Biomedical subjects

Gabriel Weinreb

Publications and source records attributed to Gabriel Weinreb.

4 recordsLinked to original sources

PubMed2005

Efficient thrombin generation requires molecular phosphatidylserine, not a membrane surface.

Activation of prothrombin to thrombin is catalyzed by a "prothrombinase" complex, traditionally viewed as factor X(a) (FX(a)) in complex with factor V(a) (FV(a)) on a phosphatidylserine (PS)-containing membrane surface, which is widely regarded as required for efficient activation. Activation involves cleavage of two peptide bonds and proceeds via one of two released intermediates or through "channeling" (activation without the release of an intermediate). We ask here whether the PS molecule itself and not the membrane surface is sufficient to produce the fully active human "prothrombinase" complex in solution. Both FX(a) and FV(a) bind soluble dicaproyl-phosphatidylserine (C6PS). In the presence of sufficient C6PS to saturate both FX(a) and FV(a2) (light isoform of FV(a)), these proteins form a tight (Kd = 0.6 +/- 0.09 nM at 37 degrees C) soluble complex. Complex assembly occurs well below the critical micelle concentration of C6PS, as established in the presence of the proteins by quasi-elastic light scattering and pyrene fluorescence. Ferguson analysis of native gels shows that the complex migrates with an apparent molecular mass only slightly larger than that expected for one FX(a) and one FV(a2), further ruling out complex assembly on C6PS micelles. Human prothrombin activation by this complex occurs at nearly the same overall rate (2.2 x 10(8) M(-1) s(-1)) and via the same reaction pathway (50-60% channeling, with the rest via the meizothrombin intermediate) as the activation catalyzed by a complex assembled on PS-containing membranes (4.4 x 10(8) M(-1) s(-1)). These results question the accepted role of PS membranes as providing "dimensionality reduction" and favor a regulatory role for platelet-membrane-exposed PS.

Arginine↗

PubMed2002

Soluble phosphatidylserine triggers assembly in solution of a prothrombin-activating complex in the absence of a membrane surface.

Factor X(a) (FX(a)) binding to factor V(a) (FV(a)) on platelet-derived membranes containing surface-exposed phosphatidylserine (PS) forms the "prothrombinase complex" that is essential for efficient thrombin generation during blood coagulation. There are two naturally occurring isoforms of FV(a), FV(a1) and FV(a2). These two isoforms differ by a 3-kDa polysaccharide chain (at Asn(2181) in human FV(a1) (Kim, S. W., Ortel, T. L., Quinn-Allen, M. A., Yoo, L., Worfolk, L., Zhai, X., Lentz, B. R., and Kane, W. H. (1999) Biochemistry 38, 11448-11454)) and have different coagulant activities. We examined the interaction of the two bovine isoforms with active site-labeled FX(a), finding no significant difference. A soluble form of PS (C6PS) bound to FV(a1) and FV(a2) with comparable affinities (K(d) = 11-12 microm) and changes in FV(a) intrinsic fluorescence. At concentrations well below its critical micelle concentration, C6PS binding to bovine FV(a2) enhanced its affinity for FX(a) in solution by nearly 3 orders of magnitude (K(d)(eff) = 40-2 nm over a C6PS range of 30-400 microm) but had no effect on the affinity of FV(a1) for FX(a) (K(d) = 1 microm). This results in a soluble complex between FX(a) and FV(a2), whose expected molecular weight was confirmed by calibrated native gel electrophoresis. This complex behaved as a normal Michaelis-Menten enzyme in its ability to produce thrombin from meizothrombin (apparent k(cat)/K(m) congruent with 10(9) m(-1) s(-1)). The ability of soluble PS to trigger formation of a soluble prothrombinase complex suggests that exposure of PS molecules during platelet activation is likely the key event responsible for the assembly of an active membrane-bound complex.

Animals↗

PubMed2002

Role of procoagulant lipids in human prothrombin activation. 1. Prothrombin activation by factor X(a) in the absence of factor V(a) and in the absence and presence of membranes.

Activation of prothrombin by factor X(a) requires proteolysis of two bonds and is commonly assumed to occur via by two parallel, sequential pathways. Hydrolysis of Arg(322)-Ile(323) produces meizothrombin (MzII(a)) as an intermediate, while hydrolysis of Arg(273)-Thr(274) produces prethrombin 2-fragment 1.2 (Pre2-F1.2). Activation by human factor X(a) of human prothrombin was examined in the absence of factor V(a) and in the absence and presence of bovine phosphatidylserine (PS)/palmitoyloleoylphosphatidylcholine (25:75) membranes. Four sets of data were collected: fluorescence of an active site probe (DAPA) was sensitive to thrombin, MzII(a), and Pre2-F1.2; a synthetic substrate (S-2238) detected thrombin or MzII(a) active site formation; and SDS-PAGE detected both intermediates and thrombin. The fluorescence data provided an internal check on the active site and SDS-PAGE measurements. Kinetic constants for conversion of intermediates to thrombin were measured directly in the absence of membranes. Both MzII(a) and Pre2-F1.2 were consumed rapidly in the presence of membranes, so kinetic constants for these reactions had to be estimated as adjustable parameters by fitting three data sets (thrombin and MzII(a) active site formation and Pre2 appearance) simultaneously to the parallel-sequential model. In the absence of membranes, this model successfully described the data and yielded a rate constant, 44 M(-1) s(-1), for the rate of MzII(a) formation. By contrast, the parallel-sequential model could not describe prothrombin activation in the presence of optimal concentrations of PS-containing membranes without assuming that a pathway existed for converting prothrombin directly to thrombin without release from the membrane-enzyme complex. The data suggest that PS membranes (1) regulate factor X(a), (2) alter the substrate specificity of factor X(a) to favor the meizothrombin intermediate, and (3) "channel" intermediate (MzII(a) or Pre2-F1.2) back to the active site of factor X(a) for rapid conversion to thrombin.

Animals↗

PubMed2002

Role of procoagulant lipids in human prothrombin activation. 2. Soluble phosphatidylserine upregulates and directs factor X(a) to appropriate peptide bonds in prothrombin.

Activation of human prothrombin to thrombin (II(a)) by factor X(a) during blood coagulation requires proteolysis of two bonds and thus involves two possible activation pathways (parallel-sequential activation model). Hydrolysis of Arg(322)-Ile(323) produces meizothrombin (MzII(a)) as an intermediate, while hydrolysis of Arg(273)-Thr(274) produces prethrombin 2-fragment 1.2 (Pre2-F1.2). A soluble lipid, dicaproylphosphatidylserine (C6PS), enhances activation by 60-fold [Koppaka et al. (1996) Biochemistry 35, 7482]. We report here that C6PS binding to factor X(a) not only enhances the rate of activation but also alters the pathway. Activation was monitored using a chromogenic substrate (S-2238) to detect both II(a) and MzII(a) active site formation and SDS-PAGE to detect Pre2-F1.2 as well as II(a) and MzII(a). Of the four kinetic constants needed to describe activation, two (MzII(a) and Pre2-F1.2 consumption) were measured directly, and two (MzII(a) and Pre2-F1.2 formation) were obtained by fitting the three time courses simultaneously to the parallel-sequential reaction model. The time courses of II(a), MzII(a), and Pre2-F1.2 formations were all well described below the C6PS critical micelle concentration (CMC) by this activation model. The rate of Arg(322)-Ile cleavage leading to MzII(a) formation increased by 150-fold, while the rate of Arg(273)-Thr cleavage leading to Pre2-F1.2 formation was inhibited slightly. At concentrations of water-soluble C6PS above its CMC, all four proteolytic reactions increased in rate by 2-5-fold at the C6PS CMC. We conclude that soluble C6PS differentially affects the rate of individual bond cleavages during prothrombin activation in solution such that activation occurs almost exclusively via MzII(a) formation. Finally, C6PS enhanced the rates of all proteolytic reactions to within a factor of 3 of the enhancement seen with PS-containing membranes. We conclude that PS-containing membranes regulate prothrombin activation by factor X(a) mainly via interaction of individual PS molecules with factor X(a).

Enzyme Activation↗