vWD Patients Research Articles

Evaluation of Two Automated Methods for Measurement of the Ristocetin Cofactor Activity of von Willebrand Factor

We have evaluated two automated methods for measuring ristocetin cofactor activity (VWF:RCo) on an automated coagulation analyzer (STAR, Diagnostica Stago Inc., Parsippany, NJ). A modification of the BC von Willebrand Reagent method (Dade Behring Inc., Newark, Delaware) was compared with a standard method using a platelet aggregometer, and an in-house automated method was developed using commercially available components. The STAR measures the change in optical density (OD) caused by agglutination of platelets. Change in OD was linear for plasma dilutions between .25 and .04 U/mL. Coefficient of variation compared favorably with that of the standard method. In 123 normal women, the correlation coefficient (r) = .905. In menorrhagia cases, r = .901. In 20 VWD patients, r = .93. The CDC in-house method was compared to the BC Reagent method in 79 additional menorrhagia cases, with r = .94. The automated methods produced results equivalent or superior to those of traditional methods of measuring VWF:RCo.

Use of ristocetin cofactor activity in the management of von Willebrand disease.

von Willebrand disease (vWD), the most common of the hereditary bleeding disorders, arises from quantitative or qualitative defects in von Willebrand factor (vWF). vWF is a multimeric plasma protein that plays a key role in primary and secondary haemostasis. In the current classification scheme, vWD is divided into six subtypes that are based on the nature of the vWF defect. Therapeutic strategies depend on the accurate identification of these subtypes. In most clinical situations, desmopressin is effective treatment for the great majority of patients with mild (type 1) disease, while replacement therapy with factor VIII/vWF concentrates that contain high levels of vWF activity is required for most type 2 and nearly all type 3 vWD patients. Several factor VIII/vWF replacement products are available, one of which (Humate P) has been approved for the treatment of vWD by the US Food and Drug Administration. Preliminary results of recent studies support the hypothesis that treatment with factor VIII/vWF concentrates based upon the content of vWF activity as reflected in the ristocetin cofactor assay is practicable, safe and efficacious. The establishment of optimal treatment regimens with respect to dose intensity and duration will require further study.

Allelic frequencies of three VNTRs in intron 40 of the human von Willebrand factor gene in Types 1, 2, and 3 von Willebrand disease patients and controls of a Brazilian population

Intron 40 of the human von Willebrand factor (vWF) gene contains a polymorphic region with three variable-number tandem repeats (VNTRs), type (ATCT) n . In the present report, we evaluated the allelic frequencies of these three VNTRs in a population constituted by 51 Brazilian Caucasian and 25 Types 1, 2, and 3 von Willebrand disease (vWD) patients, and performed segregation analysis in eight families affected by vWD Types 1 and 2. Three pairs of primers were used to amplify independently nucleotides 1640–1794 (VNTR 3), 1890–1991 (VNTR 1), and 2215–2396 (VNTR 2) from intron 40. The observed heterozygosities (0.86, 0.66, and 0.66 for VNTRs 3, 1, and 2, respectively) were in accordance with the expected heterozygosities derived from the allele frequencies (0.81, 0.64, and 0.70, respectively). Although the three VNTRs were highly polymorphic, VNTR 3 showed the highest values of heterozygosity [Haemostasis 25 (1995) 264; Hum. Mol. Genet. 1 (1992) 287.].

Gynaecological and obstetrical morbidity in women with type I von Willebrand disease: results of a patient survey

Type 1 von Willebrand disease (vWD) is generally regarded clinically as 'mild' and the obstetrical-gynaecological features have not been fully described. We administered a patient questionnaire and provider survey of the medical and quality of life aspects of childbirth and menstruation to 99 type 1 vWD patients and compared the patients presently menstruating (n=81) to a cohort of 150 menstruating females in the general population. The following measurements had a statistically higher proportion in the vWD group: number of tampons/towels used for a typical menstrual cycle (P=0. 002); percentage reporting that clothes are stained by menses (P = 0. 001); past or present history of anaemia (P = 0.001); childbirth-related bleeding (P=0.001); and childbirth-related bleeding necessitating RBC transfusion (P=0.002). Quality of life assessment of the impact of menses in both of the above cohorts was measured by a Likert scale using seven quality of life parameters. Compared to the control group, the vWD patients had a significantly higher score, with P-values of < 0.0001 for each parameter. Hormonal interventions for menorrhagia in the vWD patients were < or = 50% effective. Menorrhagia resulted in red blood cell transfusions in 6% of patients, dilatation and curettage in 17% and hysterectomy in 13%. Despite the common connotation of type 1 vWD as clinically 'mild', childbirth and the monthly challenge to haemostasis presented by menstruation result in a substantial degree of morbidity in females with type 1 vWD. These results support the rationale for ongoing international efforts to increase awareness of vWD as a cause for menorrhagia and to improve the quality of life in females with known vWD.

The Effect of DDAVP Infusion on Thrombin Generation in Platelet-rich Plasma of von Willebrand Type 1 and in Mild Haemophilia A Patients

In von Willebrand disease (vWD) type 1 and mild haemophilia A patients we studied the effect of an infusion of DDAVP (0.3 microg/kg body weight) on thrombin generation in platelet-rich plasma (PRP) and platelet-poor plasma (PPP). Baseline thrombin generation in PRP was diminished both in the haemophilia A and vWD patients. It was normal in vWD plasma when sufficient procoagulant phospholipids were present, either via adding phospholipid vesicles to PPP or via scrambling of the platelet membrane with ionomycin in PRP. In haemophilia A plasma, thrombin generation did not normalize by providing procoagulant phospholipids. Treatment with DDAVP temporarily restored thrombin generation in PRP to normal in both diseases. To investigate the individual roles of von Willebrand factor (vWF) and factor VIII, we also studied the effect of factor VIII infusion on thrombin generation in a severe haemophilia patient. It appears that at a fixed normal vWF concentration, <25% factor VIII is sufficient for normal thrombin generation in PRP. At a sufficient factor VIII concentration, however, thrombin generation is still lower than normal in vWD patients; approximately 40% of vWF is required for half-normal thrombin generation in PRP. It thus appears that vWF is also a clotting factor, in the sense that it is required for normal thrombin generation. This underlines the importance of the interaction between coagulation and the platelets in normal haemostasis. Thrombin generation in PRP appears to be a suitable test to reflect the combined function.

A Reliable and Reproducible ELISA Method to Measure Ristocetin Cofactor Activity of von Willebrand Factor

The ristocetin induced binding of vWF to GPIb, which is routinely tested in a platelet agglutination assay, can be reproducibly studied in an ELISA where plasma vWF binds to a captured rGPIb alpha-fragment (His1-Val289) in the presence of ristocetin. This binding is specific since the vWF-GPIb interaction could (i) be blocked by inhibitory anti-GPIb or anti-(vWF A1 domain) monoclonal antibodies (mAbs) and (ii) be enhanced by an anti-vWF mAb that also facilitates ristocetin induced platelet agglutination. Further studies were undertaken to determine whether the test could be used to differentiate vWF from patients with different types of von Willebrand's disease. The median vWF:RiCof activity in controls (n = 24) was 0.75 U/ml, in type 1 vWD patients (n = 17) 0.28 U/ml, in type 2A (n = 18) 0.055 U/ml, in type 2B (n = 4) 0.094 U/ml and in type 3 (n = 3) <0.0005 U/ml. Moreover, the values correlated well with those obtained from the vWF:RiCof-agglutination assay (r = 0.873). The vWF:RiCof-ELISA has several advantages: the use of a recombinant fragment instead of donor platelets results in a more reproducible test with a low inter- and intra-assay variability (<14% CV), the test can further be readily automated and for a single determination, only minimal amounts of patient plasma are required (8 microl).

Overview of platelet physiology and laboratory evaluation of platelet function.

Appropriate laboratory testing for the platelet-type bleeding disorders hinges on an adequate assessment in the history and physical examination. Patients with histories and screening laboratory results consistent with coagulation disorders (hemophilia, disseminated intravascular coagulation) are not appropriate candidates for platelet function testing. In contrast, patients with a lifelong history of platelet-type bleeding symptoms and perhaps a positive family history of bleeding would be appropriate for testing. Figure 6 depicts one strategy to evaluate these patients. Platelet morphology can easily be evaluated to screen for two uncommon qualitative platelet disorders: Bernard-Soulier syndrome (associated with giant platelets) and gray platelet syndrome, a subtype of storage pool disorder in which platelet granulation is morphologically abnormal by light microscopy. If the bleeding disorder occurred later in life (no bleeding with surgery or trauma early in life), the focus should be on acquired disorders of platelet function. For those patients thought to have an inherited disorder, testing for vWD should be done initially because approximately 1% of the population has vWD. The complete vWD panel (factor VIII coagulant activity, vWf antigen, ristocetin cofactor activity) should be performed because many patients will have abnormalities of only one particular panel component. Patients diagnosed with vWD should be classified using multimeric analysis to identify the type 1 vWD patients likely to respond to DDAVP. If vWD studies are normal, platelet aggregation testing should be performed, ensuring that no antiplatelet medications have been ingested at least 1 week before testing. If platelet aggregation tests are normal and if suspicion for an inherited disorder remains high, vWD testing should be repeated. The evaluation of thrombocytopenia may require bone marrow examination to exclude primary hematologic disorders. If future studies with thrombopoietin assays confirm preliminary results, however, the bone marrow examination of certain patients may be replaced by a thrombopoietin level.

Low Platelet α2β1 Levels in Type I von Willebrand Disease Correlate With Impaired Platelet Function in a High Shear Stress System

AbstractPlatelet adhesion to collagen-coated surfaces in whole blood under flow conditions is mediated by both von Willebrand factor (vWF)-dependent recruitment of the platelet glycoprotein Ib-IX receptor complex and collagen interaction with the integrin α2β1. In type 1 von Willebrand disease (vWD), platelet adhesive functions are impaired due to the decrease in vWF levels in plasma and platelets. There are at least three alleles of the human α2 gene, distinguishable by a cluster of silent or noncoding sequence differences within a segment of the gene. Two alleles, associated with low receptor density can be distinguished by nucleotide 807C, while the third allele associated with high receptor density, expresses nucleotide 807T. Gene frequencies of these alleles in a normal population (n = 167) are 0.58 for 807C and 0.42 for 807T. We measured the frequencies of these alleles in symptomatic patients with five types of vWD (type 1, n = 78; type 2A, n = 25, type 2B, n = 14; type 2M, n = 10; and type 3, n = 20). Compared with the normal group, no significant difference in allele frequencies was observed among individuals with types 2A, 2B, 2M, or 3 vWD. However, the frequency of the 807C allele, associated with low collagen receptor density, among type 1 vWD patients (807C = .71; 807T = .29) was significantly higher than that of the normal population (P = .007). Also, in patients with vWD type 1 and borderline to normal ristocetin-cofactor (vWF:RCo) activity values, collagen receptor density correlates inversely with closure time in a high shear stress system (platelet function analyzer [PFA-100]). We propose that low platelet α2β1 density results in less efficient primary platelet adhesion and may result in increased tendency to bleed, as evidenced by the high frequency of this polymorphism in patients with type 1 vWD compared with normal individuals. In addition, this may account for the variability between patients with similar levels of vWF antigen, but strikingly different bleeding histories.

Low Platelet 2β1 Levels in Type I von Willebrand Disease Correlate With Impaired Platelet Function in a High Shear Stress System

Platelet adhesion to collagen-coated surfaces in whole blood under flow conditions is mediated by both von Willebrand factor (vWF)-dependent recruitment of the platelet glycoprotein Ib-IX receptor complex and collagen interaction with the integrin 2β1. In type 1 von Willebrand disease (vWD), platelet adhesive functions are impaired due to the decrease in vWF levels in plasma and platelets. There are at least three alleles of the human 2 gene, distinguishable by a cluster of silent or noncoding sequence differences within a segment of the gene. Two alleles, associated with low receptor density can be distinguished by nucleotide 807C, while the third allele associated with high receptor density, expresses nucleotide 807T. Gene frequencies of these alleles in a normal population (n = 167) are 0.58 for 807C and 0.42 for 807T. We measured the frequencies of these alleles in symptomatic patients with five types of vWD (type 1, n = 78; type 2A, n = 25, type 2B, n = 14; type 2M, n = 10; and type 3, n = 20). Compared with the normal group, no significant difference in allele frequencies was observed among individuals with types 2A, 2B, 2M, or 3 vWD. However, the frequency of the 807C allele, associated with low collagen receptor density, among type 1 vWD patients (807C = .71; 807T = .29) was significantly higher than that of the normal population (P = .007). Also, in patients with vWD type 1 and borderline to normal ristocetin-cofactor (vWF:RCo) activity values, collagen receptor density correlates inversely with closure time in a high shear stress system (platelet function analyzer [PFA-100]). We propose that low platelet 2β1 density results in less efficient primary platelet adhesion and may result in increased tendency to bleed, as evidenced by the high frequency of this polymorphism in patients with type 1 vWD compared with normal individuals. In addition, this may account for the variability between patients with similar levels of vWF antigen, but strikingly different bleeding histories.

Diagnosis and management of von Willebrand disease

von Willebrand disease (vWD) is a bleeding disorder caused by quantitative or qualitative defects of von Willebrand factor (vWF). The diagnosis is based on measurements of plasma and platelet vWF, the ability of vWF to interact with its platelet receptor and the analysis of the mutlimeric composition of vWF. Due to the heterogeneity of vWF defects, a correct diagnosis of types and subtypes may be sometimes difficult but is very important for an appropriate therapy. The aim of treatment is to correct the dual defects of haemostasis, i.e. abnormal coagulation expressed by low levels of factor VIII (FVIII) and abnormal platelet adhesion expressed by a prolonged bleeding time (BT). Desmopressin is the treatment of choice in patients with type 1 vWD, who account for approximately 80% of cases, because it corrects the FVIII/vWF levels and the prolonged BT in most of these patients. In type 3 and in the majority of type 2 vWD patients, desmopressin is not effective and it is necessary to resort to plasma concentrates containing FVIII and vWF. Treated with virucidal methods, these concentrates are effective and currently safe, but the BT defect is not always corrected by them. Platelet concentrates or desmopressin can be used as adjunctive treatments when poor correction of the BT after concentrates is associated with continued bleeding.

Haemostatic treatment in connection with surgery in patients with von Willebrand disease

Haemostatic treatment in patients with von Willebrand disease (vWD) in connection with surgery aims at normalizing the haemostatic defect in order to avoid bleeding complications. Factor VIII (FVIII) levels in plasma must be normalized in connection with major surgery, whereas the bleeding time is more important for mucous membrane bleedings. Most patients respond well to treatment with desmopressin which stimulates the endogenous release of FVIII and von Willebrand factor (vWF) and shortens the bleeding time. Non-responders to desmopressin are substituted with a plasma-derived factor concentrate which contains vWF and FVIII. This paper includes a summary of retrospective data from the last 10 years on haemostatic treatment in connection with surgery from four haemophilia centres in Sweden and Denmark on 40 invasive procedures in 27 vWD patients and on one normal delivery. If a FVIII-containing concentrate is given prior to surgery a dose of 30-40 IU VIII:C kg(-1) will normalize FVIII levels in most severe cases. If a pure vWF concentrate is used, a dose of 40-50 IU RCoF kg(-1) will normalize RCoF in most cases, but FVIII levels will not be normalized until after about 12 h or later. Repeated doses of FVIII-vWF concentrate may lead to very high levels of FVIII in plasma because of the combined effect of the exogenous FVIII-substitution and the endogenous FVIII-release induces by the infused vWF. Dosage should be adjusted according to FVIII levels in plasma.

Evaluation of the PFA-100® System in the Diagnosis and Therapeutic Monitoring of Patients with von Willebrand Disease

We have evaluated platelet function at high shear with the PFA-100 system in different subtypes of von Willebrand disease (vWD), before and after the intravenous infusions of desmopressin or a factor-VIII/von Willebrand factor (vWF) concentrate. Closure times with the PFA-100 system were determined for both the collagen/ADP and the collagen/epinephrine cartridges in 52 patients with vWD (9 type 1 "platelet normal", 5 type 1 "platelet-discordant", 8 type 1 "platelet-low", 6 type 2A, 9 type 2B, 6 type 2M Vicenza. 6 type 3 and 3 acquired vWD) and 40 controls. Measurements were repeated 1 and 4 h after the i.v. infusion of desmopressin (0.3 microg/Kg) in 26 patients with types 1, type 2M Vicenza or type 2A vWD, or of a factorVIII/vWF concentrate (Alphanate HT, 60 U/Kg) in 4 patients with type 3 vWD. At all time points, vWF plasma levels and the bleeding time (Symplate II) were also determined. Baseline closure times were longer in vWD patients than in controls with both the collagen/ADP and the collagen/ epinephrine cartridges. The sensitivity of the PFA-100 system (88% and 87% with the two cartridges) was higher than that of the bleeding time (65%). Treatment with desmopressin normalized the closure times in patients with type 1 "platelet-normal" or type 2M Vicenza vWD, had no significant effects in patients with type 1 "platelet-low", type 1 "platelet-discordant" or type 2A vWD. Infusion of a factorVIII/vWF concentrate in patients with type 3 vWD slightly shortened their prolonged closure times. In general, changes in PFA-100 were paralleled by shortenings of the bleeding times and increases in plasma vWF levels. The PFA-100 test reflects vWF-dependent platelet function under high shear stress and could be useful in the diagnosis and therapeutic monitoring of patients with vWD.

Assessment of menstrual blood loss and gynaecological problems in patients with inherited bleeding disorders.

Menstrual blood loss and gynaecological problems in patients with inherited bleeding disorders were assessed in this study. One hundred and sixteen women, including 66 with von Willebrand's disease (vWD), 30 carriers of haemophilia and 20 with factor XI (FXI) deficiency were interviewed and their gynaecological history obtained. Their case records were also reviewed and menstrual loss was objectively assessed using a pictorial blood assessment chart (PBAC). Comparison with an age-matched control group (69 women) was performed. Menorrhagia (PBAC score> 100) was confirmed in 74%, 57% and 59% of women with vWD, carriers of haemophilia and FXI deficiency, respectively, in comparison with 29% in the control group (P = 0.001). PBAC scores were higher in vWD patients with a von Willebrand factor activity (vWF:Ac) of </= 30 IU dL-1 compared to those with higher levels, but the difference was not statistically significant. However, there was no relation between PBAC score and the severity of the disease in FXI deficient patients and carriers of haemophilia. Duration of menstruation was significantly longer (P = 0.001) and episodes of flooding was significantly more common (P = 0.001) in patients with inherited bleeding disorders compared to the control group. However, there was no difference in the passage of clots during menstruation. Forty-seven per cent of patients with inherited bleeding disorders had consultations with their family practitioner or gynaecologist for menorrhagia, 36% had medical treatment and 27% had surgical procedures, including 10 hysterectomies. Post-operative bleeding followed in four out of 28 cases of hysteroscopy and/or dilatation and curettage. Bleeding complications following hysterectomy were reported in five out of 10 patients. In conclusion, menorrhagia is a common and major problem in patients with inherited bleeding disorders, especially vWD. Increased awareness among gynaecologists and haematologists of the high prevalence of menorrhagia and the treatment options available is necessary for optimal management of these patients. Appropriate preoperative assessment and haemostatic control during any gynaecological procedure, however minor, and in collaboration with the local haemophilia centre is essential to minimize risks of haemorrhagic complications.

Safety and Efficacy of Continuous Infusion of a Combined Factor VIII – von Willebrand Factor (vWF) Concentrate (Haemate-P™) in Patients with von Willebrand Disease

We studied the safety and efficacy of treatment with continuous infusion of a von Willebrand factor (vWF) concentrate Haemate-P in patients with von Willebrand disease (vWD). Three patients with mild and 5 patients with severe forms of vWD, were treated with continuous infusion of Haemate-P by minipump. The indications for treatment were: to prevent bleeding during 9 surgical procedures or 1 vaginal delivery in 6 patients and to treat 2 bleeding episodes in 2 patients. The patients were monitored daily for factor VIII (FVIII:C) and ristocetin cofactor (vWF: RCo) levels and the infusion rate was adjusted to maintain the desired therapeutic level of vWF:RCo. The treatment was effective in preventing surgical bleeding and controlling bleeding episodes. All factor VIII:C and most of the vWF:RCo levels measured during the study period were above the target therapeutic levels. A significant decrease in clearance of FVIII:C and vWF:RCo was observed over the treatment period. Haemate-P consumption averaged 24.3+/-7.9 vWF:RCo U/kg/day which is approximately half the expected dose had intermittent bolus injections been used. We suggest that continuous Haemate-P infusion is superior to intermittent bolus injections for the treatment of vWD patients by virtue of its efficiency, simplicity and considerable savings.

A Novel Mutation in the D3 Domain of von Willebrand Factor Markedly Decreases Its Ability to Bind Factor VIII and Affects Its Multimerization

AbstractIn type 2N von Willebrand disease (vWD), von Willebrand factor (vWF) is characterized by normal multimeric pattern, normal platelet-dependent function, but a markedly decreased affinity for factor VIII (FVIII). In this report, we describe the case of a vWD patient who has an abnormal vWF multimers distribution associated with a markedly decreased vWF ability to bind FVIII. Sequencing analysis of patient's vWF gene showed, at heterozygous state, a G→A transition resulting in the substitution of Asn for Asp at position 116 of the mature vWF subunit and a C→T transition, changing the codon for Arg 896 into a stop codon. His sister who has a subnormal vWF level, but a normal FVIII/vWF interaction, was found to be heterozygous for the Arg896ter mutation only. Recombinant vWF (rvWF) containing the candidate (Asn116) missense mutation was expressed in COS-7 cells. The expression level of Asn116rvWF was significantly decreased compared with wild-type rvWF. The multimeric pattern of Asn116rvWF was greatly impaired as shown by the decrease in high molecular weight forms. The FVIII binding ability of Asn116rvWF was dramatically decreased. These data show that the Asp116Asn substitution is the cause of both the defective FVIII/vWF interaction and the impaired multimeric pattern observed in the patient's vWF. The monoclonal antibody 31H3 against D' domain of vWF (epitope aa 66-76) that partially inhibits the FVIII binding and recognizes only nonreduced vWF, showed a decreased ability to bind Asn116rvWF when used as capture-antibody in enzyme-linked immunosorbent assay (ELISA). This result suggests that a potential conformation change in the D' domain is induced by the Asp116Asn substitution, which is localized in the D3 domain.

A Novel Mutation in the D3 Domain of von Willebrand Factor Markedly Decreases Its Ability to Bind Factor VIII and Affects Its Multimerization

In type 2N von Willebrand disease (vWD), von Willebrand factor (vWF) is characterized by normal multimeric pattern, normal platelet-dependent function, but a markedly decreased affinity for factor VIII (FVIII). In this report, we describe the case of a vWD patient who has an abnormal vWF multimers distribution associated with a markedly decreased vWF ability to bind FVIII. Sequencing analysis of patient’s vWF gene showed, at heterozygous state, a G→A transition resulting in the substitution of Asn for Asp at position 116 of the mature vWF subunit and a C→T transition, changing the codon for Arg 896 into a stop codon. His sister who has a subnormal vWF level, but a normal FVIII/vWF interaction, was found to be heterozygous for the Arg896ter mutation only. Recombinant vWF (rvWF) containing the candidate (Asn116) missense mutation was expressed in COS-7 cells. The expression level of Asn116rvWF was significantly decreased compared with wild-type rvWF. The multimeric pattern of Asn116rvWF was greatly impaired as shown by the decrease in high molecular weight forms. The FVIII binding ability of Asn116rvWF was dramatically decreased. These data show that the Asp116Asn substitution is the cause of both the defective FVIII/vWF interaction and the impaired multimeric pattern observed in the patient’s vWF. The monoclonal antibody 31H3 against D’ domain of vWF (epitope aa 66-76) that partially inhibits the FVIII binding and recognizes only nonreduced vWF, showed a decreased ability to bind Asn116rvWF when used as capture-antibody in enzyme-linked immunosorbent assay (ELISA). This result suggests that a potential conformation change in the D’ domain is induced by the Asp116Asn substitution, which is localized in the D3 domain.

Females with von Willebrand disease: 72 years as the silent majority.

The monthly challenge of menstruation as well as the haemostatic challenge of childbirth postpartum renders more females than males symptomatic with von Willebrand disease. Among vWD patients, the obstetrical and gynaecological morbidity is certainly more pronounced in Type 2,3 patients compared to Type 1 patients, but even in the latter group there is a high proportion of menorrhagia with associated anaemia, loss of time from work/school and the use of hysterectomy for ultimate control of bleeding. Despite the well known adage of the "gestational palliation" of vWD, there is a high proportion of postpartum haemorrhage in Type 1 patients also especially after the first 24 h after delivery. This may occur despite normalization of the factor VIIIc level in the third trimester, particularly in Type 2,3 patients. With the increasing availability of intranasal/subcutaneous DDAVP that could be readily administered at home for menorrhagia, there recently has been ongoing efforts internationally to determine the prevalence of vWD in females presenting with menorrhagia with a prevalence of 17% combined from two studies of 180 patients total. Issues remain regarding the optimal dose/schedule of intranasal/subcutaneous DDAVP for menorrhagia and the relative efficacy of antifibrinolytic agents. The proper role of oral contraceptives and danazol also deserves further study in vWD patients with menorrhagia. In sum, a comprehensive care approach in females with vWD is warranted analogous to the successful model of care of male haemophiliacs with the intent to (a) reduce unnecessary surgical interventions for menorrhagia, (b) improve the quality of life during menses and (c) optimize peri-partum management.

The substantial impact of von Willebrand disease on menstruation

von Willebrand disease (vWd) is the most common of all congenital bleeding disorders with an estimated prevalence of 1–3% in the general population. However, the gynecological complications have not been thoroughly described. Objective: To compare the clinical and quality of life aspects of vWd in menstruating women in relation to a cohort of menstruating women in the general population. Methods: A patient questionnaire and provider survey of the medical and quality of life aspects of menstruation was administered to 81 menstruating vWd patients registered at four geographically linked Hemophilia Treatment Centers. The questionnaire was also administered to 150 menstruating women volunteers that comprised a control group used to determine normal coagulation levels in menstruating women. We assessed the impact on quality of life of menses in both of the cohorts by a Likert scale of 1–10 with 10 being “most significant impact” using 7 quality of life parameters with those comparisons by Wilcoxon rank sum test. Results: 88% of the vWd patients (pts) had type I vWd, the remaining Type II or unknown. The mean age of the vWd patients was 31.6 ± 10.3; the mean age of the control group was 35.5 ± 7.6. The following comparisons were made using χ 2 and Wilcoxon rank sum test: Controls vWd pts P value # of tampons/pads used for a typical menstrual cycle (mean ± SD) 20.3 ± 9.1 26.5 ± 14.8 .002 % reporting that clothes are stained by menses 20.4% 69.1% .001 Past or present history of anemia 1.3% 65.4% .001 Impact of menses on general activity (mean score) 1.1 ± 1.8 4.2 ± 3.0 <.0001 Impact of menses on ability to work or go to school (mean score) 0.5 ± 1.4 3.2 ± 3.3 <.0001 Impact of menses on family activities (mean score) 0.6 ± 1.4 3.5 ± 2.9 <.0001 Impact of menses on ability to enjoy life (mean score) 0.9 ± 1.5 3.9 ± 3.3 <.0001 Impact of menses on sleep (mean score) 1.3 ± 2.3 3.6 ± 3.1 <.0001 Impact of menses on mood (mean score) 3.5 ± 2.8 5.6 ± 3.3 <.0001 Impact of menses on over-all quality of life (mean score) 1.7 ± 1.8 4.2 ± 3.1 <.0001 Conclusions: vWd markedly diminishes the quality of life during menses. This observation warrants efforts to reduce the attendant morbidities of vWd in menstruating women.

Creating a DDAVP Releasable Pool of Factor VIII: Its Impact on the Gene Therapy of Hemophilia A † 786

The administration of DDAVP has been know for many years to result in a marked increase in plasma von Willebrand factor (vWF) and Factor VIII (FVIII). This occurs within minutes of intravenous administration and suggests a pre-existing pool of both proteins. The two cells in the body that can synthesize vWF are the endothelial cell and the megakaryocyte which respectively store vWF in the endothelial cell Weibel-Palade Body and the platelet α-granule. Most investigations have suggested that the primary source of the increased plasma vWF is the endothelial cell, but there is no recognized storage source of FVIII. In fact, the cell that produces FVIII is not definitively determined. Transplantation studies suggest that liver, spleen, and lymph nodes may be organ sources of FVIII but the tissue source is not well defined. To further define the nature of FVIII synthesis, storage and release, we studied two patients with severe hemophilia A undergoing prophylactic administration of recombinant FVIII and a type 3 von Willebrand disease (vWD) patient receiving regular infusions of vWF without significant FVIII. These individuals were administered 0.3 μg/kg DDAVP intravenously before and following the administration of the respective therapeutic product to determine if the DDAVP-releasable pool of FVIII was established. Plasma assays of vWF and FVIII demonstrated that there was no measurable release of FVIII into plasma in either disorder. In the vWD patient, the endogenous production of FVIII was at a normal level when the vWF was exogenously administered. Yet, there was no increase of FVIII following DDAVP. These studies suggested that the storage pool of FVIII was vWF dependent and that the storage reservoir of both proteins may need to be in the same cell - presumably the same cell in which they were synthesized. Since we have demonstrated that FVIII co-localizes with vWF when the gene for FVIII is inserted into an endothelial cell, we postulate that the DDAVP-releasable pool of FVIII is in a vWF containing cell. This most likely would occur in a subpopulation of endothelial cells that are not yet defined. Furthermore, gene therapy of hemophilia A may need to be targeted to the vascular endothelium to produce a physiologic storage pool that is protected from the proteolytic activity of plasma proteases.

Comparison between von Willebrand Factor (VWF) and VWF Antigen II in Normal Individuals and Patients with von Willebrand Disease

Von Willebrand disease is characterised by a quantitative (type 1) or qualitative (type 2) decrease in von Willebrand factor (vWF) a multimeric glycoprotein involved in primary haemostasis. The propeptide of von Willebrand, also named vWF antigen II (vWF:AgII), is released from platelets and endothelial cells and circulates in plasma as a glycoprotein of 100 kD. In the present study, we attempted to determine whether vWF:AgII level may provide information on the synthesis of vWF, specially in patients with von Willebrand disease (vWD). To elucidate that point, we developed an ELISA and quantify the vWF:AgII in normal individuals and in various vWD patients. The propeptide molar concentration was found to be 5 nM as compared to 31 nM for mature vWF. In normal individuals, the level of vWF:AgII was significantly decreased in females from O and A blood groups. In type 2 vWD patients the level of plasma vWF:AgII appears normal in the patients with normal level of platelet vWF. In type 2 B vWD characterised by increased affinity of mature vWF for platelet glycoprotein Ib, the vWF:AgII in contrast to the vWF antigen (vWF:Ag) was not decreased. In type 2A vWD patients the level of vWF:AgII was decreased in patients with absence of high molecular weight vWF in platelets and plasma but normal in patients with increased sensitivity to proteolysis. Finally, in type 1 vWD, some studied patients have a parallel decrease in vWF:AgII and vWF:Ag whereas in others, the vWF:Ag levels were much more affected than corresponding vWF:AgII levels, as observed in some type 2 vWD patients. Thus, in contrast to that already described, the plasma vWF:AgII level cannot discriminate type 1 from type 2 vWD patients. We conclude that the vWF:AgII measurement provides additional information on the mechanisms responsible for vWD and might also contribute to the classification of vWD patients.