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)

We have purified factor VIII from a patient with moderately severe hemophilia A (FVIII, 4 U/dL; FVIII:Ag, 110 U/dL) and subjected the protein to Western blot analysis after time course activation with thrombin. The cross reacting material-positive (CRM+) FVIII has the normal distribution of heavy and light chains before thrombin activation, and, after incubation with the enzyme, appropriate cleavages are made at positions 740 and 1689. However, the normal thrombin cleavage at position 372 in the heavy chain of this molecule does not occur. This result is consistent with the demonstration in the patient's leukocyte DNA of a C to T transition in codon 372, leading to the substitution of a cysteine for an arginine residue at the heavy chain internal cleavage site. The severely impaired functional activity of this molecule confirms that the heavy chain of FVIII must be proteolysed in order to effect full cofactor activation in vivo. However, a threefold activation was detected when this protein was incubated with thrombin. No evidence of thrombin-mediated cleavage at position 336 in the heavy chain was detected, in contrast to the variant recombinant B domainless-molecule, FVIII 372-Ile, described by Pittman and Kaufman (Proc Natl Acad Sci USA 85:2429, 1988). Using gel permeation studies of the FVIII/von Willebrand factor (vWF) complex before and after thrombin activation, we have demonstrated that the 40 Kd A2 domain of wild type FVIII dissociates from vWF after cleavage by the enzyme. In contrast, incomplete dissociation was detected in the case of FVIII 372-Cys. We conclude that the functional defect in FVIII 372-Cys is a consequence of the resistance to proteolysis of the internal scissile bond in the heavy chain.
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PMID:Purification and characterization of factor VIII 372-Cys: a hypofunctional cofactor from a patient with moderately severe hemophilia A. 210 44

Hydroxyethyl starch (HES 450.000/0.7; Hespan 6.0 g/100 ml) was compared with standard crystalloid solutions in postoperative volume replacement in 20 patients undergoing routine orthopaedic surgery. The HES group showed no clinical evidence of haemorrhage and no laboratory evidence of significant haemostatic defects as assessed by standard coagulation tests, platelet aggregation and fibrinogen concentrations. There was a slight shortening in the thrombin time and a smaller increase in post-operative FVIII RAg and FVIII RCof levels in the HES group. HES is a safe and effective volume expander for postoperative use.
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PMID:The haemostatic effects of hydroxyethyl starch (HES) used as a volume expander. 241 87

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

In vitro, concentration-dependent effects of rt-PA on a range of coagulation and fibrinolytic assays in thawed plasma samples were investigated. In absence of a fibrinolytic inhibitor, 2 micrograms rt-PA/ml blood (3.4 micrograms/ml plasma) caused prolongation of clotting time assays and decreases of plasminogen (to 44% of the control value), fibrinogen (to 27%), alpha 2-antiplasmin (to less than 5%), FV (to 67%), FVIII (to 41%) and FXIII (to 16%). Of three inhibitors tested, a specific polyclonal anti-rt-PA antibody prevented interferences in all fibrinolytic and most clotting assays. D-Phe-Pro-Arg-CH2Cl (PPACK) enabled correct assays of fibrinogen and fibrinolytic parameters but interfered with coagulometric assays dependent on endogenous thrombin generation. Aprotinin was suitable only for a restricted range of both assay types. Most in vitro effects were observed only with rt-PA plasma concentrations in excess of therapeutic values. Nevertheless it is concluded that for clinical application, collection of blood samples on either specific antibody or PPACK is essential for a correct assessment of in vivo effects of rt-PA on the haemostatic system in patients undergoing fibrinolytic therapy.
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PMID:Comparison of specific antibody, D-Phe-Pro-Arg-CH2Cl and aprotinin for prevention of in vitro effects of recombinant tissue-type plasminogen activator on haemostasis parameters. 244 90

The effects of injury with hemorrhagic shock on the clotting and fibrinolytic systems were studied serially in 22 patients receiving 21 +/- 13 transfusions and 1.26 +/- 0.58 L of fresh frozen plasma (FFP) during operation (OR). The PT, aPTT, thrombin time (TT), fibrinogen (FI), factors V (FV) and VIII (FVIII), fibrin(ogen) split products (FSP) and fibrin monomers were measured in OR and after OR at 6 and 15 hours, days 2 and 4, and at convalescence (25 days). The TT, PT, and aPTT were were prolonged in OR and reflected the low FI, FV, and FVIII, respectively. After OR, clotting times and factor levels returned toward normal. By day 4 and convalescence, FI, FV, and FVIII exceeded normal levels. FSP levels were normal in OR. After OR FSP rose progressively through day 4 when all patients had levels greater than 10 mcg/ml and most patients had levels above 40 mcg/ml. Fibrin monomers were absent until the 15-hour study after which a small number of patients had monomers through the convalescent study. The acute fall in clotting factors is likely due to increased hemostatic demands, plasma dilution from resuscitation, and extravascular relocation from shock-induced extravascular expansion. Later factor restoration likely reflects enhanced hepatic synthesis, factor half-life, capillary selectivity retaining large molecular weight factors, and intravascular relocation from abundant extravascular stores. Throughout this biphasic response, the clotting times reflect factor levels. Fibrinolysis contributes little to these changes.
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PMID:The effect of hemorrhagic shock on the clotting cascade in injured patients. 281 Apr 19

This study compares the ability of unmodified and carbohydrate-modified forms of factor VIII/von Willebrand factor (FVIII/vWF) protein to bind to platelets in the presence of ristocetin or thrombin. Treatment of intact FVIII/vWF with alpha-D-neuraminidase results in more than 95% desialylation. Asialo FVIII/vWF retains total activity in ristocetin- and thrombin-mediated binding to platelets as demonstrated by direct and competitive binding assays. Examination of its multimeric pattern by sodium dodecyl sulfate-agarose electrophoresis reveals a normal multimeric structure. Treatment of intact FVIII/vWF with beta-D-galactosidase results in the removal of 20% of galactose (agalacto FVIII/vWF) whereas 55% of galactose is released from asialo FVIII/vWF (asialo agalacto FVIII/vWF). Agalacto and asialo-agalacto FVIII/vWF are both unable to bind to platelets in the presence of ristocetin. In contrast, they still bind to thrombin-stimulated human (except thrombasthenic) platelets. Removal of either ultimate (agalacto FVIII/vWF) or ultimate and penultimate (asialo-agalacto FVIII/vWF) galactose results in the same loss of the larger molecular weight multimers and in an increase of smaller multimers. These results suggest (1) that sialic acid does not play a significant role in ristocetin- or thrombin-mediated FVIII/vWF-platelets interactions and multimeric structure of FVIII/vWF (2) that ultimate beta-linked galactose residues are essential for the maintenance of a normal multimer organization (3) that ristocetin- and thrombin-mediated binding of FVIII/vWF to platelets differ in FVIII/vWF galactose requirement.
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PMID:Effect of carbohydrate modifications of factor VIII/von Willebrand factor on binding to platelets. 286 50

Thrombin generation, as evidenced by plasma fibrinopeptide A (FPA) concentrations, was studied during blood collection from donors taking oral contraceptives (OC). 450 ml blood were drawn into Fenwal PVC bags from 26 OC users and 28 nonusers. Blood samples for determination of FPA, beta-thromboglobulin (BTG), thrombotest (TT), prekallikrein (PKK), antithrombin-III (AT-III) and factor VIII procoagulant activity (FVIII:C) were drawn from the bags immediately after ending blood donation and following storage for 24 h at 4 degrees C. The FPA concentrations following donation were significantly higher in the OC than in the control group (p less than 0.05). The levels of PKK were also higher in blood obtained from OC users (p less than 0.001), as was the FVIII:C level, the latter difference, however, was not significant (p = 0.06). No cold-promoted activation of factor VII, as evidenced from TT, was detected following storage at 4 degrees C, neither was any change observed in the FPA, PKK and AT-III levels. The BTG concentrations increased significantly during storage, most pronounced in the control group (p less than 0.05). The decay of FVIII:C was similar in the two groups, averaging 24.7%. No correlation was observed between the FPA levels and the other parameters determined. We conclude that thrombin generation is more pronounced during routine blood collection from donors taking OC.
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PMID:Thrombin generation during collection of blood from donors taking oral contraceptives. 295 72

The binding capacity of radiolabelled human alpha-thrombin (alpha-thrombin), inactivated with phenyl-methyl sulphonyl fluoride (PMSF), to factor VIII related antigen (VIII:RAg) multimers isolated by immune precipitation and resolved by sodium dodecyl sulphate (SDS) agarose gel electrophoresis has been examined. 125I-PMSF alpha-thrombin bound predominantly to four low molecular weight multimers (LMW) of VIII:RAg prepared from either normal or haemophilia A plasma. Two of these VIII:RAg multimers present in serum had lost the capacity to bind thrombin. No bands were observed when either plasma or serum from three patients with severe von Willebrand's disease (vWd) was processed in an identical manner. This binding was not dependent on the presence of an intact catalytic site on the thrombin molecule. A similar pattern of binding to VIII:RAg multimers was observed with 125I-labelled human anti-FVIII antibodies as 125I-alpha-thrombin and appears to involve the same multimers.
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PMID:Binding of human thrombin to human factor VIII:RAg. 308 49

Purified human factor FVIII (FVIII; 6000-8000 U/mg) was radiolabeled and bound to immobilized von Willebrand factor (vWF). The complex was incubated with human thrombin. Thrombin induced a release of 65% of the radioactivity initially bound. Released FVIII fragments and fragments remaining bound during incubation with thrombin were analyzed using gel electrophoresis. This led to the following observations. Released fragments largely consisted of Mr-70000 and Mr-50000 fragments; Mr-90000 and Mr-80000 fragments were only found in the fractions remaining bound to vWF and decreased with time. In contrast to these digestion products of FVIII, the Mr-42000 heavy-chain fragment remained bound to vWF, comprising the larger part of the radioactivity after a 2-h incubation. No thrombin-induced cleavages were observed in vWF. Furthermore, vWF-coated wells preincubated with thrombin were still able to bind 125I-FVIII. These results implicate a new concept for the activation of vWF-bound FVIII. Activation is a multistep process in which several cleavages are necessary to produce and release a coagulant-active FVIII molecule (FVIIIa), which is probably an Mr-50000/70000 heterodimer. Inactivation of FVIIIa is likely to be the result of a nonproteolytic dissociation due to loss of the joining divalent cation(s).
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PMID:The effect of thrombin on the complex between factor VIII and von Willebrand factor. 311 51

In the Federal Republic of Germany fresh-frozen plasma (FFP) is still the most important therapeutic agent for acquired coagulation disorders. However, thawing by waterbath (WB) requires about 30 minutes, which is too slow in emergency situations and carries the risk of bacterial contamination of the FFP. There are conflicting data about the use of microwaves for thawing. Therefore, we examined a new microwave oven (MWO; 2450 +/- 50 MHz), which was developed with our cooperation and allows thawing of FFP in 5 minutes, heating FFP to a surface temperature of 21.5 degrees C. A shaking WB (30 min, 37 degrees C) was also used in parallel for comparison. We measured activated partial thromboplastin time (aPTT), nonactivated PTT (NaPTT), fibrinogen, factors VIII:C, X, and XI, fibrinopeptide A, beta-thromboglobulin (beta-TG), thrombin-AT III-complexes, factor VIII-related antigen, C3c, C4, and the plasticizer di(2-ethylhexyl)phthalate (DEHP) in 84 units of FFP as paired samples from 42 double aphereses. Immediately after thawing there was no significant difference in the coagulation test results of FFP with low-cell contamination, regardless of the thawing procedure. Two hours later, after storage at room temperature, FFP thawed by MWO showed even less change than that thawed by WB (NaPTT, p less than 0.01; FX, p less than 0.01). The differences became more evident in comparison with FFP with higher cell contamination and could be observed immediately after thawing (FVIII:C p less than 0.001; FXI, p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Thawing of fresh-frozen plasma with a new microwave oven. 319 32


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