The activation of prothrombin by the prothrombinase complex. The contribution of the substrate-membrane interaction to catalysis

The conversion of prothrombin to thrombin requires the cleavage of two peptide bonds and is catalyzed by the prothrombinase complex composed of factors Xa and Va assembled on a membrane surface. Presteady-state kinetic studies of the effects of membranes on the proteolytic reaction were undertaken using model membranes composed of phosphatidylcholine and phosphatidylserine (PCPS). The concentration of PCPS was varied to alter the concentration of free phospholipid available for substrate binding without influencing the concentration of membrane-assembled prothrombinase. In fluorescence stopped-flow measurements, increasing concentrations of PCPS resulted in an increase in the rate of product formation. Assessment of bond cleavage by sodium dodecyl sulfate-polyacrylamide gel electrophoresis following rapid chemical quench using 125I-prothrombin revealed that the activation reaction proceeded through the ordered cleavage at Arg323-Ile324 followed by cleavage at Art274-Thr275 at all concentrations of PCPS. Increasing the PCPS concentration resulted in a large increase in the Arg323-Ile324 cleavage reaction with a much smaller effect on the subsequent cleavage at Arg274-Thr275, thereby leading to an increase in the extent of accumulation of the intermediate, meizothrombin. Fluorescence stopped-flow and rapid chemical quench measurements were also conducted using prethrombin 2 plus fragment 1.2 or meizothrombin as substrates to assess the influence of PCPS on the individual cleavage reactions. The rate of cleavage at Arg323-Ile324 by prothrombinase was increased approximately 60-fold with increasing PCPS, whereas the cleavage at Arg274-Thr275 was increased by a factor of approximately 5. These differential effects of PCPS on the two cleavage reactions adequately explain changes in the extent of accumulation of meizothrombin during prothrombin activation. Proteolytic removal of the membrane binding fragment 1 domain of the substrates, meizothrombin and prethrombin 2-fragment 1.2, had no effect on the cleavage at Arg274-Thr275 at saturating PCPS but completely eliminated the membrane-dependent rate enhancement for cleavage at Arg323-Ile324. Thus, membrane binding by the substrate is essential for the first cleavage reaction at Arg323-Ile324, which leads to the conversion of prothrombin to meizothrombin. In contrast, the substrate-membrane interaction mediated by the fragment 1 domain has no detectable effect on the second cleavage reaction at Arg274-Thr275 which is required for the conversion of meizothrombin to thrombin.     

Activation-dependent exposure of the inter-EGF sequence Leu83-Leu88 in factor Xa mediates ligand binding to effector cell protease receptor-1

Binding of factor Xa to human umbilical vein endothelial cells (HUVEC) is contributed by effector cell protease receptor-1 (EPR-1). The structural requirements of this recognition were investigated. Factor Xa or catalytically inactive 5-dimethylaminonaphthalene-1sulfonyl (dansyl) Glu-Gly-Arg-(DEGR)-chloromethylketone-factor Xa bound indistinguishably to HUVEC and EPR-1 transfectants, and inhibited equally well the binding of 125I-factor Xa to these cells. Similarly, factor Xa active site inhibitors TAP or NAP5 did not reduce ligand binding to EPR-1. A factor X peptide duplicating the inter-EGF sequence Leu83-Phe84-Thr85-Arg86-Lys87-Leu88- (Gly) inhibited factor V/Va-independent prothrombin activation by HUVEC and blocked binding of 125I-factor Xa to these cells in a dose-dependent manner (IC50 approximately 20-40 microM). In contrast, none of the other factor X peptides tested or a control peptide with the inter-EGF sequence in scrambled order was effective. A recombinant chimeric molecule expressing the factor X sequence Leu83-Leu88 within a factor IX backbone inhibited binding of 125I-factor Xa to HUVEC and EPR-1 transfectants in a dose-dependent fashion, while recombinant factor IX or plasma IXa had no effect. An antibody generated against the factor X peptide 83-88, and designated JC15, inhibited 125I-factor Xa binding to HUVEC. The JC15 antibody bound to factor Xa and the recombinant IX/X83-88 chimera in a concentration dependent manner, while no specific reactivity with factors X or IXa was observed. Furthermore, binding of 125I-factor Xa to immobilized JC15 was inhibited by molar excess of unlabeled factor Xa, but not by comparable concentrations of factors X or IXa. These findings identify the inter-EGF sequence Leu83-Leu88 in factor Xa as a novel recognition site for EPR-1, and suggest its potential role as a protease activation-dependent neo-epitope. This interacting motif may help elucidate the contribution of factor Xa to cellular assembly of coagulation and vascular injury.     

Probable regulation of factor VIIa-tissue factor and prothrombinase by factor Xa-TFPI and TFPI in vivo

Given that factor VIIa-tissue factor (TF) probably initiates coagulation in vivo, this study investigated the relationship between plasma concentrations of factor VIIa and prothrombin fragment 1 + 2 in plasma (the latter as an index of prothrombinase activity in vivo). The relationships between these two parameters and the concentrations of tissue factor pathway inhibitor (TFPI) and factor Xa-TFPI in plasma were also investigated. TFPI inactivates factor Xa in a reaction accelerated by heparin, whereas factor Xa-TFPI inactivates factor VIIa-TF and prothrombinase. Established enzyme-linked immunosorbent assays (ELISAs) were used to quantify TFPI and prothrombin fragment 1 + 2, whereas we developed an ELISA to quantify factor Xa-TFPI using affinity purified rabbit (anti-human TFPI)-IgG and chicken anti-(human factor Xa-TFPI)-IgY as the capture and detector antibodies, respectively. Plasma factor VIIa was quantified using truncated tissue factor. The concentrations of factor VIIa and prothrombin fragment 1 + 2 increased in parallel in the plasmas of up to 145 healthy adults assayed (P = 0.007), as did the concentrations of factor VIIa and TFPI (P = 0.0039), and prothrombin fragment 1 + 2 and TFPI (P = 0.013). In contrast, there was an inverse relationship between the concentrations of free factor Xa-TFPI and factor VIIa (P < 0.0001) and free factor Xa-TFPI and prothrombin fragment 1 + 2 (P = 0.0095). These results are consistent with factor Xa-TFPI regulating factor VIIa-tissue factor and prothrombinase in vivo.     

The gamma-carboxylation recognition site is sufficient to direct vitamin K-dependent carboxylation on an adjacent glutamate-rich region of thrombin in a propeptide-thrombin chimera

The propeptides of the vitamin K-dependent proteins contain a gamma-carboxylation recognition site that is required for gamma-glutamyl carboxylation. To determine whether the propeptide is sufficient to direct carboxylation, two mutant prothrombin species were expressed and characterized with regard to posttranslational gamma-carboxylation. A double point mutant, in which serine substituted for cysteines 17 and 22 disrupted a conserved loop formed by a disulfide bond, was fully carboxylated when expressed in Chinese hamster ovary cells. A propeptide/thrombin chimeric protein, constructed by deleting the Gla, aromatic amino acid stack, and kringle domains of prothrombin, has the signal peptide and propeptide juxtaposed to a glutamate-rich COOH-terminal region of prothrombin, residues 249-530. Of the 8 glutamic acid residues contained within the first 40 residues of the NH2 terminus adjacent to the propeptide, at least seven were fully carboxylated as demonstrated by direct gamma-carboxyglutamic acid analysis of the alkaline hydrolysate and by NH2-terminal sequence analysis. These results indicate that the gamma-carboxylation recognition site within the prothrombin propeptide in a prothrombin propeptide-thrombin chimeric protein is sufficient to direct gamma-carboxylase-catalyzed carboxylation of adjacent glutamic acid residues in a glutamate-rich region of thrombin that is not normally gamma-carboxylated. Furthermore, the disulfide loop in the Gla domain of prothrombin is not required for complete carboxylation.     

Protein C activation and factor Va inactivation on human umbilical vein endothelial cells

The inactivation of factor Va was examined on primary cultures of human umbilical vein endothelial cells (HUVECs), either after addition of activated protein C (APC) or after addition of alpha-thrombin and protein C (PC) zymogen. Factor Va proteolysis was visualized by Western blot analysis using a monoclonal antibody (alpha HVaHC No. 17) to the factor Va heavy chain (HC), and cofactor activity was followed both in a clotting assay using factor V-deficient plasma and by quantitation of prothrombinase function. APC generation was monitored using the substrate 6-(D-VPR)amino-1-naphthalenebutylsulfonamide (D-VPR-ANSNHC4H9), which permits quantitation of APC at 10 pmol/L. Addition of APC (5 nmol/L) to an adherent HUVEC monolayer (3.5 x 10(5) cells per well) resulted in a 75% inactivation of factor Va (20 nmol/L) within 10 minutes, with complete loss of cofactor activity within 2 hours. Measurements of the rate of cleavage at Arg506 and Arg306 in the presence and absence of the HUVEC monolayer indicated that the APC-dependent cleavage of the factor Va HC at Arg506 was accelerated in the presence of HUVECs, while cleavage at Arg306 was dependent on the presence of the HUVEC surface. Factor Va inactivation proceeded with initial cleavage of the factor Va HC at Arg506, generating an M(r) 75,000 species. Further proteolysis at Arg306 generated an M(r) 30,000 product. When protein C (0.5 mumol/L), alpha-thrombin (1 nmol/L), and factor Va (20 nmol/L) were added to HUVECs an APC generation rate of 1.56 +/- 0.11 x 10(-14) mol/min per cell was observed. With APC generated in situ, cleavage at Arg506 on the HUVEC surface is followed by cleavage at Arg306, generating M(r) 75,000 and M(r) 30,000 fragments, respectively. In addition, the appearance of two novel products derived from the factor Va HC are observed when thrombin is present on the HUVEC surface: the HC is processed through limited thrombin proteolysis to generate an M(r) 97,000 fragment, which is further processed by APC to generate an M(r) 43,000 fragment. NH2-terminal sequence analysis of the M(r) 97,000 fragment revealed that the thrombin cleavage occurs in the COOH-terminus of the intact factor Va HC since both the intact HC as well as the M(r) 97,000 fragment have the same sequence. Our data demonstrate that the inactivation of factor Va on the HUVEC surface, initiated either by APC addition or PC activation, follows a mechanism whereby cleavage is observed first at Arg506 followed by a second cleavage at Arg306. The latter cleavage is dependent on the availability of the HUVEC surface. This mechanism of inactivation of factor Va is similar to that observed on synthetic phospholipid vesicles.     

Naturally occurring Arg(-1) to His mutation in human protein C leads to aberrant propeptide processing and secretion of dysfunctional protein C

The dysfunctional protein C from a thrombophilic patient heterozygote for a G1388 to A converting the codon for Arg(-1) to His was purified from plasma and characterized. N-terminal amino acid sequence analysis of the light chain of the protein C demonstrated that the dysfunctional protein C is elongated with one amino acid, namely the mutated His. This finding is compatible with disruption by the mutated His of the original basic propeptidase recognition sequence (Arg(-5)-Ile-Arg-Lys-Arg(-1)), resulting in a shift of the cleavage site to a new position, Lys(-2)-His(-1), which follows an alternative basic amino acid propeptidase recognition sequence (Arg(-5)-Ile-Arg-Lys(-2)). Because the mutation affects the propeptide that directs the gamma-carboxylation converting Glu to Gla residues in the Gla domain, it was investigated whether the mutation impaired this reaction. Gla fragment obtained by cleavage of the dysfunctional protein C light chain with endoproteinase Asp-N was isolated by reverse-phase high-performance liquid chromatography, methylated, and subjected to N-terminal sequence analysis. The methylation step enabled the positive identification of Gla residues as well as the determination of the relative amount of Gla and Glu residues at each of the nine gamma-carboxylation sites of the Gla domain. The analysis showed that all nine potential gamma-carboxylation sites of the dysfunctional protein C were normally carboxylated. This result is compatible with the notion that position -1 is not a part of the recognition element for the gamma-carboxylase. In conclusion, evidence is provided showing that the mutation leads to aberrant propeptide processing and secretion of dysfunctional normally carboxylated protein C extended with the mutated His.     

Proteolysis of factor V by cathepsin G and elastase indicates that cleavage at Arg1545 optimizes cofactor function by facilitating factor Xa binding

The single-chain procofactor factor V is cleaved by thrombin (FVaIIa) at Arg709, Arg1018, and Arg1545 and by a variety of other proteases to generate a cofactor species with various levels of cofactor function. Having demonstrated previously that monocyte-bound forms of cathepsin G and elastase cleave and activate factor V, studies were initiated here using purified proteins to probe factor V structure/function. Electrophoretic, Western blotting, and amino-terminal sequence analyses revealed that cathepsin G cleaves factor V at several sites (Phe1031, Leu1447, Tyr1518, and potentially Tyr696), ultimately generating an amino-terminal 103 kDa heavy chain and a carboxy-terminal 80 kDa light chain (FVaCG). Elastase also cleaves factor V at several sites (Ile708, Ile819, Ile1484, and potentially Thr678), generating a cofactor species, FVaHNE, with an amino-terminal 102 kDa heavy chain and a carboxy-terminal 90 kDa light chain. Incubation of FVaIIa with either cathepsin G or elastase resulted in cleavage within the heavy chain, releasing peptides of approximately 2000 and approximately 3000 Da, respectively, generating FVaIIa/CG and FVaIIa/HNE. The functional activity of each cofactor species was assessed either by clotting assay or by employing a purified prothrombinase assay using saturating amounts of factor Xa. Significant differences in cofactor function were observed between the two assay systems. Whereas FVaIIa, FVaCG, FVaIIa/CG, FVaHNE, and FVaIIa/HNE all had similar cofactor activities in the purified prothrombinase assay, FVaCG and FVaHNE had no cofactor activity in the clotting-based assay, and FVaIIa/CG and FVaIIa/HNE had approximately 30-35% clotting activity relative to FVaIIa. These disparate results led us to examine the binding interactions of these cofactors with the various prothrombinase components. Kinetic analyses indicated that FVaIIa (Kd(app) = 0.096 nM), FVaIIa/CG (Kd(app) = 0.244 nM), and FVaIIa/HNE (Kd(app) = 0.137 nM) bound to membrane-bound factor Xa much more effectively than FVaCG (Kd(app) = 1.46 nM) and FVaHNE (Kd(app) = 0.818 nM). In contrast, studies of the activated protein C (APC)-catalyzed inactivation of each of the factor V(a) species indicated that they were all equivalent substrates for APC with no differences observed in the rate of inactivation or the cleavage mechanism, suggesting that APC interacts with the light chain at a site distinct from factor Xa. The Km values for prothrombin, as well as the kcat values for each of the FV(a) species, were all similar (approximately 0.25 microM and approximately 1900 min-1). In addition, kinetic analyses indicated that whereas FVaCG and FVaHNE exhibited a slightly reduced ability to interact with phospholipid vesicles (approximately 2-3-fold), the remaining FV(a) species assembled equally well on this surface. Collectively, these data indicate that FVaCG and FVaHNE have a diminished capacity to support factor Xa binding; however, cleavage at Arg1545 and removal of the extended B-domain in these cofactors restore near-total factor Xa binding. Thus, cleavage at Arg1545 optimizes cofactor function within prothrombinase by facilitating factor Xa binding to membrane-bound FVa.     

Regions remote from the site of cleavage determine macromolecular substrate recognition by the prothrombinase complex

The proteolytic formation of thrombin is catalyzed by the prothrombinase complex of blood coagulation. The kinetics of prethrombin 2 cleavage was studied to delineate macromolecular substrate structures necessary for recognition at the exosite(s) of prothrombinase. The product, alpha-thrombin, was a linear competitive inhibitor of prethrombin 2 activation without significantly inhibiting peptidyl substrate cleavage by prothrombinase. Prethrombin 2 and alpha-thrombin compete for binding to the exosite without restricting access to the active site of factor Xa within prothrombinase. Inhibition by alpha-thrombin was not altered by saturating concentrations of low molecular weight heparin. Furthermore, proteolytic removal of the fibrinogen recognition site in alpha-thrombin only had a modest effect on its inhibitory properties. Both alpha-thrombin and prethrombin 2 were cleaved with chymotrypsin at Trp148 and separated into component domains. The C-terminal-derived zeta2 fragment retained the ability to selectively inhibit macromolecular substrate cleavage by prothrombinase, while the zeta1 fragment was without effect. As the zeta2 fragment lacks the fibrinogen recognition site, the P1-P3 residues or the intact cleavage site, specific recognition of the macromolecular substrate by the exosite in prothrombinase is achieved through substrate regions, distinct from the fibrinogen recognition or heparin-binding sites, and spatially removed from structures surrounding the scissile bond.     

Role of the activation peptide domain in human factor X activation by the extrinsic Xase complex

The activation of factor X by the extrinsic coagulation system results from the action of an enzyme complex composed of factor VIIa bound to tissue factor on phospholipid membranes in the presence of calcium ions (extrinsic Xase complex). Proteolysis at the Arg52-Ile53 peptide bond in the heavy chain of factor X leads to the formation of the serine protease, factor Xa, and the generation of a heavily glycosylated activation peptide comprising residues 1-52 of the heavy chain. The role of the activation peptide region in mediating substrate recognition and cleavage by the extrinsic Xase complex is unclear. The protease Agkistrodon rhodostoma hydrolase gamma (ARHgamma), from the venom of the Malayan pit viper, was used to selectively cleave human factor X in the activation peptide region. Three cleavage sites were found within this region and gave products designated Xdes1-34, Xdes1-43, and Xdes1-49. The products were purified to yield Xdes 1-49 and a mixture of Xdes 1-34 and Xdes 1-43. Reversed phase high pressure liquid chromatography analysis indicated that the cleaved portion of the activation peptide was likely removed during purification. All cleaved species were inactive and could be completely activated to factor Xa by the extrinsic Xase complex or by a purified activator from Russell's viper venom. Steady state kinetic studies using tissue factor reconstituted into membranes yielded essentially equivalent kinetic constants for the activation of intact factor X and the cleaved derivatives under a wide range of conditions. Since Xdes 1-49 lacks all but three residues of the activation peptide and is devoid of the carbohydrate present in this region, the data suggest that the specific recognition of human factor X by the extrinsic Xase complex is not achieved through specific interactions with residues 1-49 of the activation peptide or with carbohydrate structures attached to these residues.     

The interaction of prothrombin with phospholipid membranes is independent of either kringle domain

Prothrombin contains two kringle domains, a structural motif common to other plasma proteins involved in hemostasis and fibrinolysis. To determine the role of the kringle domains of prothrombin, we prepared three recombinant human prothrombin forms lacking the first kringle domain (residues 63-144; PT/delta K1), the second kringle domain (residues 144-249; PT/delta K2), or both kringle domains (63-249; PT/delta K1,2). The isolated prothrombin proteins were greater than 95% pure by SDS-polyacrylamide gel electrophoresis and were well carboxylated. PT/delta K1 displayed 50% of the specific coagulant activity of plasma prothrombin, PT/delta K2 had 10% of the specific coagulant activity, and PT/delta K1,2 was inactive. Polyclonal antibodies directed against the Ca(II)-specific conformer of prothrombin bound PT/delta K1 and PT/delta K2 with the same affinity as prothrombin, indicating that the Ca(II)-induced conformational transition does not involve sites on the prothrombin kringle domains. Gel filtration studies demonstrated that radiolabeled plasma prothrombin and all of the prothrombin kringle deletion mutants bound to phospholipid vesicles in the presence of Ca(II) but not in the presence of Mg(II) or EDTA. Relative dissociation constants of 1.10 +/- 0.75 and 0.49 +/- 0.18 microM were obtained by quasielastic light scattering for the interaction of phospholipid vesicles with plasma prothrombin and PT/delta K1, respectively. These data indicate that neither the first nor the second kringle domain contain unique sites for the interaction of prothrombin with phospholipid vesicles and are not required for prothrombin-phospholipid binding.     

Selective inhibition of factor Xa in the prothrombinase complex by the carboxyl-terminal domain of antistasin

Studies of antistasin, a potent factor Xa inhibitor with anticoagulant properties, were performed wherein the properties of the full-length antistasin polypeptide (ATS-119) were compared with the properties of forms of antistasin truncated at residue 116 (ATS-116) and residue 112 (ATS-112). ATS-119 was 40-fold more potent than ATS-112 in prolonging the activated partial thromboplastin time (APTT), whereas ATS-119 inhibited factor Xa 2.2-fold less avidly and about 5-fold more slowly than did ATS-112. The decreased reactivity of ATS-119 suggests that the carboxyl-terminal domain of ATS-119 stabilizes an ATS conformation with a reduced reactivity toward factor Xa. The observation that calcium ion increases the reactivity of ATS-119 but not that of ATS-112 suggests that calcium ion may disrupt interactions involving the carboxyl terminus of ATS-119. Interestingly, ATS-119 inhibited factor Xa in the prothrombinase complex 2-6-fold more potently and 2-3-fold faster than ATS-112. These differences in affinity and reactivity might well account for the greater effectiveness of ATS-119 in prolonging the APTT and suggest that the carboxyl-terminal domain of ATS-119 disrupts interactions involving phospholipid, factor Va, and prothrombin in the prothrombinase complex. The peptide RPKRKLIPRLS, corresponding to the carboxyl domain of ATS-119 prolonged the APTT and inhibited prothrombinase-catalyzed processing of prothrombin, but it failed to inhibit the catalytic activity of isolated factor Xa. Thus, this novel inhibitor appears to exert its inhibitory effects at a site removed from the active site of factor Xa.     

Structure/function analyses of recombinant variants of human factor Xa: factor Xa incorporation into prothrombinase on the thrombin-activated platelet surface is not mimicked by synthetic phospholipid vesicles

This report describes the expression, purification, and characterization of a series of recombinant factor Xa variants bearing aspartate substitutions for each of the glutamate residues which normally undergo gamma-carboxylation. Factor X was expressed in human embryonic kidney cells and purified from conditioned media by immunoaffinity and hydroxylapatite chromatography. Factor X was activated with Russell's viper venom factor X activator, and single-chain unactivated factor X was removed from activated factor X by size-exclusion chromatography. Recombinant wild-type factor Xa had normal activity in a clotting assay, and mutants with aspartate substitutions for glas residues 16, 26, and 29 had no detectable clotting activity. In purified component assays, these gla variants had essentially no detectable activity in the prothrombinase complex assembled on synthetic phospholipid vesicles but had significant activity when the prothrombinase was assembled on thrombin-activated platelets. In addition, the gla 32 variant had normal activity in the platelet prothrombinase but diminished activity in prothrombinase assembled on synthetic PSPC vesicles. These differences were not accounted for by the total phospholipid composition of the thrombin-activated platelet membrane. We have produced fully active recombinant human factor Xa and demonstrated that gla residues 16, 26, and 29 are critical for normal activity of factor Xa. More importantly, this study provides an extensive characterization of macromolecular enzyme complex formation with gla variants of a vitamin K-dependent coagulation protein and provides evidence that prothrombinase complex assembly on thrombin-activated platelets is not equivalent to assembly on synthetic phospholipid vesicles. The data suggest that thrombin-activated platelets possess some element(s) (other than 30% phosphatidyl serine or factor Va), presumably either protein or phospholipid, that serves as a component of the factor Xa binding site.     

The structural basis of function of the TF. VIIa complex in the cellular initiation of coagulation

Cell surface tissue factor (TF), the major in vivo initiator of coagulation, activates coagulation by binding and allosteric activation of the serine protease factor. VIIa (VIIa). A graphic scheme to account for function of this initial bimolecular activation complex has emerged from the integration of structural with functional analyses. The VIIa light chain, specifically the Gla and EGF-1 domains, form extended hydrophobic contacts with TF which account for most of the free energy of binding. These contacts tether VIIa and facilitate interactions of the protease domain with TF necessary for induction of protease function. Several contact residues in the VIIa protease domain-TF interface are involved in the activation of VIIa by complex allosteric effects. Macromolecular substrate zymogens interact with both the VIIa protease domain and the carboxyl-terminal module of TF. Docking of the VIIa Gla-domain to the latter region of TF appears to contribute to substrate assembly. The current data suggest an extended embrace between TF and VIIa to form the bimolecular enzyme TF.VIIa.     

Prediction of solution structures of the Ca2+-bound gamma-carboxyglutamic acid domains of protein S and homolog growth arrest specific protein 6: use of the particle mesh Ewald method

The solution structures of the N-terminal domains of protein S, a plasma vitamin K-dependent glycoprotein, and its homolog growth arrest specific protein 6 (Gas6) were predicted by molecular dynamics computer simulations. The initial structures were based on the x-ray crystallographic structure of the corresponding region of bovine prothrombin fragment 1. The subsequent molecular dynamics trajectories were calculated using the second-generation AMBER force field. The long-range electrostatic forces were evaluated by the particle mesh Ewald method. The structures that stabilized over a 400-ps time interval were compared with the corresponding region of the simulated solution structure of bovine prothrombin fragment 1. Structural properties of the gamma-carboxyglutamic acid (Gla) domains obtained from simulations and calcium binding were found to be conserved for all three proteins. Analysis of the predicted solution structure of the Gla domain of Gas6 suggests that this domain should bind with negatively charged phospholipid surfaces analogous to bovine prothrombin fragment 1 and protein S.     

Structure of human des(1-45) factor Xa at 2.2 A resolution

The structure of a large molecular fragment of factor Xa that lacks only a Gla (gamma-carboxyglutamic acid) domain (N-terminal 45 residues) has been solved by X-ray crystallography and refined at 2.2 A resolution to a crystallographic R-value of 0.168. The fragment identity was clearly established by automated Edman degradation. X-ray structure analysis confirmed the biochemical characterization and also revealed that the N-terminal epidermal growth factor (EGF)-like domain is flexibly disordered in crystals. The second EGF module, however, is positionally ordered making contacts with the catalytic domain. The overall folding of the catalytic domain is similar to that of alpha-thrombin, excluding the insertion loops of the latter with respect to simpler serine proteinases. The C-terminal arginine of the A-chain interacts in a substrate-like manner with the S1 specificity site of the active site of a crystallographically neighboring molecule. Based on this interaction and the structure of D-PheProArg methylene-thrombin, a model of the commonly used dansylGluGlyArg methylene inhibitor-factor Xa interaction is proposed. The region of factor Xa corresponding to the fibrinogen recognition site of thrombin has a reversed electrical polarity to the anion binding fibrinogen recognition site of thrombin but possesses a site similar to the Ca2+ binding site of trypsin and other serine proteinases. The structure of the C-terminal EGF domain of factor Xa is the first to be determined crystallographically. Its folding has been comprehensively compared with similar domains determined by NMR. Although the A-chain makes 44 contacts at less than 3.5 A with the catalytic domain, only 16 involve the EGF module. In addition, the A-chain makes 30 intermolecular contacts with a neighboring catalytic domain.     

Effects of cardiopulmonary bypass and deep hypothermic circulatory arrest on the thyroid axis during and after repair of congenital heart defects: preservation by deep hypothermia?

YM-60828 was found to potently inhibit human factor Xa following oral administration. YM-60828 showed high affinity for factor Xa (Ki = 1.3 nM), but did not affect thrombin (Ki > 100 microM). YM-60828 doubled factor Xa clotting time, prothrombin time (PT) and activated partial thromboplastin time (APTT) at 0.10, 0.21, 0.24 microM, respectively. Importantly, it did not prolong thrombin time at 100 microM. YM-60828 also inhibited factor Xa in the prothrombinase complex with an IC50 value of 7.7 nM. In addition to its anticoagulant activity, YM-60828 inhibited platelet aggregation induced by various agonists (IC50 = 3 to 23 microM). Squirrel monkeys were used to study the ex vivo anticoagulant activity and pharmacokinetic properties of YM-60828. One hour after oral administration at 3 mg/kg, YM-60828 strongly prolonged PT and APTT by 4.8- and 1.9-fold, respectively, and plasma concentration reached 788 +/- 167 ng/ml. Bioavailability was calculated to be 20.3%. These results strongly suggest that YM-60828 will be a valuable orally active and potent anticoagulant agent showing potential antithrombotic activity.     

The presence of protein-bound gamma-carboxyglutamic acid in calcium-containing renal calculi

The amino acid gamma-carboxyglutamic acid (Gla) is found in four blood-clotting proteins, in a bone protein, in kidney protein, and in the protein present in various ectopic calcifications. This paper reports the presence of Gla in the EDTA-soluble, nondialyzable proteins of calcium-containing renal calculi including calcium oxalate, hydroxyapatite, and mixed stores of apatite and struvite (MgNH4PO4). Calculi composed of pure struvite and those composed of only uric acid or cystine do not contain Gla. From calcium oxalate and hydroxyapatite stontes, a protein of about 17,000 daltons was obtained which contained about 40 residues of Gla per 1,000 amino acids. The amino acid composition of this protein had no apparent relationship to the Gla-containing bone protein or to the similarly-sized F1 fragment of prothrombin which contains about 64 residues of Gla per 1,000 amino acid residues. The Gla-rich protein in calcium-containing renal stones thus may be a different Gla-containing protein. These data as well as other studies demonstrating the presence of Gla in pathologically calcified tissues not normally containing Gla suggest that the Gla-containing proteins may be of considerable pathophysiological significance.     

The contributions of individual gamma-carboxyglutamic acid residues in the calcium-dependent binding of recombinant human protein C to acidic phospholipid vesicles

The dependence on the integrity of the human protein C (PC) gamma-carboxyglutamic acid (Gla) domain on its Ca(2+)-dependent binding properties to acidic phospholipid (PL) vesicles has been examined by analysis of these interactions with recombinant (r)-PC Gla domain muteins. The concentration of Ca2+ that results in 50% saturation (C50-Ca) of PL with wild-type (wt) r-PC was not altered by more than 2-fold for the following r-variants of PC, viz. [Gla6-->Asp]r-PC, [Gla14-->Asp]r-PC, [Gla19-->Asp]r-PC, [Gla25-->Asp]r-PC, [Gla29-->Asp]r-PC, and [Gln32-->Gla]r-PC. The C50-Ca was substantially higher than that of wtr-PC for [Gla7-->Asp]r-PC (8.2 mM), [Arg15-->Leu]r-PC (4.5 mM), [Gla16-->Asp]r-PC (> 10 mM), [Gla20-->Asp]r-PC (> 10 mM), and [Gla26-->Asp]r-PC (> 10 mM), indicating that the Ca(2+)-induced conformations of these latter variants interacted poorly with these acidic PL vesicles. Titration of the PL vesicles with wtr-PC at a constant concentration of 20 mM Ca2+ leads to calculation of a concentration of PC that results in 50% saturation of the PL (C50-PC) of 0.38 microM. Essentially this same value was determined for the r-mutants, [Gla7-->Asp]r-PC and [Gln32-->Gla]r-PC. An approximate 2-fold lower C50-PC was obtained for [Gla14-->Asp]r-PC (0.14 microM), [Gla25-->Asp]r-PC (0.16 microM), and [Gla29-->Asp]r-PC (0.15 microM). Somewhat higher C50-PC values were found for [Gla6-->Asp]r-PC (1.2 microM), [Arg15-->Leu]r-PC (1.2 microM), [Gla16-->Asp]r-PC (1.2 microM), [Gla19-->Asp]r-PC (1.8 microM), [Gla20-->Asp]r-PC (1.1 microM), and [Gla26-->Asp]r-PC (1.6 microM). The results of this investigation, in conjunction with other structural and functional studies with these mutants, and the x-ray crystallographic structure of the prothrombin Gla domain-Ca2+ complex, show that Gla16 and Gla26 are the most indispensable Gla residues for maintenance of the functional Ca(2+)-dependent structure of the Gla domain of PC, whereas Gla14 is the least critical Gla residue in this regard. Of the non-Gla residues investigated, Arg15 is of great importance for maintenance of a functional Ca(2+)-dependent structure of PC, and insertion of a Gla residue at position 32, a situation that exists in the cases of some other proteins of this type, does not significantly alter these characteristics of r-PC.     

Molecular dissection of effector cell protease receptor-1 recognition of factor Xa. Assignment of critical residues involved in antibody reactivity and ligand binding

Receptor-mediated assembly of blood proteases on vascular cells maintains the hemostatic balance and initiates intracellular signal transduction. Effector cell protease receptor-1 (EPR-1) is an approximately 62-kDa vascular cell membrane receptor for the clotting protease factor Xa, participating in thrombin formation and lymphocyte activation. Here, recombinant EPR-1 fragments were engineered in the frame of intercellular adhesion molecule-1, transfected in mammalian cells, and analyzed for antibody recognition and ligand binding. Chimeric transfectants containing the EPR-1 sequence Met1-Arg60 bound the immunosuppressive anti-EPR-1 monoclonal antibody (mAb) 2E1. In contrast, transfected cells expressing the EPR-1 sequence Pro120-Ala154 were recognized by the functionally inhibitory anti-EPR-1 mAbs 9D4 and B6, bound 125I-factor Xa in a reaction quantitatively indistinguishable from that of wild-type EPR-1 transfectants, and promoted factor Xa concentration-dependent prothrombin activation in the absence of exogenous factor V/Va. Chimeric transfectants expressing the COOH terminus end of the EPR-1 extracellular domain (Ala157-Glu221) did not bind anti-EPR-1 mAbs and did not associate with factor Xa. Mutagenesis of Asn131 or Lys133 in the EPR-1 ligand recognition domain abolished factor Xa binding by 80 +/- 5.5 and 96 +/- 4%, respectively, while mutation of Lys126, Gly128, Asn129, and Asn134 was without effect. A synthetic peptide duplicating the EPR-1 sequence S123PGKPGNQNSKNEPP137 dose dependently inhibited factor V/Va-independent thrombin generation of resting endothelium (IC50 approximately 1 microM), while the adjacent EPR-1 sequence P136PKK-RERERSSHCYP150 was ineffective. These findings demonstrate that EPR-1 contains two spatially distinct functional domains implicated in lymphocyte activation (Met1-Arg60) or factor Xa binding and prothrombin activation (Pro120-Ala154). These interacting sequences may provide a novel potential target for inhibition of factor Xa-dependent vascular cell responses.     

Genetic analysis and functional characterization of prothrombins Corpus Christi (Arg382-Cys), Dhahran (Arg271-His), and hypoprothrombinemia

The structural abnormalities and functional characteristics of dysfunctional prothrombin variants in two new kindreds have been determined. Prothrombin Corpus Christi (family 1) was purified and found to have markedly reduced fibrinogen clotting activity, yet normal amidolytic and near-normal platelet aggregating activity. A transition (C to T) at nucleotide position 8885, present in the heterozygous form in affected family members, resulted in the substitution of Cys for Arg 382. This substitution results in the loss of a positive charge within the fibrinogen-binding exosite of thrombin, thus accounting for the observed functional defect. A heterozygous C to T transition was also present at position 19994 in other family members with a hypoprothrombinemic phenotype. This mutation results in the replacement of Gln 541 (CAA) by a premature stop codon (TAA). Prothrombin Dhahran (family 2) was found to have markedly reduced fibrinogen clotting activity, but normal amidolytic activity. Affected family members were found to have a G to A transition at nucleotide position 7312 resulting in the substitution of His for Arg 271. This substitution results in the abolition of a factor Xa cleavage site, yielding meizothrombin rather than thrombin, on activation of prothrombin Dhahran by factor Xa. All but one of the above mutations occur at CpG dinucleotides, thus further supporting the observation of a high incidence of CpG transitions in hereditary dysprothrombinemia. The significant bleeding tendencies of individuals homozygous for prothrombin Dhahran (prothrombin clotting activity 5% to 7%) contrast sharply with the absence of significant chronic bleeding in the proband expressing prothrombin Corpus Christi (prothrombin clotting activity 2%). Our findings underscore the capacity of thrombin to contribute to clinical hemostasis by mechanisms other than its fibrinogen clotting activity.