Clinical and laboratory variability in a cohort of patients diagnosed with type 1 VWD in the United States.

von Willebrand disease (VWD) is the most common inherited bleeding disorder, and type 1 VWD is the most common VWD variant. Despite its frequency, diagnosis of type 1 VWD remains the subject of debate. In order to study the spectrum of type 1 VWD in the United States, the Zimmerman Program enrolled 482 subjects with a previous diagnosis of type 1 VWD without stringent laboratory diagnostic criteria. von Willebrand factor (VWF) laboratory testing and full-length VWF gene sequencing was performed for all index cases and healthy control subjects in a central laboratory. Bleeding phenotype was characterized using the International Society on Thrombosis and Haemostasis bleeding assessment tool. At study entry, 64% of subjects had VWF antigen (VWF:Ag) or VWF ristocetin cofactor activity below the lower limit of normal, whereas 36% had normal VWF levels. VWF sequence variations were most frequent in subjects with VWF:Ag <30 IU/dL (82%), whereas subjects with type 1 VWD and VWF:Ag ≥30 IU/dL had an intermediate frequency of variants (44%). Subjects whose VWF testing was normal at study entry had a similar rate of sequence variations as the healthy controls (14%). All subjects with severe type 1 VWD and VWF:Ag ≤5 IU/dL had an abnormal bleeding score (BS), but otherwise BS did not correlate with VWF:Ag. Subjects with a historical diagnosis of type 1 VWD had similar rates of abnormal BS compared with subjects with low VWF levels at study entry. Type 1 VWD in the United States is highly variable, and bleeding symptoms are frequent in this population.

A longer acting rFVIII, safe and effective.

In this issue of Blood, Mahlangu et al describe a well-designed and executed phase 3 multicenter study of a recombinant factor VIII (rFVIII) product fused with the Fc fragment of immunoglobulin G1 (IgG1) in 165 patients with severe hemophilia A.

No increase in bleeding identified in type 1 VWD subjects with D1472H sequence variation.

The diagnosis of von Willebrand disease (VWD) is complicated by issues with current laboratory testing, particularly the ristocetin cofactor activity assay (VWF:RCo). We have recently reported a sequence variation in the von Willebrand factor (VWF) A1 domain, p.D1472H (D1472H), associated with a decrease in the VWF:RCo/VWF antigen (VWF:Ag) ratio but not associated with bleeding in healthy control subjects. This report expands the previous study to include subjects with symptoms leading to the diagnosis of type 1 VWD. Type 1 VWD subjects with D1472H had a significant decrease in the VWF:RCo/VWF:Ag ratio compared with those without D1472H, similar to the findings in the healthy control population. No increase in bleeding score was observed, however, for VWD subjects with D1472H compared with those without D1472H. These results suggest that the presence of the D1472H sequence variation is not associated with a significant increase in bleeding symptoms, even in type 1 VWD subjects.

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.

Emergency department visits in children with hemophilia.

BACKGROUND: The pediatric emergency department (ED) management of bleeding and other complications of hemophilia constitutes an increasingly important component of hemophilia therapy. This retrospective study examined the overall ED use by children with hemophilia in a single center, with a particular aim to investigate visits related to injury or bleeding, and those related to blood stream infection in patients with a central venous catheter (CVC). METHODS: Electronic medical records of patients with hemophilia presenting to Children's Hospital of Michigan ED were reviewed. Different categories of ED visits over a 5-year period (January 2006-December 2010) were examined. RESULTS: There were 536 ED visits from 84 male patients (median age 4 years, range 0-21) with hemophilia over the 5-year period. The reasons for ED visits were: injury or bleeding (61.2%); suspected CVC-related infection (11.8%); causes unrelated to hemophilia (19.2%); and routine clotting factor infusion (7.8%). Eighteen visits from six patients were secondary to injury or bleeding in a patient not yet diagnosed with hemophilia. An intracranial hemorrhage was detected in five visits. Overall, 5.4% of all visits represented distinct episodes of bloodstream infection. CONCLUSION: The pediatric ED is an indispensable component of the overall hemophilia care, because: (1) patients with potentially lethal problems such as ICH or CVC-related infection may present to the ED for their initial management; (2) previously undiagnosed patients with hemophilia may also present to the ED for their first bleeding episodes, initiating the diagnostic investigations; (3) the ED provides after-hours treatment service for many episodes of injury or bleeding, and also for clotting factor infusion.

Common VWF exon 28 polymorphisms in African Americans affecting the VWF activity assay by ristocetin cofactor.

The diagnosis of von Willebrand disease relies on abnormalities in specific tests of von Willebrand factor (VWF), including VWF antigen (VWF:Ag) and VWF ristocetin cofactor activity (VWF:RCo). When examining healthy controls enrolled in the T. S. Zimmerman Program for the Molecular and Clinical Biology of von Willebrand disease, we, like others, found a lower mean VWF:RCo compared with VWF:Ag in African American controls and therefore sought a genetic cause for these differences. For the African American controls, the presence of 3 exon 28 single nucleotide polymorphisms (SNPs), I1380V, N1435S, and D1472H, was associated with a significantly lower VWF:RCo/VWF:Ag ratio, whereas the presence of D1472H alone was associated with a decreased ratio in both African American and Caucasian controls. Multivariate analysis comparing race, SNP status, and VWF:RCo/VWF:Ag ratio confirmed that only the presence of D1472H was significant. No difference was seen in VWF binding to collagen, regardless of SNP status. Similarly, no difference in activity was seen using a GPIb complex-binding assay that is independent of ristocetin. Because the VWF:RCo assay depends on ristocetin binding to VWF, mutations (and polymorphisms) in VWF may affect the measurement of "VWF activity" by this assay and may not reflect a functional defect or true hemorrhagic risk.

Thromboelastography in children with coagulation factor deficiencies.

Hemophilia is traditionally classified according to the levels of the deficient coagulation factor as Severe (<1%), Moderate (1-5%) or Mild (>5%). However, it is well known that the factor activity does not necessarily correspond to the clinical bleeding manifestations. As prophylactic therapy is the best method of prevention of serious complications such as hemophilic arthropathy, a test that may predict the bleeding pattern would be extremely beneficial. Thromboelastography (TEG) uses whole blood to determine clot formation characteristics, such as initiation, propagation as well as strength of the clot, and is now being extensively studied in bleeding and thrombophilia. This study attempted to determine the TEG characteristics in 47 children with moderate hemophilia (MH) and severe hemophilia with (SHI) and without inhibitors (SH) and tried to retrospectively correlate them to the clinical bleeding patterns. TEG showed evidence of faster and better clot formation, as evidenced by a higher maximum thrombin/fibrin generation, in those with mild bleeding manifestations compared to those with severe bleeding tendency, in addition to the expected prolongation in time to formation of clot related to factor deficiency. This may be a potentially useful tool to evaluate the bleeding tendency and determine need for prophylaxis in children with hemophilia.

Decreased clearance of von Willebrand factor in a patient with type 2B von Willebrand disease following development of immune thrombocytopenia.

We report a case of concurrent type 2B von Willebrand disease (VWD) and immune thrombocytopenia (ITP). The patient had characteristic loss of von Willebrand factor (VWF) high molecular weight multimers (HMWM) but a normal platelet count in the initial 8 years after diagnosis of type 2B VWD. When he developed severe thrombocytopenia, however, both his VWD indices and VWF HMWM normalized. As his platelet count increased, he again lost the HMWM and his VWD indices decreased. These results suggest that the severe thrombocytopenia led to decreased clearance of VWF, especially the HMWM.

How do you treat bleeding disorders with desmopressin?

Desmopressin is an analog of vasopressin that exerts a substantial haemostatic effect by inducing the release of von Willebrand factor from its storage sites in endothelial cells. It has proved useful in treating or preventing bleeding episodes in patients with von Willebrand disease, haemophilia A and platelet function defects. Its efficacy in achieving a satisfactory level of haemostasis has reduced the use of blood products to treat bleeding episodes. Clinicians need to become familiar with the use of this drug that has become a home medication for many patients with inherited bleeding disorders.

Milestones in hemophilia and concepts in future clinical trial design.

Hemophilia has long been documented as a bleeding disorder that afflicts males from early childhood. While some early societies set guidelines or laws to protect affected children, true advances in the understanding of the underlying deficiency and in the treatment of hemophilia have been relatively recent and continue today. This paper presents some of the key milestones that mark the path to current knowledge on hemophilia. Given that further studies are needed to explore issues such as optimal treatment and dosage, particularly in patients with inhibitors to factor VIII or factor IX, this paper also considers potential pitfalls in the design and conduct of clinical trials in hemophilia and suggests how careful forward planning may help to avoid them.

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.

Phenotypes of allo- and autoimmune antibody responses to FVIII characterized by surface plasmon resonance.

Evidence of antibody isotype/subtype switching may provide prognostic value regarding the state of immune responses to therapeutic proteins, e.g. anti-factor VIII (FVIII) antibodies that develop in many hemophilia A patients, clinically termed "inhibitors". A sensitive, high- information-content surface plasmon resonance (SPR) assay has been developed to quantify IgG subtype distributions and the domain specificity of anti-drug antibodies. Plasma samples from 22 subjects with an allo- or auto-immune reaction to FVIII were analyzed. Pre-analytical treatment protocols were developed to minimize non-specific binding and specific matrix interference due to von Willebrand factor-FVIII interactions. The dynamic range for IgG quantification was 0.2-5 µg/ml (∼1-33 nM), allowing characterization of inhibitor-positive samples. Subtype-specific monoclonal antibodies were used to quantify the IgG subtype distribution of FVIII-specific antibodies. Most samples obtained from multiply-infused inhibitor subjects contained IgG4 antibodies. Several distinct phenotypes were assigned based on the IgG subtype distribution: IgG1, IgG4, IgG1 & IgG4, and IgG1, IgG2 & IgG4. An IgG1-only response was found in mild/moderate HA subjects during early FVIII infusions, and analysis of serial samples followed antibody class switching as several subjects' immune responses developed. Competition studies utilizing a recombinant FVIII-C2 domain indicated 40-80% of FVIII-specific antibodies in most samples were directed against this domain.

A community-based partnership to promote information infrastructure for bleeding disorders.

Specialists in rare disorders often face challenges in collecting surveillance and research data. As movement toward more fully realizing the potential of electronic health information gains momentum, practitioners who treat individuals with rare disorders are in need of public-private support to tap into the advantages offered by the developing electronic information technologies and the interoperability standards promulgated by the USDHHS. The not-for-profit American Thrombosis and Hemostasis Network (ATHN) was created in 2006 to provide stewardship of a secure, national, web-based database to support federally funded hemophilia treatment centers (HTCs) across the country. In pursuit of its mission to support clinical outcomes analysis, research, advocacy, and public health reporting in the hemostasis and thrombosis community, ATHN has established a spectrum of community-based partnerships. This paper describes the process and public health benefits of creating formal relationships with 127 of the 134 HTCs from 12 regional networks across the U.S., government agencies such as the CDC, Health Resources and Services Administration, and NIH; consumer-based organizations; and industry leaders. This community-based partnership model can be applied to other rare disorders communities with high economic and public health impact.

Fibrinolytic parameters in children with noncatheter thrombosis: a pilot study.

Although the incidence of pediatric thrombosis has increased over the last decade, noncatheter-related deep venous thrombosis (nCDVT) is rare in children. Congenital and acquired hypercoagulable states may play an important role in the pathogenesis of nCDVT. In this study, we evaluated fibrinolytic parameters by measuring individual concentrations of fibrinolytic proteins and by tissue factor initiated whole blood thromboelastography (TEG), in which a fibrin clot was lyzed by exogenously added tissue plasminogen activator (tPA). Children with nCDVT were compared with age and sex-matched controls. TAFI concentrations were significantly higher in the patient group but there was no difference in the PAI-1, tPA and lipoprotein (a) concentrations. Significantly decreased fibrinolysis was found on TEG in the patient group suggesting that hypofibrinolysis may play an important role in the pathogenesis of nCDVT in children. To our knowledge, this is the first pediatric study that has systematically evaluated the role of fibrinolysis in the pathogenesis of DVT. Given our results, the role of fibrinolysis in the pathogenesis of nCDVT in children should be further evaluated in larger studies.

Evolution of recombinant factor VIII safety: KOGENATE and Kogenate FS/Bayer.

The use of factor VIII (FVIII) concentrates in the treatment of hemophilia A has raised important safety issues, historically of pathogen transmission and increasingly of inhibitor development to FVIII treatment. While manufacturing processes of current recombinant FVIII products have been shaped entirely around preventing pathogen transmission, the same modifications that afford a greater margin of safety could affect immunogenicity of the product, consequences of which could only be seen through long-term clinical experience. This review summarizes pathogen safety and inhibitor reports from clinical trials, post-marketing surveillance studies, and study reports on KOGENATE and its successor, Kogenate FS/Bayer. Although KOGENATE and Kogenate FS/Bayer are nearly identical products, subtle manufacturing improvements to address the need for greater margins of safety from a pathogen transmission perspective have also led to a potentially improved immunogenicity profile (15% in previously untreated/minimally treated patients with severe hemophilia A for Kogenate FS/Bayer). Notably, there has been no occurrence of pathogen contamination, and minimal de novo inhibitor formation in previously treated patients throughout the use of both products. Overall, KOGENATE and Kogenate FS/Bayer have a long history of safety in a variety of clinical settings, including treatment of bleeding, surgical management, and prophylaxis therapy.

Pediatric reference values for molecular markers in hemostasis.

BACKGROUND: The hemostatic system is a developing and changing process relative to age. OBJECTIVES: To distinguish the differences in hemostatic parameters between children and adults, and to establish the normal range of these parameters in children of different age groups. DESIGN/METHODS: Blood was obtained from healthy children aged 1 to 18 years (n=70) and adults (n=26). Children were categorized into 3 age groups: 1 to 5 years, 6 to 10 years, and 11 to 18 years. Several coagulation and fibrinolysis parameters were determined. RESULTS: Children in all age groups showed no significant difference in mean levels of von Willebrand factor antigen and activity, activated partial thromboplastin time, fibrinogen, activated factor VII, tissue plasminogen activator, plasminogen activator inhibitor-1, and thrombin activatable fibrinolysis inhibitor compared with adults. However, children aged 1 to 5 years had significantly higher mean values of soluble thrombomodulin (P=0.001), prothrombin time (P=0.03), tissue factor (P<0.001), thrombin-antithrombin complex (P<0.001), and D-dimer (P=0.009) whereas they had significantly lower mean levels of protein C activity (P=0.02) than did adults. CONCLUSIONS: These data indicate physiologic differences in the hemostatic system between children and adults and should serve as a useful reference guide in interpreting test results for children with suspected bleeding disorders.

The safety and efficacy of recombinant human blood coagulation factor IX in previously untreated patients with severe or moderately severe hemophilia B.

This international clinical trial evaluated the safety and efficacy of recombinant factor IX (rFIX) in previously untreated patients (PUPs) with severe or moderately severe hemophilia B (FIX activity, < or = 3 IU/dL). Sixty-three PUPs aged younger than 1 month to 14 years received rFIX (median treatment duration, 37 months; range, 4-64 months). Mean rFIX recovery (0.68 +/- 0.27 IU/dL per IU/kg) remained constant over 5 years and was similar in infants (1 month to < 2 years) and children (2 to < 12 years). Fifty-four PUPs used rFIX (median dose, 62.7 IU/kg per infusion; range, 8.2-292 IU/kg) to treat 997 hemorrhages. Bleeding was well controlled, with 75% of hemorrhages requiring only one rFIX infusion. Response to rFIX was "excellent" or "good" in 94% of cases. Effective hemostasis was achieved in 32 PUPs receiving rFIX for routine prophylaxis, with 91% of prophylaxis responses rated "excellent." rFIX administered for 30 surgical procedures in 23 PUPs achieved hemostasis for all rated procedures. Five patients experienced allergic-type manifestations, including 2 (3%) patients who developed FIX inhibitors (both > 5 BU/dL). rFIX was well tolerated, with no associated thrombotic events or evidence of viral transmission. These data indicate that rFIX is a safe and effective treatment for PUPs with hemophilia B.

First and second generation recombinant factor VIII concentrates in previously untreated patients: recovery, safety, efficacy, and inhibitor development.

The first of the prospective multicenter studies in previously untreated patients (PUPs) with a recombinant factor VIII (FVIII) concentrate began in January 1989. Over the past 11 years, PUP studies have amassed a great deal of information concerning safety, efficacy, and inhibitor development of the two "first-generation" recombinant (r) FVIII concentrates (Kogenate and Recombinate) and of two "second-generation" products (ReFacto and Kogenate FS, which is formulated with sucrose rather than with albumin). Each of these products has proved to be safe, effective, and well-tolerated. Side effects have been rare and mild in nature. There have been no clinical reactions to hamster or murine proteins. During the course of the multinational PUP trials with Kogenate, Recombinate, and ReFacto, inhibitors developed in 29.7, 31, and 33%, respectively, of severely affected PUPs. Half of these were high titer and half were low titer. In each of these trials, several inhibitors were transient. PUPs and minimally treated patients (MTPs) in the Kogenate SF trial have not been followed long enough to determine the incidence of inhibitor development; however, the product appears to be safe and effective. Following demonstration of safety and efficacy with each rFVIII concentrate in previously treated patients with hemophilia A, studies in PUPs began. In general, the prospective trials in PUPs with each recombinant product were conducted similarly, allowing comparison of data. This article is intended to provide a review of the experience with both first- and second-generation rFVIII products in the prospective clinical trials in PUPs.

Congenital Hemorrhagic Disorders: New Insights into the Pathophysiology and Treatment of Hemophilia.

The diagnostic and treatment strategies related to hemophilia are rapidly evolving. This article focuses on some of the issues of importance. Diagnostic advances in molecular genetics are reviewed by Dr. Ginsburg in Section I, including the current state of knowledge regarding the mutations responsible for hemophilia, with reference to the potential clinical applications of DNA diagnosis and prenatal testing. Within the area of new therapeutic approaches in hemophilia, recombinant factor VIII and factor IX concentrates, their use and availability are addressed by Dr. Lusher in Section II as well as the use of so-called "primary prophylaxis" with the aim of decreasing long-term hemophilia athropathy. The use of radionuclide synovectomy as replacement for more invasive methods is also reviewed. Various approaches to the ongoing challenge of the management of hemophilia patients with inhibitors against factor VIII and factor IX are reviewed by Dr. Hedner in Section III, including the principles for immune tolerance induction and the use of recombinant factor VIIa to induce hemostasis in bleeding patients with inhibitors. In Section IV, gene therapy in hemophilia is reviewed by Dr. High, who focuses on recent developments in the rapidly moving field of gene therapy for hemophilia. Three phase I trials of gene therapy for hemophilia were initiated in 1999, and additional proposed trials are currently in the regulatory review process. Certain aspects of the pathophysiology of hemophilia make it an attractive model for a gene-based approach to treatment. These include latitude in choice of target tissue, a wide therapeutic window, the availability of small and large animal models of the disease, and the ease of determining therapeutic efficacy. Since there is very little published information regarding the ongoing trials, this section reviews the approaches being used, the published pre-clinical data, and considerations affecting clinical trial design in hemophilia gene therapy.