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Cell-based Coagulation 4-23-15.pptx
Cell-based Coagulation Coagulation System Overview And Cell-Based Coagulation Coagulation Summary George A. Fritsma, MS MLS The Fritsma Factor Your Interactive Hemostasis Resource [email protected] 1 A 3 year-old boy experienced painful joints. His hematologist ordered a prothrombin time (PT), activated partial thromboplastin time (PTT), and platelet count. Results: Platelet count PT PTT 3/22/2015 Platelets Plasma-based cascade Coagulation controls Fibrinolysis Cell-based coagulation Virtues and limitations Cell-based Coagulation RI 150–400,000/uL 12.6–14.6 sec 25–35 sec • Liver disease, vitamin K deficiency, renal disease? – No symptoms, normal diet, PT normal – Liver enzymes normal • Lupus anticoagulant? – Unlikely in child • Inherited single factor deficiency 3 PT and PTT Test Results in Inherited Coagulopathies PT Long Long Normal PTT Congenital Single Factor Deficiency (Hemophilia) Normal VII Long X, V, II, and fibrinogen1 VIII, IX, XI Long Contact factors: XII, prekallikrein, high MW kininogen2 1. PT and PTT are prolonged only when fibrinogen is < 100 mg/dL 5 2. Contact factor deficiencies affect PTT results, but do not cause bleeding www.fritsmafactor.com 2 What are the Possibilities? 3 YO Boy: Chronic Joint Pain Result 324,000/uL 12.9 sec 67 sec • • • • • • 4 A 3 Year-old Boy: Easy Bruising Single Factor Assay Results Factor VIII IX XI RI Result 12% 95% 108% 50–150% • Implications of factor VIII deficiency: hemophilia A • The mild form accounts for late onset and mild symptoms • Therapy: RICE, DDAVP, tranexamic acid, FVIII concentrate 6 1 Cell-based Coagulation Plasma-Based Coagulation Model 1964–2004 SMC Internal Elastic Lamina EC • Davie EW, Ratnoff OD. Waterfall sequence for intrinsic blood clotting. Science 1964; 145: 1310–12. • Macfarlane RG. An enzyme cascade in the blood clotting mechanism and its function as a biological amplifier. Nature 1964; 202: 498–9. • Nemerson Y. The tissue factor pathway of blood coagulation. Semin Hematol 1992;29:170–6. EC EC • • • • • • • • • • Plasminogen activator inhibitor -1 Tissue factor pathway inhibitor Tissue plasminogen activator Negative surface charge Prostacyclin (PGI2) EC Thrombomodulin Heparan sulfate ADPase (CD39) SMC Nitric oxide Urokinase EC EC Integrins bind: α1β1 : fibronectin αvβ1 : collagen α3β1 : laminin α6β1 : laminin α2β3 : vitronectin EC EC Cytokines: • IL-1 • TNF • γ-interferon EC EC Permeability RBC RBC PLT EC EC EC SMC FB EC EC SMC FB Collagen SMC FB EC EC EC EC EC EC • • • • • Collagen, fibronectin, vitronectin, laminin Membrane phospholipids Von Willebrand factor PLT Heparan sulfate Tissue factor EC EC EC Tunica Intima EC Chemokines: Adhesion Molecules: ICAM-1 ICAM-2 VCAM-1 PECAM P-selectin EC: Endothelial cell FB: Fibroblast SMC: Smooth muscle cell RBC RBC PLT WBC EC EC EC EC SMC FB SMC Monocyte chemoattractive protein IL-6 IL-8 PLT RBC 8 Tunica Media SMC Damaged intimae trigger hemostasis ry EC Tunica Intima EC WBC EC SMC EC EC EC PLT RBC PLT Internal Elastic Lamina Hypoxia Inj u EC Normal Blood Flow in Intact Vessels Injured Endothelial Cells Become Hemostatic DDAVP EC Fritsma GA. Platelet production, structure, and function. In Keohane EM, Smith LJ, Walenga JM. Rodak’s Hematology Clinical Principles and Applications. 2015, Elsevier 7 Shear Stress EC Tunica Media SMC Intact intimae suppress hemostasis: EC: Endothelial cell FB: Fibroblast SMC: Smooth muscle cell THR SMC FB EC EC SMC FB Collagen Trauma to Vessel Exposes Collagen and Tissue Factor 9 Initiation: vascular injury exposes fibroblast (FB) tissue factor (TF) P P P P P P PP FB ++ Ca Tissue Factor “Extrinsic” Pathway IX TF a VII XIa VII VIIIa IXa Xa Ca++ XI VIII P Ca++ X 10 Va Propagation on activated platelet (P) surfaces with phosphatidyl serine V P Access to tissue factor (TF) on smooth muscle cells and fibroblasts in injured vessel wall activates VII. In inflammation, TF appears on endothelial cells and monocytes. By Shape: ote Pr Thrombin as Prothrombin Fibrinogen Coagulation Fibrin ed P nk sli lot P P P P os c Cr ibrin P P f P P P P www.fritsmafactor.com e XIIIa gen o Zy m XIII Fibroblast TF VII VIIa X TF VIIa binds TF, activates X Xa r facto Co P P P 12 2 Cell-based Coagulation Tissue Factor and VIIa Activate IX of the “Common” Pathway Fibroblast VIIa By Shape: te Pro Zym oge VIIa and TF activate IX TF X n IXa Ca++ as Phospholipid e Cofa ctor X VIIIa PLT Phos Xa Ca++ VIII THR Fibrinogen 13 Fibrin Polymerization and Crosslinking E β-chain B B FPa, FPb D D D D E D E Fibrinopeptides A and B 15 P P P P P P PP FB ++ Ca a VII XI XIa VII VIIIa IXa Xa VIII P Ca++ Ca++ E Va Propagation on activated platelet (P) surfaces with phosphatidyl serine V P D D E D D D E D Fibrinogen Fibrin D E D E D E D D D Crosslinked Fibrin Polymer Thrombin activates XI IXa binds VIIIa, platelet phosphatidyl serine, and Ca++ to form the “tenase” complex that activates X XI IX THR XIa IXa VIIIa Ca++ PLT Phos Thrombin XIIIa Coagulation D D D E XIII Prothrombin E D XIIIa stabilizes fibrin polymer by forming intermolecular γ-dimers between neighboring γ-chain glutamine and lysine E D E Intrinsic Pathway IX TF X D D D D D XIIIa B B A D D D E domain γ-chain Initiation: vascular injury exposes fibroblast (FB) tissue factor (TF) E E Fibrin Polymer E D THR D domain A Crosslinked Fibrin Fibrin Fibrinogen THR A Thrombin activates XIII, XIIIa crosslinks fibrin THR XIIIa D D domain Common Pathway V PRO Thrombin Cleaves Fibrinogen α-chain Va PLT Phos XIII IXa binds VIIIa, platelet phosphatidyl serine, and Ca++ to form “tenase,” which activates factor X Xa A Thrombin activates V, a cofactor in the prothrombinase complex IX TF VII Xa binds Va, platelet phosphatidylserine, and Ca++ to form the “prothrombinase” complex that activates prothrombin, forming thrombin ed P nk sli lot P P P P os c Cr ibrin P P f P P P P www.fritsmafactor.com P P P XIa activates IX X Xa Common pathway proceeds from Xa 3 Cell-based Coagulation Vitamin K In Vitro Contact Activation XIIa binds HMWK (Fitzgerald factor) and PK (Fletcher factor) to activate factor XI HMWK XI PK Ca++ -- - XIIa NCS XIa XII Negatively charged particles or surfaces activate XII Intrinsic pathway proceeds from XIa • Necessary for normal activity of prothrombin (II) and factors VII, IX, and X • Also necessary for normal activity of control proteins C, S, and Z • Mediates γ-carboxylation of glutamic acid • Necessary for Ca++ fixation to phospholipid • Affected by oral anticoagulants 19 20 γ-carboxylation of Glutamic Acid Fibroblast TF COOH γ β HOOC CH2 ++ Ca VIIa COOH Xa CH Vitamin K VIIIa IXa Va CH2 CH2 IIa Simplified Coagulation Pathway α H2N-CH-COOH H2N-CH-COOH Fibrin polymer Gla Glu Crosslinked fibrin NH2 COOH 21 Procoagulant Concentrations and Their Plasma Half-lives Thrombin Properties Mann KG. Thrombin formation. Chest 2003; 124:4S-10S XIIIa II, VII, IX, X, PC, S, or Z os Ph PLT 12-20 Gla copies Neg-charged surface PK XIa XIIa HMWK Platelets Va XIa THR (IIa) Fibrin polymer VIIIa XIIIa TAFI Thrombomodulin www.fritsmafactor.com 23 Factor Category Half-life Plasma Hemostatic Level Level Fibrinogen (I) Substrate 4 days 280 mg/dL 50 mg/dL Prothrombin (II) Protease 60 hours 1300 µg/mL 20% V Cofactor 16 hours 680 µg/mL 25% VII Protease 6 hours 120 µg/mL 20% VIII Cofactor 12 hours 0.24 µg/mL 30% IX Protease 24 hours 5 µg/mL 30% X Protease 30 hours 10 µg/mL 25% XI Protease 2-3 days 6 µg/mL 25% XIII Transglutaminase 7-10 days 290 µg/mL 2-3% VWF Cofactor 30 hours 6 µg/mL 50% 24 4 Cell-based Coagulation The Platelet Clot or “White” Clot The Fibrin Clot or “Red” Clot Platelet Adhesion Properties Courtesy of Kathy Jacobs, Chronolog, Inc • Composed of platelets, fibrin, and RBCs • The endpoint of “secondary” hemostasis • Complete hemostasis in higher vertebrates Courtesy of Kathy Jacobs, Chronolog, Inc • Composed of platelets and von Willebrand factor • The endpoint of “primary” hemostasis • Complete hemostasis in invertebrates and lower vertebrates A 7 Year-old Girl: Elective Surgery • Platelets bind vessel wall via VWF and fibrin – Platelet receptors GP Ia/IIa, IV, and VI bind intimal collagen – Platelet receptors GP Ib/V/IX and GP IIb/IIIa adhere to VWF and fibrin – GP IIb/IIIa supports platelet aggregation • Platelets bind other adhesive proteins: thrombospondin, fibronectin • Fibrin binds platelet interior actin: clot retraction A healthy 7 year-old girl was scheduled for elective outpatient surgery. The surgeon ordered a screening platelet count, PT, and PTT. She had experienced no bleeding. Results: Result RI Platelet count 237,000/uL 150–400,000/uL PT 13.5 sec 12.6–14.6 sec PTT (APTT) 47 sec 25–35 sec 27 What are the Possibilities? A 7 Year-old Girl: Elective Surgery Mixing studies were performed to determine the cause for the prolonged PTT. Results: Test PTT patient PTT control PTT 1:1 patient/control 2h incubated control 2h incubated PTT 1:1 PTT RI Result 47 sec 29 sec 32 sec 34 sec 36.5 sec 25–35 sec www.fritsmafactor.com 28 • Lupus anticoagulant inhibitor? – No: immediate correction within 10% of control • Specific inhibitor? – No: 2h correction to within 10% of 2h control • Liver disease, vitamin K deficiency, renal? – No symptoms, PT normal, liver enzymes normal • Inherited single factor deficiency? 29 30 5 Cell-based Coagulation PT and PTT Test Results in Inherited Coagulopathies PT PTT Fibroblast Congenital Single Factor Deficiency (Hemophilia) Long Normal VII Long Long X, V, II, and fibrinogen1 VIII, IX, XI Normal Long TF PT prolonged by deficiencies of “extrinsic” VII, and “common” X, V, prothrombin (II), fibrinogen Contact factors: XII, prekallikrein, high MW kininogen2 VIIa Xa VIIIa IXa Va IIa Fibrin polymer XIIIa 1. PT and PTT prolonged when fibrinogen is < 100 mg/dL 2. Contact factor deficiencies affect PTT results, but do not cause bleeding Neg-charged surface PK XIa XIIa HMWK Crosslinked fibrin PTT prolonged by deficiencies of “Intrinsic” XII, XI, IX, VIII, and “common” X, V, prothrombin (II), fibrinogen 31 A 7 Year-old Girl: Elective Surgery Factor Assay Results True or False 1. Coagulation is triggered by exposure to tissue factor. 2. Coagulation is also triggered by the in vivo activation of factor XII. 3. Factor VIII deficiency causes bleeding. 4. Coagulation can occur without platelet phosphatidylserine. Intrinsic Pathway Factor Result Factor XI 123% Factor XII 37% PK 97% HMWK 89% RI 50–150% • Bleeding implications of factor XII deficiency: none • Incidence: 3% 33 Antithrombin Glycosaminoglycan • Linear heteropolysaccharide • Plasma antithrombin activity is upregulated by endothelial cell heparan sulfate, a long-chain glycosaminoglycan • Therapeutic heparin upregulates plasma antithrombin 1000-2000× • Antithrombin is a serine protease inhibitor (SERPIN) that neutralizes thrombin and Xa 35 www.fritsmafactor.com 34 – Disaccharide repeating unit – Chondroitin sulfate – Dermatan sulfate – Keratan sulfate – Heparan sulfate – Hyaluronic acid – Heparin 36 6 Cell-based Coagulation Unfractionated Heparin Binds Antithrombin to Thrombin Protease Binding Site AT Heparin Binding Site Active Protease Site IIa Protease Binding Site AT Heparin Binding Site Heparin Binding Site IIa AT Unfractionated Heparin Binds Antithrombin to Xa AT Xa The Protein C Control Pathway Fibroblast UFH:AT UFH:AT TF VIIa VIIIa Neg-charged surface PK XIa XIIa HMWK IXa Va UFH:AT No Heparin Binding Site UFH IIa AT Xa Xa Xa AT UFH UFH:AT Active Protease Site IIa Heparin: Antithrombin Control Points Fibrin polymer • Protein C activated by thrombomodulin-bound thrombin • Becomes serine protease specific for Va and VIIIa • Forms cell-surface complex with protein S, digests Va, VIIIa Fritsma MG, Fritsma GA. Normal Hemostasis and Coagulation. In Rodak BF, Fritsma GA, Keohane EM. Hematology Clinical Principles and Applications, 4th Edition. 2012, Elsevier XIIIa Crosslinked fibrin 40 Protein C Control Pathway Fibroblast TF Va VIIa VIIIa Endothelial Cell Thrombin EPCR-1 APC PC Xa Vi PS IIa APC is a serine protease 40% Free PS C4b-BP Bound PS PS Va, VIIIa Vi, VIIIi TM: PC: APC: PS: C4b-BP EPCR-1 Activated factors V and VIII Inactivated factors V and VIII Thrombomodulin Protein C Activated protein C Protein S C4b binding protein Endothelial protein C receptor www.fritsmafactor.com VIIIa Fibrin polymer XIIIa Crosslinked fibrin 41 IXa Va APC VIIIi APC TM Neg-charged surface PK XIa XIIa HMWK Activated Protein C Control Points Fritsma MG, Fritsma GA. Normal Hemostasis and Coagulation. In Rodak BF, Fritsma GA, Keohane EM. Hematology Clinical Principles and Applications, 4th Edition. 2012, Elsevier 7 Cell-based Coagulation True or False Fibroblast TF TFPI VIIa ZPI Xa VIIIa Neg-charged surface PK XIa XIIa HMWK IXa Va IIa Fibrin polymer XIIIa Crosslinked fibrin Tissue Factor Pathway Inhibitor and Z-dependent Protease Inhibitor Control Points Fritsma MG, Fritsma GA. Normal Hemostasis and Coagulation. In Rodak BF, Fritsma GA, Keohane EM. Hematology Clinical Principles and Applications, 4th Edition. 2012, Elsevier Virtues of the Plasma Coagulation System Model 1. Antithrombin deficiency confers a risk of thrombosis. 2. Protein S excess confers a risk of hemorrhage. 3. Deep vein thrombosis is common in 30-yearolds. 4. Though thrombin is procoagulant, it activates protein C on endothelial cells. 44 Limitations of the Plasma Coagulation System • It models coagulation as a series of amplifying proteolytic reactions – Each protease cleaves and activates the subsequent substrate zymogen in the series • It recognizes the participation of platelet anionic phospholipids, mainly phosphatidylserine – Inert although essential assembly site • It models the screening tests PT and PTT as corresponding to the “extrinsic” and “intrinsic” systems • If there is a separate tissue factor pathway, why doesn’t VIIa/ TF activate enough X to compensate for a lack of factor VIII or IX in hemophilia? • If VIII or IX deficiency both cause severe bleeding, why is XI deficiency bleeding mild and variable? • Why is no fibrin generated when the platelet count is less than 10,000/uL? • Why does aspirin reduce thrombin formation? 45 46 The Cell-Based Coagulation System 2001-Present The Answer “It just got worse” • There is no “extrinsic,” “common,” or “intrinsic” pathway • There is no plasma coagulation • Coagulation occurs only under the control of cells Hoffman H, Monroe DM. Coagulation 2006: a modern view of hemostasis. Hematol Oncol Clin North Am 2007;21:1-11 Hoffman M, Cichon LJH: Practical coagulation for the blood banker. Transfusion 53:1594-1602, 2013. 47 www.fritsmafactor.com 48 8 Cell-based Coagulation Overlapping Coagulation Phases Initiation Amplification Initiation: Tissue Factor Bearing Cell • TF constitutive on fibroblast, smooth muscle cell, or induced on monocytes or endothelial cells • Exposure of TF cleaves VII → VIIa and binds TF/VIIa • TF/VIIa cleaves X → Xa; Xa cleaves and binds V → Va • Xa/Va cleaves prothrombin (Pro, II) → thrombin (Thr, IIa) • TF/VIIa also cleaves IX → IXa Propagation – With no VIII around, IXa cannot function • Free initiation proteases are bound by TFPI, ZPI or AT • Occurs away from injury site and outside of vessels Dahlbäck B. Blood coagulation. Lancet 2000; 355: 1627-32. 49 50 Amplification: COAT Platelets Initiation: TF-bearing Cell • Amplification begins at injury • Platelets adhere to injury site collagen and VWF • Platelets contact thrombin-producing tissue factorbearing cells and become partially activated • Called “collagen and thrombin-stimulated” (COAT) platelets TF-bearing cell VII TF Va VIIa Xa V X PI TF TFPI Pro (II) Thr (IIa) Xa ZPI AT IXa AT AT IX 51 52 Amplification: COAT Platelet Amplification: COAT Platelet • Thrombin (IIa) in direct “hand-off” from TF-bearing cells escapes AT and binds nearby COAT platelets Tissue factor-bearing fibroblast delivers IIa, IXa, and Xa – Triggers release of VIII/VWF and V from α-granules – Cleaves and activates VIII → VIIIa and V → Va – Cleaves VIII from VIII/VWF – Cleaves and activates XI from plasma and PLT α-granules → XIa • IXa from TF-bearing cells binds VIIIa on COAT platelets • Xa from TF-bearing cells binds Va on COAT platelets • XIa binds COAT platelet membranes C AP VIIIa VIIIa IXa XIa IX Thr (IIa) XI Xa Va Thr (IIa) Va C AP – Cleaves and activates IX from plasma/platelets → IXa 53 www.fritsmafactor.com Thr (IIa) V COAT Platelet VIII/VWF 54 9 Cell-based Coagulation Propagation: COAT Platelet Propagation: COAT Platelet • COAT platelet continues to be activated by thrombin, collagen and VWF • Tenase and prothrombinase complexes now assemble on platelet surface IXa VIIIa Xa XI – TF/VIIa and Xa/Va are the extrinsic mechanism – XIa and IXa/VIIIa are intrinsic • The pathways now act at the platelet surface to produce high-volume thrombin • Fibrin polymerization and stabilization COAT Platelet XI XIa Va Thr (IIa) D IX E D PRO X XIII XIIIa D E D D D E D E D E D D D D E D E D E D E D D D 55 56 Cell Localization Cell Localization: Platelets • • • • • Tissue factor has to be on a cell that supports prothrombinase activity (Va/Xa) – Fibroblasts, smooth muscle cells, also macrophages, monocytes, and endothelial cells when induced • Malignant cells that make TF but don’t support prothrombinase activity are not as effective • • • • No TF on platelets, so they have to adjoin TF bearing cells No factor VII or VIIa receptor site on platelets Thrombin cleaves protease activated receptor (PAR) COAT activation moves phosphatidylserine to outer leaflet to support “tenase” and “prothrombinase” GP Ib/V/IX binds VWF, COAT platelets adhere to injury sites Glycoprotein IIb/IIIa binds fibrinogen and VWF Platelet provides surface receptors for VIIIa, Va, IX, X, XI Subsequent non-COAT platelet layer damps the reaction 57 Virtues of the Cellular Coagulation System Coagulation Control: Endothelial Cells • Thrombomodulin (TM): binds thrombin and activates protein C → APC • Endothelial protein C receptor (EPCR-1) binds APC to surface • APC binds protein S, inactivates Va and VIIIa • Heparan-like glycosaminoglycans activate AT • Cell-surface ADPase neutralizes platelet ADP 59 www.fritsmafactor.com 58 • If there is a separate tissue factor pathway, why can’t the activation of factor X by VIIa/TF compensate for a lack of factor VIII or IX in hemophiliacs? – Because the activation of X by VIIa/TF occurs on the wrong cell— the fibroblast – Free Xa is inhibited by AT and TFPI—it does not diffuse to a platelet • If VIII or IX deficiency both cause severe bleeding, why is XI deficiency bleeding variable? – Free IXa can transfer to the platelet as AT has less effect – Free IXa on platelet bypasses need for factor XI 60 10 Cell-based Coagulation Thrombosis and Cellular Coagulation Fibrinolysis: Fibrin Degradation • If thrombosis is hemostasis that occurs on endothelial cells, therapy could target the vulnerable endothelial cells • Aspirin effect on platelets also slows coagulation • Thrombocytopenia also slows coagulation Plasminogen PAI-1 D D 61 Kringle Adhesion D D E TPA: PAI-1 E D TPA: PAI-1 THR TAFI E D D D D E D E D E D E D D D Tissue plasminogen activator Plasminogen activator inhibitor-1 Thrombin (factor IIa) Thrombin activatable fibrinolysis inhibitor • PAI-1, TPA, and plasmin bind fibrin polymers through “kringle” structures • Plasmin cleaves fibrin at selected lysine and arginine residues • TAFIa cleaves fibrin C-terminal lysines that plasmin requires for their cofactor activity D D E D E THR TAFIa Plasmin TPA E TAFI Fibrinolysis Plasmin TPA α2-plasmin inhibitor PAI-1 D PAI-1 Plasmin TPA D Tissue plasminogen activator Plasminogen activator inhibitor-1 64 Wound Healing Fibrinolysis: Fibrin Degradation D E D E D D D E E D D D D D D E E E D E D D D D Plasmin D D E D D D D D D E D D E D E D D D D D D E E E D E D D E D D Y E E D X D-dimer and Fibrin Degradation Products www.fritsmafactor.com D E E • Fibrin attaches to platelet actin, clot retracts • Neutrophils that first appear are replaced by macrophages • Macrophages secrete vascular endothelial cell growth factor (VEGF), triggers neovascularization • Platelets secrete platelet derived growth factor that attracts and stimulates fibroblasts and smooth muscle cells 66 11 Cell-based Coagulation Thank You For Listening The End Questions? 67 www.fritsmafactor.com 68 12
