Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: HUMANGGP:027518 (factor Xa)
 5,167 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A decreased plasma antithrombin activity in presence or in absence of heparin was discovered in a 47-year-old patient presenting with recurrent venous thromboembolism. The immunoreactive material (AT III-IR) was normal. The same biological abnormalities were found in two relatives of the patient, leading to the diagnosis of hereditary qualitative AT III deficiency. The propositus' AT III was coeluted with normal AT III from an heparin-sepharose column. An additional step of ion-exchange chromatography on a Mono Q column using a FPLC system (Pharmacia, St-Quentin en Yvelines, France) allowed the purification of a protein which was homogenous in SDS-10% polyacrylamide electrophoresis gel (PAGE). AT III purified from propositus' plasma, normal plasma and the plasma of the patient known to have an AT III variant with defective protease binding (AT III Charleville) were compared. The specific activities measured as heparin cofactor antithrombin or factor Xa inhibition in absence of heparin were decreased to half the normal value. Kinetic studies confirmed a decreased rate of thrombin inhibition for both abnormal AT III preparations. SDS-PAGE experiments performed in purified system and immunoblots obtained from plasma showed that the two variants have different behaviour: in the case of AT III Charleville thrombin induced an apparent 5 k delta increase in molecular mass, probably due to a conformational change. AT III Avranches did not form stoechiometric complexes with thrombin, but was unmodified by the protease.
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PMID:Antithrombin III Avranches, a new variant with defective serine-protease inhibition--comparison with antithrombin III Charleville. 318 51

The antifactor Xa activities of heparin fractions are widely used as an ex vivo index of their antithrombotic efficacy. Its clinical meaning, however, remains speculative. In the study reported, we measured the effects of standard heparin, a synthetic pentasaccharide heparin (antifactor Xa activity only), and a low molecular weight heparin (LMWH) on factor Xa, factor Va, and thrombin generation in thromboplastin-activated plasma. We clearly demonstrated that the antifactor Xa activity of heparin contributed little in its anticoagulant activity. The inhibition of factor Va generation, dependent on the heparin antithrombin activity only, is of prime importance to the inhibition of thrombin generation in plasma. The inhibition of thrombin generation by the LMWH was comparable with that of standard heparin on the basis of their respective antithrombin specific activities, but not on the basis of their antifactor Xa activities.
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PMID:The limited importance of factor Xa inhibition to the anticoagulant property of heparin in thromboplastin-activated plasma. 319 77

Studies were conducted to define the location of components and sequences in heparin with respect to their distance from the peptide linkage in the native proteoglycan. A purified heparin-oligopeptide was linked via its amino terminus to a matrix containing an azo bond and an activated carboxyl group. The polysaccharide chain was maximally degraded, either with heparinase or nitrous acid, and the soluble products were removed. The heparin-oligopeptide fragments that remained on the matrix were released by reductive cleavage of the azo linkage and characterized. The fragments, as well as heparin released without prior degradation, contained serine and glycine as the principal amino acids; the ratio of galactose to xylose was 2:1. The ratio of glucosamine to serine of 33:1 in the undegraded heparin was reduced to 6:1 and 1:1 in the heparinase-treated and nitrous acid-treated products, respectively. The undegraded sample and the fragments contained phosphate in equivalent amounts, demonstrating its presence in the heparin-protein linkage region. The heparin-oligopeptide preparation was also fractionated by gel filtration and high and low molecular weight fractions thus obtained were each linked to the insoluble matrix. The products that were subsequently released were subfractionated on a molecular weight-calibrated column of Sephadex G-200, and eluates were assayed for activity in promoting the neutralization of thrombin and factor Xa by antithrombin. The results revealed a sharp decrease in specific activity in heparin-oligopeptide fractions below Mr = 15,000 indicating that the anticoagulant-conferring segment is located at about 20 disaccharide units away from the peptide linkage region.
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PMID:Location of specific oligosaccharides in heparin in terms of their distance from the protein linkage region in the native proteoglycan. 333 97

The role of 3-O- and 6-O-sulfated glucosamine residues within the heparin octasaccharide critical for biological activity, iduronic acid----N-acetylglucosamine 6-O-sulfate----glucuronic acid----N-sulfated glucosamine 3,6-di-O-sulfate----iduronic acid 2-O-sulfate----N-sulfated glucosamine 6-O-sulfate----iduronic acid 2-O-sulfate----anhydromannitol 6-O-sulfate, was determined by comparing its ability to bind antithrombin, induce a conformational change in this protease inhibitor as monitored by the enhancement of intrinsic fluorescence, and accelerate (at saturation) the interaction of this protein with human factor Xa. The octasaccharide produced a maximum 48% increase in intrinsic fluorescence at 37 degrees C and a rate of factor Xa inhibition of 6 X 10(5) M-1 s-1 as measured by stopped-flow fluorometry at 25 degrees C. The basal rate of the antithrombin-factor Xa interaction observed in the absence of oligosaccharide was 2 X 10(3) M-1 s-1. The synthetic pentasaccharide, consisting of residues 2-6, produced fluorescence enhancement and rate of inhibition equal to those of the octasaccharide. However, a similar pentasaccharide, identical in all respects except that it lacked the 3-O-sulfate on residue 4, produced less than a 5% fluorescence enhancement and a rate of factor Xa inhibition of 8 X 10(3) M-1 s-1. The tetrasaccharide consisting of residues 2-5 produced a 35% fluorescence enhancement and a rate of factor Xa inhibition of 3 X 10(5) M-1 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Contribution of 3-O- and 6-O-sulfated glucosamine residues in the heparin-induced conformational change in antithrombin III. 342 19

Four members of an Italian family (two with histories of venous thromboembolism) had a qualitative defect of antithrombin III reflected by normal antigen concentrations and half-normal antithrombin activity with or without heparin. Anti-factor Xa activities were consistently borderline low (about 70% of normal). For the propositus' plasma and serum the patterns of antithrombin III in crossed-immunoelectrophoresis with or without heparin were indistinguishable from those of normal plasma or serum. A normal affinity of antithrombin III for heparin was documented by heparin-sepharose chromatography. Affinity adsorption of the propositus' plasma to human alpha-thrombin immobilized on sepharose beads revealed defective binding of the antithrombin III to thrombin-sepharose. Hence the molecular defect of this variant appears to be at the active site responsible for binding and neutralizing thrombin, thus accounting for the low thrombin inhibitory activity.
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PMID:Characterization of an abnormal antithrombin (Milano 2) with defective thrombin binding. 356 66

Numerous investigators have postulated that a hypercoagulable state exists in humans for a period of time before the development of thrombotic episodes. A clear biochemical definition of the prethrombotic state, however, has proved elusive due in part to the lack of reliable techniques for monitoring pertinent changes in blood coagulability. Based on recent advances in our knowledge of the biochemistry of the coagulation system, a series of highly sensitive and specific immunochemical tools has been developed that can quantitate the activities of various steps of the hemostatic mechanism in vivo at the subnanomolar level. We have established assays for F1+2 and the protein C activation peptide, which measure the cleavage of the prothrombin molecule by factor Xa and the scission of protein C by the thrombin-thrombomodulin complex, respectively. Nossel and coworkers had previously constructed similar assays for fibrinopeptide A (FPA) and fragment B beta 1-42, which monitor the cleavage of fibrinogen by thrombin and the proteolysis of fibrin I by plasmin, respectively. Substantial elevations in the levels of these markers have been found in patients with disseminated intravascular coagulation and many subjects with acute deep venous thrombosis. The F1+2 and FPA assays have been used to demonstrate that significant increments in factor Xa activity but not thrombin activity regularly occur in the blood of nonanticoagulated individuals with congenital deficiencies of antithrombin or protein C. These two disorders are known to be correlated with the subsequent development of thrombosis. Patients with protein C deficiency have also been noted to have significantly reduced plasma levels of protein C activation peptide. By using the immunoassays for FPA and B beta 1-42 in studies of postoperative patients, it has been shown that an imbalance between the procoagulant action of thrombin and the anticoagulant effect of plasmin on fibrin I polymer may induce an acquired thrombotic diathesis. Finally, we have recently demonstrated that prothrombin activation as measured by the F1+2 assay is suppressed by oral anticoagulants in the blood of patients with thrombotic diatheses. These investigations suggest that these assay techniques can be used to improve our understanding of the hypercoagulable state as well as to develop more effective treatment strategies for the prevention of thromboembolic events.
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PMID:The pathophysiology of the prethrombotic state in humans: insights gained from studies using markers of hemostatic system activation. 360 75

Antithrombin III (AT III) plasma levels were investigated in 18 full term neonates and 14 healthy preterm neonates. A control group of 20 healthy adults was also studied. AT III was measured as antigen concentration (Ag) and antithrombin or anti-factor Xa heparin cofactor (H.C.) activities. Crossed immunoelectrophoresis on heparin-agarose (H-CIE) was carried out on plasma samples; moreover the distribution of isoantithrombins was investigated on whole plasma by a technique of crossed immunoelectrofocusing (CIEF). AT III plasma levels in full term infants were significantly lower as compared to the adult values. The preterm newborns group showed a further significant decrease in AT III levels as compared to the full term neonates. In all infants AT III H-CIE runs displayed a single fast moving anodal peak, so that a normal binding to heparin was demonstrated. The CIEF AT III plasma pattern of the adults as well as of all neonates displayed three major peaks at pH range 5.2-4.9, a small amount of AT III at pH 4.9-4.8 and a minor peak at pH 4.8-4.6, so that it was concluded that the isoantithrombins plasma distribution in neonatal age is identical to that of the adult subjects. Four neonates whose mothers were affected by AT III congenital defect were also investigated: diagnosis of congenital deficiency was established in three cases.
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PMID:Antithrombin III in full-term and pre-term newborn infants: three cases of neonatal diagnosis of AT III congenital defect. 366 Mar 33

We have developed a radioimmunoassay (RIA) for prethrombin 2 (Pr2), a potential intermediate in the transformation of prothrombin to thrombin. Antisera against human Pr2 were raised in rabbits and the respective immunoglobulin G fractions were chromatographed on prothrombin-Sepharose. The specific antibody population obtained was used to construct a double-antibody RIA capable of measuring as little as 0.05 nmol/L of this component. The immunoreactivity of prothrombin was approximately 40,000 times less than that of Pr2 on a molar basis. Because of nonspecific contributions of plasma constituents to the immunoreactive signal, the measurement of Pr2 in this milieu required the use of a titration curve in which Pr2 was added back to Pr2-depleted plasma. This assay was then used to determine the levels of this species in two patient populations with increased prothrombin activation as determined by the prothrombin fragment F1 + 2 RIA, a measure of the in vivo cleavage of prothrombin by factor Xa. The mean Pr2 concentrations in eight patients with disseminated intravascular coagulation and six asymptomatic individuals with congenital antithrombin deficiency not receiving antithrombotic therapy were not significantly elevated as compared with those of normal controls (0.244 nmmol/L and 0.242 nmol/L vs. 0.184 nmol/L, respectively). Our studies show that Pr2 is cleared from the plasma of dogs with a t1/2 of approximately 25 minutes. Given that the t1/2 of F1 + 2 is estimated to be approximately 90 minutes, the low plasma levels of Pr2 observed in patients with thrombophilia cannot result from rapid clearance of this component.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Development of a radioimmunoassay for quantitating prethrombin 2 in human plasma. 366 58

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.
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PMID:Analysis of the generation and inhibition of activated coagulation factor X in pure systems and in human plasma. 372 68

Heparin was isolated from Mercenaria mercenaria by ion-exchange chromatography and was fractionated into two distinct populations with immobilized antithrombin. The high-affinity glycosaminoglycan accelerated dramatically the inhibition of purified human factors IIa and Xa via purified human antithrombin. Specific anti-factor IIa and anti-factor Xa activities were 363 and 348 U.S.P. units/mg, respectively. The highly active clam heparin exhibited a molecular weight of approximately 18,000 and contained approximately 2.5 sulfate groups per disaccharide. The intrinsic fluorescence of purified human antithrombin was enhanced in the presence of the high-affinity invertebrate glycosaminoglycan to an extent comparable to the level induced by vertebrate heparin. In addition, the critical tetrasaccharides containing 3-O-sulfated glucosamine residues, which constitute part of the unique antithrombin-binding domain of mammalian heparin, were also detected in high-affinity Mercenaria heparin.
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PMID:Anticoagulantly active heparin from clam (Mercenaria mercenaria). 374 Aug 45


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