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J Jesty

Publications and source records attributed to J Jesty.

At least 37 records · Page 2Linked to original sources
PubMed1988

A comparison of phospholipid and platelets in the activation of human factor VIII by thrombin and factor Xa, and in the activation of factor X.

Two aspects of the activation of factor X by the intrinsic clotting pathway have been studied in purified human systems, in the presence of either purified phosphatidylserine:phosphatidylcholine vesicles (PS:PC) or platelets activated with ionophore A23187: (1) the activation of factor VIII by factor Xa and by thrombin, and (2) the activation of factor X by the factor IXa/VIIIa complex. Factor VIII activation by thrombin was unaffected in either rate or extent by the presence of PS:PC or activated platelets. In contrast, factor VIII activation by factor Xa required either PS:PC or platelets. The products of optimal factor VIII activation by the two enzymes, designated factor VIIIa(T) and factor VIIIa(Xa), are kinetically different in the activation of factor X by factor IXa, factor VIIIa(T) being approximately twice as active (in factor X activation) as factor VIIIa(Xa) in the presence of PS:PC or platelets. Factor VIIIa(Xa) can be converted to the more active VIIIa(T) by thrombin treatment, but the activity of factor VIIIa(T) is unchanged by factor Xa treatment. Factor X activation was also studied with optimally activated factor VIIIa(T), in the presence of PS:PC or activated platelets, as a function of factor IXa concentration in order to determine the apparent dissociation constant for the factor IXa-VIIIa interaction in the two cases. Activated platelets increased the apparent affinity more than fivefold.

Blood Coagulation↗
PubMed1986

Effect of limited modification of amino groups on the reactivity of human factor Xa.

The basis of the specificity of human coagulation factor Xa has been probed with a reagent that reacts with nucleophiles, N-succinimidylpropionate. At pH 8.0 and 0.25 mM N-succinimidylpropionate, 0.4 microM factor Xa lost approx. 90% of its activity toward prothrombin in 4 min. The decay was first-order, k = 0.64 min-1, which increased to 0.98 min-1 in 1 mM Ca2+, and the dependence of k upon pH was consistent with primary amines being the target. The rate of modification was unaffected by the presence of a tetrapeptide substrate during modification; likewise, activity toward a tripeptide p-nitroanilide was unaltered during exposure of factor Xa to N-succinimidylpropionate with or without Ca2+. In addition, inhibition by antithrombin III was retained with a somewhat enhanced rate after modification; however, the acceleration of this by heparin was significantly less. Kinetic determination of the number of residues modified gave a reaction order of 2.0, while reaction with N-succinimidyl[3H]propionate yielded labeled factor Xa containing 1.0 mol N-succinimidylpropionate/mol factor Xa and 50% normal clotting activity, or 2.0 mol N-succinimidylpropionate/mol and 1% activity, respectively. Thus, one nucleophilic group is required for the reaction of factor Xa with prothrombin but not for the hydrolysis of peptides or recognition of antithrombin III. The decay of clotting activity of the factor X zymogen in N-succinimidylpropionate was much slower though still Ca2+-dependent. Conversely, the reaction of a related compound--N-succinimidyl(4-hydroxyphenyl)propionate or Bolton-Hunter reagent--with factor Xa broadly resembled that of N-succinimidylpropionate but the decay curves indicated more complex kinetics. Therefore, the target groups vary in their accessibility to modification according to the structural characteristics of both the protein and the reagent.

Antithrombin III↗
PubMed1986

The kinetics of inhibition of alpha-thrombin in human plasma.

Methods have been developed for kinetic studies of the inhibition of exogenous unmodified thrombin in human plasma containing normal levels of fibrinogen and calcium ion. To prevent interference by other proteases, factor VIII-deficient plasma was used and contained 50 nM Phe-Phe-Arg-chloromethyl ketone and 1 kallikrein-inactivating unit/ml aprotinin; neither inhibited thrombin at these levels. Two independent assays were used. The first was the discontinuous amidolytic assay of thrombin activity, which measures both free thrombin and thrombin-alpha 2-macroglobulin complex, and was used to estimate the rates of inhibition both by "inactivating" inhibitors, such as anti-thrombin and alpha 1-protease inhibitor, and by alpha 2-macroglobulin (alpha 2M). The contribution of alpha 2M was confirmed by a second method, which measured with time the generation of amidolytic activity due to the thrombin-alpha 2M complex. The total rate of thrombin inhibition in plasma containing 4 mM free Ca2+ was of the order of 1.9 min-1, of which 0.4 min-1 was due to alpha 2M and 0.9 min-1 was due to inhibitors that were removed when plasma was passed through heparin-agarose. Thrombin inhibition was also measured in varying dilutions of plasma and confirmed that total inhibition rate is approximately linearly related to plasma (and thus inhibitor) concentration. Negatively charged phospholipid micelles had very little effect on thrombin inhibition rate, but platelets accelerated inhibition to more than 2.5 min-1.

Adenosine Diphosphate↗
PubMed1986

Analysis of the generation and inhibition of activated coagulation factor X in pure systems and in human plasma.

The overall generation and inhibition of human factor Xa have been studied in pure systems and plasma to determine the kinetic characteristics of inhibition during factor Xa generation. Generation curves were measured amidolytically in a pure system containing factor X and antithrombin, which was activated with the factor X-activating enzyme of Russell's viper venom (RVV-X). The measured change in factor Xa level with time was fitted to a 3-parameter 2-exponential model to determine apparent first-order rates of inhibition. With antithrombin at 4.5 microM, the inhibition rate constant thus obtained was very close to the known rate of inhibition of exogenous enzyme. Factor Xa generation curves were also analyzed in plasma; however, to reduce interference in the assay of thrombin, congenitally prothrombin-deficient plasma was used containing 0.5 microM D-Phe-Pro-Arg-chloromethylketone. In plasma, factor Xa generated in the presence of phospholipid and Ca2+ ions by RVV-X, factor IXa, or tissue factor was inhibited more slowly than exogenous enzyme. The reduction was particularly severe with tissue factor activation, where the rate was 0.04-0.06 min-1. This protection by tissue factor was also observed in pure systems and apparently required factor VII.

Antithrombins↗
PubMed1986

Measurement of the kinetics of inhibition of activated coagulation factor X in human plasma: the effect of plasma and inhibitor concentration.

A method has been developed for detailed kinetic studies of the inhibition of factor Xa in human plasma. Radiolabeled enzyme is not required, and the method can be used at initial factor Xa levels of 1 nM. The method is discontinuous and based on the removal of samples into an amidolytic assay done in the presence of 1% Lubrol-PX detergent. This permits the study of inhibition in mixtures containing phospholipid, platelets, or thromboplastin. The method can be used at inhibition rates in excess of 1 min-1, and by suitable analysis can be used to estimate the contribution of inhibition by alpha 2-macroglobulin, which does not itself inhibit amidolytic activity. The method is at present limited to cases where thrombin is not generated in large excess. Factor Xa inhibition has been studied in citrated plasma as a function of total plasma concentration, and--by the use of antithrombin-depleted plasma--as a function of the antithrombin concentration of the plasma. In all situations inhibition is characterized by second-order behavior: (i) total inhibition rate is proportional to plasma concentration up to 95%, giving a maximum rate in the absence of calcium of 1 min-1; (ii) inhibition in depleted plasma reconstituted with antithrombin shows inhibition rate to remain linearly related to antithrombin concentration; and (iii) the estimated rate due to alpha 2-macroglobulin is proportional to plasma concentration. It is thus confirmed that, as in pure systems, inhibition of factor Xa in whole plasma is linearly related to the concentration of each class of inhibitor.

Antithrombins↗
PubMed1986

Parlin, a general microcomputer program for parallel-line analysis of bioassays.

Commonly used manual and calculator methods for analysis of clinically important parallel-line bioassays are subject to operator bias and provide neither confidence limits for the results nor any indication of their validity. To remedy this, the authors have written a general program for statistical analysis of these bioassays for the IBM Personal Computer and its compatibles. The program has been used for analysis of bioassays for specific coagulation factors and inflammatory lymphokines and for radioimmunoassays for prostaglandins. The program offers a choice of no transform, logarithmic, or logit transformation of data, which are fitted to parallel lines for standard and unknown. It analyzes the fit for parallelism and linearity with an F test, and calculates the best estimate of the result and its 95% confidence limits. Comparison of results calculated by PARLIN with those previously obtained manually shows excellent correlation (r greater than 0.99). Results obtained using PARLIN are quickly available with current assay technics and provide a complete evaluation of the bioassay at no increase in cost.

Animals↗
PubMed1985

Altered factor VII activity in hemophilia.

Factor VII levels have been studied in hemophilia A and B plasmas and normal controls in a controlled, prospective study. Three assay methods were used: a standard clotting assay (FVIIc-A); a modified clotting assay (FVIIc-B) (Seligsohn et al, Blood 52:978-988, 1978); and a coupled amidolytic assay. By the FVIIc-B assay, the hemophilic plasmas were significantly lower than in the normal group (68.2 +/- 3.3% [SE] and 83.5 +/- 3.8%, respectively; P less than .01). The amidolytic assay, however, which measures total factor VII regardless of its activity state (factor VII or VIIa), was higher in the patient group than in the control group (126.9 +/- 9.6% and 99.4 +/- 5.7%, respectively; P less than .01). Control experiments showed that the differences in FVIIc-B activity were not caused by artifactual activation of factor VII ex vivo in the control group. The mean FVIIc-A assay of hemophilic plasmas (126.3 +/- 6.5%) agreed closely with the amidolytic assay, suggesting that the FVIIc-A method is also insensitive to the factor VII activity state. These data support the hypothesis that the FVIIc-B assay is more sensitive to the presence of factor VIIa. The increased sensitivity of the FVIIc-B assay to factor VII activation was confirmed by comparison of the two clotting assays on plasma subjected to activation in glass at 4 degrees C. The results of this study indicate that factor VII in hemophilic plasma is less activated than in normal plasma. Whether this contributes to the bleeding diathesis of hemophilia is unknown. However, it does provide evidence for the idea that factor VII in vivo is normally subject to some degree of activation by an enzyme (or enzymes) generated by a turnover of the intrinsic pathway.

Adult↗
PubMed1985

The kinetics of inhibition of thrombin by antithrombin in the presence of components of the hemostatic system.

The inhibition of human thrombin by antithrombin has been measured in pure systems in the presence of other components of the hemostatic system that might affect the kinetics of the reaction. These included fibrinogen, calcium ions, phospholipid, prothrombin, platelets (both adenosine diphosphate [ADP]-stimulated and -unstimulated), and platelet extracts. Inhibition rates were measured in each case by a discontinuous amidolytic assay over a range of antithrombin concentrations, from 0 to 4.5 mumol/L. Under all conditions, rates of inhibition were proportional to antithrombin concentration. Calcium ions at 5 mmol/L caused a small (20%) reduction in rate, but phospholipid and prothrombin had no additional effect. In contrast, both fibrinogen and platelets significantly changed the rate of inhibition. In the presence of calcium, fibrinogen at concentrations from 0 to 12 mumol/L reduced the rate of inhibition in a competitive manner, giving an apparent Kd for fibrinogen of 6.0 mumol/L. As the plasma fibrinogen level is about 8 mumol/L, one may therefore predict that variations in fibrinogen level will have a significant effect on the rate of thrombin inhibition in plasma. More unexpected was the observation that platelets increase the rate of inhibition: unstimulated platelets increased the rate constant by 40%, and ADP-stimulated platelets increased it by 55%. However, this acceleratory effect could not be mimicked with either a KCI extract or a Triton extract of platelets, and its cause remains unknown. In sum, it has been shown that the rate of inhibition of thrombin can be modulated in at least three ways-antithrombin concentration, fibrinogen concentration, and platelets; each of which can vary independently in vivo. It is well known that defects of the first lead to an increased risk of thrombosis, and it is proposed that this may be substantially caused by changes in the kinetics of inhibition such as those described. Additionally, it is suggested that changes in inhibition rate caused by other components may also be significant, for the same reason, in modulating the clotting system in vivo.

Antithrombins↗
PubMed1984

Measurement of human activated factor X-antithrombin complex by an enzyme-linked differential-antibody immunosorbent assay.

An enzyme-linked immunoabsorbent assay (ELISA) has been developed for the measurement of the complex of human antithrombin and Factor Xa. Rabbit anti-human Factor X antibodies are adsorbed to ELISA plates, and samples containing Xa-antithrombin complex are added. This is followed by the addition of F(ab')2 fragments of rabbit antibodies against human antithrombin, previously labeled with alkaline phosphatase, and subsequent measurement of the bound labeled antibody by hydrolysis of p-nitrophenylphosphate. The minimum level of complex detectable in a sample is ca. 0.1 nM. The assay has been used to follow the generation of Xa-antithrombin complex in kinetic situations by the addition of 1 microM Ile-Glu-Gly-Arg-chloro- methylketone to the ELISA sampling buffer, and it has also been used in plasma systems, where a 20-fold reduction in the sensitivity of the assay is observed. This reduction was shown to be entirely caused by the plasma Factor X. The assay has been used to follow generation of the Xa-antithrombin complex in defibrinated plasma upon activation of the clotting system with the Factor X-activating protein of Russell's Viper venom, and has been compared with the total generation of Factor Xa, measured by a radiopeptide assay of Factor X activation in the same mixtures.

Antibodies↗
PubMed1984

Tissue factor-dependent activation of tritium-labeled factor IX and factor X in human plasma.

Recent investigations have suggested that the activation of factor IX by factor VII/tissue factor may be an important alternative route to the generation of factor Xa. Accordingly, we have compared the tissue factor-dependent activation of tritium-labeled factor IX and factor X in a human plasma system and have studied the role of proteases known to stimulate factor VII activity. Plasma was defibrinated by heating and depleted of its factors IX and X by passing it through antibody columns. Addition of human brain thromboplastin, Ca2+, and purified 3H-labeled factor X to the plasma resulted, after a short lag, in burst-like activation of the factor X, measured as the release of radiolabeled activation peptide. The progress of activation was slowed by both heparin and a specific inhibitor of factor Xa, suggesting a feedback role for this enzyme, but factor X activation could not be completely abolished by such inhibitors. In the case of 3H-factor IX activation, the rate also increased for approximately 3 min after addition of thromboplastin, but was not subsequently curtailed. A survey of proteases implicated as activators of factor VII in other settings showed that both factor Xa and (to a much smaller extent) factor IXa could accelerate the activation of factor IX. However, factor Xa was unique in obliterating activation when present at concentrations greater than approximately 1 nM. Heparin inhibited the tissue factor-dependent activation of factor IX almost completely, apparently through the effect of antithrombin on the feedback reactions of factors Xa and IXa on factor VII. These results suggest that a very tight, biphasic control of factor VII activity exists in human plasma, which is modulated mainly by factor Xa. Variation of the factor IX or factor X concentrations permitted kinetic parameters for each activation to be derived. At saturation of factor VIIa/tissue factor, factor IX activation was significantly more rapid than was previously found in bovine plasma under similar conditions. The activation of factor X at saturation was slightly more rapid than in bovine plasma, despite the presence of heparin.

Animals↗
PubMed1983

The activation of Factor IX by tissue factor-Factor VII in a bovine plasma system lacking Factor X.

The activation of Factor IX by tissue factor-Factor VII has been studied in a bovine plasma system under conditions that minimize the activation of Factor VII. The plasma was defibrinated, then passed twice through a column of anti-Factor X coupled to Sepharose in order to lower the Factor X level below its limit of assay (ca. 5 ng/ml), and once through an anti-Factor IX column to remove Factor IX. Varying levels of tritium-labelled Factor IX were then added back to the plasma, permitting measurement of its activation upon the addition of tissue factor and Ca2+. Despite the absence of significant levels of Factor X in the system, the course of Factor IX activation was initially characterized by some upward curvature, which suggested activation of the plasma Factor VII during the incubation. In order to obtain linear activation of Factor IX three proteolytic inhibitors were added to the system: 1) a Factor Xa inhibitor, 1,2-bis-(5-amidinobenzimidazole)-ethane, 2) aprotinin, and 3) heparin. Under these conditions the apparent Km of non-activated Factor VII (+ tissue factor) on Factor IX was 17.3 +/- 2.5 nM (SE), and the maximum velocity was 0.12 nM/min. In parallel experiments the plasma Factor VII was activated by first treating the plasma with Factor Xa for 30 seconds before the addition of inhibitors and the final addition of substrate. Under these conditions the maximum velocity rose to 4.2 nM/min, and the Km increased to 53.3 +/- 6.0 nM (SE). This change in the Km is highly significant (P less than 0.002), and indicates that the activation of Factor IX by nonactivated plasma Factor VII cannot be due only to traces of Factor VIIa in the plasma. At least in part, activation of Factor IX in the presence of tissue factor is suggested to be a result of the action of Factor VII itself.

Animals↗
PubMed1981

The activation and inactivation of human factor VIII by thrombin: effect of inhibitors of thrombin.

The activation and inactivation of human factor VIII by thrombin have been investigated by the use of thrombin inhibitors. The addition of inhibitors to nonactivated factor VIII blocks activation by thrombin. In contrast, their addition to factor VIII activated with thrombin does not block inactivation, but causes an initial period of decay that is more rapid than in the absence of inhibitor. This effect was seen only with protease inhibitors that inhibit thrombin. After the initial decay, low levels of factor VIII coagulant activity persist in the presence of inhibitors, but an assay specific for activated factor VIII showed this to be largely a result of the persistence of nonactivated factor VIII. Only in the case of reversible inhibition is activated factor VIII present in this plateau phase. Possible mechanisms that would account for these observations were studied by iterative computer simulation of model reactions. Two classes were considered: (formula: see text). The experimental results are inconsistent with the first mechanism, which predicts that thrombin indicators should stabilize activated factor VIII (VIIIt). Alternative mechanisms were studied where activation is thrombin-dependent, but inactivation is a first-order reaction (mechanism 2). This family of mechanisms includes those where VIIIt is an VIII. thrombin complex. Simulation of the addition of thrombin inhibitors to such model systems shows the initial rapid decay of activity characteristic of the experimental observations and predicts qualitatively the different effects of reversible and irreversible inhibitors that are observed in the plateau phase. These results argue strongly against a two-cleavage model for the activation and inactivation of factor VII by thrombin and support a one-cleavage model in which inactivation is due to first-order decay. In addition, they provide a plausible mechanistic explanation for the fact that serine protease inhibitors appear to inhibit thrombin-activated factor VIII.

Blood Coagulation↗
PubMed1979

Dissociation of complexes and their derivatives formed during inhibition of bovine thrombin and activated factor X by antithrombin III.

The complexes formed by antithrombin III with activated bovine Factor X and thrombin have been studied by gel electrophoresis in dodecyl sulfate. When subjected to electrophoresis at pH 7, the complexes remain intact, whereas electrophoresis at pH 9 in the presence of Tris results in their dissociation. Dissociation of both the Factor Xa-antithrombin III complex and the thrombin-antithrombin III complex in dodecyl sulfate produces a modified form of antithrombin III which, unlike the native inhibitor, apparently consists of two chains. Gel electrophoresis of the dissociated complexes has also been used to study the sites where the complexes are cleaved by the respective enzymes. The cleavage of the Factor Xa-inhibitor complex by Factor Xa apparently results from hydrolysis of a single bond in the enzyme part of the complex and releases a 15,000-dalton NH2-terminal fragment of the heavy chain, with the light chain attached. Cleavage of the thrombin-inhibitor complex by thrombin involves several cleavages of the heavy (B) chain of the thrombin part of that complex. Neither enzyme-inhibitor complex is subject to cleavage by free enzyme in the inhibitor part of the complex under the conditions used.

Animals↗