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J Biol Chem 2001 Mar 9;276(10):7164-8 Related Articles, Books, LinkOut Regulation of platelet factor Va-dependent thrombin generation by activated protein C at the surface of collagen-adherent platelets.Briede JJ, Tans G, Willems GM, Hemker HC, Lindhout T.Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, 6200 MD Maastricht, The Netherlands.Recent studies have indicated that factor Va bound to activated platelets is partially protected from inactivation by activated protein C (APC). To explore whether this sustained factor Va activity could maintain ongoing thrombin generation, the kinetics of platelet factor Va-dependent prothrombinase activity and its inhibition by APC were studied. In an attempt to mimic physiologically relevant conditions, platelets were adhered to collagen type I-coated discs. These discs were then spun in solutions containing prothrombin and factor Xa either in the absence or presence of APC. The experiments were performed in the absence of platelet-derived microparticles, with thrombin generation and inhibition confined to the surface of the adherent platelets. APC completely inactivated platelet-associated prothrombinase activity with an overall second order rate constant of 3.3 x 10(6) m(-)1 s(-)1, which was independent of the prothrombin concentration over a wide range around the apparent K(m) for prothrombin. Kinetic studies on prothrombinase assembled at a planar phospholipid membrane composed of 25 mol % phosphatidylserine and 75 mol % phosphatidylcholine revealed a similar second order rate constant of inhibition (2.5 x 10(6) m(-1) s(-1)). Collectively, these data demonstrate that ongoing platelet factor Va-dependent thrombin generation at the surface of collagen-adherent platelets is effectively inhibited by APC. No differences were observed between the kinetics of APC inactivation of plasma-derived factor Va or platelet factor Va as part of the prothrombinase associated with, respectively, a planar membrane of synthetic phospholipids or collagen-adherent platelets.

The physiological inhibitor of tissue factor (TF)zfactor VIIa (FVIIa), full-length tissue factor pathway inhibitor (TFPIFL) in complex with factor Xa (FXa), has a high affinity for anionic phospholipid membranes. The role of anionic phospholipids in the inhibition of TFzFVIIacatalyzed FX activation was investigated. FXa generation at a rotating disc coated with TF embedded in a membrane composed of pure phosphatidylcholine (TFzPC) or 25% phosphatidylserine and 75% phosphatidylcholine (TFzPSPC) was measured in the presence of preformed complexes of FXazTFPIFL or FXazTFPI1‐161 (TFPI lacking the third Kunitz domain and C terminus). At TFzPC, FXazTFPIFL and FXazTFPI1‐161 showed similar rate constants of inhibition (0.07 3 10 8 M 21 s 21 and 0.1 3 10 8 M 21 s 21 , respectively). With phosphatidylserine present, the rate constant of inhibition for FXazTFPIFL increased 3-fold compared with a 9-fold increase in the rate constant for FXazTFPI1‐161. Incubation of TFzPSPC with FXazTFPIFL in the absence of FVIIa followed by depletion of solution FXazTFPIFL showed that FXazTFPIFL remained bound at the membrane and pursued its inhibitory activity. This was not observed with FXazTFPI1‐161 or at TFzPC membranes. These data suggest that the membrane-bound pool of FXazTFPIFL may be of physiological importance in an on-site regulation of TFzFVIIa activity.

The activation of prothrombin is catalyzed by prothrombinase, a complex of factor Xa and factor Va assembled on a negatively charged phospholipid membrane, We used a tubular flow reactor to identify the relative contributions of factor Va, prothrombin, and the negatively charged phosphatidylserine to the assembly of prothrombinase. Perfusion of phospholipid-coated capillaries with a mixture of factor Xa, factor Va, and prothrombin resulted in a steady-state rate of thrombin production that increased with (i) the phosphatidylserine content of the phospholipid bilayer, (ii) the factor Va concentration, and, most interestingly, (iii) the prothrombin concentration of the perfusion solution, Incorporation of 20 mol % phosphatidylethanolamine, a phospholipid with poor ability to promote prothrombinase activity, into a 5 mol % phosphatidylserine membrane also increased the steady-state rate of thrombin production, Direct measurements of the amount of prothrombinase in the flow reactor demonstrated that increased catalytic activities were the result of an increased steady-state amount of membrane associated prothrombinase. Thus, similar turnover numbers of prothrombin activation (3100 min(-1)) were calculated, irrespective of the phosphatidylserine content of the membrane, We established for membranes with low phosphatidylserine content (

Prothrombinase assembly was studied on macroscopic planar bilayers consisting of 20% dioleoyl-phosphatidylserine (DOPS) and 80% dioleoyl-phosphatidylcholine (DOPC). The dissociation constant for the binding of factor Xa to the bilayer, measured by ellipsometry, was K(d) = 47 +/- 8 nM (mean +/- S.D.) and this value was lowered to K(d) = 2.2 +/- 0.3 pM by preadsorption of factor Va. This latter value was determined from direct measurement of steady-state thrombin production. A comparable value of K(d) = 1.0 +/- 0.1 pM was found by repeating these experiments in suspensions of phospholipid vesicles, and it was verified that prothrombinase assembly was not influenced by the addition of prothrombin.Using a minute amount (0.094 fmol cm-2) of preadsorbed factor Va, it was found that the rate of prothrombinase assembly exceeds the rate of collisions between Xa molecules from the buffer and the sparse Va molecules on the bilayer. Apparently, factor Xa adsorbs first to the membrane and then associates rapidly with factor Va by lateral diffusion. The data indicate almost instantaneous equilibrium of this complex formation on the surface with a lower limit for the bimolecular rate constant of k(on) = 2.8 x 10(13) (mol/cm2)-1 s-1.In suspensions of small phospholipid vesicles, prothrombinase assembly is collisionally limited and the value of k(on) should be proportional to vesicle diameter. This was verified with a method for estimation of k(on) values from thrombin generation curves. Values of 0.36 x 10(9) and 1.6 x 10(9) M-1 s-1 were found for vesicles of 20-30- and 60-80-nm diameter, respectively.

Production of thrombin by phospholipid-bound prothrombinase complexes has been described as being regulated by the prothrombin concentration in the buffer (free-substrate model) as well as by the concentration of prothrombin adsorbed to the phospholipid surface (bound-substrate model). We studied simultaneous adsorption and conversion of prothrombin on planar bilayers consisting of 20% dioleoylphosphatidylserine and 80% dioleoylphosphatidylcholine. A transport limitation in the conversion of prothrombin was prevented by using a very low (0.3 fmol cm-2) amount of prothrombinase on the bilayer. The Michaelis and catalytic constants thus found were Km = 5.8 +/- 0.7 nM and kcat = 33 +/- 1 s-1 (mean +/- S.D.). The apparent bimolecular rate constant Kcat/Km = 5.7 x 10(9) M-1 s-1 exceeds the theoretically maximal value for the free-substrate model. In contrast, kcat/Km is within the range expected for a diffusion-controlled bound-substrate model. A similar mechanism for prothrombin conversion in suspensions of phospholipid vesicles would imply increasing kcat/Km values for increasing vesicle diameter. This prediction was tested and a 3-fold increase in kcat/Km values was indeed found for vesicles 60-80 nm in diameter compared to vesicles of 20-30 nm diameter. It is concluded that thrombin production is dependent on protein fluxes rather than on protein concentrations.

Vascular anticoagulant alpha (VAC alpha, annexin V) is a member of the family of calcium and phospholipid binding proteins, the annexins. The binding properties of VAC alpha to phospholipid bilayers were studied by ellipsometry. Adsorption was calcium-dependent and completely reversible upon calcium depletion. Half-maximal adsorptions to phospholipid bilayers consisting of 100, 20, 5, and 1% dioleoyl-phosphatidylserine (DOPS) supplemented with dioleoyl-phosphatidylcholine (DOPC) were reached at Ca2+ concentrations of 0.04, 0.22, 1.5, and 8.6 mM. These surfaces all showed the same maximal adsorption of 0.22 +/- 0.01 micrograms of VAC alpha/cm2 (mean +/- S.D.). The adsorption to bilayers containing more than 10% DOPS was independent of VAC alpha concentrations in the range of 0.5-100 nM. Dissociation constants for VAC alpha binding to these surfaces were estimated to be below 2 x 10(-10) M. No adsorption was observed on pure DOPC bilayers at a Ca2+ concentration of 3 mM. The ability to mediate VAC alpha binding to 20% DOPS/80% DOPC bilayers was highly specific for Ca2+. The use of other divalent cations resulted in decreased binding in the order Cd2+ greater than Zn2+ greater than Mn2+ greater than Co2+ greater than Ba2+ greater than Mg2+. Zinc ions had a synergistic effect on Ca2(+)-dependent VAC alpha binding. The Ca2+ concentration needed for half-maximal binding to cardiolipin, dioleoyl-phosphatidylglycerol, DOPS, phosphatidylinositol, phosphatidic acid, dioleoyl-phosphatidylethanolamine, and sphingomyelin increased in that order. Adsorption was independent of the overall surface charge of the phospholipid membrane.