David Lillicrap

Update on the pathophysiology and classification of von Willebrand disease: a report of the Subcommittee on von Willebrand Factor

Summary.  von Willebrand disease (VWD) is a bleeding disorder caused by inherited defects in the concentration, structure, or function of von Willebrand factor (VWF). VWD is classified into three primary categories. Type 1 includes partial quantitative deficiency, type 2 includes qualitative defects, and type 3 includes virtually complete deficiency of VWF. VWD type 2 is divided into four secondary categories. Type 2A includes variants with decreased platelet adhesion caused by selective deficiency of high‐molecular‐weight VWF multimers. Type 2B includes variants with increased affinity for platelet glycoprotein Ib. Type 2M includes variants with markedly defective platelet adhesion despite a relatively normal size distribution of VWF multimers. Type 2N includes variants with markedly decreased affinity for factor VIII. These six categories of VWD correlate with important clinical features and therapeutic requirements. Some VWF gene mutations, alone or in combination, have complex effects and give rise to mixed VWD phenotypes. Certain VWD types, especially type 1 and type 2A, encompass several pathophysiologic mechanisms that sometimes can be distinguished by appropriate laboratory studies. The clinical significance of this heterogeneity is under investigation, which may support further subdivision of VWD type 1 or type 2A in the future.

ISTH/SSC bleeding assessment tool: a standardized questionnaire and a proposal for a new bleeding score for inherited bleeding disorders

F . RODEGHIERO,* A . TOSETTO,* T . ABSH IRE , D . M . A R NO L D , B . COLLE R ,§ P . J AMES ,– C. NEUNER T** and D . L I LL ICRAP ON BEHALF OF THE I STH/SSC JOINT VWF AND PER INATAL/ PED IA TR I C HEMOSTA S IS SUB CO MMI TTEES WO RKI NG GRO U P 1 *Department of Cell Therapy and Hematology, San Bortolo Hospital, Vicenza, Italy; Blood Center of Wisconsin, Milwaukee, WI, USA; Michael G DeGroote School of Medicine, Department of Medicine, McMaster University and Canadian Blood Services, Hamilton, Canada; §Allen and Frances Adler Laboratory of Blood and Vascular Diseases, The Rockefeller University New York, NY, USA; –Department of Medicine, Queen s University, Kingston, Canada; **Department of Pediatric Hematology/Oncology, UT Southwestern Medical Center, Dallas, TX, USA; and Department of Pathology and Molecular Medicine, Richardson Laboratory Queen s University, Kingston, Canada

ISTH/SSC bleeding assessment tool

F . RODEGHIERO,* A . TOSETTO,* T . ABSH IRE , D . M . A R NO L D , B . COLLE R ,§ P . J AMES ,– C. NEUNER T** and D . L I LL ICRAP ON BEHALF OF THE I STH/SSC JOINT VWF AND PER INATAL/ PED IA TR I C HEMOSTA S IS SUB CO MMI TTEES WO RKI NG GRO U P 1 *Department of Cell Therapy and Hematology, San Bortolo Hospital, Vicenza, Italy; Blood Center of Wisconsin, Milwaukee, WI, USA; Michael G DeGroote School of Medicine, Department of Medicine, McMaster University and Canadian Blood Services, Hamilton, Canada; §Allen and Frances Adler Laboratory of Blood and Vascular Diseases, The Rockefeller University New York, NY, USA; –Department of Medicine, Queen s University, Kingston, Canada; **Department of Pediatric Hematology/Oncology, UT Southwestern Medical Center, Dallas, TX, USA; and Department of Pathology and Molecular Medicine, Richardson Laboratory Queen s University, Kingston, Canada

Tailored prophylaxis in severe hemophilia A: interim results from the first 5 years of the Canadian Hemophilia Primary Prophylaxis Study.

Summary.  Background: Prophylactic treatment for severe hemophilia A is likely to be more effective than treatment when bleeding occurs, however, prophylaxis is costly. We studied an inception cohort of 25 boys using a tailored prophylaxis approach to see if clotting factor use could be reduced with acceptable outcomes. Methods: Ten Canadian centers enrolled subjects in this 5‐year study. Children were followed every 3 months at a comprehensive care hemophilia clinic. They were initially treated with once‐weekly clotting factor; the frequency was escalated in a stepwise fashion if unacceptable bleeding occurred. Bleeding frequency, target joint development, physiotherapy and radiographic outcomes, as well as resource utilization, were determined prospectively. Results: The median follow‐up time was 4.1 years (total 96.9 person‐years). The median time to escalate to twice‐weekly therapy was 3.42 years (lower 95% confidence limit 2.05 years). Nine subjects developed target joints at a rate of 0.09 per person‐year. There was an average of 1.2 joint bleeds per person‐year. The cohort consumed on average 3656 IU kg−1year−1 of factor (F) VIII. Ten subjects required central venous catheters (three while on study); no complications of these devices were seen. One subject developed a transient FVIII inhibitor. End‐of‐study joint examination scores – both clinically and radiographically – were normal or near‐normal. Conclusions: Most boys with severe hemophilia A will probably have little bleeding and good joint function with tailored prophylaxis, while infusing less FVIII than usually required for traditional prophylaxis.

Generation and validation of the Condensed MCMDM-1VWD Bleeding Questionnaire for von Willebrand disease

Summary.  Background: Given the challenges involved in obtaining accurate bleeding histories, attempts at standardization have occurred and the value of quantifying hemorrhagic symptoms has been recognized. Patients/methods: An extensive validated bleeding questionnaire (MCMDM‐1VWD) was condensed by eliminating all details that did not directly affect the bleeding score (BS) and the correlation between the two versions was tested. Additionally, the diagnostic utility of the condensed version was prospectively tested. Results: Data on 259 individuals who were administered the questionnaire are presented here; 217 being prospectively investigated for von Willebrand disease (VWD) (group 1) and 42 previously known to have type 1, 2 or 3 VWD (group 2). Of the 217 prospectively investigated, 35 had positive BS (≥4) and 182 had negative scores. Seven individuals (all with positive BS) had laboratory results consistent with type 1 VWD. This results in a sensitivity of 100% and a specificity of 87%. The positive predictive value is 0.20 and the negative predictive value is 1. The correlation between the full MCMDM‐1VWD and condensed versions is excellent (Spearman’s 0.97, P < 0.001, linear regression r2 = 96.4). Inter‐observer reliability for the condensed version is reasonable (Spearman’s 0.72, P < 0.001 and intra‐class correlation coefficient 0.805, P < 0.001). There was a significant difference in BS between subtypes of VWD, with type 3 >> type 2 >> type 1 VWD (anovaP < 0.001). There is a strong inverse relationship between VWF:Ag level and BS (Spearman’s −0.411, P < 0.001). Conclusions: The Condensed MCMDM‐1VWD Bleeding Questionnaire is an efficient, effective tool in the evaluation of patients for VWD.

Efficient Lentiviral Transduction and Improved Engraftment of Human Bone Marrow Mesenchymal Cells

Human bone marrow (BM) mesenchymal stem/progenitor cells are potentially attractive targets for ex vivo gene therapy. The potential of lentiviral vectors for transducing BM mesenchymal cells was examined using a self‐inactivating vector that expressed the green fluorescent protein (GFP) from an internal cytomegalovirus (CMV) promoter. This vector was compared with oncoretroviral vectors expressing GFP from the CMV promoter or a modified long‐terminal repeat that had been optimized for long‐term expression in stem cells. The percentage of GFP‐positive cells was consistently higher following lentiviral versus oncoretroviral transduction, consistent with increased GFP mRNA levels and increased gene transfer efficiency measured by polymerase chain reaction and Southern blot analysis. In vitro GFP and FVIII expression lasted for several months post‐transduction, although expression slowly declined. The transduced cells retained their stem/progenitor cell properties since they were still capable of differentiating along adipogenic and osteogenic lineages in vitro while maintaining high GFP and FVIII expression levels. Implantation of lentivirally transduced human BM mesenchymal cells using collagen scaffolds into immunodeficient mice resulted in efficient engraftment of gene‐engineered cells and long‐term transgene expression in vivo. These biocompatible BM mesenchymal implants represent a reversible, safe, and versatile protein delivery approach because they can be retrieved in the event of an unexpected adverse reaction or when expression of the protein of interest is no longer required. In conclusion, efficient gene delivery with lentiviral vectors in conjunction with the use of bioengineered reversible scaffolds improves the therapeutic prospects of this novel approach for gene therapy, protein delivery, or tissue engineering.

Home management of haemophilia.

Summary.  The demonstrated benefits of home care for haemophilia include improved quality of life, less pain and disability, fewer hospitalizations, and less time lost from work or school. Although reduced mortality has not been demonstrated, the substantial increase in longevity since the early 1980s correlates with the introduction of home treatment and prophylaxis programmes. These programmes must be designed and monitored by haemophilia treatment centres (HTC), which are staffed with professionals with broad and complementary expertise in the disease and its complications. In return, patients and their families must be willing to accept the reciprocal responsibilities that come from administering blood products or their recombinant equivalents at home. Patients with inhibitors to factors VIII or IX pose special challenges, but these complications do not obviate participation in home care programmes. Home care was an essential prerequisite to the introduction of effective prophylactic factor replacement therapy. Prophylaxis offers significant improvements in quality of life, but requires a substantial commitment. The use of implantable venous access devices can eliminate some of the difficulty and discomfort of peripheral venous access in small children, but brings additional risks. The future holds the promise of factor concentrates for home use that have longer half‐lives, or can be administered by alternate routes. Knowledge of patient genotypes may allow treatments tailored to avoid complications such as inhibitor development. Gene therapy trials, which are currently ongoing, will ultimately lead to gene‐based treatments as a complement to traditional protein‐based therapy.

von Willebrand disease: advances in pathogenetic understanding, diagnosis, and therapy

von Willebrand disease (VWD) is the most common autosomally inherited bleeding disorder. The disease represents a range of quantitative and qualitative pathologies of the adhesive glycoprotein von Willebrand factor (VWF). The pathogenic mechanisms responsible for the type 2 qualitative variants of VWF are now well characterized, with most mutations representing missense substitutions influencing VWF multimer structure and interactions with platelet GPIbα and collagen and with factor VIII. The molecular pathology of type 3 VWD has been similarly well characterized, with an array of different mutation types producing either a null phenotype or the production of VWF that is not secreted. In contrast, the pathogenetic mechanisms responsible for type 1 VWD remain only partially resolved. In the hemostasis laboratory, the measurement of VWF:Ag and VWF:RCo are key components in the diagnostic algorithm for VWD, although the introduction of direct GPIbα-binding assays may become the functional assay of choice. Molecular genetic testing can provide additional benefit, but its utility is currently limited to type 2 and 3 VWD. The treatment of bleeding in VWD involves the use of desmopressin and plasma-derived VWF concentrates and a variety of adjunctive agents. Finally, a new recombinant VWF concentrate has just completed clinical trial evaluation and has demonstrated excellent hemostatic efficacy and safety.

Quantitation of bleeding symptoms in children with von Willebrand disease: use of a standardized pediatric bleeding questionnaire.

Summary.  Background: Excessive bruising and mucocutaneous bleeding are frequent presenting symptoms in childhood. A detailed bleeding history can distinguish children who may have an inherited bleeding disorder from those who are normal. There is a lack of standardization of such history taking in pediatric practise. Objectives: To assess the performance of a Pediatric Bleeding Questionnaire (PBQ), an adaptation of a standardized adult bleeding questionnaire and score that includes pediatric‐specific bleeding symptoms, in a cohort of children with von Willebrand disease (VWD). Patients/Methods: Bleeding scores were determined by interview, for children with a previous diagnosis of VWD and a control group of unaffected siblings. Results: Bleeding scores were obtained for 100 children with VWD, median age 10.9 years (range, 0.8–17.8 years), and 21 unaffected siblings. Median bleeding score in children with VWD was 7.0 (range, 0–29) and in the control group was 0 (range, −1–2). Bleeding score varied within and between each VWD type: definite type 1, n = 40, median, 9.0 (range, 2–18); possible type 1, n = 38, median, 2.0 (0–15); type 2, n = 6, median, 14.0 (3–17); and type 3, n = 16, median, 12.0 (4–29). Bleeding scores in affected children correlated with age (Spearman’s correlation coefficient, 0.35; P = 0.0004). The most frequent clinically significant bleeding symptoms were surgical bleeding, bleeding after tooth extraction and menorrhagia. Post‐circumcision bleeding, cephalohematoma, macroscopic hematuria and umbilical stump bleeding were clinically significant in 32% (of circumcised males), 4%, 4% and 3% of children, respectively. Conclusions: The PBQ provides a standardized quantitation of bleeding severity in children with VWD.

Eradication of neutralizing antibodies to factor VIII in canine hemophilia A after liver gene therapy

Inhibitory antibodies to factor VIII (FVIII) are a major complication in the treatment of hemophilia A, affecting approximately 20% to 30% of patients. Current treatment for inhibitors is based on long-term, daily injections of large amounts of FVIII protein. Liver-directed gene therapy has been used to induce antigen-specific tolerance, but there are no data in hemophilic animals with pre-existing inhibitors. To determine whether sustained endogenous expression of FVIII could eradicate inhibitors, we injected adeno-associated viral vectors encoding canine FVIII (cFVIII) in 2 strains of inhibitor hemophilia A dogs. In 3 dogs, a transient increase in inhibitor titers (up to 7 Bethesda Units [BU]) at 2 weeks was followed by continuous decline to complete disappearance within 4-5 weeks. Subsequently, an increase in cFVIII levels (1.5%-8%), a shortening of clotting times, and a reduction (> 90%) of bleeding episodes were observed. Immune tolerance was confirmed by lack of antibody formation after repeated challenges with cFVIII protein and normal protein half-life. A fourth dog exhibited a strong early anamnestic response (216 BU), with slow decline to 0.8 BU and cFVIII antigen detection by 18 months after vector delivery. These data suggest that liver gene therapy has the potential to eradicate inhibitors and could improve the outcomes of hemophilia A patients.