Prothrombinase

The prothrombinase complex consists of the serine protein, Factor Xa, and the protein cofactor, Factor Va. The complex assembles on negatively charged phospholipid membranes in the presence of calcium ions. The prothrombinase complex catalyzes the conversion of prothrombin (Factor II), an inactive zymogen, to thrombin (Factor IIa), an active serine protease. The activation of thrombin is a critical reaction in the coagulation cascade, which functions to regulate hemostasis in the body. To produce thrombin, the prothrombinase complex cleaves two peptide bonds in prothrombin, one after Arg271 and the other after Arg320.[1] Although it has been shown that Factor Xa can activate prothrombin when unassociated with the prothrombinase complex, the rate of thrombin formation is severely decreased under such circumstances. The prothrombinase complex can catalyze the activation of prothrombin at a rate 3 x 105-fold faster than can Factor Xa alone.[2] Thus, the prothrombinase complex is required for the efficient production of activated thrombin and also for adequate hemostasis.

Activation of Protein Precursors

Both Factor X and Factor V circulate in the blood as inactive precursors prior to activation by the coagulation cascade. The inactive zymogen Factor X consists of two chains, a light chain (136 residues) and a heavy chain (306 residues). The light chain contains an N-terminal γ-carboxyglutamic acid domain (Gla domain) and two epidermal growth factor-like domains (EGF1 and EGF2). The heavy chain consists of an N-terminal activation peptide and a serine-protease domain.[3][4] Factor X can be activated by both the factor VIIa-tissue factor complex of the extrinsic coagulation pathway and by the tenase complex of the intrinsic pathway. The intrinsic tenase complex is composed of both Factor IXa and Factor VIIIa.[5][6] The activation peptide is released when Factor X is activated to Factor Xa, but the heavy and light chains remain covalently linked following activation.

Factor V circulates as a single-chain procofactor which contains six domains, A1-A2-B-A3-C1-C2.[7] Thrombin activates Factor V by cleaving off the B domain. Other proteases also activate Factor Va, but this cleavage is primarily carried out by thrombin. Following cleavage, Factor Va contains a heavy chain, composed of the A1 and A2 domains and a light chain, consisting of the A3, C1, and C2 domains. The light and heavy chains of Factor Va are linked via a divalent metal ion, such as calcium.[8]

Complex Assembly

Prothrombinase assembly begins with the binding of Factor Xa and Factor Va to negatively charged phospholipids on plasma membranes. Activated Factor Xa and Factor Va bind to the plasma membranes of a variety of different cell types, including monocytes, platelets, and endothelial cells.[9] Both Factor Xa and Va bind to the membrane independently of each other, but they both bind to mutually exclusive binding sites.[10] Both Factor Xa and Factor Va associate with the membrane via their light chains, with Factor Xa binding via its Gla-domain in a calcium-dependent manner and Factor Va via its C2 and C1 domains.[11][12] Once bound to the plasma membrane, Factor Xa and Factor Va rapidly associate in a 1:1 stoichiometric ratio to form the prothrombinase complex.[13] Assembly of the prothrombinase complex is calcium dependent. When associated with the prothrombinase complex, the catalytic efficiency of Factor Xa is increased 300,000-fold compared to its efficiency alone.[2] Factor Xa and Factor Va interact tightly with each other when associated on the plasma membrane.[10] Further, membrane-bound Factor Va provides a strong catalytic advantage to the prothrombinase complex. Factor Va strengthens the affinity of Factor Xa for the membrane and also increases the kcat of Factor Xa for prothrombin.[10][14] Factor Va also decreases the Km of the reaction by enhancing the binding of prothrombin to the prothrombinase complex.[15]

Activity

The fully assembled prothrombinase complex catalyzes the conversion of the zymogen prothrombin to the serine protease thrombin. Specifically, Factor Xa cleaves prothrombin in two locations, following Arg271 and Arg320 in human prothrombin.[1] Because there are two cleavage events, prothrombin activation can proceed by two pathways. In one pathway, prothrombin is first cleaved at Arg271. This cleavage produces Fragment 1•2, comprising the first 271 residues, and the intermediate prethrombin 2, which is made up of residues 272-579. Fragment 1•2 is released as an activation peptide, and prethrombin 2 is cleaved at Arg320, yielding active thrombin. The two chains formed after the cleavage at Arg320, termed the A and B chains, are linked by a disulfide bond in active thrombin. In the alternate pathway for thrombin activation, prothrombin is first cleaved at Arg320, producing a catalytically active intermediate called meizothrombin.[16] Meizothrombin contains fragment 1•2 A chain linked to the B chain by a disulfide bond. Subsequent cleavage of meizothrombin by Factor Xa at Arg271 gives Fragment 1•2 and active thrombin, consisting of the A and B chains linked by a disulfide bond. When thrombin is generated by Factor Xa alone, the first pathway predominates and prothrombin is first cleaved after Arg271, producing prethrombin 2, which is subsequently cleaved after Arg320.[17] If Factor Xa acts as a component of the prothrombinase complex, however, the second pathway is favored, and prothrombin is first cleaved after Arg320, producing meizothrombin, which is cleaved after Arg271 to produce active thrombin.[17][18] Thus, the formation of the prothrombinase complex alters the sequence of prothrombin bond cleavage.

Inactivation

Factor Va is inactivated following cleavage by activated protein C. Activated protein C cleaves Factor Va in both its light and heavy chains. Cleavage in the heavy chain reduces the ability of Factor V to bind to Factor Xa.[19] Activated protein C interacts tightly and exclusively with the light chain of Factor Va, and this interaction is calcium independent.[20] Factor Xa can help to prevent the inactivation of Factor Va by protecting Factor Va from activated protein C.[21] It is likely that Factor Xa and activated protein C compete for similar sites on Factor Va.[9] Factor Xa is inhibited by the antithrombin III/heparin system, which also acts to inhibit thrombin.[9]

Role in Disease

Deficiencies of either protein components of the prothrombinase complex are very rare. Factor V deficiency, also called parahemophilia, is a rare autosomal recessive bleeding disorder with an approximate incidence of 1 in 1,000,000.[22] Congenital Factor X deficiency is also extremely rare, affecting an estimated 1 in 1,000,000.[23]

A point mutation in the gene encoding Factor V can lead to a hypercoagulability disorder called Factor V Leiden. In Factor V Leiden, a G1691A nucleotide replacement results in an R506Q amino acid mutation. Factor V Leiden increases the risk of venous thrombosis by two known mechanisms. First, activated protein C normally inactivates Factor Va by cleaving the cofactor at Arg306, Arg506, and Arg679.[24] The Factor V Leiden mutation at Arg506 renders Factor Va resistant to inactivation by activated protein C. As a result of this resistance, the half-life of Factor Va in plasma is increased, resulting in increased thrombin production and increased risk of thrombosis.[25] Secondly, under normal conditions, if Factor V is cleaved by activated protein C instead of thrombin, it can serve as a cofactor for activated protein C.[25] Once bound to Factor V, activated protein C cleaves and inactivates Factor VIIIa. The mutated form of Factor V present in Factor V Leiden, however, serves as a less efficient cofactor of activated protein C. Thus, Factor VIIIa is less efficiently inactivated in Factor V Leiden, further increasing the risk of thrombosis.[25] In fact Factor V Leiden is the most common cause of inherited thrombosis.[26]

Heterozygous Factor V Leiden is present in approximately 5% of the white population in the United States and homozygous Factor V Leiden is found less than 1% of this population.[27] Factor V Leiden is much more common in individuals of Northern European descent and in some Middle Eastern populations. It is less common in Hispanic populations, and rare in African, Asian, and Native American populations.[27] Factor V Leiden is an important risk factor for venous thromboembolism, that is, deep vein thrombosis or pulmonary embolism.[28] In fact, heterozygous Factor V Leiden increases one's risk of recurrent venous thromboembolism by 40%.[29]

Anticoagulant Drugs

Inhibition of Factor Xa prevents thrombin activation, thereby preventing clot formation. Thus, Factor Xa is used as both a direct and indirect target of several anticoagulant drugs. For example, the drug Fondaparinux is an indirect inhibitor of Factor Xa. Fondaparinux binds to antithrombin III and activates the molecule for Factor Xa inhibition. In fact, Fondaparinux imparts an increased affinity of antithrombin III to Factor Xa, and this increased affinity results in a 300-fold increase in the antithrombin III inhibitory effect on Factor Xa.[30] After the antithrombin III binds to Factor Xa, the Fondaparinux is released and can activate another antithrombin.[31] Another drug that indirectly inhibits Factor Xa is Idraparinux. Idraparinux also binds antithrombin III, however with a 30-fold increase in affinity as compared to Fondaparinux.[32] Idraparinux has an increased half-life as compared to Fondaparinux and can be administered on a weekly basis, whereas Fondaparinux must be subcutaneously injected daily.[33]

Rivaroxaban and Apixaban are direct Factor Xa inhibitors.[34][35][36]

Rivaroxaban and Apixaban bind to the active site of Factor Xa, regardless of whether Factor Xa is bound in the prothrombinase complex or is in its free form.[35][37] These direct Factor Xa inhibitors can be administered orally, as can dabigatran etexilate which is a direct thrombin inhibitor.

Fondaparinux, Rivaroxaban, Apixaban, Dabigatran Etexilate, and Endoxaban are currently used as an FDA approved anticoagulant drug. Development of Idraparinux was discontinued.[38]

References

  1. 1 2 Krishnaswamy S (January 2005). "Exosite-driven substrate specificity and function in coagulation". J. Thromb. Haemost. 3 (1): 54–67. PMID 15634266. doi:10.1111/j.1538-7836.2004.01021.x.
  2. 1 2 Nesheim ME, Taswell JB, Mann KG (November 1979). "The contribution of bovine Factor V and Factor Va to the activity of prothrombinase". J. Biol. Chem. 254 (21): 10952–62. PMID 500617.
  3. ↑ Di Scipio RG, Kurachi K, Davie EW (June 1978). "Activation of human factor IX (Christmas factor)". J. Clin. Invest. 61 (6): 1528–38. PMC 372679 Freely accessible. PMID 659613. doi:10.1172/JCI109073.
  4. ↑ Furie B, Furie BC (May 1988). "The molecular basis of blood coagulation". Cell. 53 (4): 505–18. PMID 3286010. doi:10.1016/0092-8674(88)90567-3.
  5. ↑ Di Scipio RG, Hermodson MA, Yates SG, Davie EW (February 1977). "A comparison of human prothrombin, factor IX (Christmas factor), factor X (Stuart factor), and protein S". Biochemistry. 16 (4): 698–706. PMID 836809. doi:10.1021/bi00623a022.
  6. ↑ Hoffman M, Monroe DM (June 2001). "A cell-based model of hemostasis". Thromb. Haemost. 85 (6): 958–65. PMID 11434702.
  7. ↑ Jenny RJ, Pittman DD, Toole JJ, Kriz RW, Aldape RA, Hewick RM, Kaufman RJ, Mann KG (July 1987). "Complete cDNA and derived amino acid sequence of human factor V". Proc. Natl. Acad. Sci. U.S.A. 84 (14): 4846–50. PMC 305202 Freely accessible. PMID 3110773. doi:10.1073/pnas.84.14.4846.
  8. ↑ Mann KG, Kalafatis M (January 2003). "Factor V: a combination of Dr Jekyll and Mr Hyde". Blood. 101 (1): 20–30. PMID 12393635. doi:10.1182/blood-2002-01-0290.
  9. 1 2 3 Mann KG, Jenny RJ, Krishnaswamy S (1988). "Cofactor proteins in the assembly and expression of blood clotting enzyme complexes". Annu. Rev. Biochem. 57: 915–56. PMID 3052293. doi:10.1146/annurev.bi.57.070188.004411.
  10. 1 2 3 Krishnaswamy S (March 1990). "Prothrombinase complex assembly. Contributions of protein-protein and protein-membrane interactions toward complex formation". J. Biol. Chem. 265 (7): 3708–18. PMID 2303476.
  11. ↑ Majumder R, Quinn-Allen MA, Kane WH, Lentz BR (January 2005). "The phosphatidylserine binding site of the factor Va C2 domain accounts for membrane binding but does not contribute to the assembly or activity of a human factor Xa-factor Va complex". Biochemistry. 44 (2): 711–8. PMID 15641797. doi:10.1021/bi047962t.
  12. ↑ Autin L, Steen M, Dahlbäck B, Villoutreix BO (May 2006). "Proposed structural models of the prothrombinase (FXa-FVa) complex". Proteins. 63 (3): 440–50. PMID 16437549. doi:10.1002/prot.20848.
  13. ↑ Krishnaswamy S, Mann KG, Nesheim ME (July 1986). "The prothrombinase-catalyzed activation of prothrombin proceeds through the intermediate meizothrombin in an ordered, sequential reaction". J. Biol. Chem. 261 (19): 8977–84. PMID 3755135.
  14. ↑ Rosing J, Tans G, Govers-Riemslag JW, Zwaal RF, Hemker HC (January 1980). "The role of phospholipids and factor Va in the prothrombinase complex". J. Biol. Chem. 255 (1): 274–83. PMID 7350159.
  15. ↑ van Rijn JL, Govers-Riemslag JW, Zwaal RF, Rosing J (September 1984). "Kinetic studies of prothrombin activation: effect of factor Va and phospholipids on the formation of the enzyme-substrate complex". Biochemistry. 23 (20): 4557–64. PMID 6498156. doi:10.1021/bi00315a008.
  16. ↑ Morita T, Iwanaga S (February 1978). "Purification and properties of prothrombin activator from the venom of Echis carinatus". J. Biochem. 83 (2): 559–70. PMID 416016.
  17. 1 2 Esmon CT, Jackson CM (December 1974). "The conversion of prothrombin to thrombin. III. The factor Xa-catalyzed activation of prothrombin". J. Biol. Chem. 249 (24): 7782–90. PMID 4430674.
  18. ↑ Krishnaswamy S, Church WR, Nesheim ME, Mann KG (March 1987). "Activation of human prothrombin by human prothrombinase. Influence of factor Va on the reaction mechanism". J. Biol. Chem. 262 (7): 3291–9. PMID 3818642.
  19. ↑ Guinto ER, Esmon CT (November 1984). "Loss of prothrombin and of factor Xa-factor Va interactions upon inactivation of factor Va by activated protein C". J. Biol. Chem. 259 (22): 13986–92. PMID 6438088.
  20. ↑ Krishnaswamy S, Williams EB, Mann KG (July 1986). "The binding of activated protein C to factors V and Va". J. Biol. Chem. 261 (21): 9684–93. PMID 3755431.
  21. ↑ Nesheim ME, Canfield WM, Kisiel W, Mann KG (February 1982). "Studies of the capacity of factor Xa to protect factor Va from inactivation by activated protein C". J. Biol. Chem. 257 (3): 1443–7. PMID 6895752.
  22. ↑ van Wijk R, Nieuwenhuis K, van den Berg M, Huizinga EG, van der Meijden BB, Kraaijenhagen RJ, van Solinge WW (July 2001). "Five novel mutations in the gene for human blood coagulation factor V associated with type I factor V deficiency". Blood. 98 (2): 358–67. PMID 11435304. doi:10.1182/blood.V98.2.358.
  23. ↑ Auerswald G (2006). "Prophylaxis in rare coagulation disorders -- factor X deficiency". Thromb. Res. 118 Suppl 1: S29–31. PMID 16574201. doi:10.1016/j.thromres.2006.01.015.
  24. ↑ Kalafatis M, Rand MD, Mann KG (December 1994). "The mechanism of inactivation of human factor V and human factor Va by activated protein C". J. Biol. Chem. 269 (50): 31869–80. PMID 7989361.
  25. 1 2 3 Rosendorff A, Dorfman DM (June 2007). "Activated protein C resistance and factor V Leiden: a review". Arch. Pathol. Lab. Med. 131 (6): 866–71. PMID 17550313. doi:10.1043/1543-2165(2007)131[866:APCRAF]2.0.CO;2.
  26. ↑ Mateo J, Oliver A, Borrell M, Sala N, Fontcuberta J (March 1997). "Laboratory evaluation and clinical characteristics of 2,132 consecutive unselected patients with venous thromboembolism--results of the Spanish Multicentric Study on Thrombophilia (EMET-Study)". Thromb. Haemost. 77 (3): 444–51. PMID 9065991.
  27. 1 2 Ornstein DL, Cushman M (April 2003). "Cardiology patient page. Factor V Leiden". Circulation. 107 (15): e94–7. PMID 12707252. doi:10.1161/01.CIR.0000068167.08920.F1.
  28. ↑ Folsom AR, Cushman M, Tsai MY, Aleksic N, Heckbert SR, Boland LL, Tsai AW, Yanez ND, Rosamond WD (April 2002). "A prospective study of venous thromboembolism in relation to factor V Leiden and related factors". Blood. 99 (8): 2720–5. PMID 11929758. doi:10.1182/blood.V99.8.2720.
  29. ↑ Marchiori A, Mosena L, Prins MH, Prandoni P (August 2007). "The risk of recurrent venous thromboembolism among heterozygous carriers of factor V Leiden or prothrombin G20210A mutation. A systematic review of prospective studies". Haematologica. 92 (8): 1107–14. PMID 17650440. doi:10.3324/haematol.10234.
  30. ↑ Olson ST, Björk I, Sheffer R, Craig PA, Shore JD, Choay J (June 1992). "Role of the antithrombin-binding pentasaccharide in heparin acceleration of antithrombin-proteinase reactions. Resolution of the antithrombin conformational change contribution to heparin rate enhancement". J. Biol. Chem. 267 (18): 12528–38. PMID 1618758.
  31. ↑ Bauer KA (December 2003). "New pentasaccharides for prophylaxis of deep vein thrombosis: pharmacology". Chest. 124 (6 Suppl): 364S–370S. PMID 14668419. doi:10.1378/chest.124.6_suppl.364S.
  32. ↑ McRae SJ, Ginsberg JS (2005). "New Anticoagulants for the Prevention and Treatment of Venous Thromboembolism". Vasc Health Risk Manag. 1 (1): 41–53. PMC 1993925 Freely accessible. PMID 17319097. doi:10.2147/vhrm.1.1.41.58936.
  33. ↑ Kearon C (August 2004). "Long-term management of patients after venous thromboembolism". Circulation. 110 (9 Suppl 1): I10–8. PMID 15339876. doi:10.1161/01.CIR.0000140902.46296.ae.
  34. ↑ Roehrig S, Straub A, Pohlmann J, Lampe T, Pernerstorfer J, Schlemmer KH, Reinemer P, Perzborn E (September 2005). "Discovery of the novel antithrombotic agent 5-chloro-N-({(5S)-2-oxo-3- [4-(3-oxomorpholin-4-yl)phenyl]-1,3-oxazolidin-5-yl}methyl)thiophene- 2-carboxamide (BAY 59-7939): an oral, direct factor Xa inhibitor". J. Med. Chem. 48 (19): 5900–8. PMID 16161994. doi:10.1021/jm050101d.
  35. 1 2 Pinto DJ, Orwat MJ, Koch S, Rossi KA, Alexander RS, Smallwood A, Wong PC, Rendina AR, Luettgen JM, Knabb RM, He K, Xin B, Wexler RR, Lam PY (November 2007). "Discovery of 1-(4-methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxamide (apixaban, BMS-562247), a highly potent, selective, efficacious, and orally bioavailable inhibitor of blood coagulation factor Xa". J. Med. Chem. 50 (22): 5339–56. PMID 17914785. doi:10.1021/jm070245n.
  36. ↑ Hauel NH, Nar H, Priepke H, Ries U, Stassen JM, Wienen W (April 2002). "Structure-based design of novel potent nonpeptide thrombin inhibitors". J. Med. Chem. 45 (9): 1757–66. PMID 11960487. doi:10.1021/jm0109513.
  37. ↑ Perzborn E, Strassburger J, Wilmen A, Pohlmann J, Roehrig S, Schlemmer KH, Straub A (March 2005). "In vitro and in vivo studies of the novel antithrombotic agent BAY 59-7939--an oral, direct Factor Xa inhibitor". J. Thromb. Haemost. 3 (3): 514–21. PMID 15748242. doi:10.1111/j.1538-7836.2005.01166.x.
  38. ↑ Gross PL, Weitz JI (March 2008). "New anticoagulants for treatment of venous thromboembolism". Arterioscler. Thromb. Vasc. Biol. 28 (3): 380–6. PMID 18296593. doi:10.1161/ATVBAHA.108.162677.

See also

coagulation cascade hemostasis

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