Measurement of plasma and platelet tissue factor pathway inhibitor, factor V and Protein S in people with haemophilia.

INTRODUCTION: Tissue factor pathway inhibitor (TFPI) is a naturally occurring anticoagulant found in plasma, where it circulates bound to lipoproteins, factor V (FV) or Protein S (PS), and in platelets. Therapeutic agents targeting TFPI are under development for the treatment of haemophilia A and haemophilia B. AIM: To begin to understand how TFPI, FV and PS interact to modulate haemophilia bleeding. METHODS: Plasma and platelet antigen concentrations of these factors were determined in 73 people with haemophilia A and 18 with haemophilia B. Using multiple regression models, these were compared to the same analytes measured in 224 male blood donors. RESULTS: There were no differences in plasma or platelet TFPI, FV or PS concentrations between haemophilia types or severities. However, compared to blood donors, people with haemophilia had approximately one-third lower plasma PS, 9% lower plasma TFPIα, 50% higher platelet FV and 26% lower platelet Protein S. CONCLUSION: Together, the presented data suggest that individuals with haemophilia may have a compensatory procoagulant response of both plasma and platelet proteins to the decreased concentrations of FVIII or FIX.

Prediction of Warfarin Dose in Pediatric Patients: An Evaluation of the Predictive Performance of Several Models.

OBJECTIVES: The objective of this study was to evaluate the performance of pediatric pharmacogenetic-based dose prediction models by using an independent cohort of pediatric patients from a multicenter trial. METHODS: Clinical and genetic data (CYP2C9 [cytochrome P450 2C9] and VKORC1 [vitamin K epoxide reductase]) were collected from pediatric patients aged 3 months to 17 years who were receiving warfarin as part of standard care at 3 separate clinical sites. The accuracy of 8 previously published pediatric pharmacogenetic-based dose models was evaluated in the validation cohort by comparing predicted maintenance doses to actual stable warfarin doses. The predictive ability was assessed by using the proportion of variance (R(2)), mean prediction error (MPE), and the percentage of predictions that fell within 20% of the actual maintenance dose. RESULTS: Thirty-two children reached a stable international normalized ratio and were included in the validation cohort. The pharmacogenetic-based warfarin dose models showed a proportion of variance ranging from 35% to 78% and an MPE ranging from -2.67 to 0.85 mg/day in the validation cohort. Overall, the model developed by Hamberg et al showed the best performance in the validation cohort (R(2) = 78%; MPE = 0.15 mg/day) with 38% of the predictions falling within 20% of observed doses. CONCLUSIONS: Pharmacogenetic-based algorithms provide better predictions than a fixed-dose approach, although an optimal dose algorithm has not yet been developed.

Efficacy and safety of rVIII-SingleChain: results of a phase 1/3 multicenter clinical trial in severe hemophilia A.

Recombinant VIII (rVIII)-SingleChain is a novel B-domain-truncated recombinant factor VIII (rFVIII), comprised of covalently bonded factor VIII (FVIII) heavy and light chains. It was designed to have a higher binding affinity for von Willebrand factor (VWF). This phase 1/3 study investigated the efficacy and safety of rVIII-SingleChain in the treatment of bleeding episodes, routine prophylaxis, and surgical prophylaxis. Participants were ≥12 years of age, with severe hemophilia A (endogenous FVIII <1%). The participants were allocated by the investigator to receive rVIII-SingleChain in either an on-demand or prophylaxis regimen. Of the 175 patients meeting study eligibility criteria, 173 were treated with rVIII-SingleChain, prophylactically (N = 146) or on-demand (N = 27). The total cumulative exposure was 14 306 exposure days (EDs), with 120 participants reaching ≥50 EDs and 52 participants having ≥100 EDs. Hemostatic efficacy was rated by the investigator as excellent or good in 93.8% of the 835 bleeds treated and assessed. Across all prophylaxis regimens, the median annualized spontaneous bleeding rate was 0.00 (Q1, Q3: 0.0, 2.4) and the median overall annualized bleeding rate (ABR) was 1.14 (Q1, Q3: 0.0, 4.2). Surgical hemostasis was rated as excellent/good in 100% of major surgeries by the investigator. No participant developed FVIII inhibitors. In conclusion, rVIII-SingleChain is a novel rFVIII molecule showing excellent hemostatic efficacy in surgery and in the control of bleeding events, low ABR in patients on prophylaxis, and a favorable safety profile in this large clinical study. This trial was registered at www.clinicaltrials.gov as #NCT01486927.

Hemostatic efficacy, safety, and pharmacokinetics of a recombinant von Willebrand factor in severe von Willebrand disease.

This phase 3 trial evaluated the safety and hemostatic efficacy of a recombinant von Willebrand factor (rVWF) for treatment of bleeds in severe von Willebrand disease (VWD). rVWF was initially administered together with recombinant factor VIII (rFVIII) and subsequently alone, as long as hemostatic factor VIII activity (FVIII : C) levels were maintained. Pharmacokinetics (PK) were evaluated in a randomized cross-over design (rVWF vs rVWF:rFVIII at 50 IU VWF:ristocetin cofactor activity [RCo]/kg). Bleed control for all treated bleeds (N = 192 bleeds in 22 subjects) was rated good or excellent (96.9% excellent; 119 of 122 minor, 59 of 61 moderate, and 6 of 7 major bleeds) on a 4-point scale (4 = none to 1 = excellent). A single infusion was effective in 81.8% of bleeds. Treatment success, defined as the number of subjects with a mean efficacy rating of <2.5, was 100%. The PK profile of rVWF was not influenced by rFVIII (mean VWF:RCo terminal half-life: 21.9 hours for rVWF and 19.6 hours for rVWF:rFVIII). FVIII : C levels increased rapidly after rVWF alone, with hemostatic levels achieved within 6 hours and sustained through 72 hours after infusion. Eight adverse events (AEs; 6 nonserious AEs in 4 subjects and 2 serious AEs [chest discomfort and increased heart rate, without cardiac symptomatology] concurrently in 1 subject) were associated with rVWF. There were no thrombotic events or severe allergic reactions. No VWF or FVIII inhibitors, anti-VWF binding antibodies, or antibodies against host cell proteins were detected. These results show that rVWF was safe and effective in treating bleeds in VWD patients and stabilizes endogenous FVIII : C, which may eliminate the need for rFVIII after the first infusion. This trial was registered at www.clinicaltrials.gov as #NCT01410227.

The effect of secondary prophylaxis versus episodic treatment on the range of motion of target joints in patients with haemophilia.

This study prospectively compared the effect of secondary prophylaxis to episodic treatment on target joint (TJ) range of motion (ROM), number of joint haemorrhages and new TJ development in patients with moderate or severe haemophilia. Two-hundred and eighty-six males, 17% in prophylaxis, 83% in episodic treatment group, participating in the Centers for Disease Control and Prevention's Universal Data Collection project, fulfilled inclusion criteria: age >2 years at enrollment, free of TJs at enrollment, developed at least one TJ after enrollment, and received either prophylaxis or episodic treatment continuously for two follow-up visits after TJ development. The outcomes of interest - percentage change in TJ ROM, number of joint haemorrhages and new TJ development, were modelled using multivariate linear, Poisson and logistic regression techniques respectively. Individuals who received secondary prophylaxis in comparison to episodic treatment were younger at TJ development (P < 0.01); there was no difference in the decrease in TJ ROM between the two groups (P = 0.9). Factors significantly associated with a higher rate of haemarthroses included episodic treatment, severe haemophilia, age >5 years at TJ development, obesity and inhibitor negative status. Secondary prophylaxis significantly decreased haemarthroses but was not associated with a significant improvement in TJ ROM or with new TJ development.

Thromboelastography in the assessment of bleeding following surgery for congenital heart disease.

BACKGROUND: Perioperative bleeding is common in pediatric cardiac surgery patients. Traditional laboratory tests do not adequately characterize coagulation derangements in patients with bleeding. We sought to establish preoperative thromboelastography parameters in children prior to cardiopulmonary bypass, to compare thromboelastography assessment with standard coagulation parameters postoperatively, and to assess thromboelastography in children with significant hemorrhage. METHODS: Sixty patients requiring cardiopulmonary bypass were enrolled in a prospective observational study of perioperative thromboelastography. Thromboelastography measures were obtained preoperatively, intraoperatively after protamine administration, upon admit to the intensive care unit, and when patients were treated for bleeding. Thromboelastography measures were not used for clinical care. Postoperative thromboelastography measurements were compared with the standard coagulation parameters. Intraoperative thromboelastography, postoperative thromboelastography, and clinical outcomes were compared among patients who did and did not have significant postoperative bleeding. RESULTS: Preoperative thromboelastography parameters were similar to other published normal values for pediatric patients. Transfusion recommendations based on thromboelastography measurements were significantly different from those based on the standard coagulation testing. Thromboelastography measures after initial protamine administration were significantly different in patients with postoperative bleeding. This difference was not present upon arrival to the intensive care unit. Patients with significant bleeding tended to cease bleeding when clinical interventions were in agreement with recommendations based on thromboelastography. CONCLUSIONS: Pediatric patients with significant postoperative bleeding after surgery are more likely to have abnormal thromboelastography early after cessation of cardiopulmonary bypass. Thromboelastography illustrates derangements in the coagulation system and may aid in the treatment of postoperative bleeding.

The pharmacokinetic diversity of two von Willebrand factor (VWF)/ factor VIII (FVIII) concentrates in subjects with congenital von Willebrand disease. Results from a prospective, randomised crossover study.

The pharmacokinetic (PK) profiles of von Willebrand factor (VWF) /factor VIII (FVIII) concentrates are important for treatment efficacy and safety of von Willebrand disease (VWD) patients. This prospective, head-to-head, randomised crossover study compared the PK profile of a new, high purity, human plasma-derived (pd)VWF/FVIII concentrate, Wilate, with the PK profile of an intermediate purity (pd)VWF/FVIII concentrate, Humate-P, in VWD patients. Subjects with inherited VWD were randomised to a single intravenous dose (40 IU/kg VWF ristocetin cofactor activity [VWF:RCo]) of Wilate or Humate-P in Period 1, and switched to the other study drug in Period 2. Each period was preceded by a washout time of ≥ 7 days. Coagulation factor parameters were analysed at multiple time-points. Of 22 randomised subjects, 20 had evaluable PK profiles, which indicated comparability for VWF antigen and VWF:RCo between Wilate and Humate-P. The reported VWF:RCo average and terminal t1/2 of 10.4 and 15.8 hours (h), respectively, for Wilate and 9.3 h and 12.8 h for Humate-P, were not statistically different. Also, the mean VWF:RCo in vivo recoveries (Wilate 1.89, Humate-P 1.99 IU/dl per IU/kg) were similar between the two replacement therapies. Wilate showed parallel decay curves for VWF:RCo and FVIII clotting activity (FVIII:C) over time, while FVIII:C of Humate-P displayed a plateau between 0 and 12-24 h. This study demonstrated bioequivalent PK properties for VWF between Wilate and Humate-P. The PK profile of Wilate, combined with the 1:1 VWF/FVIII ratio, theoretically should facilitate dosing and laboratory monitoring of VWF replacement to prevent bleeding in individuals with VWD.

Naturally occurring CD4+ T-cell epitope variants act as altered peptide ligands leading to impaired helper T-cell responses in hepatitis C virus infection.

Hepatitis C virus (HCV) has a high rate of replication and lacks RNA-proofreading capabilities, thereby leading to variant or mutant viruses circulating within the host as a quasispecies. Previous work in our laboratory identified viral variants that emerged in a class-II immunodominant epitope NS3(358-375) of the non-structural-3 (NS3) protein region of HCV, the sequence of which is based on genotype 1A, the most prevalent genotype in the United States. Further work suggested that positive immune selection pressure was driving viral variation. Paradoxically, viral variants account for only a small percentage of the circulating virus in human beings and in chimpanzees, suggesting that passive evasion is not the only means of escape by HCV. This observation suggests a unique pathogenesis for HCV as it persists in the host. In the current study, we hypothesize that viral variants are acting as altered peptide ligands (APLs). To test this hypothesis, we used cloned T cells specific for NS3(358-375) peptide, which demonstrated attenuated T-cell and interferon-γ (IFN-γ) responses to individual variant peptides, when compared with the NS3(358-375) stimulated T-cell clones. Furthermore, such variants could act as APLs, based on their ability to antagonize the IFN-γ proliferative responses of clones specific for NS3(358-375). In addition, major histocompatibility complex (MHC) class II tetramer staining demonstrated that variant peptide-MHC complexes were able to specifically bind to NS3(358-375) T-cell clones and that both the variant and NS3(358-375) tetramers were able to bind to the same CD4(+) T cells. Taken together, the results suggest that viral variants may act as APL to effectively blunt the T-cell response to an important HCV epitope.

Hepatitis C virus induces regulatory T cells by naturally occurring viral variants to suppress T cell responses.

Regulatory T cell markers are increased in chronically infected individuals with the hepatitis C virus (HCV), but to date, the induction and maintenance of Tregs in HCV infection has not been clearly defined. In this paper, we demonstrate that naturally occurring viral variants suppress T cell responses to cognate NS3(358-375) in an antigen-specific manner. Of four archetypal variants, S370P induced regulatory T cell markers in comparison to NS3(358-375)-stimulated CD4 T cells. Further, the addition of variant-specific CD4 T cells back into a polyclonal culture in a dose-dependent manner inhibited the T cell response. These results suggest that HCV is able to induce antigen-specific regulatory T cells to suppress the antiviral T cell response in an antigen-specific manner, thus contributing to a niche within the host that could be conducive to HCV persistence.

Eptifibatide-induced thrombocytopenia and thrombosis in humans require FcgammaRIIa and the integrin beta3 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.

Identification of FcgammaRIIa as the ITAM-bearing receptor mediating alphaIIbbeta3 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.

Full-length sucrose-formulated recombinant factor VIII for treatment of previously untreated or minimally treated young children with severe haemophilia A: results of an international clinical investigation.

The safety and efficacy of a full-length sucrose-formulated recombinant factor VIII product (rFVIII-FS; Kogenate FS; Kogenate Bayer) was evaluated in previously untreated (PUPs) and minimally treated (MTP) patients with severe haemophilia A (FVIII <2%). Patients (37 PUPs; 24 MTPs) aged 0.1-25.7 months were treated with rFVIII-FS for a cumulative of 9,141 exposure days (EDs), median 114 EDs (range 4-478), on prophylactic or on-demand therapy. Eighty-nine percent of all treated bleeding episodes were successfully treated with 1 (74%) or 2 (15%) infusions. Clinical response to first infusion for each bleeding episode was rated as 'excellent' in 58%, or 'good' in 33%, of all cases. Recombinant FVIII-FS was used in 27 surgical procedures, mainly catheter implantations, which were all conducted without bleeding complications. FVIII recovery mean values (approximately 2%/kg/IU) were as expected for any licensed FVIII concentrate. FVIII neutralizing antibody formation was 15% (9/60). Aside from inhibitor formation, three adverse events were rated as 'at least possibly drug-related' for a total drug-related adverse event rate of 0.14%. No viral seroconversions were observed. Overall, excellent safety and efficacy were demonstrated with rFVIII-FS for therapy of young children with severe haemophilia A.

Epitopes of the NS3 protein of hepatitis C virus: recognition in HLA-DR4 transgenic mice.

Hepatitis C virus (HCV) infects more than 180 million of the world's population and causes a persistent infection that over decades can result in cirrhosis and hepatocellular carcinoma. Treatment is only partially effective and control is likely only with the development of effective vaccines. Currently, only chimpanzees can be infected with HCV and alternative animal and tissue culture models are badly needed. We have used mice transgenic for HLA-DR and human CD4 to analyse the specificity of murine responses to the HCV NS3 antigen in an effort to determine whether the epitopes recognized correspond to those recognized by human T cells. Indeed, determinants mapped in transgenic mice overlap with those in a patient exposed to HCV through infection. This result provides hope that such an animal model may be a useful tool with which to analyse particular aspects of immune responses to HCV in vivo.