The role of thrombin in coagulation

A birds eye view of an amazing molecule's role in several biological processes.

1. The role of Thrombin in haemostasis

2. Alexander Schmidt Described that the conversion of fibrinogen to fibrin is the result of an enzymatic process Named the hypothetical enzyme “thrombin” and it’s precursor “pre-thrombin”

3. In this presentation: Haemostasis –a brief overview Basic biochemistry The moves The dance

4. A brief overview of a complex system Essential haematology 5 th edition

5. Prothrombin Zymogen Inactive precursor enzyme 72 kDa single chain protein mainly produced in liver Serine protease Enzyme that depends on the presence of serine in it’s active center to hidrolyse peptide bonds Active site Carboxyl terminal Catalytic triad of serine, aspartic acid and histidine Vitamin K dependant t1/2 = 60 hours

6. Thrombin: 36 kDa molecule formed when prothrombin is cleaved by prothrombinase complex Sodium activated allosteric enzyme Inactivated by antithrombin and removed from the circulation by the liver

7. Thrombin: Structure Two polypeptide chains B-chain Active site Functional epitopes Sodium binds between the two chains Exosite I Exosite II Di Cera E. Thrombin as proccoagulant and anticoagulant.J Thromb Hemost 2007;5(Suppl. 1):196-202

8. Thrombin structure: Exosite I Integrity required by fibrinogen for docking onto the thrombin surface Contains: Numerous hydrophobic patches and charged residues on it’s surface Provides electrostatic steering to fibrinogen on it’s approach to the active site Fast rate of complex formation Provides the locale for docking to TM Di Cera E. Thrombin as proccoagulant and anticoagulant.J Thromb Hemost 2007;5(Suppl. 1):196-202

9. Thrombin structure: Exosite II Locale for interaction with polyanionic ligands Heparin Glucoseaminoglycans Di Cera E. Thrombin as proccoagulant and anticoagulant.J Thromb Hemost 2007;5(Suppl. 1):196-202

10. Essential haematology 5 th edition

11. Thrombin generation in vivo Network of amplification and negative feedback loops Localised and limited production Two waves of different magnitude Initiation phase Picomolar [] Preparation for second larger burst Amplification phase Micromolar [] Three procoagulant enzyme complexes: Consisting of: Protease, cofactor, PL and calcium Tissue factor complex, tenase , prothrombinase

12. Coagulation in vivo: Initiation phase Initial generation of thrombin TF factor bearing cells: Mononuclear cells Endothelial cells Stromal fibroblasts TF bearing cell surface: Only displays TF once activated Factors V, IX, X activated Small amount of thrombin produced

13. Coagulation in vivo: Amplification phase The first thrombin generated prepares for the thrombin burst that occurs on the surface of activated platelets

14. Coagulation in vivo: Propagation phase Propagation occurs on the surface of activated platelets, leading to the formation of large amounts of thrombin

15. Clot stabilization Fibrinogen Produced in the liver T1/2 of 4/7 Dimer consisting of three paired chains Fibrin monomer Formed after T cleaves fibtinopeptides from a and b chains Fibrin polymer Self assembly of monomers, end to end association of D domains Crosslinked fibrin Covalent bonds between chains Resistant to fibrinolysis Essential haematology 5 th edition

16. Clot stabilization Factor XIII Converts loose fibrin into a firm organized structure Transglutaminase Catalyses crosslinking through the formation of gluatmine -lysine covalent bonds between chains Ciculates as a hetrotetramer Activation Requires T and Ca ++ Two steps Limited proteolysis by T Dissociation of B subunits in the presence of Ca ++

17. Cells responding to thrombin stimulation Cellular effects mediated by protease activated receptors (PARs) PAR G-protein Highly expressed in platelets Also found EC, Monocytes , fibroblasts, T-lymphocytes, neurons, smooth muscle cells 1,3,4 activated by thrombin

18. Cells responding to thrombin stimulation PAR Carry their own ligands Ligands remain tethered to receptor after the cleaving of the receptor by thrombin Activation irreversible Duration of activity limited by rapid internalization of receptor Used once and then discarded

19. Essential haematology 5 th edition

20. Thrombin -effects on endothelial cells Signals EC and vascular smooth muscle cells to: Control vascular resistance Amplifies the inflammatory response by modulating transendothelial movement of neutrophils and plasma proteins

21. Essential haematology 5 th edition

22. Thrombin -effects on platelets Most potent platelet activator Shape change Aggregation b.m.o integrin binding to vWF and fibrinogen Induces synthesis and sectretion of: ADP 5-HT Thromboxane A2 EDPGF

23. Thrombin -effects on platelets PAR-1 Increased intracellular [Ca 2+ ] Upregulation GP IIb/ IIIa Mobilization of P- selectin and CD40-L to platelet surface increased binding of platelets to fibrinogen and an increase in cross linking PAR-4 Modulate and stabilize interplatelet binding

24. Thrombin and physiological limitation of blood coagulation Important that the effect of thrombin is limited to the site of injury By –direct inhibition indirect inhibition

25. Thrombin and physiological limitation of blood coagulation: Tissue factor pathway inhibitor Synthesized in endothelial cells Present: plasma platelets Accumulates at the site of injury –platelet activation Inhibits: Xa VIIa via formation of the quaternary complex TF

26. Thrombin and physiological limitation of blood coagulation Antithrombin Serpin Direct inhibitor of thrombin Inactivates serine proteases by combining with them by peptide bonding –forming high mw stable complexes Stable complexes rapidly removed from the circulation in the liver

27. Thrombin and physiological limitation of blood coagulation Antithrombin Neutralization of thrombin enhanced when T is bound to TM Antithrombin activity enhanced by binding to heparans

28. Thrombin and physiological limitation of blood coagulation The protein C pathway

29. Thrombin and physiological limitation of blood coagulation The Protein C Anticoagulation Pathway Complex interaction of multiple proteins on cell surfaces Major players Thrombin, thrombomodulin, proteins C & S

30. Thrombin and physiological limitation of blood coagulation Thrombomodulin Presented by EC Binds to Thrombin at exosite I Inhibits fibrinogen binding at exosite I Slows fibrin generation Decreases the level of free thrombin available for procoagulant activity. Contributes to the activation of Prot C Promotes stereo chemical association between the active site of thrombin with the cleavage site on proteinC

31. Thrombin and physiological limitation of blood coagulation APC Inactivates Factor Va and VIIIa Promotes fibrinolysis by complexing and inactivating PAI-1

32. Thrombin and fibrinolysis

33. Fibrinolysis Thrombin Inhibits fibrinolysis by activating TAFI via the T, TM complex TAFI acts by removing the terminal lysine residues of fibrin that normally facilitate the binding action of Plasmin and tPA Promotes fibrinolysis via the protein C pathway

34. Just a reminder

35. Dancing Na + activated allosteric enzyme Slow Fast

36. Dancing: The role of Na + Activity Physiological [Na + ] not sufficient for saturation System optimally poised for allosteric regulation Na + binding required for Optimal cleaving of fibrinogen Activation of: Factor 5, 8, 9 necessary for the explosive generation of thrombin

37. Dancing: The role of Na + Promotes the prothrombotic and signalling functions by enhacing cleavage of PAR1, PAR3 and PAR4 destabilized Na + binding anticoagulant effect

38. Sources Essential Haematology Mechanisms in haematology Di Cera E. Thrombin as procoacgulant and anticoagulant. Journal of Thrombosis and Haemostasis , 5(Suppl. 1):196-202 Di Cera E. Thrombin; Molec Aspects of Medicine 2008;29(4):203-254 Huntingdon. Slow thrombin is zymogen -like. Journal of Thrombosis and haemostasis 2009; 7(S1STATE): 159-164