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Thromb Haemost 2010; 104(01): 61-70
DOI: 10.1160/TH09-09-0650
DOI: 10.1160/TH09-09-0650
Blood Coagulation, Fibrinolysis and Cellular Haemostasis
Down regulation of prothrombinase by activated protein C during prothrombin activation
Financial support: This work was supported by grant MT-9781 from the Canadian Institutes of Health Research (to MEN) and by grant HS4441 from the Heart and Stroke Foundation of Canada (to MEN). This work was also supported by a Heart and Stroke Foundation of Canada Doctoral Research Award (to PYK).
Further Information
Publication History
Received:
16 September 2009
Accepted after major revision:
11 February 2010
Publication Date:
23 November 2017 (online)
Summary
Activated protein C (aPC) proteolytically inactivates factor Va (FVa) and thereby downregulates prothrombinase. Although FVa inactivation by aPC has been studied extensively, the inactivation of prothrombinase during prothrombin activation has not. Therefore, prothrombin activation initiated both without and with aPC (5.0, 7.5 or 10.0 nM) was monitored over time by fluorescence. The experiments were performed with 0.075 nM FVa and 1.0 nM FXa, and with these concentrations reversed. The time courses of the residual prothrombinase activity with aPC, determined from the slopes of fluorescence over time, were pseudo first order with both limiting and excess FVa. With FVa limiting or in excess, the second rate constants for inactivation of prothrombinase were 1.98 ± 0.09 x 105 M-1s-1 and 2.54 ± 0.13 x 105 M-1s-1 , respectively. The former value is 101-fold smaller than that for FVa inactivation by aPC alone. Since with limiting FVa the second order rate constants for prothrombinsase inactivation and FVa inactivation are equal, FVa is protected 101-fold, presumably by both FXa and prothrombin. In contrast, with excess FVa, the calculated rate constant for FVa inactivation exceeds that for prothrombinase inactivation 17.3-fold, which reflects a loss of protection by FXa. Since the protective effects of the two proteins are theoretically multiplicative, FXa protected 17.3-fold and prothrombin protected 5.8-fold. With 150 nM protein S and limiting FVa, prothrombinase inactivation was two-fold faster, yet it was still protected 91-fold. These studies show that FVa is down-regulated by aPC during prothrombin activation, but both FXa and prothrombin protect FVa in a multiplicative way, with or without protein S.
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References
- 1 Esmon CT, Suttie JW, Jackson CM. The functional significance of vitamin K action. J Biol Chem 1975; 250: 4095-4099.
- 2 Steen M, Dahlbäck B. Thrombin-mediated proteolysis of factor V resulting in gradual B-domain release and exposure of the factor Xabinding site. J Biol Chem 2002; 277: 38424-38430.
- 3 Furie B, Furie BC. The molecular basis of blood coagulation. Cell 1988; 53: 505-518.
- 4 Mann KG, Bovill EG, Krishnaswamy S. Surface-dependent reactions in the propagation phase of blood coagulation. Ann NY Acad Sci 1991; 614: 63-75.
- 5 Mann KG, Kalafatis M. Factor V: a combination of Dr Jekyll and Mr Hyde. Blood 2003; 101: 20-30.
- 6 Nesheim ME, Taswell JB, Mann KG. The contribution of bovine factor V and factor Va to the activity of prothrombinase. J Biol Chem 1979; 254: 10952-10962.
- 7 Krishnaswamy S, Church WR, Nesheim ME. et al Activation of human pro-thrombin by human prothrombinase. Influence of factor Va on the reaction mechanism. J Biol Chem 1987; 262: 3291-3299.
- 8 Nicolaes GA, Dahlback B. Factor V and thrombotic disease: description of a janusfaced protein. Arterioscler Thromb Vasc Biol 2002; 22: 530-538.
- 9 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; 269: 31869-31880.
- 10 Kisiel W, Canfield WM, Ericsson LH. et al Anticoagulant properties of bovine plasma protein C following activation by thrombin. Biochemistry 1977; 16: 5824-5831.
- 11 Kalafatis M, Mann KG. Role of the membrane in the inactivation of factor Va by activated protein C. J Biol Chem 1993; 268: 27246-27257.
- 12 Norstrom EA, Steen M, Tran S. et al Importance of protein S and phospholipid for activated protein C-mediated cleavages in factor Va. J Biol Chem 2003; 278: 24904-24911.
- 13 Walker FJ. Regulation of bovine activated protein C by protein S: the role for the cofactor protein in species specificity. Thromb Res 1981; 22: 321-327.
- 14 Mann KG, Hockin MF, Begin KJ. et al Activated protein C cleavage of factor Va leads to dissociation of the A2 domain. J Biol Chem 1997; 272: 20678-20683.
- 15 Tran S, Norstrom E, Dahlback B. Effects of prothrombin on the individual activated protein C-mediated cleavages of coagulation factor Va. J Biol Chem 2008; 283: 6648-6655.
- 16 Fay PJ, Smudzin TM, Walker FJ. Activated protein C-catalyzed inactivation of human factor VIII and factor VIIIa identification of cleavage sites and correlation of proteolysis with cofactor activity. J Biol Chem 1991; 266: 20139-20145.
- 17 Lu D, Kalafatis M, Mann KG. et al Comparison of activated protein C / protein S mediated interaction of human factor VIII and factor V. Blood 1996; 87: 4708-4717.
- 18 Fay PJ, Haidaris PJ, Smudzin TM. Human factor VIIIa subunit structure : reconstitution of factor VIIIa from the isolated A1/A3-C1-C2 dimer and A2 subunit. J Biol Chem 1991; 266: 8957-8962.
- 19 Krishnaswamy S, Williams EB, Mann KG. The binding of activated protein C to factors V and Va. J Biol Chem 1986; 261: 9684-9693.
- 20 Yegneswaran S, Kojima Y, Nguyen PM. et al Factor Va residues 311–325 represent an activated protein C binding region. J Biol Chem 2007; 282: 28353-28361.
- 21 Segers K, Dahlback B, Rosing J. et al Identification of surface epitopes of human coagulation factor Va that are important for interaction with activated protein C and heparin. J Biol Chem 2008; 283: 22573-22581.
- 22 Kojima Y, Heeb MJ, Gale AJ. et al Binding site for blood coagulation factor Xa involving residues 311–325 in factor Va. J Biol Chem 1998; 273: 14900-14905.
- 23 Kalafatis M, Xue J, Lawler CM. et al Contribution of the heavy and light chains of factor Va to the interaction with factor Xa. Biochemistry 1994; 33: 6538-6545.
- 24 Nesheim ME, Canfield WM, Kisiel W. et al Studies of the capacity of factor Xa to protect factor Va from inactivation by activated protein C. J Biol Chem 1982; 257: 1443-1447.
- 25 Norstrom EA, Tran S, Steen M. et al Effects of factor Xa and protein S on the individual activated protein C-mediated cleavages of coagulation factor Va. J Biol Chem 2006; 281: 31486-31494.
- 26 Bock PE, Panizzi P, Verhamme IM. Exosites in the substrate specificity of blood coagulation reactions. J Thromb Haemost 2007; 05 (01) Suppl 81-94.
- 27 Yegneswaran S, Mesters RM, Fernandez JA. et al Prothrombin residues 473–487 contribute to factor Va binding in the prothrombinase complex. J Biol Chem 2004; 279: 49019-49025.
- 28 Yegneswaran S, Nguyen PM, Gale AJ. et al Prothrombin amino terminal region helps protect coagulation factor Va from proteolytic inactivation by activated protein C. Thromb Haemost 2009; 101: 55-61.
- 29 Chen L, Yang L, Rezaie AR. Proexosite-1 on prothrombin is a factor Vadependent recognition site for the prothrombinase complex. J Biol Chem 2003; 278: 27564-27569.
- 30 Kalafatis M, Beck DO, Mann KG. Structural requirements for expression of factor Va activity. J Biol Chem 2003; 278: 33550-33561.
- 31 Bakker HM, Tans G, Thomassen MC. et al Functional properties of human factor Va lacking the Asp683 – Arg709 domain of the heavy chain. J Biol Chem 1994; 269: 20662-20667.
- 32 Bukys MA, Kim PY, Nesheim ME. et al A control switch for prothrombinase: characterization of a hirudin-like pentapeptide from the COOH terminus of factor Va heavy chain that regulates the rate and pathway for prothrombin activation. J Biol Chem 2006; 281: 39194-39204.
- 33 Rosing J, Hoekema L, Nicolaes GAF. et al Effects of protein S and factor Xa on peptide bond cleavages during inactivation of factor Va and factor Va R506Q by activated protein C. J Biol Chem 1995; 270: 27852-27858.
- 34 Nesheim ME, Prendergast FG, Mann KG. Interactions of a fluorescent active-site-directed inhibitor of thrombin: dansylarginine N-(3-ethyl-1,5-pent-anediyl)amide. Biochemistry 1979; 18: 996-1003.
- 35 Bloom JW, Nesheim ME, Mann KG. Phospholipid-binding properties of bovine factor V and factor Va. Biochemistry 1979; 18: 4419-4425.
- 36 Bajzar L, Fredenburgh JC, Nesheim ME. The activated protein C-mediated enhancement of tissue-type plasminogen activator-induced fibrinolysis in a cell-free system. J Biol Chem 1990; 265: 16948-16954.
- 37 Walker JB, Nesheim ME. The molecular weights, mass distribution, chain composition, and structure of soluble fibrin degradation products released from a fibrin clot perfused with plasmin. J Biol Chem 1999; 274: 5201-5212.
- 38 Nesheim ME, Katzmann JA, Tracy PB. et al Factor V. Methods Enzymol 1981; 80: 249-274.
- 39 Bukys MA, Orban T, Kim PY. et al The interaction of fragment 1 of prothrombin with the membrane surface is a prerequisite for optimum expression of factor Va cofactor activity within prothrombinase. Thromb Haemost 2008; 99: 511-522.
- 40 Kim PY, Manuel R, Nesheim ME. Differences in prethrombin-1 activation with human or bovine factor Va can be attributed to the heavy chain. Thromb Haemost 2009; 102: 623-633.
- 41 Solymoss S, Tucker MM, Tracy PB. Kinetics of inactivation of membrane-bound factor Va by activated protein C. Protein S modulates factor Xa protection. J Biol Chem 1988; 263: 14884-14890.
- 42 Taylor Jr FB, Peer GT, Lockhart MS. et al Endothelial cell protein C receptor plays an important role in protein C activation in vivo. Blood 2001; 97: 1685-1688.
- 43 Bakker HM, Tans G, Janssen-Claessen T. et al The effect of phospholipids, calcium ions and protein S on rate constants of human factor Va inactivation by activated human protein C. Eur J Biochem 1992; 208: 171-178.
- 44 Heeb MJ, Kojima Y, Hackeng TM. et al Binding sites for blood coagulation factor Xa and protein S involving residues 493–506 in factor Va. Protein Sci 1996; 05: 1883-1889.
- 45 Heeb MJ, Mester RM, Tans G. et al Binding of protein S to factor Va associated with inhibition of prothrombinase that is independent of activated protein C. J Biol Chem 1993; 268: 2872-2877.