Joan Cox Gill

Dose effect and efficacy of rFVIIa in the treatment of haemophilia patients with inhibitors: analysis from the Hemophilia and Thrombosis Research Society Registry

Summary.  Recombinant activated factor VII (rFVIIa), licensed in 1999 for treatment of haemophilia patients with inhibitors (HI), represents an important advance in the therapeutic armamentarium. Standard bolus dosing ranges from 90 to 120 mcg kg−1 every 2–3 h until arrest of bleeding. As licensure, clinical use of rFVIIa has increased and broadened. Clinicians now use a wide dose range, 90–300 mcg kg−1. High‐dose regimens may optimize thrombin generation or burst, and may allow for prolonged dose interval. The Hemophilia and Thrombosis Research Society (HTRS) maintains a registry database to study haemophilia treatment and related disorders, particularly treatment of acute bleeding in HI, acquired haemophilia, FVII deficiency and von Willebrands disease (VWD). To assess the effect of rFVIIa dose on efficacy and safety in the treatment of acute bleeding in HI, data from the HTRS database from January 2000 through June 2002 were analysed. Bleeding episodes were grouped by bolus rFVIIa dose range: <100, 100–150, 150–200 and >200 mcg kg−1. Investigator‐reported efficacy for the first 72 h of treatment was evaluated. Thirty‐eight congenital HI patients were treated for 555 bleeding episodes. Patient age range was 1–55 years (median: 14). Bleeding episodes were spontaneous (45%), caused by trauma (38%), or because of surgery, dental, diagnostic, or medical procedures (17%); bleeding occurred in joint, muscle, and intra/extracranial sites. Treatment location included: 80% at home, 12% at other facilities (treatment centres, ER, inpatient and OR), and 8% at both home/other facilities. Median total dose given over 72 h was 360 mcg kg−1 (range: 40–4281, mean: 537). Bleeding stopped in 87% of the episodes. Bleeding cessation rate was 84% for the three lower dose groups, and 97% for the highest dose group (P < 0.001). Five patients experienced nine adverse events (AEs). AE rates were <1% for <100, 5% for 100–150, 0% for 150–200, <1% for >200 mcg kg−1 dose group. Decreased therapeutic response accounted for eight of the nine AEs. These data, which represent the most comprehensive report of rFVIIa use since the USA licensure, demonstrate that bleeding episodes in HI patients can be treated safely and effectively at home and that doses up to 346 mcg kg−1 appear to be well‐tolerated. Additionally, rFVIIa doses >200 mcg kg−1 appear to significantly increase efficacy (97% in the high‐dose group, compared with 84% in the lower dose groups). Optimal dosing remains to be determined; specifically, what the lowest effective dose is and whether a single high‐dose bolus eliminates the need for repeated dosing. Recombinant FVIIa appears to have a wide safety margin that may allow dose escalation to address these questions.

Identification of FcγRIIa as the ITAM-bearing receptor mediating αIIbβ3 outside-in integrin signaling in human platelets

Immunoreceptor tyrosine-based activation motif (ITAM)-containing proteins have recently been demonstrated in macrophages and neutrophils to be required for cell surface integrins to transmit activation signals into the cell. To identify ITAM-bearing proteins that mediate signaling via the platelet-specific integrin alphaIIbbeta3, fibrinogen binding was induced by (1) allowing platelets to spread directly on immobilized fibrinogen, or (2) activating the PAR1 thrombin receptor on platelets in suspension. Both initiated strong, ligand binding-dependent tyrosine phosphorylation of the ITAM-bearing platelet Fc receptor, FcgammaRIIa, as well as downstream phosphorylation of the protein tyrosine kinase Syk and activation of phospholipase Cgamma2 (PLCgamma2). Addition of Fab fragments of an FcgammaRIIa-specific monoclonal antibody strongly inhibited platelet spreading on immobilized fibrinogen, as well as downstream tyrosine phosphorylation of FcgammaRIIa, Syk, and PLCgamma2, and platelets from a patient whose platelets express reduced levels of FcgammaRIIa exhibited markedly reduced spreading on immobilized fibrinogen. Finally, fibrinogen binding-induced FcgammaRIIa phosphorylation did not occur in human platelets expressing a truncated beta3 cytoplasmic domain. Taken together, these data suggest that ligand binding to platelet alphaIIbbeta3 induces integrin cytoplasmic domain-dependent phosphorylation of FcgammaRIIa, which then enlists selected components of the immunoreceptor signaling cascade to transmit amplification signals into the cell.

Efficacy and safety of vapor‐heated anti‐inhibitor coagulant complex in hemophilia patients. FEIBA Study Group

The study reported here was designed to measure the efficacy and safety of a vapor‐heated anti‐inhibitor coagulant complex (FEIBA‐VH) for the treatment of bleeding episodes in patients with hemophilia A who have inhibitors to factor VIII (FVIII). FEIBA‐VH, a second‐generation complex, is vapor‐heated for 10 hours at a temperature of 60°C and a pressure of 1190 millibar (mbar) and for 1 additional hour at 80°C and 1375 mbar. The current study was performed because of concern that this vapor‐heating process would reduce the efficacy of FEIBA‐VH as compared with non‐heat‐treated FEIBA (FEIBA). Forty‐one patients received FEIBA‐VH for 106 evaluable bleeding episodes. Ninety‐ three (88%) episodes were controlled, and 13 (12%) were not. Eighty‐ three (79%) episodes were controlled within 36 hours of the first infusion. No significant toxicity was seen. These results were compared with those of an earlier study with FEIBA. FEIBA‐VH was at least as effective as FEIBA in controlling bleeding episodes and can be compared favorably to any reported treatment of bleeding episodes in hemophiliacs with inhibitors to FVIII.

Immunobiology of hepatitis C virus (HCV) infection: the role of CD4 T cells in HCV infection

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Successful treatment for patients with von Willebrand disease undergoing urgent surgery using factor VIII/VWF concentrate (Humate‐P®)

Summary.  von Willebrand disease (VWD) is characterized by insufficient von Willebrand factor (VWF) activity. It has been proposed that VWF:ristocetin cofactor (VWF:RCo) activity may be useful in evaluating the response to VWD treatment in patients who require replacement therapy. This prospective, open‐label, non‐randomized study evaluated the safety and efficacy of a factor VIII (FVIII)/VWF concentrate (Humate‐P®) used in treatment regimens based on VWF:RCo activity in subjects with VWD in situations requiring urgent and necessary surgery. This article summarizes the results for 39 subjects with 42 evaluable surgical treatment events, 100% of which were rated as excellent/good for overall efficacy (achievement of haemostasis). The median loading dose based upon VWF:RCo activity was 82.3 international units/kilogram (IU kg−1; range 32.5–216.8 IU kg−1), and the median maintenance dose per infusion was 52.8 IU kg−1 (range 24.2–196.5 IU kg−1) for a median of 3 days (range 1–50 days). The median number of infusions per event was 6 (range 1–67 infusions). Three unanticipated adverse events (peripheral oedema, extremity pain and pseudo‐thrombocytopenia) from two surgical treatment events were reported that were potentially treatment‐related. No serious drug‐related adverse events (AEs) were observed, and no thrombotic events were reported in this study. This study supports the safety and efficacy of the FVIII/VWF concentrate Humate‐P® for the prevention of surgical haemorrhage in patients with VWD when administered in doses calculated in VWF:RCo units.

Sites of initial bleeding episodes, mode of delivery and age of diagnosis in babies with haemophilia diagnosed before the age of 2 years: a report from The Centers for Disease Control and Prevention’s (CDC) Universal Data Collection (UDC) project

Summary.  Lack of detailed natural history and outcomes data for neonates and toddlers with haemophilia hampers the provision of optimal management of the disorder. We report an analysis of prospective data collected from 580 neonates and toddlers aged 0–2 years with haemophilia enrolled in the Universal Data Collection (UDC) surveillance project of the Centers for Disease Control and Prevention (CDC). This study focuses on a cohort of babies with haemophilia whose diagnosis was established before the age of two. The mode of delivery, type and severity of haemophilia, onset and timing of haemorrhages, site(s) of bleeding, provision of prophylaxis with coagulation factor replacement therapy, and the role played by the federally funded Haemophilia Treatment Centers (HTC) in the management of these infants with haemophilia were evaluated. Seventy‐five per cent of haemophilic infants were diagnosed early, in the first month of life, especially those with a family history or whose mothers were known carriers; infants of maternal carriers were more likely to be delivered by C‐section. Involvement of an HTC prior to delivery resulted in avoidance of the use of assisted deliveries with vacuum and forceps. Bleeding from the circumcision site was the most common haemorrhagic complication, followed by intra‐ and extra‐cranial haemorrhages and bleeding from heel stick blood sampling. Eight per cent of the infants were administered factor concentrate within 24 h of birth; more than half were treated to prevent bleeding. This study highlights the significant rate and the sites of initial bleeding unique to very young children with haemophilia and underscores the need for research to identify optimal evidence‐based recommendations for their management.

Eptifibatide-induced thrombocytopenia and thrombosis in humans require FcγRIIa and the integrin β3 cytoplasmic domain

Thrombocytopenia and thrombosis following treatment with the integrin alphaIIbbeta3 antagonist eptifibatide are rare complications caused by patient antibodies specific for ligand-occupied alphaIIbbeta3. Whether such antibodies induce platelet clearance by simple opsonization, by inducing mild platelet activation, or both is poorly understood. To gain insight into the mechanism by which eptifibatide-dependent antibodies initiate platelet clearance, we incubated normal human platelets with patient serum containing an alphaIIbbeta3-specific, eptifibatide-dependent antibody. We observed that in the presence of eptifibatide, patient IgG induced platelet secretion and aggregation as well as tyrosine phosphorylation of the integrin beta3 cytoplasmic domain, the platelet FcgammaRIIa Fc receptor, the protein-tyrosine kinase Syk, and phospholipase Cgamma2. Each activation event was inhibited by preincubation of the platelets with Fab fragments of the FcgammaRIIa-specific mAb IV.3 or with the Src family kinase inhibitor PP2. Patient serum plus eptifibatide did not, however, activate platelets from a patient with a variant form of Glanzmann thrombasthenia that expressed normal levels of FcgammaRIIa and the alphaIIbbeta3 complex but lacked most of the beta3 cytoplasmic domain. Taken together, these data suggest a novel mechanism whereby eptifibatide-dependent antibodies engage the integrin beta3 subunit such that FcgammaRIIa and its downstream signaling components become activated, resulting in thrombocytopenia and a predisposition to thrombosis.

Pharmacokinetics and safety of a novel recombinant human von Willebrand factor manufactured with a plasma-free method: a prospective clinical trial.

Safety and pharmacokinetics (PK) of recombinant von Willebrand factor (rVWF) combined at a fixed ratio with recombinant factor VIII (rFVIII) were investigated in 32 subjects with type 3 or severe type 1 von Willebrand disease (VWD) in a prospective phase 1, multicenter, randomized clinical trial. rVWF was well tolerated and no thrombotic events, inhibitors, or serious adverse events were observed. The PK of rVWF ristocetin cofactor activity, VWF antigen, and collagen-binding activity were similar to those of the comparator plasma-derived (pd) VWF-pdFVIII. In vivo cleavage of ultra-large molecular-weight rVWF multimers by ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13; the endogenous VWF protease) and generation of characteristic satellite bands were demonstrated. In 2 subjects with specific nonneutralizing anti-VWF-binding antibodies already detectable before rVWF infusion, a reduction in VWF multimers and VWF activity was observed. Stabilization of endogenous FVIII was enhanced following post-rVWF-rFVIII infusion as shown by the difference in area under the plasma concentration curve compared with pdVWF-pdFVIII (AUC0-∞) (P < .01). These data support the concept of administering rVWF alone once a therapeutic level of endogenous FVIII is achieved.

VWF mutations and new sequence variations identified in healthy controls are more frequent in the African-American population

Diagnosis and classification of VWD is aided by molecular analysis of the VWF gene. Because VWF polymorphisms have not been fully characterized, we performed VWF laboratory testing and gene sequencing of 184 healthy controls with a negative bleeding history. The controls included 66 (35.9%) African Americans (AAs). We identified 21 new sequence variations, 13 (62%) of which occurred exclusively in AAs and 2 (G967D, T2666M) that were found in 10%-15% of the AA samples, suggesting they are polymorphisms. We identified 14 sequence variations reported previously as VWF mutations, the majority of which were type 1 mutations. These controls had VWF Ag levels within the normal range, suggesting that these sequence variations might not always reduce plasma VWF levels. Eleven mutations were found in AAs, and the frequency of M740I, H817Q, and R2185Q was 15%-18%. Ten AA controls had the 2N mutation H817Q; 1 was homozygous. The average factor VIII level in this group was 99 IU/dL, suggesting that this variation may confer little or no clinical symptoms. This study emphasizes the importance of sequencing healthy controls to understand ethnic-specific sequence variations so that asymptomatic sequence variations are not misidentified as mutations in other ethnic or racial groups.

Gain-of-function GPIb ELISA assay for VWF activity in the Zimmerman Program for the Molecular and Clinical Biology of VWD

von Willebrand disease (VWD) is a common bleeding disorder, but diagnosis is sometimes challenging because of issues with the current von Willebrand factor (VWF) assays, VWF antigen (VWF:Ag) and VWF ristocetin cofactor activity (VWF:RCo), used for diagnosis. We evaluated 113 healthy controls and 164 VWD subjects enrolled in the T.S. Zimmerman Program for the Molecular and Clinical Biology of VWD for VWF:Ag, VWF:RCo, and a new enzyme-linked immunosorbent assay (ELISA)-based assay of VWF-glycoprotein Ib (GPIb) interactions using a gain-of-function GPIb construct (tGPIbα(235Y;239V)) as a receptor to bind its ligand VWF in an assay independent of ristocetin (VWF:IbCo ELISA). Healthy controls, type 1, 2A, 2M, and 2N subjects had VWF:RCo/VWF:Ag ratios similar to the ratio obtained with VWF:IbCo ELISA/VWF:Ag. Type 2B VWD subjects, however, had elevated VWF:IbCo ELISA/VWF:Ag ratios. Type 3 VWD subjects had undetectable (< 1.6 U/dL) VWF:IbCo ELISA values. As previously reported, VWF:RCo/VWF:Ag ratio was decreased with a common A1 domain polymorphism, D1472H, as was direct binding to ristocetin for a 1472H A1 loop construct. The VWF:IbCo ELISA, however, was not affected by D1472H. The VWF:IbCo ELISA may be useful in testing VWF binding to GPIb, discrimination of type 2 variants, and in the diagnosis of VWD as it avoids some of the pitfalls of VWF:RCo assays.