Search PubMed⌕ Search

Biomedical subjects

J Rosing

Publications and source records attributed to J Rosing.

At least 55 records · Page 3Linked to original sources
PubMed1996

Factor V enhances the cofactor function of protein S in the APC-mediated inactivation of factor VIII: influence of the factor VR506Q mutation.

Factor V and protein S are cofactors of activated protein C (APC) which accelerate APC-mediated factor VIII inactivation. The effects of factor V and protein S were quantitated in a reaction system in which plasma factor VIII was inactivated by APC and the loss of factor VIII activity was monitored in a factor X-activating system in which a chromogenic substrate was used to probe factor Xa formation. Factor V increased the rate of APC-mediated factor VIII inactivation in a dose-dependent manner in representative plasma samples with protein S or factor V deficiency, abnormal factor V (heterozygous or homozygous for factor VR506Q), or a combination of heterozygous protein S deficiency and heterozygous factor VR506Q. This effect was much less pronounced in the plasma samples with a decreased protein S level, but the impaired response in these plasmas was corrected by addition of protein S, indicating that both factor V and protein S are required for optimal inactivation of factor VIII by APC. The effects of factor V and protein S were also studied in a reaction system with purified proteins. APC-catalysed factor VIII inactivation was enhanced 3.7-fold in the presence of 1.1 nM factor V and 1.5-fold in the presence of 2.4 nM protein S. When both 1.1 nM factor V and 2.4 nM protein were present the rate enhancement was 11-fold. Factor V is a more potent cofactor than protein S, as can be concluded from the fact that 0.04 nM factor V gave the same stimulation as 2.4 nM protein S. Protein S lost its cofactor function after complexation with C4b binding protein, which indicates that it is free protein S that acts as a cofactor. To investigate the effect of the R506Q mutation in factor V on APC-mediated factor VIII inactivation, factor V was purified from the plasma of patients homozygous for factor VR506Q. In the absence of protein S, factor VR506Q did not enhance factor VIII inactivation by APC, but in the presence of 2.4 nM protein S a slight enhancement was observed. The APC cofactor activity of factor V was lost when factor V was activated with thrombin or with the factor V activator from Russell's viper venom. These data indicate that optimal inactivation of factor VIII by APC requires the presence of an intact factor V molecule and free protein S.

Arginine↗
PubMed1996

A prothrombinase-based assay for detection of resistance to activated protein C.

In this paper we present a new method for the detection of resistance to activated protein C (APC) that is based on direct measurement of the effect of APC on the cofactor activity of plasma factor Va. The factor V present in a diluted plasma sample was activated with thrombin and its sensitivity towards APC was subsequently determined by incubation with phospholipids and APC. The loss of factor Va cofactor activity was quantified in a prothrombinase system containing purified prothrombin, factor Xa and phospholipid vesicles and using a chromogenic assay for quantitation of thrombin formation. The reaction conditions were optimized in order to distinguish normal, heterozygous and homozygous APC-resistant plasmas. Maximal differences in the response of these plasmas towards APC were observed when factor Va was inactivated by APC in the absence of protein S and when the cofactor activity of factor Va was determined at a low factor Xa concentration (0.3 nM). Addition of 0.2 nM APC and 20 microM phospholipid vesicles to a 1000-fold diluted sample of thrombin-activated normal plasma resulted in loss of more than 85% of the cofactor activity factor Va within 6 min. Under the same conditions, APC inactivated approximately 60% and approximately 20% of the factor Va present in plasma samples from APC-resistant individuals that were heterozygous or homozygous for the mutation Arg506-->Gln in factor V, respectively. Discrimination between the plasma samples from normal and heterozygous and homozygous APC-resistant individuals was facilitated by introduction of the so-called APC-sensitivity ratio (APC-sr). The APC-sr was defined as the ratio of the factor Va cofactor activities determined in thrombin-activated plasma samples after 6 min incubation with or without 0.2 nM APC and was multiplied by 100 to obtain integers (APC-sr = ¿factor Va+APC square root of factor Va-APC¿ x 100). Clear differences were observed between the APC-sr of plasmas from normal healthy volunteers (APC-sr: 8-20, n = 33) and from individuals that were heterozygous (APC-sr: 35-50, n = 17) or homozygous APC resistant (APC-sr: 82-88, n = 7). There was no mutual overlap between the APC-sr of normal plasmas and plasmas from heterozygous or homozygous APC resistant individuals (p < 0.0001). In all cases our test gave the same result at the DNA-based assay. Since the test is performed on a highly diluted plasma sample there is no interference by conditions that affect APC resistance tests that are based on clotting time determinations (e.g. coagulation factor deficiencies, oral anticoagulation, heparin treatment, the presence of lupus anticoagulants, pregnancy or the use of oral contraceptives). Furthermore, we show that part of the factor Va assay can be performed on an autoanalyzer which increases the number of plasma samples that can be handled simultaneously.

Biological Assay↗
PubMed1995

Effects of protein S and factor Xa on peptide bond cleavages during inactivation of factor Va and factor VaR506Q by activated protein C.

Inactivation of membrane-bound factor Va by activated protein C (APC) proceeds via a biphasic reaction that consists of a rapid and a slow phase, which are associated with cleavages at Arg506 and Arg306 of the heavy chain of factor Va, respectively. We have investigated the effects of protein S and factor Xa on APC-catalyzed factor Va inactivation. Protein S accelerates factor Va inactivation by selectively promoting the slow cleavage at Arg306 (20-fold). Factor Xa protects factor Va from inactivation by APC by selectively blocking cleavage at Arg506. Inactivation of factor VaR506Q, which was isolated from the plasma of a homozygous APC-resistant patient and which lacks the Arg506 cleavage site, was also stimulated by protein S but was not affected by factor Xa. This confirms that the target sites of protein S and factor Xa involve Arg306 and Arg506, respectively. Factor Xa completely blocked APC-catalyzed cleavage at Arg506 in normal factor Va (1 nM) with a half-maximal effect (K1/2Xa) at 1.9 nM factor Xa. Expression of cofactor activity of factor Va in prothrombin activation required much lower factor Xa concentrations (K1/2Xa = 0.08 nM). When the ability of factor Xa to protect factor Va from inactivation by APC was determined at low factor Va concentrations during prothrombin activation much lower amounts of factor Xa were required (K1/2Xa = 0.03 nM). This indicates 1) that factor Va is optimally protected from inactivation by APC by incorporation into the prothrombinase complex during ongoing prothrombin activation, and 2) that the formation of a catalytically active prothrombinase complex and protection of factor Va from inactivation by APC likely involves the same interaction of factor Xa with factor Va. In accordance with the proposed mechanisms of action of protein S and factor Xa, we observed that the large differences between the rates of APC-catalyzed inactivation of normal factor Va and factor VaR506Q were almost annihilated in the presence of factor Xa and protein S. This observation may explain why, in the absence of other risk factors, APC resistance only results in a weak prothrombotic condition.

Amino Acid Sequence↗
PubMed1995

Peptide bond cleavages and loss of functional activity during inactivation of factor Va and factor VaR506Q by activated protein C.

Factor V was purified from the plasma of an activated protein C (APC)-resistant patient who is homozygous for the mutation Arg506-->Gln (factor VR506Q). Factor VR506Q was converted by thrombin into factor Va which was further purified yielding a factor Va preparation that had the same cofactor activity in prothrombin activation as normal factor Va. Inactivation of low concentrations of normal factor Va (< 5 nM) by 0.15 nM APC in the presence of phospholipid vesicles proceeded via a biphasic reaction that consisted of a rapid phase (k = 4.3 x 10(7) M-1s-1), yielding a reaction intermediate with reduced cofactor activity that was fully inactivated during the subsequent slow phase (k = 2.3 x 10(6) M-1s-1). Inactivation of factor VaR506Q proceeded via a monophasic reaction (k = 1.7 x 10(6) M-1s-1). Immunoblot analysis showed that APC-catalyzed inactivation of factor Va occurred via peptide bond cleavages in the heavy chain. The rapid phase of inactivation of normal factor Va was associated with cleavage at Arg506 and full inactivation of factor Va required subsequent cleavage at Arg306. The slow monophasic inactivation of factor VaR506Q correlated with cleavage at Arg306. Cleavage at Arg506 in normal factor Va resulted in accumulation of a reaction intermediate that exhibited 40% cofactor activity in prothrombin activation mixtures that contained a high factor Xa concentration (5 nM). Compared with native factor Va, the reaction intermediate retained virtually no cofactor activity at low factor Xa concentrations (0.3 nM). This demonstrates that factor Va that is cleaved at Arg506 is impaired in its ability to interact with factor Xa. Michaelis-Menten kinetic analysis showed that cleavage at Arg506 in membrane-bound factor Va was characterized by a low Km for factor Va (20 nM) and kcat = 0.96 s-1. For cleavage at Arg306 in factor VaR506Q the kinetic parameters were Km = 196 nM and kcat = 0.37 s-1. This means that differences between APC-catalyzed inactivation of factors Va and VaR506Q become much less pronounced at high factor Va concentrations. When factor VaR506Q was inactivated by APC in the absence of phospholipids, cleavage at Arg679 of the heavy chain also contributed to factor Va inactivation. Comparison of rate constants for APC-catalyzed cleavage at Arg306, Arg506, and Arg679 in the absence and presence of phospholipids indicated that phospholipids accelerated these cleavages to a different extent.(ABSTRACT TRUNCATED AT 400 WORDS)

Arginine↗
PubMed1994

Functional properties of human factor Va lacking the Asp683-Arg709 domain of the heavy chain.

A protease purified from the venom of the elapid snake Naja naja oxiana converts human blood coagulation factor Va into a molecule (factor VaNO) with greatly reduced cofactor activity. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that the venom protease cleaved a small peptide from the heavy chain of factor Va and reduced the apparent M(r) from 105,000 to 101,000. This peptide was isolated by high performance liquid chromatography on a reversed-phase column. Amino acid sequence analysis of the peptide indicated that the venom enzyme cleaved the peptide bond between His682 and Asp683, thus removing 27 amino acids from the carboxyl-terminal part of the heavy chain. The cofactor activities of factors Va and VaNO were compared by measuring their abilities to support factor Xa-catalyzed prothrombin activation in the presence of phospholipids and calcium ions. Both factor Va molecules stimulated the binding of factor Xa to negatively charged phospholipids. However, the amounts of factor Va required for half-maximal incorporation of factor Xa into the membrane-bound factor Xa-Va complex were much lower for native factor Va (0.25 nM) than for factor VaNO (2.01 nM). At saturating concentrations of factor Va or factor VaNO the kcat values for prothrombin activation were 114 s-1 for factor Va and 128 s-1 for factor VaNO. The Km values for prothrombin determined under these conditions were 0.24 and 0.83 microM for prothrombinase complexes with native factor Va and factor VaNO, respectively. Direct binding studies revealed that factors Va and VaNO bind with equal affinity to phospholipids. These data indicate that factor VaNO is impaired in its ability to interact with factor Xa and prothrombin. Together with the structural data this implies that the carboxyl-terminal Asp683-Arg709 domain of the heavy chain is required for optimal interaction of factor Va with factor Xa and prothrombin.

Amino Acid Sequence↗
PubMed1994

Activation of human factor V by meizothrombin.

A recombinant human prothrombin was prepared in which Arg155 was replaced by Ala. The recombinant prothrombin was converted into a meizothrombin derivative (R155A meizothrombin) that was resistant to autocatalytic removal of the fragment 1 domain. R155A meizothrombin appeared to be a potent factor V activator in reaction mixtures that contained negatively charged phospholipid vesicles. Factor V activation by R155A meizothrombin was characterized by second-order rate constants of 0.06 x 10(6) M-1 S-1 in the absence of phospholipid and 18 x 10(6) M-1 S-1 in the presence of 60 microM phospholipid vesicles composed of a 10:90 mol/mol mixture of phosphatidylserine (PS) and phosphatidylcholine (PC). The rate constant for thrombin-catalyzed activation of factor V was hardly affected by the presence of phospholipid vesicles and was 4.0 x 10(6) M-1 S-1. The initial rate of activation of 3 nM factor V by R155A meizothrombin was a function of the concentration of PS/PC vesicles present in the reaction mixture, and the calculated rate constant reached a plateau value at > or = 50 microM PS/PC. Gel electrophoretic analysis of factor V activation showed that R155A meizothrombin and thrombin cleaved the susceptible peptide bonds in factor V at different rates. However, both activators finally generated a factor Va molecule composed of a heavy chain with an M(r) of 104,000 and a light chain doublet with M(r) values of 74,000 and 71,000. Since meizothrombin is one of the major reaction products formed during the initial phase of prothrombin activation, these findings are indicative of a significant contribution of meizothrombin to in vivo factor V activation.

Base Sequence↗
PubMed1994

Protein S binds to and inhibits factor Xa.

Although human protein S binds to human factor Va and inhibits prothrombinase activity, this inhibition is not totally dependent on factor Va. Hence, we investigated possible interaction of protein S with human factor Xa. Factor Xa, diisopropylphospho-factor Xa and their biotin derivatives ligand blotted specifically to protein S and protein S ligand blotted specifically to factor X and factor Xa. Biotinylated factors X and Xa bound to immobilized protein S and, reciprocally, protein S bound to immobilized factor Xa with a Kd of approximately 19 nM. In fluid phase, protein S bound to factor Xa with a Kd of approximately 18 nM. Protein S at 33 nM reversibly inhibited 50% of factor Xa amidolytic activity. Protein S inhibition of prothrombin conversion to thrombin by factor Xa was phospholipid-independent and was 1.6 times stimulated by Ca2+ ions. Inhibition of prothrombinase activity by protein S was 2.3-fold more potent in the presence of factor Va, with 50% inhibition at approximately 8 nM protein S. Protein S prolonged the factor Xa one-stage clotting time of protein S-depleted plasma in a dose-dependent manner. These data demonstrate mechanisms of anticoagulant action for protein S that are independent of activated protein C and that involve direct binding to factors Xa and Va and direct inhibition of factor Xa.

Biotin↗
PubMed1994

Prothrombin activation on dioleoylphosphatidylcholine membranes.

Factor-Xa-catalyzed prothrombin activation is greatly accelerated by negatively charged phospholipids plus calcium ions. In 1990, we reported that neutral phosphatidylcholine membranes also stimulated prothrombin activation [Gerads, I., Govers-Riemslag, J.W.P., Tans, G., Zwaal, R. F. A. & Rosing, J. (1990) Biochemistry 29, 7967-7974]. In the present study, we have performed a detailed analysis of the prothrombin-converting activity of phosphatidylcholine membranes. Stimulation of prothrombin activation by phosphatidylcholine vesicles was particularly observed (a) with phosphatidylcholine molecules that contained unsaturated hydrocarbon side chains, (b) in the presence of factor Va, (c) at low ionic strength and (d) when Ca2+ were present in the reaction medium. It is unlikely that the prothrombinase activity of phosphatidylcholine preparations was due to contaminating anionic phospholipids. This is concluded from the fact that thin-layer chromatographic analysis showed that dioleoylphosphatidylcholine [(Ole)2GroPCho] contained less than 0.1 mol/100 mol anionic phospholipid, and that incorporation of such amounts of anionic lipids in (Ole)2-GroPCho membranes hardly increased their prothrombin-converting activity. At low ionic strength and in the presence of factor Va and Ca2+ (Ole)2GroPCho membranes accelerated prothrombin activation about 100-fold. At ionic strength (I) 0.06, prothrombin activation on 100 microM (Ole)2-GroPCho was characterized by a Km for prothrombin of 2 microM, a Vmax of 3020 IIa min-1.Xa-1 and a Kd for factor XaVa complex formation at the membrane surface of 7.5 nM. Prothrombin activation on (Ole)2GroPCho membranes was drastically reduced when the ionic strength was increased. The inhibition at high ionic strength could be explained by an effect on the Kd for XaVa complex formation which increased from 7.5 nM at I = 0.06 to 100 nM at I = 0.22. Prothrombin activation on (Ole)2GroPCho required Ca2+ and was dependent on the presence of gamma-carboxyglutamic acid domains in prothrombin and factor Xa. This indicates that similar interactions may account for the assembly of prothrombinase complexes on phosphatidylcholine and an anionic lipid-containing membranes.

1-Carboxyglutamic Acid↗
PubMed1993

Characterization of two forms of human factor Va with different cofactor activities.

Factor Va is an essential cofactor in factor Xa-catalyzed prothrombin activation. Purified human factor Va appears to consist of a heavy chain (M(r) approximately 105,000) and a light chain doublet with M(r) approximately 74,000 and approximately 71,000. We separated factor Va by chromatography on a Mono-S column into two fractions, designated factors Va1 and Va2. Factor Va1 contains the light chain with M(r) approximately 74,000, and factor Va2 exclusively contains the light chain with M(r) approximately 71,000. The two forms of factor Va express different cofactor activities when prothrombin is activated at low phospholipid concentrations or on membranes containing low amounts of phosphatidylserine in phosphatidylcholine. Compared with factor Va2, much higher amounts of factor Va1 are required for factor Xa. Va complex formation at the membrane surface. Once incorporated into the prothrombinase complex, factors Va1 and Va2 are equally active in prothrombin activation. This indicates that the two forms of factor Va do not differ in their ability to promote the catalytic activity of factor Xa or to interact with prothrombin. Direct binding experiments show that the different cofactor activities are explained by a greatly impaired ability of factor Va1 to bind to negatively charged membranes. Factor V is also separated into two protein peaks after chromatography on a Mono-S column. Upon incubation with thrombin, the first peak yields factor Va1 and the second peak factor Va2. The same two forms of factor Va were generated when freshly prepared plasma samples or platelet suspensions were treated with thrombin. This shows that the heterogeneity of the light chain domain is an intrinsic property of both plasma and platelet factor V. It is hypothesized that the heterogeneity is caused by small differences in the carboxylterminal C2 domain of factor V that are introduced as the result of post-ribosomal processing.

Binding Sites↗
PubMed1993

Binding of protein S to factor Va associated with inhibition of prothrombinase that is independent of activated protein C.

Since plasma protein S serves an anticoagulant function by mechanisms which are not completely understood, its possible interaction with Factor Va was investigated. Human protein S bound to immobilized human Factor Va in a calcium-dependent, saturable, and reversible manner and Factor Va bound similarly to immobilized protein S. Binding of protein S to immobilized Factor V was greatly enhanced by pretreatment of the surface-bound Factor V with increasing doses of thrombin up to 1 unit/ml. Binding of protein S to Factor Va was also demonstrated in fluid phase with a Kd of 33 +/- 9 nM. Biotin-labeled heavy chain of Factor Va bound to immobilized protein S, and this binding was reversed by a 17-fold molar excess of intact unlabeled Factor Va. Protein S competed efficiently with prothrombin for binding to immobilized Factor Va. The prothrombinase activity in a reaction mixture of purified clotting factors was inhibited by protein S and exhibited a pattern of mixed inhibition. The concentration of protein S needed for 50% inhibition of the prothrombinase activity of a mixture containing 1 nM Factor Xa, 20 pM Factor Va, and 50 microM phospholipids was about 16 nM. Since not all protein S preparations exhibited this degree of prothrombinase inhibitory activity, extensive control experiments were performed to verify that the inhibitory activity was associated with protein S during immunoaffinity chromatography and was not caused by traces of activated protein C in the protein S preparations. These data show that protein S has an anticoagulant function which is independent of activated protein C and, at least in part, that this is because of its competition with prothrombin for direct binding to Factor Va.

Binding, Competitive↗
PubMed1993

Protein C activation by an activator purified from the venom of Agkistrodon halys halys.

The protein C activator from Agkistrodon halys halys venom was purified 533-fold by ion-exchange chromatography on QAE-Sephadex A-50, affinity chromatography on aprotinin-Sepharose and Mono-Q fast protein liquid chromatography. The purified enzyme is a single chain protein with an apparent molecular weight of 36,000 that activates protein C by proteolytic removal of a small fragment from the heavy chain. The protein C activator exhibited a high amidolytic activity towards the tripeptide substrates D-Pro-Phe-Arg-pNA (S2302) and D-Phe-(pipecolyl)-Arg-pNA (S2238). The activity of the activator was not affected by thiolprotease or metalloprotease inhibitors. The activator was inhibited, however, by benzamidine, Phe-Pro-Arg chloromethyl ketone, p-nitrophenyl p-guanidinobenzoate and soy bean trypsin inhibitor, which classifies the enzyme as a serine protease. The purified protease was capable of activating both human and bovine protein C. Activation of human protein C only occurred at an appreciable rate in a calcium-free reaction medium at low ionic strength. Ca2+ ions inhibited the activation of human protein C with an apparent Ki of 0.8 mM. Addition of NaCl to the reaction medium also strongly inhibited human protein C activation (50% inhibition at 20 mM NaCl). Kinetic analysis of human protein C activation by the venom activator (in a calcium-free medium) revealed an apparent Km for protein C of 0.52 microM and a kcat of 0.17 s-1 at I = 0.05 (kcat/Km = 3.3 x 10(5) M-1 s-1). At I = 0.15 rates of human protein C activation became linear with protein C indicating a strong increase in Km with increasing ionic strength. Activation of bovine protein C was hardly affected by variation of Ca2+ and NaCl concentrations in the reaction medium. The apparent Kis for calcium ion and NaCl inhibition of bovine protein C activation were > 10 mM and 220 mM, respectively. At I = 0.1 and in the absence of Ca2+ ions bovine protein C was activated with a Km of 0.056 microM and a kcat of 0.24 s-1 (kcat/Km = 4.3 x 10(6) M-1 s-1). Our data are indicative for a rather large conformational and/or structural difference between human and bovine protein C at physiological ionic strength.

Amino Acid Sequence↗
PubMed1992

Effect of membrane fluidity and fatty acid composition on the prothrombin-converting activity of phospholipid vesicles.

Vesicles composed of phospholipids with different fatty acyl side chains have been utilized to examine the importance of the nonpolar membrane region for the prothrombin-converting activity of procoagulant phospholipid vesicles. Membranes composed of phosphatidylserine (PS) and phosphatidylcholine (PC) with unsaturated fatty acyl side chains were more active in prothrombin activation than membranes composed of phospholipids with saturated fatty acyl chains. This phenomenon was observed above the phase transition temperature, i.e., on membranes in the liquid-crystalline state. The prothrombin-converting activity of saturated phospholipids approached the activity of unsaturated phospholipids at high factor Va concentrations, which is indicative for a less favorable equilibrium constant for prothrombinase assembly on membrane surfaces composed of saturated phospholipids. The difference between saturated and unsaturated phospholipids was annulled on membranes with high mole percentages of PS. This may result from a compensating contribution of electrostatic forces to the binding equilibria involved in prothrombinase assembly. Additional effects on the prothrombin-converting activity were observed when membranes containing saturated phospholipids were studied below their phase transition temperature. In agreement with Higgins et al. [(1985) J. Biol. Chem. 260, 3604-3612], we found that the time required for the assembly of prothrombinase from membrane-bound factors Xa and Va is considerably prolonged on solid membranes. However, we also observed an effect of membrane fluidity on the steady-state rate of prothrombin activation. Kinetic experiments at saturating factor Va concentrations showed that the transition from the liquid-crystalline to the gel state caused a more than 9-fold decrease of the kcat of prothrombin activation without affecting the Km for prothrombin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

The effect of phospholipids, calcium ions and protein S on rate constants of human factor Va inactivation by activated human protein C.

Rate constants for human factor Va inactivation by activated human protein C (APC) were determined in the absence and presence of Ca2+ ions, protein S and varying concentrations of phospholipid vesicles of different lipid composition. APC-catalyzed factor Va inactivation in free solution (in the presence of 2 mM Ca2+) was studied under first-order reaction conditions with respect to both APC and factor Va and was characterized by an apparent second-order rate constant of 6.1 x 10(5) M-1 s-1. Stimulation of APC-catalyzed factor Va inactivation by phospholipids was dependent on the concentration and composition of the phospholipid vesicles. Optimal acceleration (230-fold) of factor Va inactivation was observed with 10 microM phospholipid vesicles composed of 20 mol% dioleoylglycerophosphoserine (Ole2GroPSer) and 80 mol% dioleoylglycerophosphocholine (Ole2GroPCho). At higher vesicle concentrations and at higher molar fractions of Ole2GroPSer some inhibition of APC-catalyzed factor Va inactivation was observed. Membranes that contained anionic phospholipids other than phosphatidylserine also promoted factor Va inactivation. The ability of different anionic lipids to enhance factor Va inactivation increased in the order phosphatidylethanolamine less than oleic acid less than phosphatidic acid less than phosphatidylglycerol less than phosphatidylmethanol less than phosphatidylserine. APC-catalyzed factor Va inactivation in the presence of phospholipid vesicles could be saturated with respect to factor Va and the reaction obeyed Michaelis-Menten kinetics. Both the Km for factor Va and the Vmax of factor Va inactivation were a function of the phospholipid concentration. The Km increased from 1 nM at 2.5 microM phospholipid (Ole2GroPSer/Ole2GroPCho 20:80, mol/mol) to 65 nM at 250 microM phospholipid. The Vmax increased from 20 mol factor Va inactivated.min-1.mol APC-1 at 2.5 microM phospholipid to 62 mol factor Va inactivated.min-1.mol APC-1 at 10 microM phospholipid and remained constant at higher phospholipid concentrations. Protein S appeared to be a rather poor stimulator of APC-catalyzed factor Va inactivation. Protein-S-dependent rate enhancements were only observed in reaction mixtures that contained negatively charged phospholipid vesicles. Independent of the concentration and the lipid composition of the vesicles, protein S caused a twofold stimulation of APC-catalyzed factor Va inactivation. This suggests that, in the human system, enhancement of APC binding to phospholipid vesicles by protein S is of minor importance. Considering that protein S is a physiologically essential antithrombotic agent, it is likely that other factors or phenomena contribute to the in vivo antithrombotic action of protein S.

Calcium↗
PubMed1992

Structural and functional properties of snake venom prothrombin activators.

In this review we have summarized the current knowledge about the prothrombin activating principles present in the venom of a large number of different snake species. It appears that snake venom prothrombin activators can be classified into four different groups based on their structural properties and on their functional properties in prothrombin activation. Group I activators efficiently convert prothrombin into meizothrombin and their activity is not influenced by the non-enzymatic cofactors of the prothrombinase complex (CaCl2, factor Va and phospholipid). Group II and III activators can cleave both peptide bonds in prothrombin necessary to convert prothrombin into thrombin. The prothrombin-converting activity of Group II activators is strongly stimulated by phospholipids and factor Va in the presence of CaCl2, whereas the activity of group III activators is only stimulated by CaCl2 and phospholipid. Group IV consists of snake venom proteases which do not convert prothrombin into enzymatically active products but cleave peptide bonds in prothrombin, resulting in the formation of inactive precursor forms of thrombin.

Animals↗
PubMed1992

Activation of bovine factor V by an activator purified from the venom of Naja naja oxiana.

The crude venom of many elapid snakes appeared to contain proteins that activated blood coagulation factor V. The factor V activator present in the venom of Naja naja oxiana was purified to homogeneity by chromatography on a mono-S column. The activator was a single chain protein with an apparent mol. wt of 48,000, as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and by gel permeation chromatography on Sephacryl S200. Activation of bovine factor V by the purified venom activator was accompanied by proteolytic cleavage of factor V and resulted in the formation of two major polypeptide chains with mol. wts of about 90,000 and 77,000. The final product obtained was compared with thrombin-activated factor V for its ability to function as cofactor in factor Xa-catalysed prothrombin activation in the presence of negatively charged phospholipid vesicles (5 mole% phosphatidylserine/95 mole% phosphatidylcholine). The Km for prothrombin obtained at a saturating amount of venom-activated factor Va was nine-fold higher than with thrombin-activated factor V (0.83 microM vs 0.09 microM, respectively) whereas both factor Va molecules stimulated the Vmax of thrombin formation some 6000-fold. Both forms of factor Va promoted the binding factor Xa to negatively charged phospholipid vesicles. However, the apparent Kd for factor Xa was less favorable in the presence of venom-activated factor V (0.67 x 10(-9) M) than in the presence of thrombin-activated factor V (0.043 x 10(-9) M). Thrombin cleaved a peptide bond in the 77,000 mol. wt polypeptide chain of venom-activated factor V, which resulted in the formation of a normal factor Va light chain. This peptide bond cleavage was, however, not associated with a change of cofactor activity. Venom treatment of thrombin-activated factor V, on the other hand, did remove a small fragment (mol. wt approximately 4000) from the heavy chain of factor Va (94,000), yielding a molecule with reduced cofactor activity. The diminished cofactor activity of venom-activated factor V is, therefore, likely due to the fact that a small peptide fragment, involved in the interaction with prothrombin and factor Xa, is missing from the heavy chain of venom-activated factor V.

Animals↗
PubMed1991

Meizothrombin formation during factor Xa-catalyzed prothrombin activation. Formation in a purified system and in plasma.

Meizothrombin and thrombin formation were quantitated during factor Xa-catalyzed activation of human prothrombin in reaction systems containing purified proteins and in plasma. In the purified system considerable amounts of meizothrombin accumulated when prothrombin was activated by factor Xa (with or without accessory components) under initial steady state conditions. The ratio of the rates of meizothrombin and thrombin formation was not influenced by variation of the pH, temperature, or ionic strength of the reaction medium. When 2 microM prothrombin was activated by the complete prothrombinase complex (factor Xa, factor Va, Ca2+, and phospholipid) 80-90% of the initially formed reaction product was meizothrombin. Lowering the prothrombin concentration from 2 to 0.03 microM caused a gradual decrease in the ratio of meizothrombin/thrombin formation from 5 to 0.6. When the phosphatidylserine content of the phospholipid vesicles was varied between 20 and 1 mol % and prothrombin activation was analyzed at 2 microM prothrombin the relative amount of meizothrombin formed decreased from 85 to 55%. With platelets, cephalin, or thromboplastin as procoagulant lipid, thrombin was the major reaction product and only 30-40% of the activation product was meizothrombin. We also analyzed complete time courses of prothrombin activation both with purified proteins and in plasma. In reaction systems with purified proteins substantial amounts of meizothrombin accumulated under a wide variety of experimental conditions. However, little or no meizothrombin was detected in plasma in which coagulation was initiated via the extrinsic pathway with thromboplastin or via the intrinsic pathway with kaolin plus phospholipid (cephalin, platelets, or phosphatidylserine-containing vesicles). Thus, thrombin was the only active prothrombin activation product that accumulated during ex vivo coagulation experiments in plasma.

Enzyme Activation↗