Membrane-Dependent Interaction of Factor Xa and Prothrombin with Factor Va in the Prothrombinase Complex

Abstract

Because all three protein components of prothrombinase, factors (f) Xa and Va and prothrombin, bind to negatively charged membrane phospholipids, the exact role of the membrane in the prothrombinase reaction has not been fully understood. In this study, we prepared deletion derivatives of fXa and prothrombin in which both the Gla and first EGF-like domains of the protease (E2-fXa) as well as the Gla and both kringle domains of the substrate (prethrombin-2) were deleted. The fVamediated catalytic activity of E2-fXa toward prethrombin-2 was analyzed in both the absence and presence of phospholipids composed of 80% phosphatidylcholine (PC) and 20% phosphatidylserine (PS). PCPS markedly accelerated the initial rate of prethrombin-2 activation by E2-fXa, with the cofactor exhibiting saturation only in the presence of phospholipids (apparent K d ∼60 nM). Competitive kinetic studies in the presence of the two exosite-1-specific ligands Tyr 63 -sulfated hirudin [54][55][56][57][58][59][60][61][62][63][64][65] and TM456 suggested that while both peptides are highly effective inhibitors of the fVamediated activation of prethrombin-2 by E2-fXa in the absence of PCPS, they are ineffective competitors in the presence of phospholipids. Since neither E2-fXa nor prethrombin-2 can interact with membranes, these results suggest that fVa interaction with PCPS improves the affinity of the activation complex for the proexosite-1 of the substrate. Direct binding studies employing OG 488 -EGR-labeled fXa and E2-fXa revealed that the interaction of the Gla-domain of fXa with PCPS also induces conformational changes in the protease to facilitate its high-affinity interaction with fVa.Factor X is a vitamin K-dependent serine protease zymogen in plasma that upon activation to factor Xa (fXa) 1 binds with high affinity to other components of the prothrombinase complex (fVa, negatively charged phospholipid vesicles and calcium) in order to activate prothrombin to thrombin during the blood coagulation process (1-5). The complex formation improves the catalytic efficiency of fXa by greater than 5 orders of magnitude by decreasing the apparent K m of prothrombin ∼100-fold and increasing the k cat ∼1000-fold (2,6). The decrease in K m is thought to be due to the γ-carboxyglutamic acid (Gla) dependent interaction of both fXa and prothrombin with negatively charged phospholipid vesicles which raises the local concentration of prothrombin near the enzyme complex, while the increase in k cat is largely attributed to the cofactor effect of fVa in the reaction (7,8). The mechanism by which fVa improves k cat of prothrombin activation by fXa in the prothrombinase complex is poorly understood, though it has been established that several protein-membrane and protein-protein interactions are the driving force behind the high catalytic efficiency of fXa in the prothrombinase complex (8)(9)(10)(11). Thus, a recent study has postulated that fVa binding to fXa in the prothrombinase complex exposes a new binding exosite on the protease that is a recog...

Determinants of the specificity of protease‐activated receptors 1 and 2 signaling by factor Xa and thrombin

“…It should be noted that a prothrombinase assay, conducted on supernatants obtained from HUVECs, indicated that these cells do not express prothrombin to a detectable level, thus no thrombin was generated in these assays to interfere with the interpretation of results. Results of several studies have indicated that the basic residues of FXa 162-helix (Arg-165 and Lys-169) interact with FVa in the prothrombinase complex (Rezaie, 2000; Rudolph et al, 2001; Qureshi et al, 2009). Interestingly, the Ala-substitution of these residues abrogated the activity of mutants toward PAR2-ALP, expressed on endothelial cells (data not presented).…”

Identification of Exosite Residues of Factor Xa Involved in Recognition of PAR-2 on Endothelial Cells

“…The amidolytic activities of FXa derivatives, have been previously characterized and normal activities for all of them have been observed (21–23,28), suggesting that the mutagenesis of residues under study did not adversely affect the folding or the reactivity of the catalytic triad of mutant proteases. We and others have also investigated the contribution of basic residues of both the 148-loop (21) and the 162-helix (28–30) to the catalytic activity of FXa in the prothrombinase complex. The results indicated that, similar to wild-type FXa, all three mutants of the 148-loop (R143A, R150A and R154A) interact with FVa with a similar K d(app) of 1–2 nM and exhibit a similar catalytic specificity (k cat /K m ) toward prothrombin in the prothrombinase complex (21,23).…”

“…The results indicated that, similar to wild-type FXa, all three mutants of the 148-loop (R143A, R150A and R154A) interact with FVa with a similar K d(app) of 1–2 nM and exhibit a similar catalytic specificity (k cat /K m ) toward prothrombin in the prothrombinase complex (21,23). On the other hand, the basic residues of the 162-helix (in particular Arg-165) appear to be critical for the activity of FXa and have been shown to provide recognition sites for FVa in the prothrombinase complex (28–30). Thus the mutagenesis of Arg-165 and Lys-169 significantly impaired the affinity of the FXa mutants for interaction with FVa as well as their catalytic activity toward prothrombin in the prothrombinase complex (28–30).…”

“…On the other hand, the basic residues of the 162-helix (in particular Arg-165) appear to be critical for the activity of FXa and have been shown to provide recognition sites for FVa in the prothrombinase complex (28–30). Thus the mutagenesis of Arg-165 and Lys-169 significantly impaired the affinity of the FXa mutants for interaction with FVa as well as their catalytic activity toward prothrombin in the prothrombinase complex (28–30). However, the contribution of the acidic residues of the 39-loop and the basic residues of the 60-loop to the catalytic function of FXa in the prothrombinase complex has not been investigated.…”

Leptospira Infection Interferes with the Prothrombinase Complex Assembly during Experimental Leptospirosis

“…At the final steps of coagulation, FII can generate thrombin by two pathways: (i) in the presence of FXa and calcium ions or (ii) in the presence of FXa, FVa, and calcium ions in a phospholipidic surface—the prothrombinase complex (Colman et al, 1994; Betz and Krishnaswamy, 1998; Yang et al, 2008; Qureshi et al, 2009). Formation of the full prothrombinase complex is supported by both intrinsic and extrinsic pathways of coagulation, and may enhance the conversion of FII to thrombin by 300,000-fold (Rosing et al, 1980; Betz et al, 1997).…”