Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.5 (thrombin)
33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Factor VIII/von Willebrand factor (FVIII/vWF) is a glycoprotein with a molecular weight greater than one-million daltons. Two activities are associated with this large molecule: FVIII procoagulant activity and vWF activity. Incubation of FVIII/vWF with proteolytic enzymes causes rapid inactivation of the FVIII procoagulant activity but has little effect on the vWF activity or antigenicity. In an attempt to gain insight into the structural features required for these two activities, antisera were raised in rabbits to normal, thrombin-inactivated, and plasmin-inactivated FVIII/vWF. All of these proteolytically modified forms of FVIII/vWF cross-reacted with each of the rabbit antisera; each blocked the ability of a human inhibitor to inactivate native active FVIII/vWF. Each of the antisera was a potent inhibitor of vWF activity and inactivated vWF activity at the same titer. The antisera were much less potent inhibitors of FVIII activity than of vWF activity. Antibodies to thrombin-inactivated FVIII/vWF or normal FVIII/vWF had about the same ability to inactivate FVIII procoagulant activity. Surprisingly, those to plasmin-inactivated FVIII/vWF still retained about 50% of this inhibitory capacity. A comparison of the three types of antigens by polyacrylamide gel electrophoresis in sodium dodecyl sulfate-6 M urea demonstrated that the structure of thrombin-inactivated FVIII/vWF was indistinguishable from that of normal FVIII/vWF, while plasmin-inactivated FVII/vWF was completely cleaved to lower molecular weight fragments. Some of the reported variations in the ability of rabbit antibodies to inhibit procoagulant activity may be due to partial degradation of the starting antigen. The retention by FVIII/vWF protein of its immunologic properties even after extensive proteolytic degradation suggests that under nondenaturing conditions, the conformation of the native and degraded molecules are very similar.
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PMID:Immunologic studies of native and modified human factor VIII/von Willebrand factor. 8 37

The presence of specific Factor VIII/von Willebrand factor (FVIII/vWF) binding sites on human platelets has been demonstrated by using 125I-FVIII/vWF and washed human platelets. Binding is ristocetin-dependent and increases in proportion to the concentration of ristocetin from 0.2 to 1 mg/ml. Binding of 125I-FVIII/vWF to platelets can be competitively inhibited by unlabeled human or bovine FVIII/vWF, but not by human thrombin, fibrinogen, alpha 2-macroglobulin, equine collagen, or a lectin of Ricinus communis. Scatchard analysis of binding data indicated that the dissociation constant of FVIII/vWF receptors is 0.45--0.5 nM. There are 31,000 binding sites per platelet at 1 mg/ml of ristocetin concentration. The optimal pH range for binding is from 7.0 to 7.5. At a concentration of 2 mM, EGTA inhibits 86% of the binding; however, 20 mM of Ca++, Mg++, or EDTA have little effect. Binding sites for FVIII/vWF were found only on platelets, and no significant binding was detected with human erythrocytes or polymorphonuclear leukocytes.
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PMID:Demonstration and characterization of specific binding sites for factor VIII/von Willebrand factor on human platelets. 10 91

Present methods for assay of platelet aggregating agents use freshly prepared platelets. Much time is spent in daily preparation of platelets and standardization presents problems. The preparation of fixed washed platelets (FWP) and their use in two bioassays are described in this report. Washed human platelets were fixed for 48 hours with 4 per cent paraformaldehyde, washed twice in phosphate buffer, pH 6.4, and stored at 4 degrees C. Aggregation of FWP was studied with a macroscopic test and a light absorbance measurement. FWP did not aggregate with adenosine diphosphate, collagen, adrenalin, and thrombin. FWP aggregated with bovine or porcine plasma, poly-L-lysine, and ristocetin with normal human plasma but not with von Willebrand's disease plasma. These observations confirm the direct aggregating effect of these agents. Macroscopic aggregation times were dependent on the amount of aggregating agent (bovine plasma, normal human plasma). A quantitative assay for bovine platelet aggregating factor (PAF) and von Willebrand factor (vWF) with FWP was developed. The ability of FWP to aggregate remained unchanged after 1 month of storage at 4 degrees C. Ristocetin alone caused a decrease in light transmission of FWP suspensions, depending upon the concentration of ristocetin, but did not cause aggregation. FWP constitute a stable reagent suitable for quantitative measurement of PAF and vWF.
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PMID:Platelets fixed with paraformaldehyde: a new reagent for assay of von Willebrand factor and platelet aggregating factor. 23 99

A platelet-aggregating activity was found in many snake venoms, predominantly those of the genus Bothrops, that is apparent only in the presence of the platelet-aggregating von Willebrand factor of plasma. It is designated "venom coagglutinin." The coagglutinin can be largely separated from the thrombin-like enzyme of the venoms by ion-exchange chromatography. The venom factor acts on formaldehyde-fixed platelets and is effective with decalcified, heparinized, and afibrinogenemic plasmas but not with severe von Willebrand disease plasmas or with normal plasmas in which the von Willebrand factor has been neutralized by specific antibodies. Use of this coagglutinin permits the assay of von Willebrand factor without the many disadvantages of the ristocetin test. The coagglutinin is active with human, dog, pig, and bovine plasmas and with platelets of any one of these species. This broad-spectrum activity without regard to species contrasts with the ristocetin-resistance of many combinations of plasma and platelets from various species. The assay provides a procedure for studying human, porcine, and canine von Willebrand disease. The lack of species specificity of the coagglutinin suggests that it may be a universal activator of the von Willebrand factor-platelet reaction.
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PMID:Venom coagglutinin: an activator of platelet aggregation dependent on von Willebrand factor. 30 34

When Factor VIII/von Willebrand factor (FVIII/vWF) protein is rechromatographed on 4% agarose in 0.25 M CaCl(2), the protein and vWF activity appear in the void volume, but most of the FVIII procoagulant activity elutes later. Recent evidence suggests that the delayed FVIII procoagulant activity is a proteolytically modified form of FVIII/vWF protein that filters anomalously from agarose in 0.25 M CaCl(2). To test whether or not thrombin is the protease involved, the effect of 0.25 M CaCl(2) on FVIII/vWF and its reaction with thrombin was examined. About 30% of the FVIII procoagulant activity was lost immediately when solutions of FVIII/vWF protein were made 0.25 M in CaCl(2). When FVIII in 0.15 M NaCl was activated with 0.04 U thrombin/ml and then made 0.25 M in CaCl(2), the procoagulant activity of a broad range of FVIII/vWF protein concentrations remained activated for at least 6 h. But, in 0.25 M CaCl(2), the increase in FVIII procoagulant activity in response to thrombin was much more gradual and once activated, the procoagulant activity was stabilized by 0.25 M CaCl(2). When thrombin-activated FVIII/vWF protein was filtered on 4% agarose in 0.15 M NaCl, there was considerable inactivation of FVIII procoagulant activity; however, the procoagulant activity that did remain eluted in the void volume. In contrast, when thrombin-activated FVIII/vWF protein was filtered in 0.25 M CaCl(2), the FVIII procoagulant activity eluted well after the void volume and remained activated for 6 h. The procoagulant peak isolated by filtering nonthrombin-activated FVIII/vWF protein on agarose in 0.25 M CaCl(2) was compared to that isolated from thrombin-activated FVIII/vWF protein. Both procoagulant activity peak proteins had about the same specific vWF activity as the corresponding void volume protein. Before reduction, the sodium dodecyl sulfate gel patterns for the two procoagulant activity peak proteins were the same. After reduction, the gel pattern for the nonthrombin-activated procoagulant activity peak protein contained bands of 195,000, 148,000-120,000, 79,000, 61,000, 51,000, and 18,000 daltons whereas the pattern for the reduced thrombin-activated procoagulant activity peak protein always lacked the higher molecular weight bands, but consistently showed the four lower molecular weight bands to be well resolved. Taken together, these results imply that thrombin generates the FVIII procoagulant activity that is stabilized by 0.25 M CaCl(2) and elutes aberrantly from 4% agarose in that solvent.
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PMID:Some effects of calcium on the activation of human factor VIII/Von Willebrand factor protein by thrombin. 40 79

The mechanism of activation of platelets by collagen was examined. Hirudin interfered with the initial collagen-platelet interaction and both hirudin and heparin inhibited collagen-induced release of platelet granular contents. Hirudin completely inhibited the release of both [3H]5HT and beta-glucuronidase whereas heparin completely inhibited release of beta-glucuronidase but only partly inhibited release of [3H] 5HT. beta-Glucuronidase and maximal [3H] 5HT were only released when plasma was present. The results are compatible with an essential intermediary role for thrombin in collagen activation of platelets. Evidence was also obtained that von Willebrand factor may participate in this reaction.
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PMID:Platelet activation in haemostasis: role of thrombin and other clotting factors in platelet-collagen interaction. 87 Mar 98

Thimerosal, a sulphydryl inhibitor, induces aggregation of normal platelet rich plasma over a wide range of concentrations. Low doses induce a monophasic response preceded by a lag phase, high doses produce an immediate biphasic response. Thimerosal induces platelet aggregation through its binding by sulphydryl groups. Thimerosal induced aggregation is not mediated by ADP, it is not influenced by fibrinogen, von Willebrand factor, calcium, and magnesium ions of the medium. Thimerosal induced platelet aggregation is normal in patients affected by thrombocytopathia (defect of ADP release) but not in patients affected by Glanzmann's thrombasthenia. Mercaptopropionglycine, a substance which tends to preserve SH groups, inhibits platelet aggregation induced by thimerosal, thrombin, collagen, and ADP. A mechanism is proposed for thimerosal induced aggregation and the role of SH groups also in ADP, thrombin and collagen induced aggregation is indicated.
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PMID:Platelet aggregation by thimerosal: role of ADP and SH groups. 98 91

When human, canine, or bovine factor VIII preparations are chromatographed on 4% agarose at ionic strength 0.2, the factor VIII activity elutes as a single peak in the void volume with slight tailing. Incubation of such preparations with dilute (0.01 U/ml) highly purified thrombin results in some activation of factor VIII. Chromatography of such incubation mixtures, under the same conditions as before, results in elution of two peaks of factor VIII activity one in the void volume and one much later with marked tailing. The void volume peak has most of the protein and some factor VIII activity. These void volume fractions also contain all the von Willebrand factor activity of thrombin-treated bovine preparations. Longer treatment with thrombin, or treatment with stronger thrombin, appears to shift much more of the procoagulant activity to the later eluting peak. Also, when the peak of factor VIII activity, found in the void volume after thrombin treatment, was again incubated with dilute thrombin, an increase in factor VIII activity occurred. Chromatography of this incubation mixture demonstrated only a small amount of activity in the void volume, while the bulk of the activity was present in the second peak. On the other hand, thrombin treatment of factor VIII activity from peak 2 caused a rapid decline of activity instead of a further increase. It is proposed that the residual factor VIII activity found in the void volume represents unreacted factor VIII, while the late eluting peak represents thrombin-activated material that is of smaller apparent size. The late eluting peak differs from the small active factor VIII fragment obtained by Ca2+ dissociation, as the latter can be activated by thrombin. A similar set of experiments was performed using ultracentifugation of bovine factor VIII preparations on sucrose density gradients. Results of these experiments agreed completely with those obtained with get chromatography. Preparations made from human hemophilic plasma, by the procudure employed in the purification of human factor VIII, were also incubated with thrombin and chromatographed. von Willebrand factor was again found only in the void volume fractions, but there was no factor VIII activity in any fractions eluted. In other control experiments, activated and unactivated factor VIII fractions did not clot fibrinogen and contained no assayable factor IX or X. The thrombin-modified factor VIII of small size was inactivated by both a naturally occurring human inhibitor to factor VIII and the gamma globulin fraction of a rabbit antisera produced against the calcium-dissociated small active factor VIII fragment.
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PMID:Effects of thrombin treatment of preparations of factor VIII and the Ca2+-dissociated small active fragment. 108 Apr 90

In 18 insulin-dependent diabetics (6 without retinopathy, 6 with proliferative retinopathy and 6 with proliferative retinopathy treated by hypophysectomy) matched for age and duration of diabetics, in vitro haemostasis was studied using ADP induced platelet aggregation, ristocetin induced platelet aggregation which allows von Willebrand factor (VIII VWF) assay, and determination of antihemophilic factor procoagulant activity (VII AHF). Using gel filtration-isolated platelets, the ADP induced hyperaggregation previously reported in diabetics with severe retinopathy untreated by hypophysectomy appeared to be related to a platelet and not a plasma factor; the normal results of thrombin induced aggregation suggests that the presumed abnormal platelet factor is related to the platelet plasma membrane. High level of plasma VII VWF was observed in diabetics with proliferative retinopathy while the VII AHF level was within normal limits.
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PMID:Platelet hyperaggregation and increased plasma level of Von Willebrand factor in diabetics with retinopathy. 108 63

When purified antihemophilic factor (Factor VIII) was rechromatographed on 4% agarose in 0.15 M NaCl or 1.0 M NaCl, a single protein peak, containing both procoagulant activity and von Willebrand factor activity, as defined by ristocetin-induced platelet aggregation, was eluted in the void volume. Purified Factor VIII immediately lost about 30% of its procoagulant activity when dissolved in 0.25 M CaCl2, and when rechromatographed on 4% agarose in 0.25 M CaCl2, the protein peak and von Willebrand factor activity remained coincident in the void volume; however, most of the remaining procoagulant activity was eluted after the void volume. The elution position of Factor VIII procoagulant activity from 4% agarose in 0.25 M CaCl2, and hence its apparent molecular weight, varied with the protein concentration applied to the column; at low protein concentrations it was eluted close to the inner volume. Yet on Sephadex G-200 in 0.25 M CaCl2, the protein and procoagulant activity were eluted together in the void volume. These observations suggested that the Factor VIII procoagulant activity was not eluting according to size or shape, but was adsorbing to some extent to the agarose. Isolated activity peak material from the 0.25 M CaCl2 columns contained protein and had a typical ultraviolet spectrum. Even at high concentrations, the protein contained no thrombin, Factors IX, X, or Xa activity, or detectable phospholipid. In addition to Factor VIII procoagulant activity, which could be inactivated by a human antibody to Factor VIII, the activity peak protein also contained von Willebrand factor activity. Like native Factor VIII and the void volume protein, the activity peak contained protein that did not enter a sodium dodecyl sulfate 5% polyacrylamide gel in the absence of reducing reagent. After reduction of disulfide bonds, several subunits ranging from 195,000 to 30,000 daltons were observed. These results indicate that the protein in the shifted Factor VIII procoagulant activity peak is large and that its anomalous elution pattern from 4% agarose in 0.25 M CaCl2 results from interaction with the agarose. The Factor VIII-like properties of the activity peak protein and its electrophoretic pattern on sodium dodecyl sulfate gels suggest that it is a species of Factor VIII modified by proteolytic cleavage. These results allow an interpretation that is different from the recently proposed "carrier protein-small active subunit" hypotheses for the structure-function relationships of the Factor VIII molecule.
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PMID:Studies on human antihemophilic factor. Evidence for a covalently linked subunit structure. 108 90


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