Activation of factor V during intrinsic and extrinsic coagulation. Inhibition by heparin, hirudin and d-Phe-Pro-Arg-Ch2Cl

Abstract

The validity of the hypothesis that Factor Xa activates Factor V in heparinized plasma was examined by establishing the temporal relationships between Factor V proteolysis and prothrombin consumption in plasma. Factor V was cleaved into Factor Va heavy chain (approx. 110 kDa) and an intermediate (approx. 230 kDa) 30 s after CaCl2 was added to contact-activated plasma (CAP). The larger fragment was converted into Factor V activation peptide (approx. 150 kDa) and Factor Va light chain (approx. 80 kDa) 15 s later. Heparin (approx. 0.05 microM) delayed Factor V proteolysis in CAP by at least 30 s. On supplementing CAP with 1 nM-Factor Xa or 1 nM-thrombin, Factor V was activated 15 s later. Heparin prolonged by 15 s and 45 s the time required to demonstrate Factor V activation in CAP supplemented with Factor Xa and thrombin respectively. Factor V was activated 20 s after tissue factor and CaCl2 were added to plasma, both in the absence and in the presence of approx. 0.05 microM-heparin. In contrast, hirudin and D-Phe-Pro-Arg-CH2Cl (two thrombin inhibitors more effective than heparin) delayed Factor V activation in this plasma by at least 30 s. The fragments of Factor V obtained in heparinized CAP suggest thrombin escapes inhibition and contributes to Factor V activation in that plasma. Production of Factor Va heavy chain and the 230 kDa Factor V fragment invariably preceded efficient prothrombin activation. These observations suggest that heparin, hirudin and D-Phe-Pro-Arg-CH2Cl delay Factor V activation by inhibiting thrombin. The availability of Factor Xa markedly moderates the ability of heparin to inhibit Factor V activation.

Modulation of the Enzymatic Activity of α-Thrombin by Polyanions: Consequences on Intrinsic Activation of Factor V and Factor VIII

“…Inhibi tion of factor V and factor VIII activation by endogenous a-thrombin is better achieved when the enzyme is specifically inactivated at its catalytic center. Such an inhibitory action can be readily achieved with Z)-PhePro-ArgCHiCl [5,8,9,23], hirudin [9], hep arin [5][6][7][8][9] and lactobionic acid. Relative to factor V and factor VIII, the higher Km the a-thrombin and fibrinogen reaction [28][29][30] is consistent with the ability of hirugen and the pentasaccharide to inhibit fibrinopeptide A release without being able to inhibit signif icantly factor V and factor VIII activation by the enzyme.…”

“…Effects of heparin (a) and hirugen (b) on intrinsic prothrom bin activation (consumption). CaCl2 was added to contact-acti vated plasma and prothrombin consumption at the various incuba tion times quantitated using the ELISA procedure reported pre viously [8,9,23]. See figure la, b for an explanation of the symbols used.…”

“…The procedures used to immunoquantitate prothrombin activation have also been reported [8,9,23]. Figure 3 compares the effects of heparin and hirugen on intrinsic prothrombin activation.…”

“…The first method determined when factor Va heavy and light chains, and factor V activation peptide (Mr ~ 150,000) were first produced after CaCL was added to contact-activated plasma. The electropho retic procedures used to demonstrate the in trinsic pathway proteolysis of factor V in human plasma have been reported pre viously [9,23]. Table 1 summarizes the rela tive effectiveness of the four polyanions as inhibitors of intrinsic factor V proteolysis resulting from CaCh addition to contactactivated plasma.…”

“…Factor Va is the protein cofactor for efficient prothrombin activation, and factor Xa in prothrombinase catalyzes the conversion of prothrombin to thrombin at a rate ~ 300,000-fold factor than factor Xa alone [3,4], Endogenous a-thrombin plays a cru cial role in its own generation in plasma as it is the major enzyme which catalyzes factor V and factor VIII activation [5][6][7][8][9]. During in trinsic prothrombin activation, factor VIII activation is necessary to optimize factor X activation by factor IXa, as factor Villa is the cofactor for optimizing factor X activa tion [10], probably in a manner analogous to factor Va during prothrombin activation.…”