at the edges of vessels making them ideally placed to rapidly response to vessel damage Primary haemostasis Platelets: Platelet adhesion: Platelets adhere to the exposed subendothelial matrix (directly or indirectly via vWF). Platelet activation: Once platelets adhere, they then become activated and recruit (and activate) additional platelets to the injured site Platelet plug formation: Fibrinogen forms bridges between activated platelets to form the platelet plug  Platelet adhesion Platelet adhesion to Circulating VWF is collagen exposed on the immobilized to the collagen sub endothelium  Platelet rolling Once tethered via the initial vWF- GPIb interaction, blood flow forces This promotes further the platelet to roll over the initial VWF-GPIb exposed collagen interactions Platelet adhesion & activation GPIb – vWF – collagen GPIa – collagen GPIIbIIIa – vWF – collagen GPVI - collagen GPIa GPVI GPIIbIIIa Primary haemostasis https://youtu.be/R8JMfbYW2p4 Secondary haemostasis Platelet plug alone is not enough to stem blood loss, reinforcement is required! This is where the coagulation pathways come into play Secondary haemostasis (coagulation) is usually initiated simultaneously with primary haemostasis upon endothelial damage Result of activation of coagulation pathways is that soluble fibrinogen is converted to insoluble fibrin  Coagulation factors Zymogens (proenzyme) which interact in a complex integrated system of enzyme activation Coag factors that act as enzymes are serine proteases X Va Some require presence of cofactors to maximise rate of substation activation, e.g. TF-FVIIa or FVa Activation reactions occur mostly at membrane surfaces as coag factors & precursors have specific binding sites for phospholipids, Ca2+ or other specific receptors on cell surface Allows for localisation of haemostatic response Coagulation pathway The Coagulation cascade Circulating inactive zymogens X marks the are sequentially activated to the spot! active enzyme forms Intrinsic pathway – requires enzymes and factors all presentin the plasma Extrinsic pathway- requires enzymes and factors present in the plasma as well as an activator Both pathways converge on a common pathway to generate the fibrin clot  Extrinsic pathway Endothelial injury exposes tissue factor (TF) which binds its circulating ligand in plasma, the pro-enzyme FVII Binding to TF activates FVII TF and promotes activity TF The TF-FVIIa complex then activates the pro-enzyme FX FXa is able to cleave its substrate prothrombin (FII) to thrombin Only a small amount of FXa is generated by this reaction Extrinsic → common pathway FXa + Fva Need phospholipids (PL) and Ca2+ ions Fxa + Fva + PL + Ca2+ =Extrinsic Prothombinase complex It is able to cleave its substrate prothrombin (FII) to thrombin Remember only small amounts of thrombin are generated by this reaction FXIIIa acts upon fibrin to form a crosslinked mesh Amplification stage Small amount of thrombin (FIIa) generated enters the AMPLIFICATION STAGE Thrombin activates: - FV - FVIII - Platelets FVa is a co-factor of Fxa FIXa migrates to surface of activated platelets The availability of FVa & FVIIIa helps to generate MORE thrombin!  Intrinsic pathway 12 11 9 10 8 TENET of haematology Propagation stage FVIIIa is formed during the amplification phase FIXa + FVIIIa + FX = Tenase complex (major activator of FX) 50x more efficient than TF-FVIIa during the activation phase >90% Fxa is produced by intrinsic Tenase complex FXa + FVa (cofactor) = Prothrombinase complex This generates an explosive burst of thrombin from prothrombin  Common pathway Roles of Thrombin include: Intrinsic Extrinsic - Causes proteolysis of fibrinogen - Activation of FV, FVII, FVIII, FXI and FXIII - Activation of platelets via protease- activated receptor-1 (PAR-1) FX - Activation of Protein C FV - Activation of thrombin-activatable fibrinolysis inhibitor (TAFI) FII - Chemokine production to attract WBCs to site of injury FI - Tissue repair and development  Fibrin formation Thrombin converts soluble fibrinogen to insoluble fibrin Fibrinogen was the first biological macromolecule to be visualised by electron microscopy Glycoprotein synthesised by hepatocytes in the liver, & present in high concentration in the plasma (1.8 - 3.5 g/L) Platelets take up fibrinogen from the plasma by endocytosis and store it in their alpha granules Fibrin is mechanically stronger through the action of FXIIIa as it catalyses formation of cross-links FXIIIa also binds to α2 –antiplasmin which inhibits plasmin from degrading clots Haemostasis recap Secondary haemostasis https://youtu.be/cy3a__OOa2M Naturally occurring inhibitors Naturally occurring inhibitors of coagulation prevent initiation and inappropriate activation of the clotting cascade Inhibitors also prevent amplification to limit coagulation If these were not present, the body may undergo multiple clotting events which can result in multiple vascular occlusions A number of endogenous (internal) inhibitors exist including; - Antithrombin (AT) - Protein C (PC) - Tissue factor pathway inhibitor (TFPI)  Antithrombin (AT) 432 αα long, produced by hepatocytes in the liver Serpin protease inhibitor – serine protease inhibitors Can inhibit all serine proteases - thrombin, IXa, Xa, XIa, XIIa, kallikrein and plasmin Forms stable 1:1 complexes with its target enzymes Inefficient in the absence of co-factors Cofactor activity is provided by heparin sulphate (HS) which ↑s the reaction x1000 ! Medicinal Heparin (in surgery) binds to AT, increasing its inhibitory action Binds & inhibits free FXa & thrombin  Protein C Activated protein C (APC) is a serine protease that inhibits FVa and FVIIIa Protein C and its co-factor protein S are vitamin K dependent proteins Synthesised in the liver Thrombin forms on the vessel wall at the site of injury Thrombomodulin (TM) on the endothelial cell forms a complex with thrombin, stopping it binding coagulation factors This complex activates PC, which then in association with free protein S (PS) inactivates FVa and FVIIIa. Tissue Factor Pathway Inhibitor TFPI inhibits the FVIIa-TF complex Kunitz-type inhibitor Therefore inhibits the extrinsic pathway Protein S (PS) serves as a co-factor for TFPI’s inhibition of Xa i.e. TFPI rapidly downregulates FXa The microvascular endothelium is the major source of TFPI Most TFPI binds to heparin on the surface of endothelial cells Rest circulates in blood bound to low density lipoprotein Haemostatic mechanism Haemostatic mechanism is three distinct phases: Primary haemostasis interactions between blood vessels, platelets and von Willebrand factor Secondary haemostasis pathways of coagulation to generate a fibrin strand Fibrinolysis biochemical system that degrades the fibrin clot  Fibrinolysis Fibrin clots are degraded through proteolysis Fibrin degraded by the enzyme plasmin Degradation by plasmin forms fibrin degradation products (FDPs) = D-Dimers ! Fibrinolysis regulation  Further reading Moore, G., Knight, G., Blann, A., (2010). “Haematology” Fundamentals of Biomedical Science, Oxford UK, Oxford University Press, p435-476. Hoffbrand, AV., Moss, PAH.,(2015). “Hoffbrand’s Essential Haematology’, 7th Edition, Wiley Press. Amazing book! →