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From Proteopedia

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Introduction

is the serine protease that catalyzes the penultimate step in blood coagulation. It is activated from its zymogen, prothrombin, at the site of tissue injury by FXa and its cofactor FVa in the presence of phospholipid membrane and calcium. Thrombin is then able to catalyze the cleavage of fibrinogen to insoluable fibrin which spontaneously polymerizes to form a stable clot.^[1][2] Thrombin also acts as a procoagulant by:

• Activating platelets through their protease activated receptors (PARs)^[2]
• Preventing VWF processing by cleaving ADAMTS13 ^[3][4]
• Enhancing its own production through FIX activation ^[4]
• Activating the fibrin crosslinking transglutaminase FXIII ^[5]
• Activation of thrombin activatable fibrinolysis inhibitor (TAFI) ^[6]

Activity of thrombin is regulated physiologically by the serpin inhibitors:

• Antithrombin ^[4][7][8]
• Heparin cofactor II ^[4][9][10]
• Protein C inhibitor ^[4][11][12]
• Protease nexin 1 ^[4][13][14]

Generation of thrombin is decreased when thrombin reaches endothelial lining and interacts with thrombomodulin which significantly increases activity of thrombin in activating protein C (APC).^[15] This enzyme will go on to inactivate FVa^[16][17] and FVIIIa^[18] , cofactors for activation of prothrombin^[19] and FXa^[20], respectively.

By balancing substrate specificity, activity, and inhibition thrombin plays a central role in the blood coagulation cascade. ^[4]

spinqualitylabelsall models Thrombin (1PPB) viewed in cartoon representation colored by secondary structure. Active site residues Ser195, Asp102, and His57 are viewed in ball and stick form. Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image Contents 1 Thrombin Life Cycle 2 Prothrombin Activation 3 Thrombin Structure and Function 4 Thrombin Allostery Thrombin Life Cycle Upon tissue damage tissue factor (TF) is released by subendothelial cells. This interacts with circulating FVIIa, a zymogen-like serine protease, significantly increasing its activity. The FVIIa-TF complex activates FVII, FIX, and FX. The now active FXa cleaves prothrombin bound to membranes through its Gla domain, activating it to thrombin. The later will interact with platelet membrane protein GpIbα and subsequently cleave protease activated receptor-1 (PAR1) causing a signaling cascade which leads to platelet α-granule release and membrane flipping exposing the negatively charged phosphatidylserine. The platelet alpha granules contain the physiologically relevant pool of FVa. Thrombin also leads to activation of FIX, through FXI cleavage, and FIII which form the Xase complex to activate FX. FVa and FXa form the prothrombinase complex in the presence of calcium and phospholipid. It causes rapid activation of prothrombin to thrombin. This increase in thrombin allows sufficient fibrinogen to be cleaved to fibrin which is able to polymerize to form a stable blood clot. Further supporting coagulation, thrombin activates FXIII, a transglutaminase that crosslinks fibrin at lysine residues. The structure of thrombin facilitates its inactivation. Once the endothelial lining is reached thrombin binds heparin glycosaminoglycans. This facilitates its inactivation by serpin inhibitors antithrombin and heparin cofactor II. In addition, thrombin will interact with thrombomodulin which significantly increases its catalytic efficiency activating protein C. Activated protein C inactivates FVIIa and FVa thus down regulating thrombin generation. Thrombin plays a critical dual role in blood coagulation. It must be both promiscuous and specific so that it may complete both procoagulant and anticoagulant functions. The structure of thrombin facilitates its important physiologic functions. Prothrombin Activation Image:Prothrombin activation scheme.png Activation scheme of in vivo activation of prothrombin by the prothrombinase complex in presence of calcium and a phospholipid bilayer Prothrombin is the zymogen form of thrombin. From N-terminal to C-terminal it consists of a Gla domain, two kringle domains, and a catalytic domain. Prothrombin is activated by prothrombinase which consists of FXa, FVa, calcium, and a phospholipid surface. In vivo the first cleavage occurs at the R320-I321 bond, corresponding to residues 15-16 in thrombin which is the N-terminus of the B chain, producing meizothrombin.[21] Subsequent cleavage at R271-T272 yields thrombin.[22] The initial cleavage can also occur at R271 resulting in prothrombin-2 which will then be cleaved at R320 to produce thrombin.[23] After cleavage by prothrombinase the new B chain N-terminus (Ile16) folds into the core protease domain and forms a salt bridge with Asp194.[24] This leads to stabilization of regions of the 180s-loop, Na+ binding loop, and γ-loop (zymogen activation domains). These changes provide the correct conformation for the S1 pocket and oxyanion hole for catalysis.[25][26] Thrombin Structure and Function Thrombin (1PPB) overlayed with electrostatic surface. Structural features 60-loop, γ-loop, exosite I, and exosite II labeled Thrombin is a α/β heterodimer composed of a 36 amino acid A chain and 259 amino acid B chain connected by a bridge between Cys1 and Cys122, in addition to 3 other intrachain disulfide bonds.[27] Its overall fold is similar to trypsin and chymotrypsin and it belongs to the peptidase S1 protease family[28]. It is an overall spherical protein with approximate dimensions of 45 X 45 X 50 Å^3.[29] Important structural features include: A prominent active site cleft flanked by the 60- and γ-loops A sodium binding loop Two surface patches referred to as exosite I and exosite II. The is mostly helical and is wound around the B chain and shaped like a boomerang. It is bound to the B chain mostly through side chain interactions including a salt bridge and H-bond cluster at residues D14, E8, and E14c.[30] Furthermore the C-terminus region forms a short amphipathic helix with hydrophobic side chains interacting with the B chain.[31] The contains the active site of the protein and has numerous notable structural features. The active site is formed at the rims of two interacting 6 stranded (N-terminal barrel in red and C-terminal barrel in orange) which are surrounded by 4 helical regions and many turns. The serine protease , based on chymotrypsin numbering, are Ser195, His57, and Asp102. As is common with serine proteases, an hole is formed by backbone amides of Ser195 and Gly193.[32] This has the functional role of stabilizing the oxyanion intermediate involved in the serine protease mechanism by hydrogen bonding to the oxygen of the P1 residue (traditional substrate-protein nomeclature [33]. In addition, since thrombin cleaves after Arg/Lys the , formed by the 180s- and 220s- loops, has Asp189 at the base to form a salt bridge with the incoming substrate. Furthermore, the S4 binding pocket accommodates hydrophobic substrate residues. The active site cleft rims are formed by the hydrophobic and rigid (residues L60, Y60a, P60b, P60c, W60d, D60e, K60f, N60g, F60h, T60i, and N60g) and the (residues T147, W147a, T147b, A147c, N147d, and V147f) while the base is mostly hydrophilic negatively charged amino acids. The cleft is deep compared to more promiscuous serine proteases, consequently substrates must either have a large loop that is cleaved or have favorable interactions with the insertion loops [34]. Many other loops project out of the B chain but most are rigid due to proline and tryptophan residues. Exosite I is located on the B chain and had both basic and hydrophobic character. It is important in binding fibrinogen, platelet activated receptors, and thrombomodulin. Exosite II is also part of the B chain, and derived from numerous basic amino acids, this is the site of heparin binding through the sulfate groups on the glycosaminoglycan. It is also the site of GpIα on the platelet surface. The is formed by the 180s- and 220s- loops. Na+ is bound by the backbone oxygens of Arg221a and Lys224 in addition to four water molecules in a classic octahedral geometry[35]. Through the covelent disulfide linkage between Cys220 and Cys 191 the sodium binding site is linked to Ser195 and the oxyanion hole. Thrombin Allostery Binding of thrombin by sodium or at exosite I stabilizes a form of thrombin that improves substrate recognition.[36] This occurs due to energetic linkage between these sites to the S1 binding pocket and oxyanion hole. Rapid kinetic analysis suggests that thrombin is in a dynamic equilibrium that consists of a fast, slow, and inactive state[37]. There is question as to the physiologic relevance of the inactive state. Regardless, there will be a proportion of fast:slow thrombin and sodium binding to the fast form stabilizes that conformation. Indeed, mutation of the residues involved in sodium binding diminishes the activity of thrombin.[38] It should be restated, that current data suggest that sodium binding does not induce a conformation change, rather, it stabilizes a conformation of thrombin that has greater activity.

References

1. ↑ Fenton JW 2nd. Thrombin specificity. Ann N Y Acad Sci. 1981;370:468-95. PMID:7023326
2. ↑ ^{2.0 2.1} Coughlin SR. Thrombin signalling and protease-activated receptors. Nature. 2000 Sep 14;407(6801):258-64. PMID:11001069 doi:http://dx.doi.org/10.1038/35025229
3. ↑ Crawley JT, Lam JK, Rance JB, Mollica LR, O'Donnell JS, Lane DA. Proteolytic inactivation of ADAMTS13 by thrombin and plasmin. Blood. 2005 Feb 1;105(3):1085-93. Epub 2004 Sep 23. PMID:15388580 doi:http://dx.doi.org/10.1182/blood-2004-03-1101
4. ↑ ^{4.0 4.1 4.2 4.3 4.4 4.5 4.6} Lane DA, Philippou H, Huntington JA. Directing thrombin. Blood. 2005 Oct 15;106(8):2605-12. Epub 2005 Jun 30. PMID:15994286 doi:http://dx.doi.org/10.1182/blood-2005-04-1710
5. ↑ Takagi T, Doolittle RF. Amino acid sequence studies on factor XIII and the peptide released during its activation by thrombin. Biochemistry. 1974 Feb 12;13(4):750-6. PMID:4811064
6. ↑ Miljic P, Heylen E, Willemse J, Djordjevic V, Radojkovic D, Colovic M, Elezovic I, Hendriks D. Thrombin activatable fibrinolysis inhibitor (TAFI): a molecular link between coagulation and fibrinolysis. Srp Arh Celok Lek. 2010 Jan;138 Suppl 1:74-8. PMID:20229688
7. ↑ Huntington JA. Natural inhibitors of thrombin. Thromb Haemost. 2014 Apr 1;111(4):583-9. doi: 10.1160/TH13-10-0811. Epub 2014 Jan, 30. PMID:24477356 doi:http://dx.doi.org/10.1160/TH13-10-0811
8. ↑ Huntington JA. Thrombin inhibition by the serpins. J Thromb Haemost. 2013 Jun;11 Suppl 1:254-64. doi: 10.1111/jth.12252. PMID:23809129 doi:http://dx.doi.org/10.1111/jth.12252
9. ↑ Huntington JA. Natural inhibitors of thrombin. Thromb Haemost. 2014 Apr 1;111(4):583-9. doi: 10.1160/TH13-10-0811. Epub 2014 Jan, 30. PMID:24477356 doi:http://dx.doi.org/10.1160/TH13-10-0811
10. ↑ Huntington JA. Thrombin inhibition by the serpins. J Thromb Haemost. 2013 Jun;11 Suppl 1:254-64. doi: 10.1111/jth.12252. PMID:23809129 doi:http://dx.doi.org/10.1111/jth.12252
11. ↑ Huntington JA. Natural inhibitors of thrombin. Thromb Haemost. 2014 Apr 1;111(4):583-9. doi: 10.1160/TH13-10-0811. Epub 2014 Jan, 30. PMID:24477356 doi:http://dx.doi.org/10.1160/TH13-10-0811
12. ↑ Huntington JA. Thrombin inhibition by the serpins. J Thromb Haemost. 2013 Jun;11 Suppl 1:254-64. doi: 10.1111/jth.12252. PMID:23809129 doi:http://dx.doi.org/10.1111/jth.12252
13. ↑ Huntington JA. Natural inhibitors of thrombin. Thromb Haemost. 2014 Apr 1;111(4):583-9. doi: 10.1160/TH13-10-0811. Epub 2014 Jan, 30. PMID:24477356 doi:http://dx.doi.org/10.1160/TH13-10-0811
14. ↑ Huntington JA. Thrombin inhibition by the serpins. J Thromb Haemost. 2013 Jun;11 Suppl 1:254-64. doi: 10.1111/jth.12252. PMID:23809129 doi:http://dx.doi.org/10.1111/jth.12252
15. ↑ Esmon CT. The regulation of natural anticoagulant pathways. Science. 1987 Mar 13;235(4794):1348-52. PMID:3029867
16. ↑ Kalafatis M, Rand MD, Mann KG. The mechanism of inactivation of human factor V and human factor Va by activated protein C. J Biol Chem. 1994 Dec 16;269(50):31869-80. PMID:7989361
17. ↑ Lu D, Kalafatis M, Mann KG, Long GL. Comparison of activated protein C/protein S-mediated inactivation of human factor VIII and factor V. Blood. 1996 Jun 1;87(11):4708-17. PMID:8639840
18. ↑ Lu D, Kalafatis M, Mann KG, Long GL. Comparison of activated protein C/protein S-mediated inactivation of human factor VIII and factor V. Blood. 1996 Jun 1;87(11):4708-17. PMID:8639840
19. ↑ Duga S, Asselta R, Tenchini ML. Coagulation factor V. Int J Biochem Cell Biol. 2004 Aug;36(8):1393-9. PMID:15147718 doi:http://dx.doi.org/10.1016/j.biocel.2003.08.002
20. ↑ Saenko EL, Shima M, Sarafanov AG. Role of activation of the coagulation factor VIII in interaction with vWf, phospholipid, and functioning within the factor Xase complex. Trends Cardiovasc Med. 1999 Oct;9(7):185-92. PMID:10881749
21. ↑ Lechtenberg BC, Freund SM, Huntington JA. An ensemble view of thrombin allostery. Biol Chem. 2012 Sep;393(9):889-98. doi: 10.1515/hsz-2012-0178. PMID:22944689 doi:http://dx.doi.org/10.1515/hsz-2012-0178
22. ↑ Lechtenberg BC, Freund SM, Huntington JA. An ensemble view of thrombin allostery. Biol Chem. 2012 Sep;393(9):889-98. doi: 10.1515/hsz-2012-0178. PMID:22944689 doi:http://dx.doi.org/10.1515/hsz-2012-0178
23. ↑ Tijburg PN, van Heerde WL, Leenhouts HM, Hessing M, Bouma BN, de Groot PG. Formation of meizothrombin as intermediate in factor Xa-catalyzed prothrombin activation on endothelial cells. The influence of thrombin on the reaction mechanism. J Biol Chem. 1991 Feb 25;266(6):4017-22. PMID:1995649
24. ↑ Lechtenberg BC, Freund SM, Huntington JA. An ensemble view of thrombin allostery. Biol Chem. 2012 Sep;393(9):889-98. doi: 10.1515/hsz-2012-0178. PMID:22944689 doi:http://dx.doi.org/10.1515/hsz-2012-0178
25. ↑ Lechtenberg BC, Freund SM, Huntington JA. An ensemble view of thrombin allostery. Biol Chem. 2012 Sep;393(9):889-98. doi: 10.1515/hsz-2012-0178. PMID:22944689 doi:http://dx.doi.org/10.1515/hsz-2012-0178
26. ↑ Bobofchak KM, Pineda AO, Mathews FS, Di Cera E. Energetic and structural consequences of perturbing Gly-193 in the oxyanion hole of serine proteases. J Biol Chem. 2005 Jul 8;280(27):25644-50. Epub 2005 May 12. PMID:15890651 doi:http://dx.doi.org/10.1074/jbc.M503499200
27. ↑ Bode W, Mayr I, Baumann U, Huber R, Stone SR, Hofsteenge J. The refined 1.9 A crystal structure of human alpha-thrombin: interaction with D-Phe-Pro-Arg chloromethylketone and significance of the Tyr-Pro-Pro-Trp insertion segment. EMBO J. 1989 Nov;8(11):3467-75. PMID:2583108
28. ↑ Page MJ, Di Cera E. Evolution of peptidase diversity. J Biol Chem. 2008 Oct 31;283(44):30010-4. doi: 10.1074/jbc.M804650200. Epub 2008 , Sep 3. PMID:18768474 doi:http://dx.doi.org/10.1074/jbc.M804650200
29. ↑ Bode W, Mayr I, Baumann U, Huber R, Stone SR, Hofsteenge J. The refined 1.9 A crystal structure of human alpha-thrombin: interaction with D-Phe-Pro-Arg chloromethylketone and significance of the Tyr-Pro-Pro-Trp insertion segment. EMBO J. 1989 Nov;8(11):3467-75. PMID:2583108
30. ↑ Bode W, Mayr I, Baumann U, Huber R, Stone SR, Hofsteenge J. The refined 1.9 A crystal structure of human alpha-thrombin: interaction with D-Phe-Pro-Arg chloromethylketone and significance of the Tyr-Pro-Pro-Trp insertion segment. EMBO J. 1989 Nov;8(11):3467-75. PMID:2583108
31. ↑ Bode W, Mayr I, Baumann U, Huber R, Stone SR, Hofsteenge J. The refined 1.9 A crystal structure of human alpha-thrombin: interaction with D-Phe-Pro-Arg chloromethylketone and significance of the Tyr-Pro-Pro-Trp insertion segment. EMBO J. 1989 Nov;8(11):3467-75. PMID:2583108
32. ↑ Lechtenberg BC, Freund SM, Huntington JA. An ensemble view of thrombin allostery. Biol Chem. 2012 Sep;393(9):889-98. doi: 10.1515/hsz-2012-0178. PMID:22944689 doi:http://dx.doi.org/10.1515/hsz-2012-0178
33. ↑ Schechter I, Berger A. On the size of the active site in proteases. I. Papain. 1967. Biochem Biophys Res Commun. 2012 Aug 31;425(3):497-502. doi:, 10.1016/j.bbrc.2012.08.015. PMID:22925665 doi:http://dx.doi.org/10.1016/j.bbrc.2012.08.015
34. ↑ Huntington JA. Molecular recognition mechanisms of thrombin. J Thromb Haemost. 2005 Aug;3(8):1861-72. PMID:16102053 doi:http://dx.doi.org/10.1111/j.1538-7836.2005.01363.x
35. ↑ Zhang E, Tulinsky A. The molecular environment of the Na+ binding site of thrombin. Biophys Chem. 1997 Jan 31;63(2-3):185-200. PMID:9108691
36. ↑ Lechtenberg BC, Freund SM, Huntington JA. An ensemble view of thrombin allostery. Biol Chem. 2012 Sep;393(9):889-98. doi: 10.1515/hsz-2012-0178. PMID:22944689 doi:http://dx.doi.org/10.1515/hsz-2012-0178
37. ↑ Lechtenberg BC, Freund SM, Huntington JA. An ensemble view of thrombin allostery. Biol Chem. 2012 Sep;393(9):889-98. doi: 10.1515/hsz-2012-0178. PMID:22944689 doi:http://dx.doi.org/10.1515/hsz-2012-0178
38. ↑ Lechtenberg BC, Freund SM, Huntington JA. An ensemble view of thrombin allostery. Biol Chem. 2012 Sep;393(9):889-98. doi: 10.1515/hsz-2012-0178. PMID:22944689 doi:http://dx.doi.org/10.1515/hsz-2012-0178