Hubert Metzner

In vitro inhibition of factor XIII retards clot formation, reduces clot firmness, and increases fibrinolytic effects in whole blood.

BACKGROUND: Thrombelastography has received renewed interest in the perioperative setting. The main determinants of thrombelastographic results are coagulation factor concentrations (various zymogens and fibrinogen) and platelet count; thus, platelet inhibition renders these assays mainly coagulation factor dependent. Assays with and without platelet inhibition are thus increasingly used to trigger and monitor replacement therapy with blood products. In this study, we evaluated the effect of factor XIII inhibition and additional glycoprotein (GP) IIb/IIIa blockade on (platelet-inhibited) whole blood thrombelastography and whether a modified routine assay (using factor XIII antibody) can be used to detect factor XIII deficiency. METHODS: Normal whole blood was incubated with increasing amounts of a nonspecific antibody, an anti-GPIIb/IIIa antibody, or a neutralizing anti-factor XIII antibody; samples were analyzed with a tissue factor-activated and platelet-inhibited whole blood thrombelastographic assay. Clotting time, clot formation time, maximum clot firmness, and clot lysis at 60 min were evaluated in triplicate. Also, 25 whole blood routine samples were evaluated for factor XIII deficiency using a new thrombelastographic assay incorporating a factor XIII antibody and using a standard factor XIII assay for comparison. RESULTS: Although GPIIb/IIIa inhibition did not alter the results of the platelet-inhibited whole blood thrombelastography, factor XIII inhibition significantly reduced maximum clot firmness (P = 0.020) and increased clot formation time (P = 0.025) and clot lysis (P = 0.007), leaving clotting time unchanged; a ceiling effect seemed to be present with increasing antibody concentrations in whole blood (but not plasma). The thrombelastographic assay for factor XIII deficiency (<70% activity) had a 90% sensitivity and negative predictive value (area under receiver operating characteristic curve 0.803, P = 0.0015); for a deficiency <60%, sensitivity and negative predictive value were 100% (area under receiver operating characteristic curve 0.84, P = 0.0037). CONCLUSION: Factor XIII has significant impact on platelet-inhibited activated whole blood thrombelastography. This phenomenon should be considered when interpreting thrombelastographic results in the bleeding patient, especially when the results trigger procoagulant therapy. Antibody-mediated factor XIII inhibition can be used to establish thrombelastography-based assays to detect factor XIII deficiency.

Preclinical efficacy and safety of rVIII-SingleChain (CSL627), a novel recombinant single-chain factor VIII

INTRODUCTION The preclinical efficacy and safety of rVIII-SingleChain (CSL627), a novel recombinant single-chain factor VIII, was assessed in a series of animal studies. MATERIALS AND METHODS In the tail-clip bleeding model, hemophilia A mice were injected with escalating doses (1-150 IU/kg) of rVIII-SingleChain, B-domain deleted (BDD) rFVIII (ReFacto AF(®)), or full-length rFVIII products (Advate(®), Helixate(®)). Total blood loss and the percentage of animals in which hemostasis occurred were assessed in this observer-blinded, randomized study. In a second non-randomized study in hemophilia A mice, thromboelastographic analysis, thrombin generation, and activated partial thromboplastin time assays were performed. General safety and toxicity were assessed in three animal species, including determination of the prothrombotic potential of rVIII-SingleChain in a rabbit venous thrombosis model. RESULTS Under acute bleeding conditions, the effect of rVIII-SingleChain on total blood loss and hemostasis was indistinguishable from BDD and full-length rFVIII. rVIII-SingleChain and full-length rFVIII (both 20 IU/kg) corrected thromboelastographic parameters, activated partial thromboplastin time, and thrombin generation to a similar degree in hemophilia A mice. In a thrombosis model, the effect of rVIII-SingleChain on thrombus incidence was non-significant and comparable to BDD rFVIII at doses up to 500 IU/kg. Treatment with rVIII-SingleChain did not cause anaphylactic reaction or local intolerance in safety and toxicity studies, and demonstrated an excellent overall safety profile. CONCLUSIONS rVIII-SingleChain showed convincing hemostatic efficacy and excellent tolerability in animal studies, warranting continued investigation in human Phase I/III trials (AFFINITY).

Non-clinical pharmacokinetics and pharmacodynamics of rVIII-SingleChain, a novel recombinant single-chain factor VIII

INTRODUCTION rVIII-SingleChain (CSL627), a novel recombinant coagulation factor VIII (FVIII), is under investigation in a phase I/III clinical programme (AFFINITY) for the treatment of haemophilia A. Non-clinical studies were conducted to investigate the pharmacokinetic/pharmacodynamic profile of rVIII-SingleChain in comparison with full-length recombinant FVIII. MATERIALS AND METHODS Binding affinity of rVIII-SingleChain for von Willebrand factor was investigated by surface plasmon resonance analysis. The pharmacokinetic profile of rVIII-SingleChain was compared with a marketed full-length recombinant FVIII concentrate (Advate(®)) in haemophilia A mice, von Willebrand factor knock-out mice, Crl:CD (SD) rats, rabbits and cynomolgus monkeys. Systemic FVIII activity or antigen levels were recorded. Procoagulant activity was measured in an FeCl3-induced arterial occlusion model and by recording thrombin generation activity (ex vivo) after administration of 200-250 IU/kg rVIII-SingleChain or full-length FVIII to haemophilia A mice. RESULTS rVIII-SingleChain displayed a high affinity for von Willebrand factor (KD=44 pM vs. 139 pM for full-length recombinant FVIII). In all animal species tested, rVIII-SingleChain had more favourable pharmacokinetic properties than full-length recombinant FVIII: clearance was decreased and area under the curve and terminal half-life were enhanced vs. full-length recombinant FVIII, while in vivo recovery and volume of distribution were equivalent. rVIII-SingleChain showed a prolonged thrombin generation potential and prolonged procoagulant activity vs. full-length recombinant FVIII in an FeCl3-induced arterial occlusion model. CONCLUSIONS rVIII-SingleChain had a higher affinity for von Willebrand factor than full-length recombinant FVIII and displayed favourable pharmacokinetic/pharmacodynamic properties in non-clinical models.

Physicochemical characterisation of rVIII-SingleChain, a novel recombinant single-chain factor VIII

rVIII-SingleChain is a novel recombinant single-chain factor VIII (FVIII) construct, comprising covalently bonded heavy and light chains. Post-translational modifications of FVIII affect physicochemical parameters, including hydrophobicity and charge. The most relevant post-translational modifications of FVIII products are N-glycosylation of asparagine residues and tyrosine sulphations. Here, the physicochemical properties, thrombin cleavage products and post-translational modifications of rVIII-SingleChain were investigated and compared against commercially available recombinant FVIII (rFVIII) products with a predominant two-chain structure (B-domain deleted rFVIII and full-length rFVIII). rVIII-SingleChain was expressed in Chinese hamster ovary (CHO) cells and purified by chromatographic methods. Physicochemical properties of rVIII-SingleChain or thrombin-derived cleavage products were assessed using size-exclusion chromatography, reversed-phase chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis. Analysis of the respective carbohydrate structures was performed after release of N-glycans by PNGase F followed by fluorescence labelling and high-performance liquid chromatography. Proteolysis by trypsin generated the corresponding peptides, which were analysed for sulphated tyrosines by liquid chromatography-electrospray ionisation time of flight-mass spectrometry. rVIII-SingleChain was shown to be of high purity and homogeneity, and presented a well-defined single-chain molecule with predominant β-sheet conformation. The coagulation-relevant thrombin-activation products of rVIII-SingleChain were comparable with those obtained by activation of commercially available rFVIII products. rVIII-SingleChain post-translational modifications were similar to other CHO cell-derived rFVIII products for N-glycopattern and tyrosine sulphation. In conclusion, rVIII-SingleChain is of high homogeneity and purity, and provides an expected cleavage pattern on activation, setting the basis for optimal efficacy in the patient.