摘要:

A recent report hypothesized that an Arg79±Gln mutation in the first epidermal growth factor-like domain of human factor VII is the molecular basis for a severe (< 1%) factor VII functional deficiency. In the present study, a site-specific mutant human factor VII cDNA (Arg79±GIn) was constructed, subcloned and expressed in baby hamster kidney cells. Mutant factor VII was purified to homogeneity and characterized with respect to -γ-carboxyglutamic acid content, ability to activate, tissue factor-dependent amidolytic activity and expression of factor VIIa proteolytic activity on tissue factor-bearing cells. Mutant factor VII was fully carboxylated and exhibited the same molecular weight and coagulant activity as plasma factor VII. Mutant factor VII was activated by factor Xa at the same rate, and to the same extent, as plasma factor VII. In the presence of tissue factor, mutant factor VII was converted to factor VIIa in an autocatalytic manner at a rate indistinguishable from that observed with plasma factor VII. In addition, the amidolytic activities of mutant factor VIIa and plasma factor VIIa towards S-2288 in the presence of relipidated tissue factor were identical. Finally, following complex formation with cell surface tissue factor, mutant factor VIIa activated factor X at essentially the same rate as plasma factor VIIa under comparable conditions. These results are not consistent with the notion that the arginine-79 residue in the first epidermal growth factor-like domain of human factor VII is essential for the expression of tissue factor-dependent factor VIIa proteolytic activity.

ISSN:0957-5235    

出版商:OVID    

年代:1992

数据来源: OVID

摘要:

Previous epitope mapping studies of human factor VIII (FVIII) inhibitor antibodies with heavy chain specificity localized epitopes to the amino-terminal half of the FVIII A2 domain. In this report we have used unidirectional deletion analysis and site-directed mutagenesis to identify a minimum length polypeptide and amino acid residues that contribute to the FVIII conformation recognized by these antibodies. Bacterial expression plasmids were exploited to demonstrate that a FVIII polypeptide of approximately 150 residues is required to generate a common heavy chain epitope(s). Another series of plasmids was constructed that synthesize: a FVIII polypeptide containing an internal deletion; four polypeptides with single residue substitutions; two polypeptides with triple residue changes; and a quadruple amino acid replacement within one polypeptide. The relative reactivities of the wild-type and mutant FVIII polypeptides were tested by immunoblotting, inhibitor neutralization assays and ELISA with a variety of human FVIII inhibitor auto- and alloantibodies. These techniques illustrate that the internal deletion mutant and one of the relatively conservative amino acid substitution triple mutants, mutant 389, resulted in significantly decreased immunoreactivity. The data identify FVIII Glu389,390,391as three critical components of an epitope for human FVIII inhibitor antibodies and identify a major inhibitory epitope involved in the immune response to FVIII.

ISSN:0957-5235    

出版商:OVID    

年代:1992

数据来源: OVID

摘要:

Each of three distinct, concentric layers of human arterial thrombi, was analysed immunochemically for the plasminogen and fibrin content, and for theex vivosusceptibility to thrombolysis by various thiombolytic agents in a saline or plasma milieu. The age of the thrombus layer determined: (a) the plasminogen content; (b) the fibrin content, inferred from the recovery of fibrin degradation products after complete lysis and from the binding of a monoclonal anti-fibrin antibody in a perfusion System, and (c) the lysibility of the thrombus. Plotting concentration of the various thrombolytic agents against percentage of lysis at several time points allows for reading of equivalent potencies of the respective units. Undiluted solutions of APSAC and rt-PA, prepared according to the manufacturer's directions, were less effective than diluted solutions, which has consequences for local therapy. Ail agents were at least as effective in saline as in a plasma milieu. We conclude that the plasminogen content of aged arterial thrombus sufficient for complete and rapid thrombolysis. Only after several months do fibrin and plasminogen become so far degraded or replaced that the thrombi become resistant to thrombolysis.

ISSN:0957-5235    

出版商:OVID    

年代:1992

数据来源: OVID

摘要:

The non-vitamin K-dependent coagulation factors fibrinogen, factor V, von Willebrand factor and factor XIII were measured in plasma of patients receiving oral anticoagulant treatment. Levels of fibrinogen, von Willebrand factor and factor XIII were above the upper normal limit in 60.0%, 58.6% and 53.4% of patients respectively. Factor V levels were below the lower normal limit in 56.3% of patients. Compared with a control group we found significant differences in the anticoagulated group for fibrinogen (P< 0.0001), factor V (P< 0.0001), von Willebrand factor (P< 0.0001) and factor XIII (P< 0.0001). It appears that the down-regulation of factors II, VII, IX and X during oral anticoagulation is accompanied by a subsequent up-regulation of fibrinogen, von Willebrand factor and factor XIII. This up-regulation or its absence could explain the thrombotic and bleeding complications seen in some patients on anticoagulant therapy and might also reduce the beneficial effect of oral anticoagulation in cardiovascular disease.

ISSN:0957-5235    

出版商:OVID    

年代:1992

数据来源: OVID

摘要:

α-polymer formation, as opposed to -γ-chain dimerization has been considered a relatively late event in factor XIII-induced fibrin stabilization. Recently it has been shown, however, that plasma from healthy individuals and from patients with fibrinaemia contains small amounts of soluble fibrin/fibrinogen oligomers interlinked through dimerized γ-chains as well as cross-linked α-chains. The present work was carried out to see if these early α-chain polymers also arise during coagulation of plasmain vitro.Plasma samples from healthy individuals, prepared by immediate centrifugation of blood collected without anticoagulant, were allowed to clot spontaneously for varying periods. The plasma clots were solubilized in SDS-urea-mercaptoethanol and samples were subjected to SDS-PAGE and Western blotting using polyclonal antibodies to human fibrinogen, or monoclonal antibodies specific either for Aα/α-chains, for fibrinopeptide A-containing chains, for the N-terminus of the fibrin β-chain or for the γ-chains. Fibrin/fibrinogen oligomers were seen to form long before visible gelation of plasma. These oligomers were cross-linked through -γ-chain dimerization, but also through Aα- or α-chain polymerization. The number and amount of α-polymers containing Aα-chains increased immediately after clot formation, but these disappeared about 20 min later, due to complete removal of fibrinopeptide A (FPA) by thrombin. It is concluded that α-polymer formation is a very early event during plasma coagulationin vitro, and that both Aα- and α-chains are involved.

ISSN:0957-5235    

出版商:OVID    

年代:1992

数据来源: OVID

摘要:

Studies were conducted to assess the effect of the serine protease inhibitor aprotinin on platelet adherence to both thrombin-stimulated and unstimulated human umbilical vein endothelial cells. Aprotinin treatment reduced significantly the adherence of platelets to endothelium pretreated or not with thrombin. In addition, aprotinin similarly reduced the adherence of platelets to plastic or collagen-coated tissue culture wells suggesting that the main site of action of the drug in this system is on the platelets. The role of endothelium-derived relaxing factor (EDRF; nitric oxide) in these platelet-endothelium reactions was investigated by prior incubation of both platelets and endothelial cells with NG-monomethyl-L-arginine (L-NMMA) which prevents the production of nitric oxide. The results demonstrated that nitric oxide was a significant inhibitor of the thrombin-induced platelet adherence in this assay system. Treatment with aprotinin in the presence or absence of L-NMMA reduced adherence of platelets to equivalent levels suggesting that aprotinin acts directly on the platelets via a mechanism that is EDRF-independent, to inhibit adherence.

ISSN:0957-5235    

出版商:OVID    

年代:1992

数据来源: OVID

摘要:

Citrated samples from 100 patients on i.v. heparin and 20 normal patients were tested with three batches each of three activated partial thromboplastin time (APTT) reagents: Thrombosil I (Ortho); Automated APTT (Organon Teknika) and Actin FSL (Baxter). The ratio of APTT over the geometric mean normal APTT for each heparinized sample was calculated. One batch of reagent arbitrarily chosen as a reference gave the ratios APTRREF(y). The remaining reagents to be standardized against the reference system gave the ratios APTRTEST(x). The best correlation between systems was given bylog y vs log x.Standard curves were prepared from the APTT ratios of the 20 normal patients and 65 of the heparinized samples. On plottinglogAPTRTESTvs log APTRREFthey intercept was close to zero soxwas expressed in terms of y using;log x= HSLlog y, where HSI (Heparin Sensitivity Index) = slope. The APTRTESTresults of the remaining 35 heparinized samples were transformed using; APTRTEST= (APTRTEST)HSI. APTRTRANSwas then compared to APTRREFto determine whether the transformation brought the results closer to the reference. We conclude that although some improvement was found by using the transform, it was not possible to mathematically relate APTT results due to a high degree of variation between results using different reagents. A standard APTT reagent for the monitoring of heparin therapy is recommended. A separate APTT reagent may be required for the screening of factor deficiencies and lupus anticoagulants.

ISSN:0957-5235    

出版商:OVID    

年代:1992

数据来源: OVID

摘要:

During a 3-year period we studied 393 adult patients (382 of whom were unrelated) with a history of acute venous thromboembolism. A congenital deficiency state known to predispose to thrombosis was found in 27.2%. Of these, most were due to deficiencies of protein C (9.2%), protein S (7.6%), antithrombin III (5%) or to increased plasma PAI-1 concentration (3.1%) which, in the absence of any known factor that predisposes towards thrombosis, results in a diminished fibrinolytic activity. There was a characteristic pattern between the age of onset (mean 34 years) of thrombosis and individual protein deficiency. Thrombosis appeared spontaneously in 73% of cases with recurrence in 80%. In contrast, in the remaining unrelated patients, 138 (35.1%) in whom venous thromboembolism was secondary and occurred at a mean age of 43 years, and in the other 140 (35.6%) who suffered thromboembolism spontaneously at a later age (mean age 55), there was no permanent protein deficiency state or alteration in fibrinolytic activity and thrombosis recurrence was lower (53.6% and 20.7% respectively). Of the 393 patients, deep vein thrombosis was the most common manifestation; however, in congenital thrombophilia, thrombosis of visceral vessels and Raynaud's syndrome (6%) were also detected.

ISSN:0957-5235    

出版商:OVID    

年代:1992

数据来源: OVID

摘要:

Verapamil, an inhibitor of calcium channels, was shown to inhibit PAF-induced platelet activation. In the presence of 50 μM Verapamil both thromboxane (Tx) formation and release of ATP from dense granules induced by 100 nM PAF was completely inhibited. This concentration of Verapamil only produced partial inhibition of PAF-induced aggregation. It also reduced the size of the PAF-induced calcium (Ca2+) transient demonstrated in Fura-2 loaded platelets. In the absence of extracellular Ca2+, following chelation by EGTA, PAF was still able to induce a Ca2+transient confirming the requirement of both intra and extracellular Ca2+for PAF-induced platelet activation. 100 μM Verapamil was able to completely abolish the calcium transient induced by low doses of PAF. These results further suggest that Verapamil is able to inhibit PAF-induced platelet activation by mechanisms apart from blocking Ca2+channels.

ISSN:0957-5235    

出版商:OVID    

年代:1992

数据来源: OVID

摘要:

We studied the natural inhibitors (NI) of blood coagulation and fibrinolysis in 50 patients with lupus anticoagulant (LA), in order to identify possible alterations of these NI, that could favour thrombotic manifestations. We found no statistically significant difference in antithrombin III, protein C and α2-antiplasmin between controls and patients with LA, irrespective of their clinical manifestations. We found an increase of plasminogen activator inhibitor (PAI,P< 0.001) and a decrease of free protein S (PS1,P< 0.001) and total protein S (PSt, 0.01 <P< 0.05) in the patients with LA when compared with the control group. We found no difference in the levels of NI between patients with thrombosis (n= 19) and without thrombosis (n= 31) nor between patients with (n= 25) or without thrombosis and/or foetal loss (n= 25). In contrast, we observed a decrease of PSfin women with foetal loss (n= 10) as compared with women without foetal loss (n= 22, 0.01 <P< 0.05) and a decrease of PS, when comparing 19 patients with systemic lupus erythematosus (SLE) with 31 patients without SLE (0.01 <P< 0.05). These findings show that the patients with LA had several abnormalities in the NI system, but there was no significant association between levels of PAI, PSf, PStand a history of thrombosis.

ISSN:0957-5235    

出版商:OVID    

年代:1992

数据来源: OVID