Evolutionary Adaptations in Pseudonaja Textilis Venom Factor X Induce Zymogen Activity and Resistance to the Intrinsic Tenase Complex

Abstract

The venom of the Australian snake Pseudonaja textilis comprises powerful prothrombin activators consisting of factor X (v-ptFX)- and factor V-like proteins. While all vertebrate liver-expressed factor X (FX) homologs, including that of P. textilis, comprise an activation peptide of approximately 45 to 65 residues, the activation peptide of v-ptFX is significantly shortened to 27 residues. In this study, we demonstrate that exchanging the human FX activation peptide for the snake venom ortholog impedes proteolytic cleavage by the intrinsic factor VIIIa–factor IXa tenase complex. Furthermore, our findings indicate that the human FX activation peptide comprises an essential binding site for the intrinsic tenase complex. Conversely, incorporation of FX into the extrinsic tissue factor–factor VIIa tenase complex is completely dependent on exosite-mediated interactions. Remarkably, the shortened activation peptide allows for factor V-dependent prothrombin conversion while in the zymogen state. This indicates that the active site of FX molecules comprising the v-ptFX activation peptide partially matures upon assembly into a premature prothrombinase complex. Taken together, the shortened activation peptide is one of the remarkable characteristics of v-ptFX that has been modified from its original form, thereby transforming FX into a powerful procoagulant protein. Moreover, these results shed new light on the structural requirements for serine protease activation and indicate that catalytic activity can be obtained without formation of the characteristic Ile16–Asp194 salt bridge via modification of the activation peptide.

Crassostrea gigas-Based Bioactive Peptide Protected Thrombin-Treated Endothelial Cells against Thrombosis and Cell Barrier Dysfunction

“…Activated FX (FXa) can further form prothrombinase with FVIIa and PCPS. 45 The results can also explain the plasma clotting time prolongation of preconditioning group (thrombin and P-2-CG) because of the decreased thrombin generation amount and prolonged thrombin generation time.…”

“…The FXa generation amount was also significantly lower in the adherent HUVECs group (0.21 nM FXa), compared with that of the thrombin group (0.27 nM) ( p < 0.001, Figure C). Activated FX (FXa) can further form prothrombinase with FVIIa and PCPS . The results can also explain the plasma clotting time prolongation of preconditioning group (thrombin and P-2-CG) because of the decreased thrombin generation amount and prolonged thrombin generation time.…”

Site‐specific functional roles of the Factor X activation peptide in the intrinsic tenase‐mediated Factor X activation

“…The intrinsic tenase activation of FX is driven by exosite binding [7], and as previous studies have suggested that the FX‐AP contributes to activator specificity and prevents the promiscuous activation of FX [8,11,19], the FX‐AP may be an important exosite. This was demonstrated in a competitive inhibition study, where a chimeric FX construct containing human FX and the AP of venom‐derived Pseudonaja textilis was a significantly weaker competitor of the intrinsic tenase‐mediated activation of human FX, compared with a nonactivable FX construct containing a Gln substitution of the Arg residue at the P1 site [12]. This suggested that the exosite interactions that drive the intrinsic tenase‐mediated activation of human FX are located both in the FX‐AP and at extended sites on the FX surface outside the FX‐AP.…”

“…An emerging proposition for the distinctive substrate specificity of intrinsic tenase is that exosite interactions, rather than recognition of sequences flanking the scissile bond, is a major determinant of biomolecular substrate specificity [7]. The FX-AP is considered to be important for the specificity of the intrinsic tenase complex towards FX [8][9][10][11][12], but details of the molecular role of the FX-AP remain a major question in coagulation biochemistry.…”

Activity Sensing of Coagulation and Fibrinolytic Proteases

“…Activated FX catalyzes the conversion of prothrombin into thrombin (FIIa) by cleavage at the R271−T272 and R320−I321 residues [87,88] . FXa forms the prothrombinase complex with protein cofactor FVa and Ca 2+ assembled on the PL membrane [89] . Other naturally occurring substrates of FXa include FV, FVII, FVIII, protein S, and FX upon autoactivation (residues are summarized in Figure 5).…”