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 5th edition

6. 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

7. 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

8. 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

9. 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 TMDi Cera E. Thrombin as proccoagulant and anticoagulant.J Thromb Hemost
2007;5(Suppl. 1):196-202

10. 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

12. Essential haematology 5th edition

13. 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

14. 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

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

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

17. 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 5th edition

18. 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++

20. 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

21. 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

22. Essential haematology 5th edition

23. 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

24. Essential haematology 5th edition

25. 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

26. Thrombin -effects on platelets
• PAR-1
▫ Increased intracellular [Ca2+]
▫ 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

27. 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

28. 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

29. 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

30. 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

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

32. 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

33. 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

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

35. Thrombin and fibrinolysis

36. 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

37. Just a reminder

38. Dancing
• Na+ activated allosteric
enzyme
• Slow Fast

39. 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

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

44. 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