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.     

The phosphatidylserine binding site of the factor Va C2 domain accounts for membrane binding but does not contribute to the assembly or activity of a human factor Xa-factor Va complex

Factors V(a) and X(a) (FV(a) and FX(a), respectively) assemble on phosphatidylserine (PS)-containing platelet membranes to form the essential "prothrombinase" complex of blood coagulation. The C-terminal domain (C2) of FV(a) (residues 2037-2196 in human FV(a)) contains a soluble phosphatidylserine (C6PS) binding pocket flanked by a pair of tryptophan residues, Trp(2063) and Trp(2064). Mutating these tryptophans abolishes FV(a) membrane binding. To address both the roles of these tryptophans in C6PS or membrane binding and the role of the C2 domain lipid binding site in regulation of FV(a) cofactor activity, we expressed W(2063,2064)A mutants of the recombinant C2 domain (rFV(a2)-C2) and of a B domain-deleted factor V light isoform (rFV(a2)) in Hi-5 and COS cells, respectively. Intrinsic fluorescence showed that wild-type rFV(a2)-C2 binds to C6PS and to 20% PS/PC membranes with apparent K(d) values of 2.8 microM and 9 nM, respectively, while mutant rFV(a2)-C2 does not. Equilibrium dialysis confirmed that mutant rFV(a2)-C2 does not bind to C6PS. Mutant rFV(a2) binds to C6PS (K(d) approximately 37 microM) with an affinity comparable to that of wild-type rFV(a2) (K(d) approximately 20 microM), although it does not bind to PS/PC membranes to which wild-type rFV(a2) binds with native affinity (K(d) approximately 3 nM). Both wild-type and mutant rFV(a2) bind to active site-labeled FX(a) (DEGR-X(a)) in the presence of 400 microM C6PS with native affinity (K(d) approximately 3-4 nM) to produce a solution rFV(a2)-FX(a) complex of native activity. We conclude that (1) the C2 domain PS site provides all but approximately 1 kT of the free energy of FV(a) membrane binding, (2) tryptophans lining the C2 lipid binding pocket are critical to C6PS and membrane binding and insert into the bilayer interface during membrane binding, (3) occupancy of the C2 lipid binding pocket is not necessary for C6PS-induced formation of the FX(a)-FV(a) complex or its activity, but (4) another PS site on FV(a) does have a regulatory role.     

Modulation of prothrombinase assembly and activity by phosphatidylethanolamine

Constituents of platelet membranes regulate the activity of the prothrombinase complex. We demonstrate that membranes containing phosphatidylcholine and phosphatidylethanolamine (PE) bind factor Va with high affinity (K(d) = ～10 nm) in the absence of phosphatidylserine (PS). These membranes support formation of a 60-70% functional prothrombinase complex at saturating factor Va concentrations. Although reduced interfacial packing does contribute to factor Va binding in the absence of PS, it does not correlate with the enhanced activity of the Xa-Va complex assembled on PE-containing membranes. Instead, specific protein-PE interactions appear to contribute to the effects of PE. In support of this, soluble C6PE binds to recombinant factor Va(2) (K(d) = ～6.5 μm) and to factor Xa (K(d) = ～91 μm). C6PE and C6PS binding sites of factor Xa are specific, distinct, and linked, because binding of one lipid enhances the binding and activity effects of the other. C6PE triggers assembly (K(d)(app) = ～40 nm) of a partially active prothrombinase complex between factor Xa and factor Va(2), compared with K(d)(app) for C6PS ～2 nm. These findings provide new insights into the possible synergistic roles of platelet PE and PS in regulating thrombin formation, particularly when exposed membrane PS may be limiting.     

Soluble phosphatidylserine binds to a single identified site in the C2 domain of human factor Va.

Factor V(a) (FV(a)) is a cofactor for the serine protease factor X(a) that activates prothrombin to thrombin in the presence of Ca(2+) and a membrane surface. FV(a) is a heterodimer composed of one heavy chain (A1 and A2 domains) and one light chain (A3, C1, and C2 domains). We use fluorescence, circular dichroism, and equilibrium dialysis to demonstrate that (1) the FV C2 domain expressed in Sf9 cells binds one molecule of C6PS with a k(d) of approximately 2 microM, (2) stabilizing changes occur in the FV C2 domain upon C6PS binding, (3) the C6PS binding site in the FV C2 domain is located near residue Cys(2113), which reacts with DTNB, and (4) binding to a PS-containing membrane is an order of magnitude tighter than that to soluble C6PS. Coupled with a recently published crystal structure of the C2 domain, these results support a model for the mechanism of C2-membrane interaction.     

Cooperative roles of factor V(a) and phosphatidylserine-containing membranes as cofactors in prothrombin activation

Activation of prothrombin, as catalyzed by the prothrombinase complex (factor X(a), enzyme; factor V(a) and phosphatidylserine (PS)-containing membranes, cofactors), involves production and subsequent proteolysis of two possible intermediates, meizothrombin (MzII(a)) and prethrombin 2 plus fragment 1.2 (Pre2 & F1.2). V(max), K(m), or V(max)/K(m) for all four proteolytic steps was determined as a function of membrane-phospholipid concentration. Proteolysis was monitored using a fluorescent thrombin inhibitor, a chromogenic substrate, and SDS-PAGE. The kinetic constants for the conversion of MzII(a) and Pre2 & F1.2 to thrombin were determined directly. Pre2 & F1.2 conversion was linear in substrate concentration up to 4 microm, whereas MzII(a) proteolysis was saturable. First order rate constants for formation of MzII(a) and Pre2 & F1.2 could not be determined directly and were determined from global fitting of the data to a parallel, sequential model, each step of which was treated by the Michaelis-Menten formalism. The rate of direct conversion to thrombin without release of intermediates from the membrane-V(a)-X(a) complex (i.e. "channeling") also was adjusted because both the membranes and factor V(a) have been shown to cause channeling. k(cat), K(m), or k(cat)/K(m) values were reported for one lipid concentration, for which all X(a) was likely incorporated into a X(a)-V(a) complex on a PS membrane. Comparing previous results, which were obtained either with factor V(a) (Boskovic, D. S., Bajzar, L. S., and Nesheim, M. E. (2001) J. Biol. Chem. 276, 28686-28693) or with membranes individually (Wu, J. R., Zhou, C., Majumder, R., Powers, D. D., Weinreb, G., and Lentz, B. R. (2002) Biochemistry 41, 935-949), with results presented here we conclude that both factor V(a) and PS-containing membranes induce similar rate increases and pathway changes. Moreover, we have determined: 1) factor V(a) has the greatest effect in enhancing rates of individual proteolytic events; 2) PS-containing membranes have the greatest role in increasing the preference for the MzII(a) versus Pre2 pathway; and 3) PS membranes cause approximately 50% of the substrate to be activated via channeling at 50 microm membrane concentration, but factor V(a) extends the range of efficient channeling to much lower or higher membrane concentrations.     

The Gla domain of human prothrombin has a binding site for factor Va

The role of the Gla domain of human prothrombin in interaction with the prothrombinase complex was studied using a peptide with the sequence of the first 46 residues of human prothrombin, PT-(1-46). Intrinsic fluorescence measurements showed that PT-(1-46) undergoes a conformational alteration upon binding calcium; this conclusion is supported by one-dimensional (1)H NMR spectroscopy, which identifies a change in the chemical environment of tryptophan 41. PT-(1-46) binds phospholipid membranes in a calcium-dependent manner with a K(d) of 0.5 microm and inhibits thrombin generation by the prothrombinase complex with a K(i) of 0.8 microm. In the absence of phospholipid membranes, PT-(1-46) inhibits thrombin generation by factor Xa in the presence but not absence of factor Va, suggesting that PT-(1-46) inhibits prothrombin-factor Va binding. The addition of factor Va to PT-(1-46) labeled with the fluorophore sulfosuccinimidyl-7-amino-4-methylcoumarin-3-acetic acid (PT-(1-46)AMCA) caused a concentration-dependent quenching of AMCA fluorescence, providing direct evidence of a PT-(1-46)-factor Va interaction. The K(d) for this interaction was 1.3 microm. These results indicate that the N-terminal Gla domain of human prothrombin is a functional unit that has a binding site for factor Va. The prothrombin Gla domain is important for interaction of the substrate with the prothrombinase complex.     

Residues 88-109 of factor IXa are important for assembly of the factor X activating complex.

Activated platelets and phospholipid vesicles promote assembly of the intrinsic factor X (FX) activating complex by presenting high-affinity binding sites for blood coagulation FIXa, FVIIIa, and FX. Previous reports suggest that the second epidermal growth factor (EGF)-like domain of FIXa mediates assembly of the FX activating complex (Ahmad, S. S., Rawala, R., Cheung, W. F., Stafford, D. W., and Walsh, P. N. (1995) Biochem. J. 310, 427-431; Wong, M. Y., Gurr, J. A., and Walsh, P. N. (1999) Biochemistry 38, 8948-8960). To identify important residues, we prepared several chimeric FIXa proteins using homologous sequences from FVII: FIXa(FVIIEGF2) (FIX Delta 88-124,inverted Delta FVII91-127), FIXa(loop1) (FIX Delta 88-99,inverted Delta FVII91-102), FIXa(loop2) (FIX Delta 95-109,inverted Delta FVII98-112), FIXa(loop3) (FIX Delta 111-124,inverted Delta FVII114-127), and point mutants (FIXaR94D and FIXa(loop1)G94R). In the presence and absence of FVIIIa, a 2- to 10-fold reduced V(max) of FX activation (nm FXa min(-1)) was observed for FIXa(FVIIEGF2), FIXa(loop1), FIXa(loop2), and FIXa(loop1)G94R, whereas FIXa(loop3) and FIXaR94D were normal. For all of the FIXa proteins, K(m)((app)) values were normal as were EC(50) values for interactions with FVIIIa. However, K(d)((app)) (in nm) for the FX activating complex assembled on phospholipid vesicles was increased for FIXa(FVIIEGF2) (43.3 +/- 2.70), FIXa(loop1)(10.9 +/- 2.8), FIXa(loop2) (70.5 +/- 1.60), and FIXa(loop1)G94R (17.1 +/- 2.90) relative to FIXa(N) (3.9 +/- 0.11), FIXa(WT) (4.6 +/- 0.17), FIXa(loop3) (4.5 +/- 0.20), and FIXaR94D (2.2 +/- 0.09) suggesting that reduced V(max) is a result of impaired complex assembly. These data indicate that residues 88-109 (but not Arg(94)) are important for normal assembly of the FX activating complex on phospholipid vesicles.     

Notecarin D binds human factor V and factor Va with high affinity in the absence of membranes

Notecarin D (NotD) is a prothrombin (ProT) activator in the venom of the tiger snake, Notechis scutatus, and a factor Xa (FXa) homolog. NotD binds specifically to the FXa binding site expressed on factor V (FV) upon activation to factor Va (FVa) by thrombin. NotD active site-labeled with 5-fluorescein ([5F]FFR-NotD) binds FV and FVa with remarkably high affinity in the absence of phospholipids (K(D) 12 and ≤ 0.01 nm, respectively). In the presence of membranes, the affinity of [5F]FFR-NotD for FVa is similar, but increased ～55-fold for FV. Binding of FXa active site-labeled with Oregon Green to FV and FVa in the presence of phospholipids is ～5,000- and ～80-fold weaker than [5F]FFR-NotD, respectively. NotD reports FVa and not FV binding by a 3-fold increase in tripeptide substrate hydrolysis, demonstrating allosteric regulation by FVa. The NotD·FVa·membrane complex activates ProT with K(m)((app)) similar to prothrombinase, and ～85-fold weaker without membranes. Active site-blocked NotD exhibits potent anticoagulant activity in plasma thrombin generation assays, representing inhibition of productive prothrombinase assembly and possible disruption of FXa inhibition by the tissue factor pathway inhibitor. The results show that high affinity binding of NotD to FVa is membrane-independent, unlike the strict membrane dependence of FXa for high affinity FVa binding.     

The contribution of amino acid region ASP695-TYR698 of factor V to procofactor activation and factor Va function

There is strong evidence that a functionally important cluster of amino acids is located on the COOH-terminal portion of the heavy chain of factor Va, between amino acid residues 680 and 709. To ascertain the importance of this region for cofactor activity, we have synthesized five overlapping peptides representing this amino acid stretch (10 amino acids each, HC1-HC5) and tested them for inhibition of prothrombinase assembly and function. Two peptides, HC3 (spanning amino acid region 690-699) and HC4 (containing amino acid residues 695-704), were found to be potent inhibitors of prothrombinase activity with IC(50) values of approximately 12 and approximately 10 microm, respectively. The two peptides were unable to interfere with the binding of factor Va to active site fluorescently labeled Glu-Gly-Arg human factor Xa, and kinetic analyses showed that HC3 and HC4 are competitive inhibitors of prothrombinase with respect to prothrombin with K(i) values of approximately 6.3 and approximately 5.3 microm, respectively. These data suggest that the peptides inhibit prothrombinase because they interfere with the incorporation of prothrombin into prothrombinase. The shared amino acid motif between HC3 and HC4 is composed of Asp(695)-Tyr-Asp-Tyr-Gln(699) (DYDYQ). A pentapeptide with this sequence inhibited both prothrombinase function with an IC(50) of 1.6 microm (with a K(D) for prothrombin of 850 nm), and activation of factor V by thrombin. Peptides HC3, HC4, and DYDYQ were also found to interact with immobilized thrombin. A recombinant factor V molecule with the mutations Asp(695) --> Lys, Tyr(696) --> Phe, Asp(697) --> Lys, and Tyr(698) --> Phe (factor V(2K2F)) was partially resistant to activation by thrombin but could be readily activated by RVV-V activator (factor Va(RVV)(2K2F)) and factor Xa (factor Va(Xa)(2K2F)). Factor Va(RVV)(2K2F) and factor Va(Xa)(2K2F) had impaired cofactor activity within prothrombinase in a system using purified reagents. Our data demonstrate for the first time that amino acid sequence 695-698 of factor Va heavy chain is important for procofactor activation and is required for optimum prothrombinase function. These data provide functional evidence for an essential and productive contribution of factor Va to the activity of prothrombinase.     

Sodium binding site of factor Xa: role of sodium in the prothrombinase complex

The nature of residue 225 on a consensus loop in serine proteases determines whether a protease can bind Na(+). Serine proteases with a Pro at this position are unable to bind Na(+), but those with a Tyr or Phe can bind Na(+). Factor Xa (FXa), the serine protease of the prothrombinase complex, contains a Tyr at this position. Na(+) is also known to stimulate the amidolytic activity of FXa toward cleavage of small synthetic substrates, but the role of Na(+) in the prothrombinase complex has not been investigated. In this study, we engineered a Gla-domainless form of FX (GDFX) in which residue Tyr(225) was replaced with a Pro. We found that Na(+) stimulated the cleavage rate of chromogenic substrates by FXa or GDFXa approximately 8-24-fold with apparent dissociation constants [K(d(app))] of 37 and 182 mM in the presence and absence of Ca(2+), respectively. In contrast, Na(+) minimally affected the cleavage rate of these substrates by the mutant, and no K(d(app)) for Na(+) binding to the mutant could be estimated. Unlike the wild-type enzyme, the reactivity of the mutant with antithrombin was independent of Na(+) and impaired approximately 32-fold. Ca(2+) improved the reactivity of the mutant with antithrombin approximately 5-fold. Affinity of the mutant for binding to factor Va was weakened and its ability to activate prothrombin was severely impaired. Further studies with the wild-type prothrombinase complex revealed that FXa binds to factor Va with a similar K(d(app)) of 1. 1-1.8 nM in the presence of Na(+), K(+), Li(+), Ch(+), and Tris(+) and that the catalytic efficiency of prothrombinase is enhanced less than 1.5-fold by the specific effect of Na(+) in the reaction buffer. These results suggest that (1) the loop including residue 225 (225-loop) is a Na(+) binding site in FXa, (2) the Na(+)- and Ca(2+)-binding loops of FXa are allosterically linked, and (3) the Tyr conformer of the 225-loop is critical for factor Xa function; however, both Na(+)-bound and Na(+)-free forms of factor Xa in the prothrombinase complex can efficiently activate prothrombin.     

Proexosite-1-dependent recognition and activation of prothrombin by taipan venom

An activator complex from the venom of Oxyuranus scutellatus scutellatus (taipan venom) is known to rapidly activate prothrombin to thrombin. To determine whether, similar to prothrombinase, taipan venom utilizes proexosite-1 on prothrombin for a productive complex assembly, the activation of proexosite-1 mutants of prethrombin-1 by the partially purified venom was studied. It was discovered that basic residues of this site (Arg(35), Lys(36), Arg(67), Lys(70), Arg(73), Arg(75), and Arg(77)) are also crucial for recognition and rapid activation of the substrate by taipan venom. This was evidenced by the observation that the K(m) and k(cat) values for the activation of the charge reversal mutants of prethrombin-1 (in particular K36E, R67E, and K70E) were markedly impaired. Competitive kinetic studies with the Tyr(63)-sulfated hirudin(54-65) peptide revealed that although the peptide inhibits the activation of the wild type zymogen by taipan venom with a K(D) of approximately 2 microm, it is ineffective in inhibiting the activation of mutant zymogens (K(D) > 4-30 microm). Interestingly, an approximately 50-kDa activator, isolated from the taipan venom complex, catalyzed the activation of prothrombin in a factor Va-dependent manner and exhibited identical activation kinetics toward the substrate in the presence of the hirudin peptide. These results suggest that, similar to prothrombinase, proexosite-1 is a cofactor-dependent recognition site for taipan venom.     

HD1, a thrombin-directed aptamer, binds exosite 1 on prothrombin with high affinity and inhibits its activation by prothrombinase

Incorporation of prothrombin into the prothrombinase complex is essential for rapid thrombin generation at sites of vascular injury. Prothrombin binds directly to anionic phospholipid membrane surfaces where it interacts with the enzyme, factor Xa, and its cofactor, factor Va. We demonstrate that HD1, a thrombin-directed aptamer, binds prothrombin and thrombin with similar affinities (K(d) values of 86 and 34 nm, respectively) and attenuates prothrombin activation by prothrombinase by over 90% without altering the activation pathway. HD1-mediated inhibition of prothrombin activation by prothrombinase is factor Va-dependent because (a) the inhibitory activity of HD1 is lost if factor Va is omitted from the prothrombinase complex and (b) prothrombin binding to immobilized HD1 is reduced by factor Va. These data suggest that HD1 competes with factor Va for prothrombin binding. Kinetic analyses reveal that HD1 produces a 2-fold reduction in the k(cat) for prothrombin activation by prothrombinase and a 6-fold increase in the K(m), highlighting the contribution of the factor Va-prothrombin interaction to prothrombin activation. As a high affinity, prothrombin exosite 1-directed ligand, HD1 inhibits prothrombin activation more efficiently than Hir(54-65)(SO(3)(-)). These findings suggest that exosite 1 on prothrombin exists as a proexosite only for ligands whose primary target is thrombin rather than prothrombin.     

The preferred pathway of glycosaminoglycan-accelerated inactivation of thrombin by heparin cofactor II

Thrombin (T) inactivation by the serpin, heparin cofactor II (HCII), is accelerated by the glycosaminoglycans (GAGs) dermatan sulfate (DS) and heparin (H). Equilibrium binding and thrombin inactivation kinetics at pH 7.8 and ionic strength (I) 0.125 m demonstrated that DS and heparin bound much tighter to thrombin (K(T(DS)) 1-5.8 microm; K(T(H)) 0.02-0.2 microm) than to HCII (K(HCII(DS)) 236-291 microm; K(HCII(H)) 25-35 microm), favoring formation of T.GAG over HCII.GAG complexes as intermediates for T.GAG.HCII complex assembly. At [GAG] < K(HCII(GAG)) the GAG and HCII concentration dependences of the first-order inactivation rate constants (k(app)) were hyperbolic, reflecting saturation of T.GAG complex and formation of the T.GAG.HCII complex from T.GAG and free HCII, respectively. At [GAG] > K(HCII(GAG)), HCII.GAG complex formation caused a decrease in k(app). The bell-shaped logarithmic GAG dependences fit an obligatory template mechanism in which free HCII binds GAG in the T.GAG complex. DS and heparin bound fluorescently labeled meizothrombin(des-fragment 1) (MzT(-F1)) with K(MzT(-F1)(GAG)) 10 and 20 microm, respectively, demonstrating a binding site outside of exosite II. Exosite II ligands did not attenuate the DS-accelerated thrombin inactivation markedly, but DS displaced thrombin from heparin-Sepharose, suggesting that DS and heparin share a restricted binding site in or nearby exosite II, in addition to binding outside exosite II. Both T.DS and MzT(-F1).DS interactions were saturable at DS concentrations substantially below K(HCII(DS)), consistent with DS bridging T.DS and free HCII. The results suggest that GAG template action facilitates ternary complex formation and accommodates HCII binding to GAG and thrombin exosite I in the ternary complex.     

Murine macrophage P2X7 receptors support rapid prothrombotic responses

Non-apoptotic externalization of phosphatidylserine (PS) can act as a reactive surface for the efficient assembly of the prothrombinase complex leading to thrombin generation and coagulation. Here we show that extracellular ATP, acting at the macrophage P2X(7) receptor, drives the rapid Ca(2+)-dependent formation and release of PS-rich microvesicles that enhance the assembly of the prothrombinase complex and subsequent formation of thrombin. Incubation with P2X(7) receptor antagonists (KN-62 and Brilliant Blue G) attenuates ATP induced prothrombotic responses. Consistent with the hypothesis that exposed PS enhances prothrombinase activity; pre-incubation with annexin V blocks the increase in thrombin formation. The rapid translocation of PS and formation of pro-thrombotic microvesicles occurs in the absence of cell lysis. These data demonstrate that the pro-inflammatory P2X(7) receptor can also support and propagate rapid increases in thrombin formation.     

Contribution of regions distal to glycine-160 to the anticoagulant activity of tissue factor pathway inhibitor

The functions of the first two Kunitz domains of tissue factor pathway inhibitor (TFPI) are well defined as active site-directed inhibitors of factor VIIa and factor Xa. The anticoagulant properties of the third Kunitz domain and C-terminal region were probed using altered forms of TFPI. TFPI-160 contains the first two Kunitz domains. K1K2C contains the first two Kunitz domains and the basic C-terminus. Neither TFPI-160 nor K1K2C contains the third Kunitz domain. In amidolytic assays containing calcium, TFPI-160 is a less potent inhibitor of factor Xa than TFPI. However, addition of the C-terminus in K1K2C nearly restores inhibitory activity to that of TFPI, indicating that the third Kunitz domain is not required for direct inhibition of factor Xa. When compared in assays containing phospholipids and factor Va, K1K2C and TFPI-160 are poor inhibitors compared to TFPI, demonstrating that the third Kunitz domain is required for the full anticoagulant activity of TFPI. TFPI was further characterized in amidolytic assays performed with Gla-domainless factor Xa and in prothrombin activation assays using submicellar concentrations of short-chain phospholipids (C6PS). TFPI and K1K2C are worse inhibitors of Gla-domainless factor Xa, compared to wild-type factor Xa, while TFPI-160 inhibits both forms of factor Xa equally, suggesting a C-terminus/Gla domain interaction. TFPI is a potent inhibitor of thrombin generation by prothrombinase assembled with C6PS, while TFPI-160 and K1K2C are not. Conversely, TFPI does not inhibit prothrombin activation by prothrombinase assembled on a two-dimensional lipid bilayer. Together, the data indicate that the region between Gly-160 and the end of the third Kunitz domain contributes to TFPI function by orienting the second Kunitz domain so that it can bind the active site of phospholipid-associated factor Xa prior to prothrombinase assembly and/or by slowing formation of the prothrombinase complex.     

Effects of prothrombin on the individual activated protein C-mediated cleavages of coagulation factor Va

The factor Va (FVa) inactivation by activated protein C (APC), mediated by cleavages at Arg306 and Arg506 in FVa, is inhibited by both factor Xa (FXa) and prothrombin. Although FXa is known to specifically inhibit the Arg506 cleavage, the effect of prothrombin has not been confined to one cleavage site. We used recombinant FV variants, FV:R506Q/R679Q and FV:R306Q/R679Q, to investigate the effect of prothrombin on the individual cleavage sites. The APC-mediated FVa inhibition was monitored by a prothrombinase-based FVa assay, and apparent first order rate constants were calculated for each of the cleavage sites both in the presence and absence of prothrombin. Prothrombin impaired cleavages at both Arg306 and Arg506 and the inhibition correlated with a delayed appearance of proteolytic products on Western blots. Almost complete inhibition was obtained at around 3 microm prothrombin, whereas half-maximal inhibition was obtained at 0.7 microm prothrombin. After cleavage of prothrombin by thrombin, the inhibitory activity was lost. The inhibitory effect of prothrombin on APC-mediated inhibition of FVa was seen both in the presence and absence of protein S, but in particular for the Arg306 sites, it was more pronounced in the presence of protein S. Thus, prothrombin inhibition of APC inactivation of FVa appears to be due to both impaired APC function and decreased APC cofactor function of protein S. In conclusion, FVa, being part of the prothrombinase complex, is protected from APC by both FXa and prothrombin. Release of products of prothrombin activation from the prothrombinase complex would alleviate the protection, allowing APC-mediated inactivation of FVa.     

Proexosite-1 on prothrombin is a factor Va-dependent recognition site for the prothrombinase complex

Although the contribution of basic residues of exosite-1 to the catalytic function of thrombin has been studied extensively, their role in the specificity of prothrombin recognition by factor Xa in the prothrombinase complex (factor Xa, factor Va, phosphatidylcholine/phosphatidylserine vesicles, and Ca2+) has not been examined. In this study, we prepared several mutants of prethrombin-1 (prothrombin lacking Gla and Kringle-1 domains) in which basic residues of this site (Arg35, Lys36, Arg67, Lys70, Arg73, Arg75, and Arg77 in chymotrypsinogen numbering) were individually substituted with a Glu. Following expression in mammalian cells and purification to homogeneity, these mutants were characterized with respect to their ability to function as zymogens for both factor Xa and the prothrombinase complex. Factor Xa by itself exhibited similar catalytic activity toward both the wild type and mutant substrates; however, its activity in the prothrombinase complex toward most of mutants was severely impaired. Further kinetic studies in the presence of Tyr63-sulfated hirudin-(54-65) peptide suggested that although the peptide inhibits the prothrombinase activation of the wild type zymogen with a KD of 0.5-0.7 microm, it is ineffective in inhibiting the activation of mutant zymogens (KD = 2-30 microm). These results suggest that basic residues of proexosite-1 on prothrombin are factor Va-dependent recognition sites for factor Xa in the prothrombinase complex.     

Efficacy of soluble phospholipids in the prothrombinase reaction

The prothrombinase complex is comprised of an enzyme, factor Xa, and a cofactor, factor Va, that each bind peripherally to membranes containing phosphatidylserine (PS) and activate the substrate, prothrombin. The mechanism by which the membrane contributes to enhanced catalytic efficacy of prothrombinase is not precisely known but is generally attributed to some aspect of enzyme and substrate assembly on the multisite surface of the membrane. A recent proposal has suggested a radically different role in which individual phospholipid molecules, either in the membrane or as single soluble molecules, act by an entirely allosteric mechanism that does not involve the multisite feature of the membrane [Zhai, X., Srivastava, A., Drummond, D. C., Daleke, D., and Lentz, B. R. (2002) Biochemistry 41, 5675-5684]. Our study measured prothrombinse activity in the presence of phospholipids such as short-chain phosphatidylserine and lysophosphatidylserine (lyso-PS). Both enhanced prothrombinase activity, and the increase was consistent with the requirement for extended bilayer structure. Even then, prothrombinase activity was low when compared with activity on bilayer membranes of mixed PS and phosphatidylcholine (PC). Lyso-PS approached the activity of PS/PC membranes only when it was mixed with PC bilayers. The results suggest that the two-dimensional membrane bilayer surface is necessary for the support of full prothrombinase activity.     

Nematode anticoagulant protein c2 reveals a site on factor Xa that is important for macromolecular substrate binding to human prothrombinase

The binding of recombinant nematode anticoagulant protein c2 (NAPc2) to either factor X or Xa is a requisite step in the pathway for the potent inhibition of VIIa tissue factor. We have used NAPc2 as a tight binding probe of human Xa to investigate protein substrate recognition by the human prothrombinase complex. NAPc2 binds with high affinity (K(d) approximately 1 nm) to both X and Xa in a way that does not require or occlude the active site of the enzyme. In contrast, NAPc2 is a tight binding, competitive inhibitor of protein substrate cleavage by human Xa incorporated into prothrombinase with saturating concentrations of membranes and Va. By fluorescence binding studies we show that NAPc2 does not interfere with the assembly of human prothrombinase. These are properties expected of an inhibitor that blocks protein substrate recognition by targeting extended macromolecular recognition sites (exosites) on the enzyme complex. A weaker interaction (K(d) = 260-500 nm) observed between NAPc2 and bovine X was restored to a high affinity one in a recombinant chimeric bovine X derivative containing 25 residues from the COOH terminus of the proteinase domain of human X. This region implicated in binding NAPc2 is spatially adjacent to a site previously identified as a potential exosite. Despite the weaker interaction with bovine Xa, NAPc2 was a tight binding competitive inhibitor of protein substrate cleavage by bovine prothrombinase as well. Extended enzymic surfaces elucidated with exosite-directed probes, such as NAPc2, may define a unique region of factor Xa that is modulated following its assembly into prothrombinase and in turn determines the binding specificity of the enzyme complex for its protein substrate.     

Staphylococcus aureus alpha-toxin attack on human platelets promotes assembly of the prothrombinase complex

alpha-Toxin, the major cytolysin of Staphylococcus aureus, promotes blood coagulation by its attack on human platelets (Bhakdi S., Muhly, M., Mannhardt, U., Hugo, F., Klapettek, K., Mueller-Eckhardt, C., and Roka, L. (1988) J. Exp. Med. 168, 527-542). In the present study we demonstrate that toxin attack on gel-filtered human platelets initiates the assembly of prothrombinase complexes at rates up to 10-fold of controls. Treatment of platelets with 0.1 microgram/ml alpha-toxin resulted in generation of 1.4 units of thrombin/10(8) platelets. A similar rate of thrombin generation was noted when platelets were subjected to three cycles of freezing and thawing. However, the alpha-toxin-induced prothrombinase activity was not due to platelet lysis, since less than 1% of total cellular lactate dehydrogenase was released by this alpha-toxin concentration. Two distinct and dissociable processes contributed to enhanced prothrombinase assembly. First, alpha-toxin promoted the exocytotic release of factor V from alpha-granules, which was accompanied by co-secretion of platelet factor 4. This process was calcium-dependent. Second, toxin-treated platelets exhibited an enhanced capacity to bind external factor V(a), a phenomenon that was not linked to Ca2(+)-dependent factor V secretion. Assembly of prothrombinase complexes via these two mechanisms together accounts for the procoagulant action of S. aureus alpha-toxin.