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)

A chromogenic factor IX assay is developed which requires only two time-dependent steps. Diluted plasma is mixed with a reagent containing factors VIII and X. The reaction is started by addition of a reagent containing factor XIa, thrombin, CaCl2, and phospholipids. Then factor XIa activates factor IX if present, thrombin activates factor VIII, and subsequently the complete factor X activating complex (factor IXa, factor VIIIa, Ca ions, and phospholipids) rapidly activates factor X. Finally, ethylenediaminetetraacetic acid plus a chromogenic substrate are added to stop the reaction and to measure formed factor Xa. Factor Xa formation is proportional to the plasma factor IX concentration (from 0 to 140%). The two reagents needed for the assay are stable at room temperature during a whole working day and for 3 h at 37 degrees C. A new isolation procedure for factor VIII is described. Factor VIII is purified from bovine plasma in a few steps with a yield of 20% and a 8,000-fold purification.
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PMID:Development of a sensitive and rapid chromogenic factor IX assay for clinical use. 212 37

The influence of low molecular weight (LMW) heparin (Braun 21-23, Mulsungen, West Germany) and unfractionated standard heparin (SH) on blood clotting and other routine laboratory parameters was investigated in a 30 week cross-over study in 30 hemodialysis patients. The LMW heparin dose necessary (anti FXa-activity) for effective anticoagulation was two thirds of the standard heparin dose. Using these doses, both substances displayed identical antithrombotic effects. Complications were not seen in either group. PTT and thrombin time were only marginally effected by LMW heparin, whereas they were markedly prolonged by SH heparin. Factor VIII activity was significantly lower in the LMW heparin group as compared to the standard heparin group after 18, 24, and 30 weeks. Antithrombin III, fibrinogen, fibrin monomers, plasminogen, and alpha 2-antiplasmin were comparable in both groups. Creatinine, urea, hemoglobin, and hematocrit were also unchanged, excluding differences in dialysis efficacy or occult blood loss. Equal numbers of blood transfusions were necessary, but bleeding complications did not occur in either group. In conclusion, safe and effective dialysis can be performed using this low molecular weight heparin for anticoagulation in hemodialysis and hemofiltration. The possible benefits of LMW heparin (reduced frequency of bleeding, alleviation of hypertriglyceridemia) were not, however, apparent, possibly because of the short observation period and the low incidence of hemorrhagic complications in routine dialyses.
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PMID:Low molecular weight heparin versus standard heparin. A long-term study in hemodialysis and hemofiltration patients. 215 17

The human Factor VIII procoagulant protein (VIII:C) purified from commercial Factor VIII concentrate consisted of a polypeptide doublet of 80,000 mol wt, a 92,000-mol wt polypeptide, and additional polypeptides of up to 188,000 mol wt. Thrombin digests contained a doublet of 72,000 mol wt, as well as 54,000- and 44,000-mol wt fragments. Proteolysis studies of purified VIII:C using thrombin and activated protein C have suggested that the 92,000- and 80,000 (or 72,000)-mol wt polypeptides comprise activated VIII:C. We have now used seven monoclonal antibodies raised against purified VIII:C to construct a preliminary epitope map of these VIII:C polypeptides. The specific VIII:C polypeptides with which the monoclonal antibodies reacted were determined by immunoblotting of VIII:C onto nitrocellulose sheets after reduced NaDodSO4-polyacrylamide gel electrophoresis. A minimum of five distinct epitopes were defined by these monoclonal anti-VIII:C antibodies. Identification of polypeptides bearing these epitopes allowed localization of distinct thrombin cleavage sites to the 92,000- and 80,000-mol wt chains, helped define polypeptide chain precursor-product relationships, and suggested that both the 92,000- and 80,000-mol wt polypeptides are necessary for VIII:C function. These data and their interpretation are consistent with the published description of the complete primary structure of VIII:C and its thrombin cleavage products. The 92,000- and 80,000-mol wt chains have been located at the amino- and carboxy-terminal ends of the molecule, respectively.
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PMID:Human factor VIII procoagulant protein. Monoclonal antibodies define precursor-product relationships and functional epitopes. 241 Apr 56

Thrombomodulin (TM) is a newly described endothelial cell-associated protein that functions as a potent natural anticoagulant by converting thrombin from a procoagulant protease to an anticoagulant. Various vascular tumors were characterized with immunoperoxidase staining with the use of a polyclonal anti-TM serum. The staining patterns of TM were compared with those of Factor VIII-related antigen (FVIII-RAG) and Ulex europaeus agglutinin-I (UEA-I), which have been used as markers for endothelial cells. The results showed that TM is a specific and a highly sensitive marker for angiosarcomas in comparison with FVIII-RAG or UEA-I. In contrast, UEA-I is more sensitive for benign vascular tumors than TM or FVIII-RAG. The other mesenchymal tumors of nonvascular origin showed negative staining for three endothelial markers. These results indicate that TM is a new specific and sensitive tool for the diagnosis of angiosarcomas.
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PMID:Thrombomodulin as a marker for vascular tumors. Comparative study with factor VIII and Ulex europaeus I lectin. 244 96

Hydroxyethyl starch (HES) is a recently developed synthetic volume expander. Forty patients undergoing coronary artery surgery were randomized to receive either HES or plasma protein fraction (PPF) as non-blood volume replacement according to standard haemodynamic criteria. The two groups were comparable in all respects. The median colloid use in the first 24 h was 950 ml (range 500-1500) in the HES group and 975 ml (350-2000) in the PPF group (not significant). There was no difference in blood use, urine output or blood loss between the two groups. Tests of coagulation showed the postoperative changes usual in cardiac surgical patients. There was no difference between the two groups in thrombin time, prothrombin time, activated partial thromboplastin time, or fibrinogen concentration. Similarly, tests of platelet function and Factor VIII and von Willebrand Factor activity showed no difference between the two groups. We conclude that HES is a safe and effective volume expander, and its relative lack of expense and ease of availability make its routine use after cardiac surgery an attractive proposition.
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PMID:Hydroxyethyl starch: an alternative to plasma for postoperative volume expansion after cardiac surgery. 245 59

The epitopes of four monoclonal antibodies against coagulation Factor VIII were mapped with the use of recombinant DNA techniques. Full-length Factor VIII cDNA and parts thereof were inserted into the vector pSP64, permitting transcription in vitro with the use of a promoter specific for SP6 RNA polymerase. Factor VIII DNA inserts were truncated from their 3'-ends by selective restriction-enzyme digestion and used as templates for 'run-off' mRNA synthesis. Translation in vitro with rabbit reticulocyte lysate provided defined radiolabelled Factor VIII fragments for immunoprecipitation studies. Two antibodies are shown to be directed against epitopes on the 90 kDa chain of Factor VIII, between residues 712 and 741. The 80 kDa chain appeared to contain the epitopes of the other two antibodies, within the sequences 1649-1778 and 1779-1840 respectively. The effect of antibody binding to these sequences was evaluated at two distinct levels within the coagulation cascade. Both Factor VIII procoagulant activity and Factor VIII cofactor function in Factor Xa generation were neutralized upon binding to the region 1779-1840. The antibodies recognizing the region 713-740 or 1649-1778, though interfering with Factor VIII procoagulant activity, did not inhibit in Factor Xa generation. These findings demonstrate that antibodies that virtually inhibit Factor VIII in coagulation in vitro are not necessarily directed against epitopes involved in Factor VIII cofactor function. Inhibition of procoagulant activity rather than of cofactor function itself may be explained by interference in proteolytic activation of Factor VIII. This hypothesis is in agreement with the localization of the epitopes in the proximity of thrombin-cleavage or Factor Xa-cleavage sites.
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PMID:Inhibition of human coagulation factor VIII by monoclonal antibodies. Mapping of functional epitopes with the use of recombinant factor VIII fragments. 248 38

An influence of the ABO blood group on von Willebrand and Factor VIII:C levels is known. Von Willebrand factor interacts with at least two platelet membrane receptors, but the effect of ABO group on platelet function is an unstudied area. The authors examined platelet function in 40 plateletpheresis donors using an impedance lumi-aggregometer. Aggregation responses to collagen, adenosine diphosphate (ADP), and ristocetin were measured and the adenosine triphosphate (ATP) release to thrombin. Twenty donors were Group O and 20 were Group A. Measurements of von Willebrand factor antigen (vWf:Ag), Factor VIII: C, and ristocetin co-factor (RiCoF) in the same group showed reduced levels of vWf:Ag and Factor VIII:C in Group O, as previously reported. The aggregation response to collagen and ADP and the release of ATP did not differ. The aggregation response to ristocetin, however, was better in Group O than in Group A despite the lower vWf:Ag levels. The explanation for this is unclear, but the data suggest an influence of blood group antigens on the interaction between von Willebrand's factor and platelets.
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PMID:Platelet function and ABO blood group. 249 26

Factor VIII (fVIII) is synthesized as a single chain having a domainal sequence A1-A2-B-A3-C1-C2. Analysis of the proteolyic cleavage of fVIII by thrombin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) identifies three fragments designated fVIIIA1, fVIIIA2, and fVIIIA3-C1-C2 with fragment(s) derived from the B domain being difficult to visualize. The appearance of these fragments is associated with the development of coagulant activity, but the activity is labile without further apparent proteolysis. In this study, porcine fVIII was reacted with thrombin until peak coagulant activity was obtained and then subjected to cation-exchange (Mono S) high-pressure liquid chromatography. Coagulant activity was recovered in a single peak that contained all three fragments and was stable for weeks at 20 degrees C in 0.65 M NaCl/0.01 M His-HCl/0.005 M CaCl2 at pH 6.0. Analytical ultracentrifugation of activated fVIII was done to test whether all three fragments were associated. The apparent molecular weight of activated fVIII from equilibrium sedimentation increased from 148,000 to 161,000 as the loading concentration was increased from 0.06 to 0.16 mg/mL. This agrees well with the summed apparent molecular weights of fVIIIA1, fVIIIA2, and fVIIIA3-C1-C2 calculated from SDS-PAGE analysis (148,000) or from the amino acid sequence of human fVIII (159,000). This establishes the major species in the preparation as a fVIIIA1/A2/A3-C1-C2 heterotrimer and additionally indicates either weak self-association of the trimer and/or incomplete association of the individual subunits to form the trimer. Velocity sedimentation of activated fViii revealed a single boundry (S020,w = 7.2 S).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Subunit structure of thrombin-activated porcine factor VIII. 249 50

We have purified the factor VIII from a CRM+ Hemophilia A plasma (90 U/dL VIII:Ag but 0 U/dL VIII:C) and analyzed the protein before and after thrombin activation by Western blotting with monoclonal antibodies (MoAbs). Normal or patient citrated plasma was ultracentrifuged, cryo-ethanol-precipitated and chromatographed on Sepharose 6B. The void volume fractions were reduced and subjected to ion exchange chromatography yielding material of specific activity approximately 1,000 U/mg protein (VIII:C or VIII:Ag). Factor VIII purified in this way from normal plasma is fully activatable by thrombin with proteolytic fragmentation as previously described by F. Rotblat et al (Biochemistry 24: 4294, 1985). Factor VIII 1,689-Cys has the normal distribution of factor VIII light and heavy chains prior to thrombin activation. After exposure to thrombin the heavy chain polypeptides were fully proteolysed but the light chain was totally resistant to cleavage. This is consistent with the demonstration in the patient's leucocyte DNA of a C to T transition in codon 1,689 converting Arg to Cys at the light chain thrombin cleavage site as previously described by J. Gitschier et al (Blood 72:1022, 1988). Uncleaved light chain of Factor VIII 1,689-Cys is not released from von Willebrand factor (vWF) by thrombin, but this is not the sole cause of the functional defect since the protein purified free of vWF has no coagulant activity. We conclude that the functional defect in factor VIII 1,689-Cys is a consequence of failure to release the acidic peptide from the light chain upon thrombin activation.
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PMID:Purification and characterization of factor VIII 1,689-Cys: a nonfunctional cofactor occurring in a patient with severe hemophilia A. 249 63

Factor VIII was purified 1200-fold from commercial concentrates (Centre National de Transfusion Sanguine) by immunoaffinity chromatography using an anti-(80-kDa light chain) monoclonal antibody. The different molecular forms isolated were subsequently separated and analyzed using Fast Protein Liquid Chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis. The different factor-VIII forms obtained, consisted of 80-kDa light chains, each being associated with one more-or-less fragmented heavy chain ranging over 90-210 kDa. The specific activity of these different forms was 7000 U/mg. At different stages of activation of factor VIII by thrombin, various forms were separated and identified. Activated complexes were found to result from the association of the 70-kDa light chain (generated from the 80-kDa light chain) with heavy chains ranging over 90-210 kDa. Two different thrombin activation steps were characterized. The first step corresponding to the cleavage of the 80-kDa light chain led to a sixfold increase in the procoagulant activity, and yielded a stable activated intermediate form. Compared with normal factor VIII, the ratio of von Willebrand activity to factor-VIII activity, measured in the activated fractions, decreased, indicating that von Willebrand factor dissociates from factor VIII after proteolysis of the light chain by thrombin. In the second step, the 90-kDa heavy chain was cleaved into two polypeptides of 45 kDa and 50 kDa, which were associated with the 70-kDa proteolyzed light chain, generating the final activated complex (45-50-70 kDa). The new intermediate forms we described imply a new scheme for the multistep activation process of factor VIII.
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PMID:Isolation and characterization of different activated forms of factor VIII, the human antihemophilic A factor. 250 2


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