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) had been deleted. The fVa-mediated 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) of approximately 60 nM). Competitive kinetic studies in the presence of the two exosite-1-specific ligands Tyr(63)-sulfated hirudin(54-65) and TM456 suggested that while both peptides are highly effective inhibitors of the fVa-mediated 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 interaction of fVa with PCPS improves the affinity of the activation complex for 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.

Figure 1

Concentration dependence of fVa-mediated prethrombin-2 activation by E2-fXa in the absence and presence of PCPS vesicles. The activation of prethrombin-2 (5 μM) by E2-fXa (2 nM) was monitored as a function of increasing concentrations of fVa in the absence (●) or presence (○) of 20 μM PCPS vesicles in TBS/Ca²⁺. Following 30 min activation at room temperature, EDTA was added to a final concentration of 20 mM and the rate of thrombin generation was measured from the cleavage rate of S2238 as described under “Materials and Methods”. Solid lines in the presence of PCPS vesicles are nonlinear regression fits of kinetic data to a hyperbolic equation yielding apparent K_d of 61.7 ± 13.1 nM (n =3) for fVa in the prethrombin-2 activation reaction.

Figure 2

FVa-dependent inhibitory effects of exosite-1 specific ligands on the E2-fXa activation of prethrombin-2 in the absence and presence of PCPS vesicles. A. The inhibitory effect of increasing concentrations of TM456 (x-axis) on the fVa (50 nM)-mediated prethrombin-2 (1 μM) activation by E2-fXa (1 nM) in the absence (○) or presence (●) of PCPS vesicles (20 μM) was monitored in TBS/Ca²⁺ at room temperature. The initial rate of thrombin generation was measured by an amidolytic activity assay using S2238 and the data were normalized to % of activity at each concentration of the inhibitor (100% in the absence of the inhibitor) as described under “Materials and Methods”. B. The same as A except that the inhibitory effect of Hir^54-65(SO^-₃) on the fVa-mediated prethrombin-2 activation by E2-fXa was monitored in the absence (○) or presence (●) of PCPS vesicles. Nonlinear regression analysis of data in the absence of PCPS vesicles according to a competitive binding equation (13) yielded apparent K_d values of 0.7± 0.2 μM (n =3) for the TM456 (panel A) and 0.9 ± 0.2 μM (n =3) for the hirudin C-terminal peptide (panel B) inhibition of the activation reactions.

Figure 3

Enhancement in the anisotropy of the OG₄₈₈-EGR labeled fXa derivatives upon interaction with fVa on PCPS vesicles. A. Fixed concentrations of the OG₄₈₈-EGR labeled wild-type fXa (○) (10 nM) or OG₄₈₈-EGR labeled fXa-R165A (●) (4 nM) were titrated with increasing concentrations of human fVa (0-50 nM) on PCPS vesicles (10 μM) in TBS/Ca²⁺ at 25 °C. K_D values of (1.5 ± 0.6 nM, n =3) and (10.3 ± 3.5 nM, n =3) for wild-type fXa and fXa-R165A, respectively, were calculated for fVa from the saturable changes in the anisotropy (Δr) of the labeled proteins according to a quadratic binding equation as described under “Materials and Methods”. B. The same as A except that OG₄₈₈-EGR labeled E2-fXa (10 nM) was used in the fVa titration yielding a K_D of (62 ± 4 nM, n =3) for the interaction of the mutant protease with the cofactor. C. The same as above except that OG₄₈₈-EGR labeled fXa-des-Gla (10 nM) was used in the fVa titration yielding a K_D of (58 ± 9 nM, n =3) for the interaction of the mutant protease with the cofactor.

Figure 4

Concentration dependence of the fVa-mediated prethrombin-1 activation by wild-type fXa and fXa-des-Gla on PCPS vesicles. A. The activation of prethrombin-1 (2 μM) by fXa (○) (0.1 nM) was monitored as a function of increasing concentrations of fVa on PCPS vesicles (20 μM) in TBS/Ca²⁺. Following 2 min activation at room temperature, EDTA was added to a final concentration of 20 mM and the rate of thrombin generation was measured from the cleavage rate of S2238 as described under “Materials and Methods”. B. The same as A except that fXa-des-Gla (2 nM) (●) was used as the protease in the substrate activation reaction. Solid lines in both panels are nonlinear regression fits of kinetic data to a hyperbolic equation yielding apparent K_d values of 0.5 ± 0.06 nM (n =3) fVa for wild-type fXa and 48.6 ± 6.2 nM (n =3) fVa for fXa-des-Gla.

Figure 5

PCPS-mediated changes in the fluorescence of OG₄₈₈-EGR labeled fXa derivatives. The OG₄₈₈-EGR labeled fXa (○) or fXa-R165A (●) (50 nM each) was titrated with increasing concentrations of PCPS vesicles (x-axis) in TBS/Ca²⁺ at 25 °C. The changes in the emission intensity of the fluorescence probe in the active-site pocket of both proteases, upon their interaction with PCPS vesicles, were recorded at excitation and emission wavelengths of 490 nm and 520 nm, respectively, as described under “Materials and Methods”.