PubMed Health⌕ Search

Biomedical subjects

P L Turecek

Publications and source records attributed to P L Turecek.

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↗
PubMed2004

Measurement of von Willebrand factor cleaving protease (ADAMTS-13): results of an international collaborative study involving 11 methods testing the same set of coded plasmas.

BACKGROUND: ADAMTS-13 is a von Willebrand factor (VFW)-cleaving protease. Its congenital or acquired deficiency is associated with thrombotic thrombocytopenic purpura (TTP) and more rarely with the hemolytic uremic syndrome. We report on a survey evaluating 11 methods for ADAMTS-13 measurement performed in different labs. DESIGN: Two plasmas, one normal and one from a patient with familial TTP, were mixed at the co-ordinating center to prepare 6 plasmas with 0%, 10%, 20%, 40%, 80% and 100% ADAMTS-13 levels. Each plasma was aliquoted and assembled into sets of 60 (coded from 1 to 60), each containing 10 copies of the original 6 plasmas. Plasmas were frozen and shipped in dry ice to 10 labs with a common frozen reference plasma. Laboratories were asked to measure ADAMTS-13 with their methods. Results were sent to the coordinating center for statistical analysis. RESULTS: Of the 10 methods performed under static conditions 9 were quantitative and one was semiquantitative. One method performed under flow conditions evaluated the extent of cleavage of endothelial cell-derived ultralarge VWF string-like structures and expressed results as deficient, normal, or borderline. Linearity (expected-vs-observed levels), assessed as the squared correlation coefficient, ranged from 0.98 to 0.39. Reproducibility, expressed as the coefficient of variation for repeated measurements, ranged from < 10% to 83%. The majority of methods were able to discriminate between different ADAMTS-13 levels. The majority were able to detect the plasma with 0% level and some of them to discriminate between 0% and 10%. Overall the best performance was observed for three methods measuring cleaved VWF by ristocetin cofactor, collagen binding, and immunoblotting of degraded multimers of VWF substrate, respectively. The poor interlaboratory agreement of results was hardly affected by the use of the common standard. The method performed under flow conditions identified the plasmas with 0%, 10%, 20% and 40% activity as deficient in 7, 5, 1 and 3 of the 10 replicate measurements. The plasmas with 80% and 100% were identified as normal in all of the 10 replicate measurements. CONCLUSIONS: The survey shows varied performance, but supports an optimistic view about the reliability of current methods for ADAMTS-13.

ADAM Proteins↗
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↗
PubMed2002

Massive postoperative intramuscular bleeding in acquired von Willebrand's disease.

We describe a case of acquired von Willebrand's disease (vWD) associated with monoclonal gammopathy with undetermined significance (MGUS) in a 54-year-old man who was admitted with hemarthrosis and extensive thigh muscle hematoma following arthroscopic surgery and postoperative prophylaxis with low molecular weight heparin. Coagulation tests were compatible with acquired vWD: prolonged activated partial thromboplastin time (aPTT) (56.1 s), decreased levels of factor VIII coagulant activity (23%), low concentrations of von Willebrand's factor (vWF) antigen (13%), and undetectable ristocetin cofactor activity (<10%). Infusion of a vWF-containing factor VIII concentrate failed to normalize the plasma levels of vWF-related parameters. Only additional intravenous administration of immunoglobulins led to a transient normalization of ristocetin cofactor activity, vWF antigen, and factor VIII coagulant activity. While the spontaneous bleeding tendency in this case was mild, surgery and administration of prophylactic doses of low molecular weight heparin led to life-threatening bleeding.

Arthroscopy↗
PubMed2001

Preparation and properties of an alpha-1-protease inhibitor concentrate with high specific activity.

BACKGROUND AND OBJECTIVES: Because the current demand for alpha-1-protease inhibitor (A1PI) exceeds the available supply, we aimed to develop a process for purification of A1PI from plasma which would achieve the highest possible degree of purity, specific activity and yield. MATERIALS AND METHODS: A1PI was purified from Cohn fraction IV-1,4 using ethanol precipitation and Q-Sepharose chromatography. Ceramic hydroxyapatite chromatography was used as a final purification step. Two independent virus-inactivation procedures (chemical and vapour heating) were applied. RESULTS: The resulting A1PI had an unprecedented high specific activity. In addition, the process led to the discovery of a new isoform of A1PI in isoelectric focusing gels. CONCLUSION: The high specific activity of the A1PI preparation achieved with this process should allow a reduction of the A1PI total protein load necessary to achieve clinically relevant effects.

Blood Proteins↗
PubMed2001

Immunochemical determination of cellular prion protein in plasma from healthy subjects and patients with sporadic CJD or other neurologic diseases.

BACKGROUND: Creutzfeldt-Jakob disease is thought to be caused by conversion of cellular prion protein (PrP) from its soluble form (PrP(sen)) to a pathologic form (PrP(res)). The occurrence of a new variant of CJD has increased the demand for a rapid assay capable of detecting a theoretical risk of transmission of the disease by blood or plasma. STUDY DESIGN AND METHODS: A quantitative sandwich ELISA for routine screening was developed for analysis of PrP levels in plasma. The time-resolved dissociation-enhanced fluorescence technology allowed a detection limit in plasma samples of approximately 50 pg/mL. Levels of PrP(sen) were tested in plasma from 31 patients with CJD, from 11 patients with other neurodegenerative diseases, and from a control group of 200 healthy subjects. RESULTS: The assay recognized both PrP(sen) and pathologic PrP(res), but did not differentiate between the two isoforms. PrP(sen) levels were higher in plasma from both patient groups than in plasma from the control group: 27 of the 31 (87%) CJD patients and all patients with other neurodegenerative diseases had higher levels than the highest concentration found in the control group. No correlation was found between age and PrP level. No signal could be detected in the CJD samples after protease K digestion, indicating that all detected PrP was protease-sensitive and therefore not pathologic. CONCLUSION: These data suggest that soluble PrP(sen) in plasma samples might be useful as a surrogate marker for a broad spectrum of neurologic diseases as well as for CJD.

Adolescent↗
PubMed2001

Recombinant von Willebrand factor: preclinical development.

Von Willebrand factor (vWF) is a multimeric glycoprotein (GP) that attracts platelets to the site of vascular injury, mediates platelet-platelet interaction, and stabilizes factor VIII (FVIII) in the circulation. Quantitative and qualitative defects of vWF result in von Willebrand disease (vWD), manifested by modest to severe bleeding episodes. Substitution therapy, with plasma-derived FVIII/vWF complex concentrates, is used for patients suffering the more severe forms of vWD. Efficacy of these preparations is often unsatisfactory because inadvertent proteolytic degradation during the manufacturing process causes them to lack the hemostatically most active high-molecular-weight multimers. In contrast, recombinant vWF (r-vWF), which is constitutively expressed at high yields in Chinese hamster ovary (CHO) cells and secreted into the conditioned medium under perfusion fermentation in "protein-free" medium, has high-molecular-weight multimers of extraordinary structural integrity. Functional analysis has shown that r-vWF promotes ristocetin cofactor-mediated platelet aggregation, collagen interaction and FVIII binding, and platelet-collagen adhesion under shear stress. Infusing vWF-deficient animals with r-vWF corrected vWF concentration and reduced blood loss, subsequently stabilizing endogenous FVIII associated with the reduction of bleeding time. Compared with plasma-derived vWF preparations, r-vWF was found to have a prolonged half-life, further enhancing the potential value of r-vWF as a therapeutic agent for treating patients suffering from vWD.

Animals↗
PubMed2001

Blockade of CD40/CD40 ligand interactions prevents induction of factor VIII inhibitors in hemophilic mice but does not induce lasting immune tolerance.

Patients with severe hemophilia A frequently develop neutralizing anti-factor VIII antibodies after replacement therapy with factor VIII (FVIII). In a search for new strategies to induce immune tolerance against FVIII in these patients, we used a murine model of hemophilia A to investigate the importance of CD40/CD40 ligand (CD40L) interactions for the initiation of the anti-FVIII immune response. We focused our attention in particular on the induction of neutralizing anti-FVIII antibodies and the Th1/Th2 polarization of FVIII-specific T cells. The development of anti-FVIII antibodies was analyzed by ELISA systems (detection of total anti-FVIII antibodies) and Bethesda assays (determination of neutralizing anti-FVIII antibodies). Factor VIII-specific T cells were characterized by multiparameter flow cytometry and cytokine ELISAs for the detection of cytokine production in splenic CD4+ T cells after in vitro restimulation with FVIII. Hemophilic mice received four doses of FVIII and anti-CD40L antibody MR1 (24 h before FVIII). Subsequently mice received four doses of FVIII only. The induction of neutralizing anti-FVIII antibodies in hemophilic mice after treatment with human FVIII could be prevented completely by a blockade of CD40/CD40L interactions using MR1. Furthermore, FVIII-specific T-cell responses that included both Th1 and Th2 cells were suppressed when mice were treated with FVIII and MR1. The initial blockade of CD40/CD40L interactions was, however, not sufficient to induce a lasting immune tolerance against FVIII. The immune suppression was abolished and both neutralizing anti-FVIII antibodies and FVIII-specific T cells developed when treatment with FVIII was continued after the omission of MR1. In addition, there were no alterations in the Th1/Th2 polarization induced by the initial blockade of CD40/CD40L interactions.

Animals↗
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↗
PubMed2000

Involvement of low-density lipoprotein receptor-related protein (LRP) in the clearance of factor VIII in von Willebrand factor-deficient mice.

Factor VIII is tightly noncovalently linked to von Willebrand factor (vWF) in plasma with a stoichiometry of 1:50, and vWF deficiency results in secondary factor VIII deficiency, with accelerated clearance of factor VIII from the circulation. We used a murine model of severe von Willebrand disease (vWF knockout mice) to study the effect of a recombinant vWF/pro-vWF preparation (rpvWF) on factor VIII survival and to investigate whether low-density lipoprotein receptor-related protein (LRP) might be involved in the in vivo clearance of factor VIII in the absence of vWF. vWF-deficient mice received 70 U/kg rpvWF in the first series of experiments, and in a second series, 80 mg/kg receptor-associated protein (RAP) as a recombinant fusion protein to block the action of LRP. Factor VIII levels were measured at time 0, or 1 or 3 hours after administration of rpvWF or RAP. RAP induced a sustained rise in factor VIII levels comparable to that induced by rpvWF. In a third series, the preadministration of RAP resulted in a slower disappearance of factor VIII antigen (measured by an enzyme-linked immunosorbent assay specific for human factor VIII) after infusion of recombinant factor VIII. These findings suggest that the accelerated clearance of factor VIII seen in the absence of vWF may be a result of the involvement of LRP in factor VIII metabolism. (Blood. 2000;95:1703-1708)

Animals↗
PubMed2000

Sensitive single-stage PCR using custom-synthesized internal controls.

A new approach for an internally controlled PCR was developed using a custom-synthesized oligonucleotide as the internal control. Three different PCR setups demonstrated the usefulness of this approach: (i) the addition of the respective internal control to samples containing ssDNA virus Parvo B19; (ii) the co-extraction of plasma samples and the respective internal control for the detection of the ssDNA virus TTV; and (iii) the addition of the respective internal control to a crude lysate of tail pieces for the genotyping of FVIII knockout mice, demonstrating that this approach is also applicable for dsDNA.

Animals↗
PubMed2000

Deoxyribonucleic acid preparation in polymerase chain reaction genotyping of transgenic mice.

BACKGROUND AND PURPOSE: In an attempt to find a rapid and reproducible method for routine polymerase chain reaction genotyping of transgenic mice, two novel approaches were developed. METHODS: One approach allowed reproducible amplification from crude lysates of tail snips, using a thermally activated polymerase. In a second approach, for situations in which non-invasive techniques are necessary, oral swab specimens were amplified after DNA extraction by use of an isolation kit. Samples from 10 transgenic factor VIII knockout mice were genotyped after processing by use of these and other methods. RESULTS: False-negative results were not obtained by use of the two novel approaches. Despite their relative simplicity, both approaches yielded results comparable to those obtained by use of procedures known to be reliable, such as organic extraction and a DNA extraction kit. CONCLUSION: Both approaches are useful for PCR-amplification of DNA from mammalian sources.

Animals↗
PubMed2000

von Willebrand disease in a pediatric-based population--comparison of type 1 diagnostic criteria and use of the PFA-100 and a von Willebrand factor/collagen-binding assay.

Definitive diagnosis of type 1 von Willebrand Disease (VWD) remains a problem. Provisional consensus guidelines for the diagnosis of definite and possible type 1 VWD were prepared by the Scientific Subcommittee on von Willebrand factor (VWF) of the Scientific and Standardization Committee (SSC) of the International Society on Thrombosis and Haemostasis (ISTH) during the 1996 annual meeting for the specific purpose of further evaluation in retrospective and prospective studies by a Working Party on Diagnostic Criteria (1996 Annual Report of the SSC/ISTH Subcommittee on VWF). In the first phase of this study, we compared 2 definitions of type 1 VWD. each with 3 criteria: significant bleeding history, laboratory investigations, and family history. Using the ISTH consensus guidelines for type 1 VWD definition, significantly fewer patients were diagnosed with definite type 1 disease as compared to our "in house" Hospital for Sick Children (HSC) criteria (4 vs. 31). While we recognize that the provisional ISTH consensus guidelines were not intended for clinical use, we believe that the results of our studies are of interest and will assist in any future refinements to the ISTH guidelines. In the second phase of this study, we investigated the utility of 2 new tests, a laboratory screening test and a functional test, for VWD in our well characterized, pediatric-based population. The Platelet Function Analyzer (PFA-100) provides an in vitro measure of primary hemostasis under conditions of high shear, using disposable cartridges containing collagen and either epinephrine or ADP. All tested subjects with types 2 or 3 VWD had prolonged PFA-100 closure times (CTs) with both cartridge types (n = 17) and prolonged bleeding times (n = 14). In subjects with definite type 1 VWD, 20/24 (83%) had prolonged CTs with the collagen/ADP cartridge (19/24 (79%) with collagen/epinephrine), compared with 7/26 (27%) with prolonged bleeding times. In subjects with definite types 1, 2, or 3 VWD, collagen/ADP CTs were abnormal in 37/41 subjects, giving an overall sensitivity of 90%. With this high sensitivity, the PFA-100 is a better screening test for VWD than the bleeding time. We also tested a VWF collagen-binding assay (VWF:CBA) as a functional test for VWF, in comparison with the more routinely-used ristocetin cofactor assay (VWF:RC0). The VWF:CBA is based on an ELISA technique, which has the potential to be more reproducible than the VWF:RC0. We found that the VWF:CBA detected 43/49 (88%) subjects with definite types 1, 2, or 3 VWD, performing as well as the VWF:RC0, that detected 42/48 (88%). We also showed that, used in conjunction with VWF antigen levels, the VWF:CBA may be useful in classification of VWD subtypes.

Adolescent↗
PubMed2000

Characterization of antibodies induced by human factor VIII in a murine knockout model of hemophilia A.

To investigate the usefulness of factor VIII (FVIII) knockout mice as an animal model of hemophilia A, we characterized the antibody response in FVIII knockout mice to recombinant human FVIII, administered intravenously or subcutaneously with or without adjuvant, and compared results to those in normal mice. Anti-factor VIII antibodies were detected after both intravenous and subcutaneous administration, with the highest titers after subcutaneous administration plus adjuvant. Depending on the administration strategy. knockout mice formed antibodies more rapidly and developed higher titers of inhibitory antibodies (Bethesda) than normal mice, suggesting differences in epitope specificity. Blotting thrombin cleavage products separated by gel electrophoresis showed that both strains developed antibodies against the nonfunctional B domain as well as against functional domains of factor VIII. The antibodies were mainly of the IgG1 subclass and resembled type I antibodies in hemophilia A.

Animals↗