K. John Pasi

Phase 3 study of recombinant factor VIII Fc fusion protein in severe hemophilia A

This phase 3 pivotal study evaluated the safety, efficacy, and pharmacokinetics of a recombinant FVIII Fc fusion protein (rFVIIIFc) for prophylaxis, treatment of acute bleeding, and perioperative hemostatic control in 165 previously treated males aged ≥12 years with severe hemophilia A. The study had 3 treatment arms: arm 1, individualized prophylaxis (25-65 IU/kg every 3-5 days, n = 118); arm 2, weekly prophylaxis (65 IU/kg, n = 24); and arm 3, episodic treatment (10-50 IU/kg, n = 23). A subgroup compared recombinant FVIII (rFVIII) and rFVIIIFc pharmacokinetics. End points included annualized bleeding rate (ABR), inhibitor development, and adverse events. The terminal half-life of rFVIIIFc (19.0 hours) was extended 1.5-fold vs rFVIII (12.4 hours; P < .001). Median ABRs observed in arms 1, 2, and 3 were 1.6, 3.6, and 33.6, respectively. In arm 1, the median weekly dose was 77.9 IU/kg; approximately 30% of subjects achieved a 5-day dosing interval (last 3 months on study). Across arms, 87.3% of bleeding episodes resolved with 1 injection. Adverse events were consistent with those expected in this population; no subjects developed inhibitors. rFVIIIFc was well-tolerated, had a prolonged half-life compared with rFVIII, and resulted in low ABRs when dosed prophylactically 1 to 2 times per week.

AAV5–Factor VIII Gene Transfer in Severe Hemophilia A

Background Patients with hemophilia A rely on exogenous factor VIII to prevent bleeding in joints, soft tissue, and the central nervous system. Although successful gene transfer has been reported in patients with hemophilia B, the large size of the factor VIII coding region has precluded improved outcomes with gene therapy in patients with hemophilia A. Methods We infused a single intravenous dose of a codon‐optimized adeno‐associated virus serotype 5 (AAV5) vector encoding a B‐domain–deleted human factor VIII (AAV5‐hFVIII‐SQ) in nine men with severe hemophilia A. Participants were enrolled sequentially into one of three dose cohorts (low dose [one participant], intermediate dose [one participant], and high dose [seven participants]) and were followed through 52 weeks. Results Factor VIII activity levels remained at 3 IU or less per deciliter in the recipients of the low or intermediate dose. In the high‐dose cohort, the factor VIII activity level was more than 5 IU per deciliter between weeks 2 and 9 after gene transfer in all seven participants, and the level in six participants increased to a normal value (>50 IU per deciliter) that was maintained at 1 year after receipt of the dose. In the high‐dose cohort, the median annualized bleeding rate among participants who had previously received prophylactic therapy decreased from 16 events before the study to 1 event after gene transfer, and factor VIII use for participant‐reported bleeding ceased in all the participants in this cohort by week 22. The primary adverse event was an elevation in the serum alanine aminotransferase level to 1.5 times the upper limit of the normal range or less. Progression of preexisting chronic arthropathy in one participant was the only serious adverse event. No neutralizing antibodies to factor VIII were detected. Conclusions The infusion of AAV5‐hFVIII‐SQ was associated with the sustained normalization of factor VIII activity level over a period of 1 year in six of seven participants who received a high dose, with stabilization of hemostasis and a profound reduction in factor VIII use in all seven participants. In this small study, no safety events were noted, but no safety conclusions can be drawn. (Funded by BioMarin Pharmaceutical; ClinicalTrials.gov number, NCT02576795; EudraCT number, 2014‐003880‐38.)

Targeting of Antithrombin in Hemophilia A or B with RNAi Therapy

Background Current hemophilia treatment involves frequent intravenous infusions of clotting factors, which is associated with variable hemostatic protection, a high treatment burden, and a risk of the development of inhibitory alloantibodies. Fitusiran, an investigational RNA interference (RNAi) therapy that targets antithrombin (encoded by SERPINC1), is in development to address these and other limitations. Methods In this phase 1 dose‐escalation study, we enrolled 4 healthy volunteers and 25 participants with moderate or severe hemophilia A or B who did not have inhibitory alloantibodies. Healthy volunteers received a single subcutaneous injection of fitusiran (at a dose of 0.03 mg per kilogram of body weight) or placebo. The participants with hemophilia received three injections of fitusiran administered either once weekly (at a dose of 0.015, 0.045, or 0.075 mg per kilogram) or once monthly (at a dose of 0.225, 0.45, 0.9, or 1.8 mg per kilogram or a fixed dose of 80 mg). The study objectives were to assess the pharmacokinetic and pharmacodynamic characteristics and safety of fitusiran. Results No thromboembolic events were observed during the study. The most common adverse events were mild injection‐site reactions. Plasma levels of fitusiran increased in a dose‐dependent manner and showed no accumulation with repeated administration. The monthly regimen induced a dose‐dependent mean maximum antithrombin reduction of 70 to 89% from baseline. A reduction in the antithrombin level of more than 75% from baseline resulted in median peak thrombin values at the lower end of the range observed in healthy participants. Conclusions Once‐monthly subcutaneous administration of fitusiran resulted in dose‐dependent lowering of the antithrombin level and increased thrombin generation in participants with hemophilia A or B who did not have inhibitory alloantibodies. (Funded by Alnylam Pharmaceuticals; ClinicalTrials.gov number, NCT02035605.)

Fish as Bioreactors: Transgene Expression of Human Coagulation Factor VII in Fish Embryos

A plasmid containing human coagulation factor VII (hFVII) complementary DNA regulated by a cytomegalovirus promoter was microinjected into fertilized eggs of zebrafish, African catfish, and tilapia. The active form of hFVll was detected in the fish embryos by various assays. This positive expression of human therapeutic protein in fish embryos demonstrates the possibility of exploitation of transgenic fish as bioreactors.

Simple shifts in redox/thiol balance that perturb blood coagulation

The biological chemistry that underlies and regulates the blood coagulation cascade is not fully understood. To begin to understand this, we performed clotting assays under various redox conditions. By varying the amount of oxidant and/or antioxidant in these assays, we observed that both the intrinsic/tenase complex and the extrinsic pathways were susceptible to shifts in the thiol/redox balance. We established a dichotomy where blood clotting via the intrinsic pathway was sensitive to oxidation whereas the tissue factor or extrinsic pathway was more sensitive to reduction. These differential inhibitory effects present a conceptual mechanism for selective modulation of the activities of clotting factors specific for the respective pathways. These data also suggest that blood clotting may be influenced by unidentified redox or thiol equilibria.

Hemostasis and thrombosis protocols

Part I. Introduction. Hemostasis: Components and Processes K. John Pasi Part II. Basic Techniques. Isolation of DNA and RNA David J. Perry Amplification of DNA and RNA by PCR David J. Perry Direct Sequencing of PCR Products David J. Perry Solid-Phase Sequencing of Biotinylated PCR Products with Streptavidin-Coated Magnetic Beads David J. Perry Automated DNA Sequencing Helen L. Devereux Detection of DNA by Silver Staining David J. Perry and Flora Peyvandi Promoter Studies in Hemostasis Peter R. Winship and Jonathan R. K. Spray Part III. Methods of Mutational Analysis. Detection of Mutations and Polymorphisms in Clotting Factors by Denaturing Gradient Gel Electrophoresis Rainer Schwaab and Winfried Schmidt Screening for Mutations in DNA by Single-Stranded Conformation Polymorphism (SSCP) Analysis David J. Perry Screening for DNA Heteroduplexes in the Factor VII Gene Using Ethylene Glycol Gel Electrophoresis of Solvent-Treated 32P-Labeled PCR Products Peter M. Baker Detection of Mutations Causing Hemophilia A Using an In Vitro Coupled Transcription and Translation System Chike Ononye and P. Vincent Jenkins Screening for Mutations in the Human Antithrombin Gene by Hydrolink D-5000(TM) and MDE(TM) Gel Electrophoresis David J. Perry Part IV. Methods for Analyzing Inherited/Acquired Disorders of Hemostasis Detection of Mutations In Hemophilia A Patients by Chemical Cleavage of Mismatch Method Naushin H. Waseem, Richard Bagnall, Peter M. Green, and Francesco Giannelli Inversion Mutation Analysis in Hemophilia A by Restriction Enzyme Analysis and Southern Blotting Chike Ononye and P. Vincent Hemophilia B Mutational Analysis Peter M. Green Screening for Candidate Mutations Causingvon Willebrands Disease (vWD) P. Vincent Jenkins Use of Intron 40 VNTR I in vWD Gene Tracking Mohammed S. Enayat and Gurcharan J. Surdhar Multimeric Analysis of von Willebrand Factor (vWF) Mohammed S. Enayat Identification of Mutations in the Human Factor VII Gene Peter M. Baker Molecular Analysis in Factor XI Deficiency Karen M. Johnson and John H. McVey Mutational Analysis in Antithrombin Deficiency David J. Perry Ectopic Transcript Analysis in Human Antithrombin Deficiency David J. Perry Mutational Analysis of the Human Protein C Gene Roger Luddington Analysis of the Protein S Gene in Protein S Deficiency Nuria Sala and Yolanda Espinosa-Parrilla Screening for the G to A Transition at Position 20210 in the 3-Untranslated Region (UTR) of the Prothrombin Gene Karen P. Brown Screening for the Factor V Leiden Mutation Karen P. Brown Multiplex PCR for Detection of the Prothrombin 3 UTR (C20210A) Polymorphism and the Factor V Leiden Mutation Gillian Mellars, P. Vincent Jenkins, and David J. Perry Isoelectric Focusing and Immunodetection of Plasma Antithrombin Martina Daly Characterization of Heparin Binding Variants of Antithrombin by Crossed Immunoelectrophoresis in the Presence of Heparin Martina Daly The Determination of Amino Acid Sequence Abnormalities in Proteins by HPLC Peptide Analysis David Williamson Part V. Platelet and Megakaryocyte Analysis Molecular Biological Identification and Characterization of Inherited Platelet Receptor Disorders Ramesh B. Basani, Mark Richberg, and Mortimer Poncz In Vitro Expansion of Megakaryocytes from Peripheral Blood Hematopoietic Progenitors Michael A. Thornton and Mortimer Poncz Molecular Biology Studies with Primary Megakaryocytes Yaping

Residual Factor VIII-like cofactor activity of thioredoxin and related oxidoreductases

BACKGROUNDnFactor VIII is the cofactor for Factor X activation by Factor IXa. Activated Factor X, Factor Xa, in turn activates prothrombin in a sequence that leads to fibrin clot formation at the site of vascular injury. Although the biochemistry of the cascade has been well studied, the molecular mechanism underlying the cofactor role of Factor VIII is not understood.nnnMETHODSnWe screened a bacterial peptide display library with Factor IXa and Factor X co-immobilized on tosylactivated Dynabeads which were then used as platelet surrogates. Validation of peptide selection procedure and comparison of Factor VIII-like cofactor activity of oxidoreductases was performed using COATEST assays. Determination of Factor VIII as a folding catalyst with potential disulphide isomerase activity was determined using the RNase A renaturation assay.nnnRESULTSnWe set out to identify the cofactor requirements of the Factor IXa/Factor X procoagulant complex by random peptide display, and isolated a peptide with the active-site sequence, CGPC, of thioredoxin. This peptide was able to activate Factor X in a Factor IXa-dependent manner. Redox catalysts or oxidoreductases with homologous active-site vicinal cysteines such as PDI and DsbA also mimicked Factor VIII in their requirement of Factor IXa in Factor X activation. However, the cofactor activity of these peptides was up to a 1000-fold lower than that of Factor VIII and they were therefore unable to catalyse blood coagulation. Factor X activation by PDI and by Factor VIII was abolished by oxidation in an isolated system, which implies a possible role for thiol-disulphide exchange in the activity of the tenase complex. Using scrambled RNase A as a surrogate substrate, we also found that Factor VIII could renature this enzyme.nnnCONCLUSIONnOur findings suggest that Factor VIII may be a specialized folding catalyst with disulphide isomerase activity. We suggest that it is this activity that may underlie its cofactor function in Factor X activation, and that this function is interchangeable with classical oxidoreductases.nnnGENERAL SIGNIFICANCEnThe possible involvement of thiol-disulphide interchange as a mechanism underlying Factor VIII cofactor activity may provide some insight into the biochemistry of the intrinsic tenase complex.