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)

The receptor for Factor VIII/von Willebrand factor (F. VIIIVWF) is readily available on circulating platelets. We have found that the stimulation of platelets with traces of thrombin at concentrations that are generated physiologically (0.008 U-0.05 U/ml) induced concentration-dependent binding of 125I-labeled F. VIIIVWF in a steady-state system. The binding induced by thrombin was specific because it was inhibited by a 100-fold molar excess of unlabeled F. VIIIVWF factor, by rabbit monospecific antibody against Factor VIII, and was not inhibited by an excess of fibrinogen or fibronectin. Binding induced by thrombin required metabolically active platelets, in contrast to a system with ristocetin that also prompted binding to glutaraldehyde-treated platelets. The thrombin effects on binding of 125I-F. VIIIVWF was not observed when platelets were washed with EDTA-containing buffers; EDTA and EGTA both inhibited thrombin-induced binding. Platelet membrane glycoproteins were required because enzymatic stripping od them from the platelet surface with chymotrypsin reduced binding 2.5-5.0-fold. Prostacyclin, in the concentration range of 1 to 50 nM, had two distinct effects on the receptor for F. VIIIWVF: (a) it prevented exposure of this receptor when added 10 min before thrombin, and (b) it reversed the binding of 125I-F. VIIIVWF to the platelet receptor when added 30 min after thrombin and the ligand, ie., when binding was at equilibrium. The dual effect of prostacyclin on the receptor for F. VIIIVWF was reproduced by dibutyryl cyclic AMP.
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PMID:Thrombin-induced exposure and prostacyclin inhibition of the receptor for factor VIII/von Willebrand factor on human platelets. 628 32

Human umbilical vein endothelial cells (HUV-EC) were isolated and maintained in pure culture on a fibronectin matrix with hypothalamic derived endothelial cell growth factor included in the culture medium. HUV-EC maintained under these conditions displayed only slight alterations in morphological appearance and continued to produce Factor VIII antigen. The cells showed an unaltered growth response to serum and added growth factors up to passage 16 (greater than 30 population doublings). We found that epidermal growth factor (EGF) was potently mitogenic for HUV-EC, but only in the presence of endothelial cell growth factor. Also in contrast to a previous report, we were unable to demonstrate a potentiation of this response by human alpha-thrombin. Because of these discrepancies, we performed studies to determine if they might be explained by a difference in the interaction of our HUV-EC with EGF. In studies utilizing 125I-EGF as tracer probe, we determined that our HUV-EC have an EGF receptor number of 13,000 sites/cell with an apparent Kd-4.0 X 10(-9) M. In addition, receptor-bound 125I-EGF was rapidly internalized and degraded presumably by a lysosomally mediated pathway since degradation was complete to the amino acid level. These results are in agreement with those previously published and thus do not provide a basis on which to resolve discrepancies regarding the growth response of HUV-EC to various growth factors.
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PMID:A reevaluation of the response of human umbilical vein endothelial cells to certain growth factors. 631 1

Blood interaction with the subendothelium of rabbit aorta was investigated in an annular perfusion chamber using patients with von Willebrand's disease, hemophilia, and afibrinogenemia. The vessels were exposed to nonanticoagulated blood for a range of flow conditions (wall shear rates of 650-3,300 s-1) and exposure times (1.5-10 min). The resultant platelet and fibrin interaction was quantified by the use of several morphometric techniques, one of which was developed to measure more precisely the dimensions (height and volume) of platelet thrombi attached to the subendothelium. A major finding was that under flow conditions in which little or no defect in platelet adhesion was observed in von Willebrand's disease, platelet thrombus height and volume in this disorder were significantly reduced as compared with normal controls or patients with hemophilia. Thus, Factor VIII/von Willebrand factor (VIII/VWF) may mediate not only the adhesion of platelets to subendothelium but also platelet-platelet attachments necessary for normal thrombus development. The level of Factor VIII:coagulant activity (VIII: C) was also observed to influence the resultant thrombus height and volume deposited on subendothelium, presumably through the generation of thrombin or some other procoagulant factor preceding fibrin formation, since normal values of thrombus dimensions were always observed in a patient with a fibrinogen deficiency. The influence of VIII:C became greater as shear rate was reduced, whereas as shear rate was increased, VIII/VWF was more dominant in determining the resultant platelet deposition on subendothelium. Thus, the deficiencies of VIII:C and VIII/VWF in hemophilia and von Willebrand's disease can lead to various abnormalities in platelet and fibrin association with subendothelium. The importance of a particular deficiency will depend strongly on the local blood flow conditions.
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PMID:Platelet interaction with rabbit subendothelium in von Willebrand's disease: altered thrombus formation distinct from defective platelet adhesion. 633 2

In order to assess the true incidence of haemostatic disorders in cirrhotic gastro-intestinal haemorrhage, a comparative prospective study of primary haemostasis, coagulation and fibrinolysis was carried out in 37 patients distributed into two groups: cirrhotics with gastro-oesophageal varices that had never bled (Group A = 22), and cirrhotics who had had an intestinal bleed from "ruptured" gastro-oesophageal varices (Group B = 15). Combination of thrombocytopenia (less than 100 10(9)/l) and a bleeding time greater than 8 mn was more frequent in Group B (80%) than in Group A (45%) (p = less than 0.05). On the other hand, no significant difference between the two groups was found in the activated cephalin time, thrombin time, prothrombin complex factors (II, V, VII-X), fibrinogen, antithrombin III, Factor VIII complex factors, FDP levels or plasminogen. In conclusion, these results suggest that disorders of primary haemostasis may be involved in bleeding from gastro-intestinal varices in cirrhosis. However, coagulation disorders and anomalies of fibrinolysis would not seem to play a determining role.
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PMID:[Importance of disorders of primary hemostasis in the occurrence of upper digestive hemorrhage in cirrhosis]. 633 45

Factor VIII procoagulant protein (VIII:C) purified from commercial factor VIII concentrate contained multiple polypeptides ranging in mol wt from 79,000 to 188,000, all of which were removed from solution by a monoclonal anti-VIII:C antibody specific for a thrombin-sensitive epitope. In a time-course digest of the purified VIII:C using a trace amount of purified human alpha-thrombin, changes occurred in all VIII:C polypeptides during the activation and inactivation of VIII:C activity. The generation and destruction of a mol wt 92,000 polypeptide paralleled the increase and decrease in VIII:C activity, suggesting that this polypeptide represents an activated form. These results provide the basis for a working hypothesis for the mechanism of thrombin activation of VIII:C.
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PMID:Thrombin proteolysis of purified factor viii procoagulant protein: correlation of activation with generation of a specific polypeptide. 640 80

Loss of Factor VIII procoagulant activity (VIII:C) following blood collection is a major problem in providing sufficient amounts for therapeutic use and biochemical analyses. We have examined the effects of inhibition of plasma proteases and maintenance of physiological calcium ion on plasma VIII:C stability. The addition of protease inhibitors such as benzamidine, phenylmethylsulfonyl fluoride (PMSF), aprotinin, or soybean trypsin inhibitor (SBTI) to CPD plasma provided no significant protection against decay of VIII:C activity. Neither the rate of decay in the first 24 hours nor the final VIII:C activity observed after storage for 48-72 hours were significantly altered. On the other hand, addition of DFP or heparin to CPD plasma resulted in a marked improvement in VIII:C stability over 24 hours. This demonstrated that these two inhibitors are effective in preventing VIII:C degradation during storage. In addition to protease inhibition, the importance of maintaining physiological calcium ion was demonstrated by 100% stabilization of VIII:C in heparin plasma. Plasma obtained from CPD plus heparin blood could also be stabilized provided free calcium ion levels were restored to physiological concentrations. The inactivation of VIII:C in CPD plus heparin plasma was completely reversible up to 4 hours after collection. Studies on the recovery of activity after recalcification of CPD plus heparin plasma provided kinetic data which support a renaturation process of VIII:C rather than one due to enzymatic activation. The use of a thrombin-specific chromogenic substrate revealed that after recalcification and during the recovery of VIII:C activity, there was no significant thrombin activity. Although the data suggest that proteolytic degradation plays some part in VIII:C decay, only the maintenance of physiologic calcium ion levels under cover of an effective non-chelating anticoagulant and protease inhibitor allows preservation of VIII:C activity.
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PMID:Stability of VIII:C in plasma: the dependence on protease activity and calcium. 640 38

The interaction of human Factor VIII with platelets has been studied using discontinuous albumin gradient centrifugation. When purified Factor VIII complex was mixed with released platelets about 10% of the recovered Factor VIII activity became associated with the platelets. In the corresponding experiment with thrombin-activated Factor VIII about half of the Factor VIII activity was bound to the platelets. This binding was reversible; dilution of the platelet suspension containing bound Factor VIII resulted in dissociation of part of the Factor VIII activity. With non-released platelets very little binding of Factor VIII activity occurred. A mechanism for the in vivo formation of the Factor X activator complex is suggested.
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PMID:Binding of native and thrombin activated factor VIII to platelets. 641 25

Human Factor VIII associated von Willebrand factor (VIII:vWF) binds to human platelets in vitro only in the presence of a mediator such as ristocetin, thrombin or ADP. Studies reported here were designed to determine if human platelets will adhere to solid-phase VIII:vWF. Human VIII:vWF was purified from a phosphate precipitate of A1(OH)3 absorbed plasma using 4% agarose and DEAE cellulose. Purified VIII:vWF (90 units of VIII:vWF activity/mg) was coated on dialysis membranes using ultrafiltration (final concentration of 0.4 units/cm2). Membranes (0.5 cm2) were held stationary in human citrated PRP suspension or washed platelet suspensions and stirred continuously for 5 minutes at 37 degrees C. The membranes were then rinsed in phosphate buffered saline, fixed, stained, and examined by light and scanning electron microscopy. Abundant normal platelets adhered to VIII:vWF-coated membranes, while minimal adhesion was seen on uncoated membranes and membranes coated with albumin. Adhesion occurred without ristocetin, thrombin, ADP or other agonist and in the presence of Ca+2/Mg+2 ions. Preincubation of the VIII:vWF coated membranes with monospecific rabbit anti-VIII:vWF inhibited the adhesion reaction. However, preincubation of VIII:vWF coated membranes with naturally occurring human anti-FVIIIc antibodies failed to interfere with platelet adhesion. Platelets from a patient with Bernard-Soulier Syndrome (BSS) which did not bind human VIII:vWF in the presence of ristocetin or aggregate with bovine cryoprecipitate also did not adhere to VIII:vWF-coated membranes.
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PMID:Adhesion of human platelets to purified solid-phase von Willebrand factor: studies of normal and Bernard-Soulier platelets. 641 73

Heparin, sodium butyrate, and the absence of platelet derived factors in the nutrient medium are able to inhibit the decrease of the content of myosin in cultivated arterial smooth muscle cells. Factor VIII and thrombin have no significant influence on this process.
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PMID:[Effect of blood factors on the myosin content of cultured vascular smooth muscle cells]. 642 Oct 75

We have characterized Factor VIII coagulant protein, present in normal human plasma, that reacts with a specific human 125I-labeled anti-human VIII:C antigen Fab antibody fragment. Two major Factor VIII coagulant antigen populations were present. The first, approximately 85% of the total antigen, was bound to von Willebrand factor and when tested in a standard one-stage assay had Factor VIII coagulant activity. The second antigenic population, eluting near fibrinogen when plasma was gel filtered, was not bound to von Willebrand protein, did not have Factor VIII coagulant activity unless activated, but did block anti-VIII:C Fab neutralization of clotting activity. The two antigenic populations were separable by cryoprecipitation and agarose gel electrophoresis. Although the two antigenic populations differed in their Factor VIII coagulant activity and in their binding to von Willebrand factor, the principal member of both populations is of mol wt 2.4 X 10(5). Both antigens, when proteolyzed by thrombin, were quickly converted to a 1 X 10(5)-mol wt form in association with the appearance of VIII:C activity. The 1 X 10(5)-mol wt antigen was further slowly degraded to an 8 X 10(4)-mol wt form while Factor VIII coagulant activity declined. These results demonstrate the presence of an inactive Factor VIII coagulant protein in plasma, not associated with von Willebrand factor, that can react with thrombin to yield Factor VIII coagulant activity.
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PMID:Two distinct forms of Factor VIII coagulant protein in human plasma. Cleavage by thrombin, and differences in coagulant activity and association with von Willebrand factor. 642 75


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