Safety and initial clinical efficacy of three dose levels of recombinant activated factor VII (rFVIIa): results of a phase I study.

The safety and efficacy of recombinant DNA-produced factor VIIa (rFVIIa) was investigated in 15 haemophilic patients in non-bleeding states and during bleeding episodes (mild to moderate joint bleed). Patients with severe haemophilia A without inhibitors (n = 4), haemophilia A with inhibitors (n = 10), and haemophilia B with inhibitor (n = 1) received one or more doses of rFVIIa during 32 non-bleeding study episodes and 23 bleeding episodes. The study was an open, uncontrolled, dose-escalation (17.5 micrograms/kg, 35 micrograms/kg, 70 micrograms/kg) trial. Physical evaluation, laboratory assessment, and immunology testing were conducted at baseline, monthly for 3 months and every 3 months thereafter. The immediate safety of rFVIIa was assessed by monitoring of D-dimer, fibrinogen, platelet count, antithrombin III, thrombin-antithrombin complex, and alpha 2-antiplasmin 5 min before and at multiple times throughout the following 24 h. Prothrombin time (PT) and activated partial thromboplastin time (aPTT) values were also obtained. Pain, swelling, joint circumference, and range of motion were recorded before administration of the initial dose of rFVIIa in bleeding patients and at 6, 12, and 24 h. Haemostatic response to rFVIIa was observed in patients with severe VIII and IX deficiency with and without inhibitors. Therapy with rFVIIa was judged effective in 19 of the 22 evaluable bleeding episodes at one or more time points. The 35 micrograms/kg and 70 micrograms/kg doses were associated with higher response rates at 6 and 12 h compared to the 17.5 micrograms/kg dose level. A second dose of rFVIIa was administered in 20 of the 22 bleeding episodes.(ABSTRACT TRUNCATED AT 250 WORDS)

A Large Drop in Atmospheric 14C/12C and Reduced Melting in the Younger Dryas, Documented with 230Th Ages of Corals.

Paired carbon-14 ((14)C) and thorium-230((230)Th) ages were determined on fossil corals from the Huon Peninsula, Papua New Guinea. The ages were used to calibrate part of the (14)C time scale and to estimate rates of sea-level rise during the last deglaciation. An abrupt offset between the (14)C and (230)Th ages suggests that the atmospheric (14)C/(12)C ratio dropped by 15 percent during the latter part of and after the Younger Dryas (YD). This prominent drop coincides with greatly reduced rates of sea-level rise. Reduction of melting because of cooler conditions during the YD may have caused an increase in the rate of ocean ventilation, which caused the atmospheric (14)C/(12)C ratio to fall. The record of sea-level rise also shows that globally averaged rates of melting were relatively high at the beginning of the YD. Thus, these measurements satisfy one of the conditions required by the hypothesis that the diversion of meltwater from the Mississippi to the St. Lawrence River triggered the YD event.

Management of factor VIII inhibitors: evolution and current status.

Management of patients with factor VIII (and IX) inhibitors includes management of acute bleeds and methods to induce immune suppression and tolerance and to detect patients at risk of developing inhibitors. The methods used over the years to treat acute bleeding have been more or less successful. The best method is to raise factor VIII levels by human or porcine factor VIII concentrate, but this is not usually possible. Prothrombin complex concentrates, activated or non-activated, have enjoyed some success as factor VIII by passing agents, but the development of recombinant activated factor VII represents a new and promising method of inducing haemoslasis at the site of bleeding whilst minimizing the risk of disseminated intravascular coagulation. Alternatively, the use of tissue factor is under consideration to exploit the extrinsic system. Methods to induce immunological tolerance by use of the 'Bonn' regime or by the introduction of immunomodulation with the 'Malmö' regime of extracorporeal immunodepletion, cyclophosphamide, and intravenous immunoglobulin continue to be attempted with significant but variable success. Gradually the inhibitor problem is being contained, but it is still an important complication of haemophilia therapy.

The relationship of HIV-1 viral sequences detected by the polymerase chain reaction in haemophilic patients to clinical and other markers of infection.

A nested primer polymerase chain reaction (PCR) of pol gene sequences of human immunodeficiency virus-1 (HIV-1) was applied to whole blood of 31 haemophiliacs who were, or had been, positive for HIV p24 antibody (HIVAb) by enzyme linked immunosorbent assay (ELISA) and samples from 22 persistently HIVAb negative haemophiliacs who had been at risk of contracting HIV from treatment. The results were compared with those of p24 HIV antigen determination, T4 cell counts beta 2 Microglobulin (beta 2M) levels and clinical evidence of progression of HIV disease. There was no discrepancy between the PCR results and past or present seropositivity for HIVAb. The qualitative PCR was more sensitive than the p24 antigen assay but the presence of the latter was predictive of progression of infection as determined clinically and by falling T4 cell counts and rising levels of beta 2M. The results of the PCR are reassuring for HIVAb negative haemophiliacs at risk from treatment and to HIVAb negative sexual contacts of HIVAb positive persons.

von Willebrand factor: clinical features of inherited and acquired disorders.

The physiologic mechanisms that influence plasma levels of von Willebrand factor (vWF) are poorly understood but include race, blood group, age, pregnancy, exercise, and adrenergic and neurohumoral stimuli. Inherited abnormalities in von Willebrand's disease (vWD) are associated with a defect of the vWF gene on chromosome 12, but in some cases, coexistence of impaired response of plasminogen activator and telangiectasia suggests the presence of a regulatory defect or more extensive endothelial perturbation. Three broad types of vWD are recognized; in addition, a platelet-type vWD (pseudo-vWD) is due to an abnormal platelet receptor for vWF. The prevalence of vWD, which is difficult to determine because of variations in severity even within a kindred, is reportedly as high as 1%. In a survey of European patients, the prevalence of treated vWD varied from 4.5 to 24 per million. Preliminary results of an international survey of vWD indicate that about 3% of treated patients have seroconversion to human immunodeficiency virus, 50% of whom have symptoms. Inhibitor of vWF occurs in type III vWD after treatment and is associated with the presence of gene deletions. Acquired vWD may complicate lymphoproliferative and autoimmune disorders, and proteolytic degradation of vWF complicates myeloproliferative disorders. The level of vWF is increased during pregnancy and in vascular and other disorders; it may be involved in the pathogenesis of atherosclerosis. High-molecular-weight multimers of vWF and a cofactor are thought to promote the formation of microthrombi in thrombotic thrombocytopenic purpura and the hemolytic uremic syndrome. Thus, study of vWD has shed light on pathogenetic mechanisms in a wide range of disorders.

Oliver Memorial Lecture 1989. The evolution and future of haemophilia therapy.

Lane (1840) first noted the beneficial effect of blood transfusion in controlling haemophilic bleeding following ophthalmic surgery. The appreciation that haemophilia is due to a plasma defect, at first thought to be prothrombin, coincided with the discovery of blood groups. This led in due course to the discovery of citrate anti-coagulant and the ability to store blood. The beneficial effect of citrated plasma in haemophilia led to the exploitation of stored blood, fresh frozen plasma, and the subsequent development of cryoprecipitate and factor concentrates. All this would not have been possible, however, without the selfless contribution of blood donors and the development of an organized blood transfusion service. In the United Kingdom, P. L. Oliver pioneered the development of blood donor panels, the London Blood Transfusion Service and the British Red Cross Society Blood Transfusion Service leading directly to the National Blood Transfusion Service; recognized as the World's senior service. The development of haemophilia therapy owes much, therefore, to Oliver's energetic and pioneering work and it is entirely appropriate that the first Oliver Memorial Lecture be directed to the evolution and future of haemophilia therapy. It is indeed an honour to be invited to deliver this Oliver Memorial Lecture at the combined meeting of the British Blood Transfusion Society and the British Society for Haematology here in Wembley.

Human recombinant DNA-derived antihemophilic factor (factor VIII) in the treatment of hemophilia A. recombinant Factor VIII Study Group.

BACKGROUND: Current treatment of hemophilia A, a hereditary disorder affecting approximately 1 in 10,000 males, relies on plasma-derived factor VIII concentrates. We tested the safety and efficacy of a recombinant factor VIII preparation for the treatment of this disorder. METHODS: We conducted the investigation in three stages: comparing the pharmacokinetics of plasma-derived and recombinant factor VIII, assessing the efficacy of recombinant factor VIII for home therapy, and assessing its efficacy for major surgical procedures and hemorrhage. A total of 107 subjects with hemophilia, 20 of whom had not been treated previously, enrolled in the investigation. RESULTS: The in vivo recovery and elimination half-lives of recombinant factor VIII equaled or exceeded those of plasma-derived factor VIII. Seventy-six subjects participated in a home-treatment program, using recombinant factor VIII for 69 to 807 days (median, 618); home diaries of 56 subjects treated for 5 months were analyzed. Of 540 bleeding episodes, 399 (73.9 percent) required only one treatment with recombinant factor VIII. The projected annual consumption of recombinant factor VIII was similar to that of plasma-derived factor VIII concentrate. Twenty-six subjects received recombinant factor VIII for 22 surgical procedures and 10 serious hemorrhages; hemostasis was excellent in all cases. De novo formation of inhibitors occurred in only 1 of 85 previously treated subjects. Inhibitor antibodies also developed in 6 of 21 children, 20 of whom had not previously been treated; 5 had low levels (less than or equal to 7.5 Bethesda units) despite continued treatment with recombinant factor VIII. There was no evidence of new formation of antibody to foreign proteins, and recombinant factor VIII was well tolerated. CONCLUSIONS: Recombinant factor VIII has biologic activity comparable to that of plasma factor VIII and is safe and efficacious for the treatment of hemophilia A.

Family studies in von Willebrand's disease by analysis of restriction fragment length polymorphisms and an intragenic variable number tandem repeat (VNTR) sequence.

We have previously identified a microsatellite variable number tandem repeat region of the nucleotide sequence ATCT within intron 40 of the von Willebrand factor (vWF) gene. By polymerase chain reaction (PCR) amplification of this region, eight major alleles have been demonstrated in the South Wales population, with an overall heterozygosity rate of 75%. Direct sequencing has shown that the alleles correspond to lengths of between six and 14 ATCT repeats. In the present study we describe the use of this variable repeat sequence and previously reported restriction fragment length polymorphisms (RFLP) to study inheritance patterns in families with type I, IIA and severe type III von Willebrand's disease (vWD). The results confirm that analysis of this precisely localized intragenic locus provides a highly informative marker for gene tracking studies in the major forms of vWD.

Family studies and prenatal diagnosis in severe von Willebrand disease by polymerase chain reaction amplification of a variable number tandem repeat region of the von Willebrand factor gene.

We have previously demonstrated within intron 40 of the von Willebrand factor (vWF) gene a region of ATCT repeats that was shown to vary in length between two different DNA clones from unrelated individuals. The polymerase chain reaction (PCR) was used to examine the variability in length of this variable number tandem repeat (VNTR) in 53 normal individuals, using primers to DNA sequence flanking the repeat region. Overall, eight different length allelic bands were seen. These were individually sequenced and shown to contain from 6 to 14 ATCT repeats (a nine-repeat band was not seen). Seventy-five percent of individuals were shown to be heterozygous for this vWF.VNTR, and family studies showed Mendelian inheritance with allelic frequencies from 1% (vWF.VNTR [8] and vWF.VNTR [14]) to 39% (vWF.VNTR [7]). In the family of a patient with type III severe von Willebrand disease (vWD), vWF.VNTR results mirrored the phenotypic data and results with previously reported intragenic vWF restriction fragment length polymorphisms (RFLP). The patient was shown to be a compound heterozygote. In a family with a child with severe type III vWD, prenatal diagnosis by vWF.VNTR analysis on DNA obtained by chorionic villus sampling at 10 weeks gestation during a subsequent pregnancy indicated a severely affected fetus. This diagnosis was confirmed by fetal blood sampling at 18 weeks.

A mutation adjacent to the beta cleavage site of factor IX (valine 182 to leucine) results in mild haemophilia Bm.

The genetic basis of a mild form of haemophilia Bm has been investigated. The patient under investigation has a mild bleeding disorder and has never experienced spontaneous bleeds. Factor IX coagulant activity (FIX:C) was 0.15 units/ml and factor IX antigen (FIX:Ag) 1.32 units/ml. The prothrombin time performed with an ox brain thromboplastin was 65 s (normal plasma 31 s). Studies of the abnormal factor IX protein in this patient showed a normal molecular weight and normal calcium binding properties. Activation of the mutant factor IX with factor XIa showed normal proteolytic cleavage. DNA sequence from the eight factor IX exons and flanking introns was amplified from this patient using the polymerase chain reaction. The amplified material was subjected to direct chain termination nucleotide sequencing. The only nucleotide sequence alteration found was a G----C transversion at nucleotide 20,524, changing the amino acid encoded at residue 182 from valine to leucine. This residue is one amino acid removed from the beta cleavage site of factor IX. This residue is highly conserved in other vitamin K dependent serine proteases and we propose that its alteration in this patient is responsible for his mild haemophilic phenotype, and for the abnormal interaction of this factor IX protein with the extrinsic system of coagulation.

Physiology of blood coagulation.

Blood coagulation forms part of an integrated series of haemostatic reactions, involving plasma, platelet, and vascular components. Platelets adhere to damaged endothelium or to subendothelium under the influence of adhesive proteins, and when activated they aggregate and expose binding sites for coagulation factors. Platelets, therefore, act as vehicles to concentrate and potentiate coagulation reactions on the damaged vessels. Following interaction of the 'contact' factors XII and XI, the coagulation protease zymogens undergo sequential activation, resulting in the generation of thrombin, the conversion of fibrinogen to fibrin, and the formation of a platelet-fibrin haemostatic plug. The fibrinolytic system interacts to regulate fibrin deposition and removal during healing. Central to coagulation is the generation of thrombin. It is involved in promoting haemostatic reactions as well as a number of protective functions. The activities of thrombin and other serine proteases are modulated by the serine protease inhibitors (serpins), including antithrombin III and heparin cofactor II which are important in regulating the physiological anticoagulant action of glycosaminoglycans at the endothelium and the pharmacological action of heparin. Reduction of the formation or function of thrombin and other serine proteases is one of the primary aims of anticoagulant therapy.

Factor IX Cardiff: a variant factor IX protein that shows abnormal activation is caused by an arginine to cysteine substitution at position 145.

Crude barium chloride eluates prepared from 12 unrelated patients with cross-reacting material positive (CRM+) haemophilia B were activated with celite eluate, the reaction products resolved after reduction by 13% SDS-PAGE, and factor IX antigenic material detected by probing with radiolabelled immunopurified rabbit anti-factor IX antiserum followed by autoradiography. Out of the 12, one sample showed faulty activation with the production of a stable reaction product with a MW compatible with that of a putative light chain-activation intermediate. In order to confirm this, two oligonucleotide primers that bracketed exon 6 of the factor IX gene were constructed and used to prime a polymerase chain reaction on DNA isolated from the patient's peripheral blood leucocytes. A single 489 nucleotide DNA fragment was obtained, gel purified, subcloned into M13, and DNA sequencing carried out on both strands. A single C to T transition was discovered that changed the Arg residue at position 145, the first residue of the first bond in the activation peptide, to a Cys, a result that confirmed the inferences drawn from the activation studies.

Defective propeptide processing and abnormal activation underlie the molecular pathology of factor IX Troed-y-Rhiw.

Affected members of a South Wales haemophilia B family (from Troed-y-Rhiw) were shown by Western blotting and immunoperoxidase detection to have a factor IX molecule of higher than normal molecular weight which also shows impaired calcium binding. Gene cloning and DNA sequencing revealed the same arginine to glutamine mutation at position -4 of the propeptide that has been found in two previously described factor IX variants which circulate with the propeptide still attached. The mutation also abolishes a HaeIII restriction enzyme recognition site. A potential carrier was shown to be normal both by Western blotting and DNA studies. The way in which the attached propeptide interferes with normal factor IX function was investigated by activation studies with crude normal and patient factor IX Troed-y-Rhiw preparations using Western blotting and detection with iodinated immunopurified polyclonal antifactor IX serum. We demonstrate that the -4 mutation appears to block cleavage between the Arg145-Ala146 peptide bond in the activation peptide, thus preventing the normal activation of factor IX Troed-y-Rhiw. A small amount of normally processed factor IX is produced, implying that the -4 mutation does not completely prevent propeptide cleavage, thus accounting for the low levels of factor IX activity measured in the plasma of affected family members.