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Biomedical subjects

K Váradi

Publications and source records attributed to K Váradi.

At least 19 recordsLinked to original sources
PubMed2004

FEIBA: mode of action.

FEIBA (factor eight inhibitor bypassing activity) has a history of more than 30 years of successful use in controlling bleeding in haemophilic patients who have developed inhibitory antibodies against factor (F)VIII or FIX. Recently it was shown that FEIBA contains the proenzymes of the prothrombin complex factors, prothrombin, FVII, FIX and FX, but only very small amounts of their activation products, with the exception of FVIIa, which is contained in FEIBA in greater amounts. FEIBA controls bleeding by induction and facilitation of thrombin generation, a process for which FV is crucial. A number of biochemical in vitro and in vivo studies have shown that FXa and prothrombin play a critical role in the activity of FEIBA. Consequently, they are considered to be key components of this product. The prothrombinase complex has been found to be a major target site for FEIBA. Apart from prothrombin and FXa, FEIBA contains other proteins of the prothrombin complex, which could also facilitate haemostasis in haemophilia patients with inhibitors.

Animals↗
PubMed2004

Thrombin generation assay and other universal tests for monitoring haemophilia therapy.

Factor VIII bypassing agents have different multiple modes of action, but share the common feature of inducing or facilitating thrombin generation. The information obtained from most overall assays to measure the haemostatic response to inhibitor-bypassing agents is limited to the initial phase of blood coagulation (clotting time measurement) or the formation of a solidifying clot followed by fibrinolysis (thrombelastography), and excludes the real endpoint of thrombin generation. Thrombin generation assays (TGAs) measure the whole kinetics of thrombin generation even after the clot formation, and thus assess all activating and inactivating systems of coagulation. Therefore, TGAs are not only becoming a universal tool to improve understanding of haemostasis and to investigate biochemical principles of the haemostatic system, but are also evolving as a powerful prognostic and monitoring tool for inhibitor bypassing therapies.

Blood Coagulation Factors↗
PubMed2003

Monitoring the bioavailability of FEIBA with a thrombin generation assay.

BACKGROUND: Hemophilia A patients with inhibitors are generally treated with preparations containing activated coagulation factors to achieve hemostasis by bypassing factor (F)VIII. OBJECTIVES: We developed an assay for monitoring the kinetic of thrombin generation in human FVIII inhibitor plasma reconstituted in vitro with activated prothrombin complex concentrate, FEIBA, and in plasma samples from hemophilia A patients taken after FEIBA treatment. PATIENTS AND METHODS: For pharmacokinetic studies three patients with severe hemophilia A and with a high-titer inhibitor received a single dose of FEIBA. Repeated FEIBA treatment was monitored in one patient with acquired hemophilia A. Coagulation was triggered in citrated plasma by adding a low concentration of tissue factor/phospholipid complex and CaCl2 in the presence of a fluorogenic thrombin substrate. The intensity of the fluorescence signal (FU) was continuously monitored, and the rate of increase in the fluorescence signal for every time point, which reflects the actual thrombin concentrations, was calculated. RESULTS: The maximum rate of substrate conversion, which indicates the highest thrombin concentration, was approximately 1900 FU min(-1) in a normal plasma pool. Practically no thrombin generation was observed in the FVIII inhibitor plasma, but when it was spiked with FEIBA, the rate and the peak of thrombin generation increased dose-dependently to close to normal. Plasma samples from FVIII inhibitor patients treated with a single dose of FEIBA had an improved thrombin maximum within an hour after treatment, which gradually returned to baseline values with a half-life of 4-7 h. Changes in the characteristic parameters of thrombin generation coincided with the repeated administration of FEIBA in a patient with acquired hemophilia A. CONCLUSIONS: This assay enables the pharmacodynamic and pharmacokinetic properties of bypassing therapies to be monitored, thus helping to optimize treatment.

Adult↗
PubMed2003

Factor VIII inhibitor-bypassing agents act by inducing thrombin generation and can be monitored by a thrombin generation assay.

Factor VIII (FVIII)-bypassing agents have complex modes of action but all control bleeding in inhibitor patients by triggering the generation of thrombin. No routine test is available for monitoring this therapy in patients with inhibitors against FVIII. We present an assay that records FEIBA- or FVIIa-mediated changes in thrombin generation (TG) in FVIII inhibitor plasma samples. In plasma samples spiked with FEIBA TG was normalized above 0.4 U/ml, while for recombinant FVIIa (rFVIIa) more than 12.5 microg/ml were required to induce TG in the absence of tissue factor (TF). Addition of TF increased the TG potential of rFVIIa in vitro. This assay seems suitable for monitoring the pharmacokinetics of inhibitor bypassing agents during treatment and possibly for predicting responses to treatment.

Autoantibodies↗
PubMed2001

Thrombin-mediated in vitro processing of pro-von Willebrand factor.

Von Willebrand factor (vWF) is synthesized in endothelial cells as pre-provWF and processed intracellularly to propeptide (vWFpp) and mature vWF. Building on previous studies indicating that recombinant provWF when infused into animals can also be processed extracellularly in vivo, we investigated the processing of provWF in vitro. Incubation of a recombinant provWF (rpvWF) preparation with canine and human vWF-deficient plasma induced a time-dependent decrease in provWF antigen and an increase in vWFpp antigen without changing total vWF antigen or collagen-binding activity. Multimer analysis showed the gradual transformation of the provWF multimers to mature vWF multimers and cleaved vWFpp was visualized on autoradiograms of SDS-polyacrylamide electrophoresis gels using 125-labeled provWF. Processing was facilitated by CaCl2 but prevented by a thrombin inhibitor and did not occur in prothrombin-depleted plasma. When recombinant provWF was incubated with increasing amounts of purified thrombin, the extent of provWF processing was dose-dependent. The specific cleavage of vWFpp was confirmed by immunoblots using an anti-vWFpp antibody and by amino terminal amino-acid analysis. Binding of provWF to collagen decreased the thrombin concentration necessary for propeptide removal to a concentration in the range of that found during blood clotting. Meizothrombin, an intermediate of prothrombin activation, was also able to induce dose-dependent removal of the propeptide from rpvWF. Hirudin preconditioning of vWF-deficient mice attenuated processing of infused rpvWF suggesting that thrombin plays a part in the processing events in vivo.

Animals↗
PubMed1999

Phospholipid-bound tissue factor modulates both thrombin generation and APC-mediated factor Va inactivation.

Hemostasis is initiated by tissue factor (TF) exposed on cellular phospholipid (PL) membranes, leading to thrombin generation. The binding of thrombin to thrombomodulin (TM), activates the protein C pathway, resulting in the inactivation of factors Va and VIIIa by activated protein C (APC) and a negative feedback effect on thrombin generation. A new assay system was developed for simultaneous measurement of thrombin and APC generation in defibrinated plasma induced by large unilamellar PL vesicles complexed with full-length recombinant TF (TF:PL). TF:PL preparations with a low TF concentration induced an initial rate of thrombin generation below 100 nM/min, and resulted in less thrombin formation in the presence of TM than in its absence. In contrast, TF:PL preparations with a high concentration of TF induced a higher rate of thrombin generation, and APC-mediated feedback inhibition did not occur, despite maximal APC generation. We used the same TF:PL surfaces to study factor Va inactivation by APC in a non-plasma reaction system, and found an inverse correlation between TF surface density and the rate of factor Va inactivation. This observation suggests a previously unrecognized hemostatic effect of TF, namely a non-enzymatic surface density-based inhibition of the anticoagulant effect of APC. In this model, high concentrations and surface density of TF exert complementary effects by promoting the regular procoagulant cascade and by inhibiting the protein C pathway, thereby maximizing hemostasis after vascular injury.

Animals↗
PubMed1996

Factor V enhances the cofactor function of protein S in the APC-mediated inactivation of factor VIII: influence of the factor VR506Q mutation.

Factor V and protein S are cofactors of activated protein C (APC) which accelerate APC-mediated factor VIII inactivation. The effects of factor V and protein S were quantitated in a reaction system in which plasma factor VIII was inactivated by APC and the loss of factor VIII activity was monitored in a factor X-activating system in which a chromogenic substrate was used to probe factor Xa formation. Factor V increased the rate of APC-mediated factor VIII inactivation in a dose-dependent manner in representative plasma samples with protein S or factor V deficiency, abnormal factor V (heterozygous or homozygous for factor VR506Q), or a combination of heterozygous protein S deficiency and heterozygous factor VR506Q. This effect was much less pronounced in the plasma samples with a decreased protein S level, but the impaired response in these plasmas was corrected by addition of protein S, indicating that both factor V and protein S are required for optimal inactivation of factor VIII by APC. The effects of factor V and protein S were also studied in a reaction system with purified proteins. APC-catalysed factor VIII inactivation was enhanced 3.7-fold in the presence of 1.1 nM factor V and 1.5-fold in the presence of 2.4 nM protein S. When both 1.1 nM factor V and 2.4 nM protein were present the rate enhancement was 11-fold. Factor V is a more potent cofactor than protein S, as can be concluded from the fact that 0.04 nM factor V gave the same stimulation as 2.4 nM protein S. Protein S lost its cofactor function after complexation with C4b binding protein, which indicates that it is free protein S that acts as a cofactor. To investigate the effect of the R506Q mutation in factor V on APC-mediated factor VIII inactivation, factor V was purified from the plasma of patients homozygous for factor VR506Q. In the absence of protein S, factor VR506Q did not enhance factor VIII inactivation by APC, but in the presence of 2.4 nM protein S a slight enhancement was observed. The APC cofactor activity of factor V was lost when factor V was activated with thrombin or with the factor V activator from Russell's viper venom. These data indicate that optimal inactivation of factor VIII by APC requires the presence of an intact factor V molecule and free protein S.

Arginine↗
PubMed1995

A chromogenic assay for activated protein C resistance.

Resistance to activated protein C (APC) diagnosed on the basis of prolongation of clotting time in an activated partial thromboplastin time (aPTT) assay is now considered a major cause of inherited thrombophilia. The majority of patients with APC resistance carry a factor V molecule with a point mutation at one APC cleavage site (Arg506Gln) which prevents the optimal inactivation of activated factor V by APC. To overcome the limitations of aPTT-based assays in the diagnosis of APC resistance, we have developed a chromogenic assay which is based on the capacity of APC to limit the generation of factor Xa by inactivating factor VIIIa in plasma. The ratio of the factor Xa amidolytic activity in a sample without APC to its factor Xa activity with the addition of APC reflects the response of the plasma coagulation system to APC. The normal range in 44 healthy individuals was 1.62-2.06. APC response ratios as measured by the chromogenic assay correlated with ratios measured by the aPTT assay and were below the normal range in 23/24 individuals with Arg506Gln mutant factor V from three different families with familial thrombosis and from 11 unrelated asymptomatic individuals. In reconstitution experiments, purified factor V corrected the decreased APC response in plasma samples from patients with the Arg506Gln mutation as well as with factor V deficiency, and increased the APC response in normal plasma, whereas the addition of activated factor V had no enhancing effect.

Anticoagulants↗
PubMed1994

[Menkes disease].

The Menkes or kinky hair disease is a rare, sex-linked systemic disorder of the copper metabolism. It is lethal in the first three years due to cerebral and cerebellar degeneration. Apropos of their case the authors summarise the knowledge on the diagnosis, pathogenesis and therapy.

Brain↗
PubMed1994

Activation and inactivation of human protein C by plasmin.

Transient procoagulant states resulting in failure of recanalization or rethrombosis of the reperfused artery during thrombolytic therapy might be due to an inhibitory effect of plasmin on the anticoagulant properties of protein C. We therefore studied the effect of plasmin on protein C (PC) and activated protein C (APC) using purified human proteins. Incubation of 70 nM purified human PC with 40-400 nM human plasmin resulted in rapid activation and subsequent inactivation of PC as measured by amidolytic and anticoagulant assays. The rates of activation and inactivation were dependent on the concentration of plasmin. Lower concentrations of plasmin resulted in higher peaks of generated APC and more sustained activity, while at higher concentrations, both activation and inactivation were more rapid. Anticoagulant activity appeared more sensitive to inactivation by plasmin than amidolytic activity; e. g., while amidolytic activity reached a maximum of 13.8 nM in 6 min and declined to approximately 6 nM after 30 min, anticoagulant activity reached its maximum of only 1.4 nM within 30 s and completely disappeared within 90 s. Plasmin rapidly destroyed both the anticoagulant and amidolytic activity of purified APC, with second order rate constants of 2.8 x 10(5) M-1 s-1 and 1.2 x 10(4) M-1 s-1, respectively, for 70 nM APC. The rates of activation and subsequent inactivation were slowed by the presence of CaCl2. The second order rate constant of inactivation of APC amidolytic activity decreased to 6.6 x 10(3) M-1 s-1 in the presence of 5 mM CaCl2.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium↗
PubMed1987

Formation and functioning of the factor IXa-VIII complex on the surface of endothelial cells.

The formation and functioning of the factor X activating complex on the surface of cultured human venous endothelial cells (HVEC) were investigated. To the HVEC monolayer human factors IXa, VIII, X, CaCl2, and S-2222 were added, and a gradually increasing activation of factor X was observed. The maximum activity of 88 nmol/L Xa/min was reached after a 12-minute lag phase. In the presence of thrombin-activated factor VIII (VIIIt) the same maximum activity developed in eight minutes, which suggests that VIIIt accelerates the formation of the IXa-VIII complex but does not influence its factor X-activating potential. Anti-VIII IgG did not affect the activity of the full-fledged complex. When anti-VIII IgG was added to the reaction mixture before factor VIII or during the lag phase of the reaction, it induced a concentration-dependent decrease of factor X activation. These results indicate that endothelial cells provide a binding surface for the IXa-VIII complex and that in the HVEC-bound complex factor VIII is protected from the effect of a specific antibody. However, the relatively slow development of the maximum activity indicates that HVEC only partially satisfy the surface criteria for the optimal assembly of the IXa-VIII complex.

Endothelium↗
PubMed1982

Protection of platelet surface bound factors IXa and VIII against specific inhibitors.

The rate of inactivation of factors IXa and VIII as well as of the complex formed by them on the surface of platelets, induced by specific inhibitors has been studied. After the formation of complex both factors became more protected against high molecular weight inhibitors. As evidenced by the apparent first order inactivation rate constants, after complex formation the velocity of IXa-antithrombin III + heparin reaction decreased to 1/20. Enhanced resistance toward the high molecular weight inhibitors is presumably due to a steric change, since the low molecular weight phenylmethyl sulfonyl fluoride and diethyl pyrocarbonate inhibited free and complexed factor IXa to the same extent.

Antibodies↗
PubMed1982

Effect of granulocyte proteases on human coagulation factors IX and X. The protective effect of calcium.

The inactivation of the zymogen and active forms of Factors IX and X by the effect of granulocyte proteases was investigated. As deduced from the apparent first-order inactivation rate constants, Factors IXa and Xa were about ten times as sensitive to proteolytic inactivation as the zymogen factors. In the presence of 2.5mM CaCl2 the rate of inactivation of zymogens decreased to one half. With the active factors, calcium caused complete resistance to proteolysis, prevailing for 15 min. The analysis of resistance to proteolysis, prevailing for 15 min. The analysis of resistance to proteolysis led us to the conclusion that the observed phenomenon was related to the calcium binding ability of the factors studied and can probably be explained by the protection of the active sites of enzymes.

Calcium Chloride↗
PubMed1980

Inactivation of human Factor VIII by granulocyte proteases.

The effect of human granulocyte proteases and related pancreatic enzymes on the procoagulant (VIII:C) and the von Wille-brand (VIIIR:WF) activities of factor VIII was investigated. VIII:C appeared to be several hundred times more sensitive towards granulocyute enzymes than VIIIR:WF. The elastase-like protease (ELP) decreased both activities of factor VIII more effectively than the chymotrypsin-like enzyme (CLP). ELP increased co-operatively the inactivating effect of CLP. In the presence of a CLP-ELP mixture in a ratio of 3:1, the rate of inactivation of VIII:C was seven times as high as that with CLP alone. Factor VIII was more resistant towards the damaging effect of pancreatic enzymes. The rate of inactivation of VIII:C was about a thousand times and that of VIIIR:WF about a hundred times lowere than those measured with the related granulocyte enzymes of the same proteolytic activity. The sensitivity of VIII:C towards proteolysis was not as pronounced with pancreatic enzymes as with granulocyte proteases. Our data suggest that, even though CLP and ELP are called "-like# enzymes, the specificity of granulocyte proteases is not identical with that of the pancreatic enzymes. The extreme sensitivity of VIII:C for granulocyte proteases may bring about blood coagulation disorders in certain pathological conditions.

Blood Coagulation↗