The Influence of Lepirudin, Bivalirudin and Dabigatran on the Calibrated Automated Thrombogram (CAT)

In a two centre study (laboratories in Diagnostica Stago and Biomnis) we compared the in vitro effect on thrombin generation (TG) of Dabigatran and Bivalirudin (reversible direct anti-IIa inhibitors) with that of Lepirudin (an irreversible direct anti-IIa inhibitor) spiked into normal pool plasma. The effect of Lepirudin, Bivalirudin and Dabigatran were evaluated in both centres using the CAT (Diagnostica Stago, France) TG method in a concentration ranges up to 5, 20 and 1 μg/mL respectively. Testing was done in triplicate and repeated over 2 days. To reduce assay variability both centres used the same reagents lots and the same normal pool plasma (George King, USA). The range of each drug tested extended well above the therapeutic range concentrations normally found in patient plasma (0.5 to 1.0 μg/mL, 5 to 10 μg/mL and 0.1 to 0.3 μg/mL respectively for Lepirudin, Bivalirudin and Dabigatran). To see the effect of increasing activation forces, TG was performed at 3 different final concentrations of Tissue Factor (TF) - 1, 5 and 20 pM. All reagents were used as recommended by the manufacturer (Thrombinoscope, The Netherlands). A prolongation in the lag time (LT) is observed with all 3 drugs with all 3 concentrations of TF, but this is more marked for Lepirudin and Bivalirudin than it is for Dabigatran. In the therapeutic range Dabigatran (at 5pM TF) shows both an increase in LT and a decrease in peak thrombin and the ETP. At low concentration of Bivalirudin or Lepirudin, there is a paradoxical increase in peak height, which is even more pronounced at low TF concentration. At 1pM TF, this paradoxical peak increase is also observed with Dabigatran. Results obtained in both laboratories are similar and complement our previous results and those reported elsewhere (1–4). The effect of Lepirudin and Bivalirudin on TG is different from that of Dabigatran. We also note that at lower TF concentration the anticoagulant effect on TG initiation is more intense but the test becomes less reproducible.

A Detailed Comparison of the Performance of the Standard versus the Nijmegen Modification of the Bethesda Assay in Detecting Factor VIII:C Inhibitors in the Haemophilia A Population of Canada

SummaryThe Bethesda assay is widely used to monitor the development and progress of Factor VIII:C inhibitors. Factor VIII stability in the substrate plasma (normal pool) is compromised by pH shift and reduction in protein concentration. Preliminary study, by Verbruggen and colleagues (8), suggested a reduction in spuriously positive assay results may result from buffering the normal pool plasma substrate with imidazole to pH 7.4 and substituting Factor VIII deficient plasma for imidazole buffer in the control incubation mix. These laboratory findings have now been confirmed by the performance of both the standard and the modified Bethesda assays in parallel on 877 patient samples screened during the Factor VIII:C Inhibitor Surveillance Program instituted following the conversion of all Canadian haemophilia A patients to recombinant Factor VIII. Although this study does not address the question of the clinical significance of spurious positive assays, these laboratory findings do support the conclusions of Verbruggen and the modified assay has recently been endorsed by the Factor VIII/IX Subcommittee of the SSC.

Crossed Immunoelectrofocusing Of Antithrombin III In Healthy And In Congenital And Acquired Deficiency

Isoelectrofocusing was carried out in LKB Multiphor apparatus with pH 4-6.5 carrier ampholites using poly- acrilamide gels. Specimens of human purified antithrombin, normal plasma pool, plasma with congenital and acquired AT- III deficiency were isoelectric focused. Purified Antithrombin showed two peaks. The isoelectric points of these antithrombin peaks were 4.6 and 4.9. Next the poly- acrilamide gel slabs were placed on glass plates coated with agarose containing 4% anti human antithrombin antibody; crossed electrophoresis was then carried out. Normal pool plasma exhibits two peaks, which correspond to the isoelectric focusing position of the purified AT-III. Plasmas of two families with congenital AT-III deficiency exhibit only one peak at pH lower. On the contrary plasmas of patients with acquired AT-III deficiency (hepatic cirrhosis, asparaginase therapy) exhibit two peaks. Considering that microheterogeneity is fundamentally due to a difference in glycosylation (Borsodi and Nerasimhan, Brit. J. Haemat. 1978, 39,121) it is possible to hypothesize a defect in glycosylation in congenital defect.

Binding of Ristocetin to Platelets

Ristocetin induces platelet agglutination in the presence of von Willebrand factor (VIIIR:WF), part of the factor VIII complex. The role of ristocetin in platelet agglutination is not yet. Clear. To follow the interaction of ristocetin with platelets and/or VIIIR:WF, ristocetin was labelled with[3H] by reductive methylation. [3H]-Ristocetin agglutinates platelets in the presence of VIIIR:WF in a manner indistinguishable from unlabelled ristocetin. The binding of[3H]-ristocetin to normal and chymotrypsin-treated platelets (which ore not agglutinated by ristocetin/VIIIR:WF) was studied in the presence and absence of VIIIR:WF (normal pool plasma and plasmas from patients with von Willebrand’s disease) and at non-agglutinating and agglutinating concentrations of ristocetin. Virtually no difference in binding was seen whether VIIIR:WF was present or not. Chymotrypsin-treated platelets do not bind less ristocetin than control platelets. A pronounced direct relationship was found between [3H]-ristocetin bound by normal platelets and total ristocetin concentration. This implies that at the higher concentrations of ristocetin either more binding sites are exposed or that aggregation of ristocetin occurs.