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G Tans

Publications and source records attributed to G Tans.

At least 73 records · Page 4Linked to original sources
PubMed1989

Amidolytic detection of prothrombin activation products after SDS-gel electrophoresis.

In this paper we report a method via which enzymatically active products formed during prothrombin activation can be detected by simple photographic means after SDS-gel electrophoresis, blotting onto a nitrocellulose membrane and visualization with the chromogenic substrate, S2238. After amidolytic detection the same nitrocellulose membrane can also be used for immunologic detection of prothrombin activation products, thus allowing a complete description of product formation during prothrombin activation. The detection limit of the so-called "amidoblot" is approximately 3 ng thrombin per gel sample which is comparable to the sensitivity of immunoblotting. It is further shown that the amidoblot technique can also be applied to other coagulation factors for which a suitable chromogenic substrate is available (factor XIIa, kallikrein, factor XIa, factor Xa, plasmin and activated protein C).

Blood Coagulation Tests↗
PubMed1988

The heparin-catalysed inhibition of human factor XIa by antithrombin III is dependent on the heparin type.

The effect of various well-characterized heparin preparations on the inactivation of human Factor XIa by human antithrombin III was studied. The heparin preparations used were unfractionated heparin and four heparin fractions obtained after anion-exchange chromatography. Inactivation of Factor XIa was monitored with S2366 as chromogenic substrate and followed pseudo-first-order reaction kinetics under all reaction conditions tested. Enhancement of the rate of inhibition of Factor XIa in the presence of unfractionated heparin correlated to the binding of antithrombin III to heparin. From the kinetic data a binding constant of 0.1 microM was inferred. The maximum rate enhancement, achieved at saturating heparin concentrations, was 30-fold. The rate enhancement achieved in the presence of each of the heparin fractions could also be correlated to the binding of antithrombin III to the heparin. The binding constant inferred from the kinetic data varied from 0.10 to 0.28 microM and the number of binding sites for antithrombin III varied from 0.06 to 0.74 site per heparin molecule. The maximum rate enhancements, achieved at saturating heparin concentrations, were strongly dependent on the type of heparin used and varied from 7-fold for fraction A to 41-fold for fraction D. Therefore, although the stimulation of Factor XIa inactivation by antithrombin III could be quantitatively correlated to the binding of antithrombin III to heparin, the heparin-catalysed inhibition of Factor XIa is dependent not only upon the degree of binding of antithrombin III to heparin but also upon the type of heparin to which antithrombin III is bound.

Antithrombin III↗
PubMed1988

Calcium-independent activation of prothrombin on membranes with positively charged lipids.

The activation of prothrombin by factor Xa is strongly accelerated by negatively charged phospholipids plus calcium ions. In this paper we report that positively charged membranes can also stimulate prothrombin activation provided that the activation reaction is carried out in the absence of calcium ions. Membranes composed of a mixture of phosphatidylcholine (PC) and positively charged lipids like stearylamine, sphingosine, or hexadecyltrimethylammonium bromide caused a more than 1000-fold increase of the rate of prothrombin activation. Prothrombin activation by the factor Xa-factor Va complex was also considerably stimulated by such membranes. Stimulation of prothrombin activation by positively charged membranes was suppressed at high ionic strength. This suggests that electrostatic attraction of negatively charged proteins by positively charged membranes is the major driving force in the association of prothrombin and factor Xa with the lipid surface. Calcium ions strongly inhibited prothrombin activation on vesicles composed of PC and stearylamine (80/20 M/M), which indicates that the regions of prothrombin and/or factor Xa containing gamma-carboxyglutamic acid (gla) are important for the interaction of these proteins with positively charged membranes. The importance of the gla domain was confirmed by the observation that PC/stearylamine vesicles had much less effect on the reactions between proteins that lack gla residues [gla-domainless (des-1-45) prothrombin, prethrombin 1, prethrombin 2, or gla-domainless (des-1-44) factor Xa]. The efficiency of prothrombin and prothrombin derivatives to act as substrate decreased in the order prothrombin greater than des-1-45-prothrombin = prethrombin 1 greater than prethrombin 2, while prothrombin activation by gla-domainless (des-1-44) factor Xa was hardly stimulated by positively charged membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Carboxyglutamic Acid↗
PubMed1987

Sulfatide bilayers as a surface for contact activation in human plasma.

Using a combination of gel permeation chromatography and freeze fracture electron microscopy it was found that the ability of sulfatides to promote contact activation in human plasma is associated with the ability of sulfatides to form bilayer structures. When sulfatides were dispersed in buffer at 70 degrees C by vortexing, large liposomes were found ranging in size between 2-5 micron diameter. Contact activation promoting activity was associated with these particles. Sonication resulted in the formation of small vesicles and stacked vesicles of 400-2000 A diameter and biological activity was associated with these vesicles. Homogeneous preparations of small unilamellar vesicles (500 A diameter) can be obtained by chromatography over Sepharose 2B.

Blood Coagulation↗
PubMed1987

Structural and functional characterization of a prothrombin activator from the venom of Bothrops neuwiedi.

A prothrombin activator from the venom of Bothrops neuwiedi was purified by gel filtration on Sephadex G-100, ion-exchange chromatography on DEAE-Sephacel and affinity chromatography on a Zn2+-chelate column. The overall purification was about 200-fold, which indicates that the prothrombin activator comprises about 0.5% of the crude venom. The venom activator is a single-chain protein with an apparent molecular weight of 60 kDa. It readily activated bovine prothrombin with a Km of 38 microM and a Vmax of 120 mumol prothrombin activated per min per mg of venom activator. Venom-catalyzed prothrombin activation was not accelerated by the so-called accessory components of the prothrombinase complex, phospholipids plus Ca2+ and Factor Va. Gel-electrophoretic analysis of prothrombin activation indicated that the venom activator only cleaved the Arg-323-Ile-324 bond of bovine prothrombin, since meizothrombin was the only product of prothrombin activation. The activator did not hydrolyze commercially available p-nitroanilide substrates and its prothrombin-converting activity was not inhibited by benzamidine, phenylmethylsulfonyl fluoride, dansyl-Glu-Gly-Arg-chloromethyl ketone and soy-bean trypsin inhibitor. However, chelating agents such as EDTA, EGTA and o-phenanthroline rapidly destroyed the enzymatic activity of the venom activator. The activity of chelator-treated venom activator could be partially restored by the addition of an excess CaCl2. These results indicate that the venom activator remarkably differs from Factor Xa and that the enzyme is not a serine proteinase, but likely belongs to the metalloproteinases. The structural and functional properties of the venom prothrombin activator from B. neuwiedi are similar to those reported for the venom activator from Echis carinatus.

Animals↗
PubMed1987

Autoactivation of human plasma prekallikrein.

Incubation of purified human plasma prekallikrein with sulfatides or dextran sulfate resulted in spontaneous activation of prekallikrein as judged by the appearance of amidolytic activity toward the chromogenic substrate H-D-Pro-Phe-Arg-p-nitroanilide. The time course of generation of amidolytic activity was sigmoidal with an apparent lag phase that was followed by a relatively rapid activation until finally a plateau was reached. Soybean trypsin inhibitor completely blocked prekallikrein activation whereas corn, lima bean, and ovomucoid trypsin inhibitors did not. The Ki of the reversible inhibitor benzamidine for autoactivation (240 microM) was identical to the Ki of benzamidine for kallikrein. Thus, spontaneous prekallikrein activation and kallikrein showed the same specificity for a number of serine protease inhibitors. This indicates that prekallikrein is activated by its own enzymatically active form, kallikrein. Immunoblotting analysis of the time course of activation showed that, concomitant with the appearance of amidolytic activity, prekallikrein was cleaved. However, prekallikrein was not quantitatively converted into two-chain kallikrein since other polypeptide products were visible on the gels. This accounts for the observation that in amidolytic assays not all prekallikrein present in the reaction mixture was measured as active kallikrein. Kinetic analysis showed that prekallikrein activation can be described by a second-order reaction mechanism in which prekallikrein is activated by kallikrein. The apparent second-order rate constant was 2.7 X 10(4) M-1 s-1 (pH 7.2, 50 microM sulfatides, ionic strength I = 0.06, at 37 degrees C). Autocatalytic prekallikrein activation was strongly dependent on the ionic strength, since there was a considerable decrease in the second-order rate constant of the reaction at high salt concentrations. In support of the autoactivation mechanism it was found that increasing the amount of kallikrein initially present in the reaction mixture resulted in a significant reduction of the lag period and a rapid completion of the reaction while the second-order rate constant was not influenced. Our data support a prekallikrein autoactivation mechanism in which surface-bound kallikrein activates surface-bound prekallikrein.

Dextran Sulfate↗
PubMed1987

Inhibition of factor XIa by antithrombin III.

The inactivation of human factor XIa by human antithrombin III was studied under pseudo-first-order reaction conditions (excess antithrombin III) both in the absence and in the presence of heparin. The time course of inhibition was followed by using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. After electrophoresis, proteins were blotted onto nitrocellulose and stained either for glycoprotein or for antithrombin III using antibodies against antithrombin III. Concomitant with factor XIa inactivation, two new slower migrating bands, one of which represented the intermediate complex consisting of one antithrombin III complexed with factor XIa, appeared as a transient band. Complete inactivation resulted in a single band representing the complex of factor XIa with two antithrombin III molecules. Quantitative analysis of the time course of inactivation was accomplished by measurement of the disappearance of factor XIa amidolytic activity toward the chromogenic substrate S2366. Pseudo-first-order reaction kinetics were observed throughout. The rate constant of inactivation was found to be 10(3) M-1 s-1 in the absence of heparin and 26.7 X 10(3) M-1 s-1 in the presence of saturating amounts of heparin. From the kinetic data, a binding constant (Kd) of 0.14 microM was inferred for the binding of antithrombin III to heparin. The time course of inactivation and the distribution of the reaction products observed upon gel electrophoresis are best explained assuming a mechanism of inactivation in which the two active sites present in factor XIa are inhibited in random order (i.e., independent of each other) with the same rate constant of inhibition.

Antithrombin III↗
PubMed1987

Studies on the effect of serine protease inhibitors on activated contact factors. Application in amidolytic assays for factor XIIa, plasma kallikrein and factor XIa.

Amidolytic assays have been developed to determine factor XIIa, factor XIa and plasma kallikrein in mixtures containing variable amounts of each enzyme. The commercially available chromogenic p-nitroanilide substrates Pro-Phe-Arg-NH-Np (S2302 or chromozym PK), Glp-Pro-Arg-NH-Np (S2366), Ile-Glu-(piperidyl)-Gly-Arg-NH-Np (S2337), and Ile-Glu-Gly-Arg-NH-Np (S2222) were tested for their suitability as substrates in these assays. The kinetic parameters for the conversion of S2302, S2222, S2337 and S2366 by beta factor XIIa, factor XIa and plasma kallikrein indicate that each active enzyme exhibits considerable activity towards a number of these substrates. This precludes direct quantification of the individual enzymes when large amounts of other activated contact factors are present. Several serine protease inhibitors have been tested for their ability to inhibit those contact factors selectively that may interfere with the factor tested for. Soybean trypsin inhibitor very efficiently inhibited kallikrein, inhibited factor XIa at moderate concentrations, but did not affect the amidolytic activity of factor XIIa. Therefore, this inhibitor can be used to abolish a kallikrein and factor XIa contribution in a factor XIIa assay. We also report the rate constants of inhibition of contact activation factors by three different chloromethyl ketones. D-Phe-Pro-Arg-CH2Cl was moderately active against contact factors (k = 2.2 X 10(3) M-1 s-1 at pH 8.3) but showed no differences in specifity. D-Phe-Phe-Arg-CH2Cl was a very efficient inhibitor of plasma kallikrein (k = 1.2 X 10(5) M-1 s-1 at pH 8.3) whereas it slowly inhibited factor XIIa (k = 1.4 X 10(3) M-1 s-1) and factor XIa (k = 0.11 X 10(3) M-1 s-1). Also Dns-Glu-Gly-Arg-CH2Cl was more reactive towards kallikrein (k = 1.6 X 10(4) M-1 s-1) than towards factor XIIa (k = 4.6 X 10(2) M-1 s-1) and factor XIa (k = 0.6 X 10(2) M-1 s-1). Since Phe-Phe-Arg-CH2Cl is highly specific for plasma kallikrein it can be used in a factor XIa assay selectively to inhibit kallikrein. Based on the catalytic efficiencies of chromogenic substrate conversion and the inhibition characteristics of serine protease inhibitors and chloromethyl ketones we were able to develop quantitative assays for factor XIIa, factor XIa and kallikrein in mixtures of contact activation factors.

Amino Acid Chloromethyl Ketones↗
PubMed1987

Structural and functional characterization of factor XII.

In this article we have reviewed the current knowledge regarding the involvement of Factor XII in contact activation. Clearly in the past decade an overwhelming amount of data and hypotheses have been published regarding the central role of this zymogen in the initiation and further propagation of contact activation reactions. Therefore we feel that it will be helpful to conclude this article with a figure that summarizes those interactions and reactions that are generally believed to reflect the major molecular events occurring during surface-dependent contact activation. The contact factors are capable of very efficient interation with each other, provided a suitable negatively charged surface is present. Such surfaces are thought to stimulate the interactions between the contact factors through binding of the proteins and thus bringing the proteins together. Factor XII readily binds to the negatively charged surface, but for the binding of prekallikrein and Factor XI, the cofactor HMW kininogen is likely to be necessary. Bound at the surface, the zymogens Factor XII and prekallikrein are thought to be involved in a so-called reciprocal activation mechanism in which Factor XIIa activates prekallikrein to kallikrein, which in turn converts Factor XII to Factor XIIa. The formation of Factor XIIa is further promoted by the fact that surface-bound Factor XII is likely more susceptible to proteolytic cleavage and by the fact that the activated Factor XIIa is capable of auto-activating its own zymogen Factor XII. However, the latter effect, although undoubtedly contributing to the formation of Factor XIIa at the surface, seems to be of less importance than the reciprocal activation mechanism. This is underscored by the fact that Factor XII activation is rather slow in prekallikrein-deficient plasma. Surface-bound Factor XIIa is then responsible for the activation of Factor XI to Factor XIa, thereby propagating the initial trigger. Presumably, Factor XIa must leave the surface in order to be able to become involved in the activation of blood coagulation Factor IX.

Amino Acid Sequence↗
PubMed1986

Formation of meizothrombin as intermediate in factor Xa-catalyzed prothrombin activation.

The conversion of prothrombin into thrombin by Factor Xa requires the cleavage of two peptide bonds in prothrombin. Dependent on the order of cleavage, prethrombin 2 or meizothrombin occurs as intermediate. Since prethrombin 2 has as yet been the only observed intermediate, prothrombin activation is generally considered to proceed via prethrombin 2. In this paper we present new methods that allow differentiation between meizothrombin and thrombin formed during the initial phase of prothrombin activation. These methods, which make use of the different reactivities of meizothrombin and thrombin toward fibrinogen and antithrombin III plus heparin, enabled us to show the generation of considerable amounts of meizothrombin during Factor Xa-catalyzed prothrombin activation. Both meizothrombin and thrombin incorporated the active site-directed fluorescent chloromethyl ketone 5-dimethylaminonaphthalene-1-sulfonyl-Glu-Gly-Arg-CH2Cl. Gel electrophoretic analysis of chloromethyl ketone-treated aliquots of prothrombin activation mixtures confirmed meizothrombin formation. These observations demonstrate that prothrombin may also be converted into thrombin via meizothrombin.

Animals↗
PubMed1986

The function of the heavy and light chain of human plasma kallikrein in the activation of factor XII.

In this paper we report the effect of sulfatides on rate constants of Factor XII activation by kallikrein and its isolated light chain. In free solution kallikrein and the light chain were equally effective in activating Factor XII and both enzymes had their pH optimum at pH 7.0 (k1 = 1.6 X 10(3) M-1 s-1). Sulfatides greatly stimulate Factor XII activation. When sulfatides were present kallikrein was, however, much more effective than its light chain. At 330 microM sulfatides and pH 7.0 the rate constants of Factor XII activation were 5.3 X 10(6) M-1 s-1 and 4.2 X 10(4) M-1 s-1 for kallikrein and its light chain, respectively. In the presence of sulfatides, Factor XII activation by kallikrein had its pH optimum at 6.3 and the rate constant increased considerably at lower ionic strength. Light chain-dependent Factor XII activation in the presence of sulfatides, was optimal at pH 7.0 and was not affected by variation of the ionic strength. Binding studies revealed that kallikrein, Factor XII and the heavy chain of kallikrein bind to the sulfatide surface, whereas no binding of the light chain of kallikrein was detectable. Since the effects of pH and ionic strength on sulfatide-dependent Factor XII activation by kallikrein can be explained by effects on kallikrein binding to sulfatides we conclude that surface-bound Factor XII is activated by surface-bound kallikrein. Our data suggest that sulfatides stimulate Factor XII activation via two distinct mechanisms: a) by making Factor XII more susceptible to proteolytic cleavage and b) by promoting the formation of the enzyme-substrate complex through surface binding of both kallikrein and factor XII.

Enzyme Activation↗
PubMed1986

The effect of platelets in the activation of human blood coagulation factor IX by factor XIa.

We report here the effect of activated human platelets on the activation of human factor IX by human factor XIa. Factor IXa formed during activation was determined via its ability to activate bovine factor X. To increase sensitivity, phospholipids and bovine factor VIIIa were present in the assay. The kinetic parameters of the factor IX activation were determined in the presence of 10 mmol/L CaCl2. The Km for factor IX was 0.30 mumol/L and kcat was 2.4 s-1. Activated human platelets inhibited factor IX activation by factor XIa in a dose-dependent manner, whereas unstimulated platelets had no effect. Factor IX activation was inhibited for more than 90% at a platelet concentration of 4 X 10(8)/mL, whereas concentrations of less than 10(6)/mL had no influence. The inhibitory effect could be induced by thrombin, collagen, calcium ionophore A 23187, and adrenalin. The appearance of inhibitory activity could be blocked by the addition of the prostacyclin analogue ZK 36374 at any time during platelet activation. Stirring during platelet activation was not necessary. These results suggest that the inhibition is caused by a release reaction. This was confirmed by centrifugation experiments that showed that the inhibitory activity could be recovered from the supernatant of the activated platelets. The inhibitory activity was destroyed upon boiling and was susceptible to trypsin digestion. Passage of platelet supernatant over ACA 22 showed that the inhibitory activity eluted with an apparent molecular weight of less than 1,200,000 but greater than 669,000. The inhibition of factor XIa was reversible. These data suggest that platelets release an antiprotease of factor XIa that reversibly inhibits factor XIa. Lineweaver-Burk analysis showed that the inhibitor caused both an increase in Km for factor IX and a decrease in kcat of factor IXa formation by factor XIa.

Blood Platelets↗
PubMed1985

Protein-protein interactions in contact activation of blood coagulation. Binding of high molecular weight kininogen and the 5-(iodoacetamido) fluorescein-labeled kininogen light chain to prekallikrein, kallikrein, and the separated kallikrein heavy and light chains.

Binding of the 5-(iodoacetamido)fluorescein (IAF)-labeled high molecular weight (HMW) kininogen light chain to prekallikrein and D-Phe-Phe-Arg-CH2Cl-inactivated kallikrein was monitored by a 0.040 +/- 0.002 increase in fluorescence anisotropy. Indistinguishable average dissociation constants and stoichiometries of 14 +/- 3 nM and 1.1 +/- 0.1 mol of prekallikrein/mol of IAF-light chain and 17 +/- 3 nM and 0.9 +/- 0.1 mol of kallikrein/mol of IAF-light chain were determined for these interactions at pH 7.4, mu 0.14 and 22 degrees C. Prekallikrein which had been reduced and alkylated in 6 M guanidine HCl lost the ability to increase the fluorescence anisotropy of the IAF-kininogen light chain, suggesting that the native tertiary structure was required for tight binding. The kallikrein heavy and light chains were separated on the basis of the affinity of the heavy chain for HMW-kininogen-Sepharose, after mild reduction and alkylation of kallikrein under nondenaturing conditions. Under these conditions, alkylation with iodo [14C]acetamide demonstrated that only limited chemical modification had occurred. Binding of the IAF-kininogen light chain to the isolated alkylated kallikrein heavy chain, when compared to prekallikrein and kallikrein, was characterized by an indistinguishable increase in fluorescence anisotropy, average dissociation constant of 14 +/- 3 nM, and stoichiometry of 1.2 +/- 0.1 mol of kallikrein heavy chain/mol of IAF-light chain. In contrast, no binding of the D-Phe-Phe-Arg-CH2Cl-inactivated kallikrein light chain was detected at concentrations up to 500 nM. Furthermore, 300 nM kallikrein light chain did not affect IAF-kininogen light chain binding to prekallikrein, kallikrein, or the kallikrein heavy chain. The binding of monomeric single chain HMW-kininogen to prekallikrein, kallikrein, and the kallikrein heavy and light chains was studied using the IAF-kininogen light chain as a probe. Analysis of the competitive binding of HMW-kininogen gave average dissociation constants and stoichiometries of 12 +/- 2 nM and 1.2 +/- 0.1 mol of prekallikrein/mol of HMW-kininogen, 15 +/- 2 nM and 1.3 +/- 0.1 mol of kallikrein/mol of HMW-kininogen, 14 +/- 3 nM and 1.4 +/- 0.2 mol of kallikrein heavy chain/mol of HMW-kininogen, and no detectable effect of 300 nM kallikrein light chain on these interactions. We conclude that a specific, nonenzymatic interaction between sites located exclusively on the light chain of HMW-kininogen and the heavy chain of kallikrein or prekallikrein is responsible for the formation of 1:1 noncovalent complexes between these proteins.

Binding, Competitive↗
PubMed1985

Surface-dependent activation of human factor XII (Hageman factor) by kallikrein and its light chain.

In this paper we report the effect of sulfatides on the rate constants of factor XII activation by kallikrein and its isolated light chain (the domain of kallikrein that contains the active site of the enzyme). In the absence of sulfatides, kallikrein and the light chain were equally effective in factor XII activation (k1 = 1.57 X 10(3) M-1 s-1 at pH 7.0). The pH optima were the same (pH 7.0) and the reaction was not affected by variation of the ionic strength. Sulfatides strongly increased the rate constants of factor XIIa formation. In the presence of sulfatides kallikrein was, however, much more active than its light chain. At 330 microM sulfatides, pH 7.0 and 100 mM NaCl the rate constants of factor XII activation were 5.34 X 10(6) M-1 s-1 and 4.17 X 10(4) M-1 s-1 for kallikrein and its light chain, respectively. The pH optimum of factor XII activation by kallikrein in the presence of sulfatides was shifted to pH 6.3, and the reaction became highly ionic-strength-dependent. The rate constant increased considerably at decreasing NaCl concentrations. The optimum pH for light-chain-dependent factor XII activation in the presence of sulfatides remained unaltered and the reaction was not affected by the ionic strength. Binding studies revealed that both kallikrein and factor XII bind to the sulfatide surface, whereas no binding of the light chain of kallikrein was detectable. The isolated heavy chain of kallikrein had the same binding properties as kallikrein, which indicates that the heavy-chain domain contains the functional information for kallikrein binding to sulfatides. Since the effects of pH and ionic strength on the rate constants of kallikrein-dependent factor XII activation in the presence of sulfatides correlated with effects on the binding of kallikrein, it is concluded that under these conditions surface-bound factor XII is activated by surface-bound kallikrein. Our data suggest that sulfatides stimulate kallikrein-dependent factor XII activation by two distinct mechanisms: by making factor XII more susceptible to peptide bond cleavage by kallikrein and by promoting the formation of the enzyme-substrate complex through surface binding of kallikrein and factor XII.

Electrochemistry↗
PubMed1985

Purification and properties of a prothrombin activator from the venom of Notechis scutatus scutatus.

The prothrombin activator present in the venom of the mainland tiger snake (Notechis scutatus scutatus) was purified to homogeneity by gel chromatography on Sephadex G-200 followed by ion-exchange chromatography on SP-Sephadex. The venom activator has an apparent molecular weight of 54,000. It consists of a heavy chain (Mr = 32,000) and a light chain (Mr = 23,000) held together by one or more disulfide bridges. The active site is located at the heavy chain region of the molecule. The venom activator contains 8 gamma-carboxyglutamic acid residues/molecule. Gel electrophoretic analysis of prothrombin activation indicates that the venom activator is capable of cleaving both the Arg 274-Thr 275 and Arg 323-Ile 324 bonds of bovine prothrombin. The order of bond cleavage appears to be random since prethrombin-2 and meizothrombin occur as intermediates during prothrombin activation. Prothrombin activation by the venom activator alone is very slow. This is explained by the unfavorable kinetic parameters for the reaction (Km for prothrombin = 105 microM, Vmax = 0.0025 nmol of prothrombin activated per min/microgram of venom activator). Phospholipids plus Ca2+ and Factor Va greatly stimulate venom-catalyzed prothrombin activation. In the presence of 50 microM phospholipid vesicles composed of 20 mol % phosphatidylserine and 80 mol % phosphatidylcholine, the Km drops to 0.2 microM, whereas there is hardly any effect on the Vmax. Factor Va causes a 3,500-fold increase of the Vmax (8.35 nmol of prothrombin activated per min/microgram of venom activator) and a 10-fold decrease of the Km (9.5 microM). The most favorable kinetic parameters are observed in the presence of both 50 microM phospholipid and Factor Va (Km = 0.16 microM, Vmax = 27.9 nmol of prothrombin activated per min/microgram of venom activator). These changes of the kinetic parameters explain the stimulatory effects of Factor Va and phospholipid on venom-catalyzed prothrombin activation. The venom activator slowly converts the Factor Xa-specific chromogenic substrates CH3SO2-D-leucyl-glycyl-L-arginine-p-nitroanilide and N-benzoyl-L-isoleucyl-L-glutamyl-(piperidyl)-glycyl-L-arginyl-p-nitroani lide hydrochloride. Factor Va causes a 7-fold stimulation of chromogenic substrate conversion by the venom activator. This stimulation appears to be the result of the formation of a tight 1:1 complex between the venom activator and Factor Va.

1-Carboxyglutamic Acid↗
PubMed1983

Sulfatide-dependent autoactivation of human blood coagulation Factor XII (Hageman Factor).

When purified human blood coagulation Factor XII (Hageman factor) is incubated with sulfatides at 37 degrees C, activation of Factor XII occurs as judged by the appearance of amidolytic activity towards the chromogenic substrate H-D-Pro-Phe-Arg-p-nitroanilide. Polyacrylamide gel electrophoresis studies using 125I-Factor XII as a marker show that the appearance of amidolytic activity correlates with Factor XII cleavage, that activation goes to completion and that virtually all Factor XIIa formed is present as the two-chain, 80,000 Mr form, alpha-Factor XIIa. Rigorous analysis of kinetic data establishes that, between 0.02 and greater than 90% of the reaction, the activation of Factor XII is described by a mechanism of autoactivation of Factor XII by Factor XIIa. The rate of autoactivation increases with increasing Factor XII concentrations at constant sulfatide levels but decreases with increasing sulfatide concentrations at constant levels of Factor XII. These findings suggest that the concentrations of Factor XII and Factor XIIa bound to the sulfatide surface determine the rate of autoactivation. Soybean trypsin inhibitor, Trasylol, and anti-prekallikrein antibodies have no influence on the rate of sulfatide-dependent autoactivation of Factor XII. Benzamidine inhibits autoactivation with an inhibitor constant, Ki, of 1.9 mM which is similar to the Ki of 1.5 mM for the enzyme, alpha-Factor XIIa. Thus, sulfatide-dependent activation of purified Factor XII is not due to contaminating proteases and is described by a second order mechanism of autoactivation due to the action of surface-bound Factor XIIa on surface-bound Factor XII.

Benzamidines↗