Dr. Suzana Voiculescu

Transcription

1 Dr. Suzana Voiculescu

2 Overview of hemostasis Initiated when trauma, surgery or disease disrupts the vascular endothelium, exposing blood to the subendothelial connective tissue Achieved by: 1)Vasconstriction + collateral dilation 2)Formation of a platelet plug 3)Formation of a blood clot as a result of blood coagulation Secondary hemostasis Primary hemostasis

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4 Vascular response to injury Local vasoconstriction: 1) local myogenic spasm -initiated by direct damage to the vascular wall 2) thromboxane A2 (TXA2) and serotonin release by activated platelets; endothelin-1(et-1) produced by the endothelium in response to thrombin 3)nervous reflexes-nociceptor reflex Compression of injured vessels by extravasation of blood into surrounding tissues Vasodilation of the collateral vessels- redirecting flow

5 Primary hemostasis In a normal individual, hemostasis is initiated within 20 seconds after an injury occurs to the blood vessel damaging the endothelial cells. Platelets immediately form a haemostatic plug at the site of injury. This is called primary hemostasis. This is the only phase in cappilaries

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7 Plateles What is a platelet? Small 2-3 µm Anuclear Reddish-purple granules Fragments of megakaryocyte cytoplasm Life span 9-10 days Normal Range x 10 9 /L

8 Thrombopoiesis - production in BM by fragmentation of megakariocytes - megakariocytes mature by endomitotic synchronous nuclear replication: no. of nuclear lobes increase in multiples of two together with enlargement of the cytoplasmic volume at variable stage in development (mature polyploid cell, ~ 8 nucleus stage), nuclear replication & cytoplasm growth cease

9 cytoplasm becomes granular (form. of microvesicles) platelets are released (~4,000 platelets/megakariocyte) - time interval from differentiation of the stem cell to the production of platelets (thrombocytopoiesis) ~ 10 days - control by thrombopoietin, IL-6, IL-3, Vit B12, GM-CSF (effects: in no. of megakariocytes and in mean volume or nuclear units)

10 Nuclear Cytoplasmic Platelets replication granulation

11 Platelet anatomy ext int Peripheral zone- glycocalix + membrane Sol gel zone Organelle zone

12 Peripheral zone Plasma membrane- normal lipid bilayer On the outer membrane- neutral PLphosphatidilcholine, sphingomyelin On the inner membrane- negative PL: Phosphatidilinositol supports platelet activation arachidonic acid eicosanoids PG+TxA2 (platelet activator + vasoconstrictor) Phosphatidilserine flips to the outer membrane when platelets get activated PL surface on which coagulation enzymes asemble

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14 Peripheral zone Contains glycoprotein receptors: GP Ib-IX-V complex binds von Willebrand s factor needed for platelet adhesion to collagen GP Ia/IIa= ligand collagen GP IV= ligand collagen GP VI= ligand collagen GP Ic/IIa= ligand laminin and fibronectin GPIIb/IIIa binds fibrinogen needed for aggregation G- protein coupled receptor- P2Y12- for ADP Receptors for thrombin, epinephrine- platelet activators

15 Peripheral zone Glycocalix Platelet mb surface- adsorbs albumins, fibrinogen Thicker than other cells Negative charge- repels other cells (other platelets, other blood cells, endothelial cells) Surface connected canalicular system (SCCS)- plasma membrane invades interior- GP receptors and route for granule secretion upon platelet activation

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17 Structural or Sol-Gel zone Beneath the peripheral zone Responsible for platelet retraction/contraction functions and platelet shape Maintainance of discoid shape (when inactive)- microtubules Contractile system- mainly actin, also myosin- upon activation- shape change, pseudopod extensions, internal contraction, granule release

18 Organelle zone Responsible for storage and platelet release functions Granules Dense bodies Alpha granules Lysosomes Mitochondria Glycogen Dense tubular system (DTS)- condensed RER- no proteic synthesis function, but Ca++ storage and enzymes (PLA2, COX, TxA2 synthetase, PLC)

19 Granules Dense (SAC)- Serotonin, ADP/ATP, Calcium Alpha adhesive proteins- fibrinogen, fibronectin, vwf growth promoting substances- PDGF, PF4, TGF Coagulation factors- V, HMWK, IX, plasminogen activator inhibitor-1

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22 Activation Happens at the site of vascular injury PHYSIOLOGICAL ACTIVATORS THROMBIN ADP COLLAGEN EPINEPHRINE TXA2

23 Activation pathways

24 Thrombin dependent activation Mediated by G protein coupled protease activated receptors (PAR 1 and 4) Thrombin cleaves the external domain of PAR transmembranary signalling

25 Thrombin activation is mediated by G protein-coupled proteaseactivated receptor (PAR). Thrombin cleaves the NH2-terminal exodomain of the PAR, exposing a new NH2 terminus, which then serves as a tethered ligand to bind intramolecularly to the body of the receptor to initiate transmembrane signaling.

26 ADP dependent activation Requires the coordinated activation of two G protein coupled receptors P2Y1 and P2Y12 Ligand receptor binding G protein mediated signal PLA 2 activation TXA2 camp supression

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28 Platelets activation factors

29 After platelets are activated, they undergo significant morphologic changes, producing elongated pseudopods. They also become extremely adhesive. The functional response of activated platelets involves four distinct processes: adhesion (deposition of platelets on subendothelial matrix); aggregation (cohesion of platelets); secretion (release of platelet granule proteins); and procoagulant activity (enhancement of thrombin generation).

30 Platelets activation and secretion 1. Cytoskeletal and morphological changes - formation of fingerlike pseudopodia+ presenting polar phospholipids on the surface (phosphatidilserine) 2. Prostaglandin Pathway- TXA2 formation from Arachidonic acid 3. Direct Granular Release contraction of actin and myosin SPPS exocitosis ADP and serotonin: stimulate and recruit additional platelets PDGF proliferation of smooth muscle cells and tissue repair Factor V: receptor on platelet surface for factor Xa & prothrombin initiation of coagulation 4. GPIIb/IIIa activation (heterodimer) platelet plug

31 Morphological changes

32 Eicosanoid pathway activation

33 Platelets adhesion In venules and veins (low shear rate)- TR adhere directly to collagen- GP Ia/IIa, GP IV, GP VI In capillaries and arterioles (shear rate high)- the site of the injury is first carpeted with vwf and platelet adhesion is primarily mediated by the binding of platelet surface receptor glycoprotein (GP) Ib/IX/V complex to the adhesive protein von Willebrand factor (vwf) bound to collagen Deficiency of Ib/IX/V: Bernard-Soulier syndrome AR

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35 Von Willebrand factor Multimeric globulin Synthesis by endothelial cells + megacariocytes (it is found in the dense TR granules) million Da Injury unrolls into fibrills and coats lesion (collagen) Functions Adhesion Stabilizes the procoagulant factor VIII (FVIII)

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37 Von Willebrand disease Inherited bleeding disorder 3 types, depending on the severity of vwf deficit (type III is the most severe) 1,2- mild manageable bleeding only manifests in trauma/invasive procedures Acquired Very rare vwf antibodies- hypothyroidism

38 Why not adhere on normal endoth cells? Normally, endothelial cells exhibit antiplatelet, anticoagulant, and fibrinolytic properties. ANTIPLATELET Secrete NO and PGI2- inhibit TR adhesion and aggregation Produce ADPase- it degrades ADP (which is a powerful TR activator)

39 Aggregation Requires the active conformation of GPIIb/IIIa integrin Normally, it dimerizes due to ligand mediated platelet activation (inside-out signalling) Active GPIIb/IIIa binds fibrinogen

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41 Platelets aggregation Chemical changes cause platelets to aggregate and stick to one another Newly arriving platelets become activated by agonists Exposure of GPIIb/IIIa sites bind fibrinogenfibrinogen bridges between TR= PLATELET PLUG

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44 Interactions between activated TR and clotting cascade Activation of TR anionic lipids exposure on surface Factor V is released from TR granules binds in the phospholipids activation of fv Va Va acts as an assembly site for the binding of Xa (active enzyme) and prothrombin (substrate)= PROTHROMBINASE COMPLEX Platelet plug keeps thrombin localized Simillar enzyme complex assembly applies to the activation of X by VIIIa and IXa amplification COAGULATION PURPOSE- TURN FIBRINOGEN IN THE PLUG INTO FIBRIN CLOT

45 Secondary hemostasis= coagulation Composed of 14 coagulation factors (serine proteases) which are interdependent (Factors I through XIII there is no Factor VI plus PK and HMWK) Inactive form of each is an enzyme precursor which is usually designated by a Roman numeral but also given a name Ex. Factor I fibrinogen. Numbers correspond to order of discovery NOT order in cascade. Active forms are usually designated by the letter a after the Roman numeral and may also have a different name Ex. Ia Fibrin Cofactors are needed for many reactions in the cascade Ex. Calcium, membrane phospholipids

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48 Secondary hemostasis Contact Activation pathway (Intrinsic Pathway) Tissue Factor pathway (Extrinsic pathway) It was previously thought that the coagulation cascade consisted of two pathways of equal importance joined to a common pathway. It is now known that the primary pathway for the initiation of blood coagulation is the Tissue Factor pathway. The pathways are a series of reactions, in which a zymogen (inactive enzyme precursor) of a serine protease and its glycoprotein co-factor are activated to become active components that then catalyze the next reaction in the cascade

49 Secondary hemostasis Coagulation proteins work in concert to generate prothrombin activator or prothrombinase (step 1) Prothrombinase acts on prothrombin, a plasma protein, to form thrombin (step 2) Thrombin converts fibrinogen to fibrin (step 3); fibrin consolidates the platelet plug made in primary hemostasis such that a fibrin clot (secondary hemostatic plug) is formed

50 INTRINSIC PATHWAY The contact activation pathway begins with formation of the primary complex on collagen by highmolecular-weight kininogen (HMWK), prekallikrein, and FXII (Hageman factor) FXII becomes FXIIa in contact with the negatively charged membrane; HMWK is an anchor Prekallikrein is converted to kallikrein- positive feed back on XII factor activation FXIIa converts FXI into FXIa.

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52 Intrinsic pathway Factor XIa activates FIX into IXa Factor VIII is activated by thrombin Thrombin is activated downstream and has a positive feed back on the coagulation cascade!! FVIIIa + FIXa+ Ca ions + PL= tenase complex, which activates FX to FXa.

53 CLASSIC THEORY The contact of blood with the vascular endothelial collagen or with the surface of an activated platelet triggers the intrinsic pathway or contact phase of coagulation; the trigger is inside the vascular system The interaction of blood with material from damaged cell membranes (tissue factor) activates the extrinsic pathway or tissue factor dependent pathway; the trigger is outside the vascular system

54 PL PL

55 EXTRINSEC PATHWAY Following damage to the blood vessel, FVII leaves the circulation and comes into contact with tissue factor (TF) expressed on tissue-factor-bearing cells (stromal fibroblasts and leukocytes), forming an activated complex (TF-FVIIa). This takes place on the non-vascular surface TF-FVIIa + Ca ions = tenase like complex. activation of FX into Xa

56 Tissue factor Synonim- thromboplastin, factor III Expressed on the membrane of non-vascular cells After a vessel injury, it comes into contact with VII, seves as an anchor at the surface of the cells Activation on VII VIIa needs also Ca++ from TR granules

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58 COMMON PATHWAY FXa + F Va+ PL + Ca ions= PROTHROMBINASE COMPLEX TURNES PROTHROMBIN INTO THROMBIN Thrombin acts on fibrinogen fibrin monomers fibrin (XIII factor stabilizes the clot)

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60 Thrombin Thrombin primary role is the conversion of fibrinogen to fibrin monomers, the building block of a clot monomers polymerize spontaneusely Activates factor XIII XIIIa- it is found in the TR granules crosslinkage of fibrin polymers insoluble stable mesh of fibin Activates Factors VIII and V and their inhibitor protein C (in the presence of thrombomodulin)

61 Thrombin Positive feedback- following activation by the contact factor or tissue factor pathways, the coagulation cascade is maintained in a prothrombotic state by the continued activation of - IX -X -XI -XII

62 Thrombin Binds with thrombomodulin on endothelial cell surfaces to form a complex which activates protein C Stimulates endothelial cells to produce NO, PGI2, ADP, vwf, tissue plasmin activator Activates platelets Activates plasmin- fibrinolysis

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64 Fibrinogen

65 Fibrinogen GP 3 paires of polypeptides Aa, Bb and Gg All 6 NH2- terminal ends toward the center (E domain) 2 globular ends (D domains) Thrombin- cleaves the N-terminal ends of Aa and Bb (known as fibrinopeptides A and B) fibrin monomer

66 Fibrin- polymers One E domain interacts with 4 D domains on other fibrin molecules Polymers crosslink due to XIIIa factor activity that cross- links glutamine residues on one fibrin molecule to the lys residues on another molecule (between C term ends- Aa protuberances)

67 Fibrin clot

68 Clot Fibrin mesh Thrombocytes Entrapped blood cells

69 Classic view- unanswered questions Is useful in blood test interpretation But it is not accurate- patients with a severe fxii deficit don t bleed initiation coagulation begins with the extrinsic pathway in vivo Generation/ exposure to tissue factor at the wound site, is the primary physiological event that initiates clotting Patients with VIII and IX dificits- very severe bleeding (TF- VIIa is inhibited by TFPI sustained activation of X via intrinsic pathway is critical for normal hemostasis)

70 Modern theory Initiation of coagulation VII to become VIIa F VIIa activates X directly/ indirectly via the activation of factor IX, dual pathway for fx activation- necessary because Limited amount of TF generated in vivo Presence of tissue factor pathway inhibitor (TFPI) when complexed with Xa inhibitis TF- VIIa complex (TFPI inhibits Xa and TF-VIIa complex)

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72 Plasmatic factors Secreted in the liver, except for the vwf (endoth cell and megacariocytes) Vitamin K dependent procoagulants- prothrombin, VII, IX and X Vitamin K dependent anticoagulants- protein C and S

73 Cellular theory Initiation phase- TF complexed with VIIa initiates coagulation at the site of injury small amounts of thrombin are generated and activate platelets Amplification phase-coagulation factors form complexes on TR surfaces Propagation phase- very large amounts of thrombin are formed to convert fibrinogen to fibrin which reinforces platelet plug

74 Initiation phase

75 Amplification phase

76 Propagation phase

77 Clot retraction The end process Fibrin fibres are attached to damaged blood wall Clot begins to contract by using the contractile apparatus of the TR Clot si more tight and stable In the process- the liquid that is expressed= serum (lacks most of the coagulation factors)

78 Clotting regulation Dillution of procoagulants in flowing blood Removal if activated factors through reticuloendothelial system (liver mostly) Antithrombotic natural pathways- 7 systems Antithrombin III Protein C Protein S Tissue factor pathway inhibitos (TFPI) Prostacyclin and NO- modulate vascular and platelet reactivity ADP-ase- inhibits platelet recruitment Fibrinolysis- removes the clot

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80 ATIII- heparan sulfate system ATIII- circulating plasma protease inhibitor Inhibits thrombin and fxa- key enzymes inhibits XIIa and Xia activated by heparan sulfate on the surface of the endoth cells- binds to a site on ATIII- conformational change thrombin instant and irreversable inhibition But... endoth cells are coated with already active ATIII!basis for the therapeutic use of heparin

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82 Protein C/S/thrombomodulin system Thrombomodulin- integral protein on the luminal surface of the endoth Thrombin binds to thrombomodulin thrombin inactivates and it activates protein C in plasma Protein S is a cofactor for protein C activation Activated protein C inhibits Va and VIIIa

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84 Tissue factor pathway inhibitor (TFPI) Circulating plasma protease inhibitor synth by endothelium Very low plasma concentrations Inhibits Xa TFPI-Xa factor complex inhibits TF/VIIa complex TFPI depletion disseminated intravascular coagulation (CID)

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86 Von Willebrand s disease the most common hereditary coagulation abnormality qualitative or quantitative deficiency of von Willebrand factor (vwf) Defective platelet adhesion Varying degrees of bleeding Types Hereditary- most common Aquired- autoantibodies Platelet type- inherited (affects receptors in platelets)

87 Hemophilia group of hereditary genetic disorders that impair the body's ability to control blood clotting or coagulation Excessive bleeding, minor injury Sex- linked Type A- VIII factor deficit Type B- IX factor deficit Not sex- linked Type C- XI factor deficit Severity varies due to level of active clotting factor (<1 %- severe)