Reduced Plasma Von Willebrand Factor Research Articles

Coagulation factor VIII regulates von Willebrand factor homeostasis invivo

BackgroundCoagulation factor VIII (FVIII) and von Willebrand factor (VWF) circulate as a noncovalent complex, but each has its distinct functions. Binding of FVIII to VWF results in a prolongation of FVIII’s half-life in circulation and modulates FVIII’s immunogenicity during hemophilia therapy. However, the biological effect of FVIII and VWF interaction on VWF homeostasis is not fully understood. ObjectivesTo determine the effect of FVIII in VWF proteolysis and homeostasis in vivo. MethodsMouse models, recombinant FVIII infusion, and patients with hemophilia A on a high dose FVIII for immune tolerance induction therapy or emicizumab for bleeding symptoms were included to address this question. ResultsAn intravenous infusion of a recombinant B-domain less FVIII (BDD-FVIII) (40 and 160 μg/kg) into wild-type mice significantly reduced plasma VWF multimer sizes and its antigen levels; an infusion of a high but not low dose of BDD-FVIII into Adamts13+/- and Adamts13-/- mice also significantly reduced the size of VWF multimers. However, plasma levels of VWF antigen remained unchanged following administration of any dose BDD-FVIII into Adamts13-/- mice, suggesting partial ADAMTS-13 dependency in FVIII-augmented VWF degradation. Moreover, persistent expression of BDD-FVIII at ∼50 to 250 U/dL via AAV8 vector in hemophilia A mice also resulted in a significant reduction of plasma VWF multimer sizes and antigen levels. Finally, the sizes of plasma VWF multimers were significantly reduced in patients with hemophilia A who received a dose of recombinant or plasma-derived FVIII for immune tolerance induction therapy. ConclusionOur results demonstrate the pivotal role of FVIII as a cofactor regulating VWF proteolysis and homeostasis under various (patho)physiological conditions.

Coagulation Factor VIII Enhances the Cleavage of Von Willebrand Factor By ADAMTS13 In Vivo

Introduction: Coagulation factor VIII (FVIII) and von Willebrand factor (VWF) circulate as a noncovalent complex. This interaction helps increase the half-life of circulating FVIII and modulates FVIII immunogenicity. Our previous studies demonstrate that FVIII accelerates VWF proteolysis by ADAMTS13 in vitro under fluidic shear or under certain pathophysiological conditions. However, the physiological relevance of the FVIII-enhanced VWF proteolysis remains questionable. Here, we sought to determine if addition of recombinant FVIII at various concentrations to mice with or without circulating ADAMTS13 or to patients with hereditary thrombotic thrombocytopenic purpura (TTP) affects plasma VWF multimer distribution and its antigen levels. Methods: Recombinant human B-domain deleted FVIII (rFVIII) was intravenously administered into wild-type (WT),Adamts13+/-(A13+/-) and Adamts13-/- (A13-/-) mice. Blood samples were collected prior to and 24 hours following rFVIII infusion. In addition, FVIII was stably expressed in fviii-/-mice via administration of AAV8-BDDFVIII. Moreover, plasma was collected from patients with hereditary TTP who were treated with plasma derived FVIII concentrates. Plasma VWF multimers and antigen levels were determined by SDS-agarose gel electrophoresis followed by Western blotting and enzyme-linked immunoassay (ELISA), respectively. Paired T and Mann Whitney tests were performed to determine the statistical significance of the difference between two groups. Results: Our results demonstrated that an infusion of rFVIII at a low dose (40 ug/kg) and a high dose (160 ug/kg) into WT mice resulted in a dramatic reduction in plasma VWF multimer sizes (Fig. 1) and VWF antigen levels (not shown). FVIII at high dose but not at low dose could also significantly reduce the sizes of VWF multimers in Adamts13+/- and Adamts13-/- mice (Fig. 1). The enhanced degradation of VWF multimers in Adamts13-/- mice suggests that other VWF-cleaving enzymes may participate in the degradation of VWF-FVIII complex. In either Adamts13+/- or Adamts13-/- mice, administration of rFVIII at both low and high doses didn't result in significant changes in VWF antigen levels (not shown). Furthermore, a simultaneous infusion of both rFVIII and recombinant murine ADAMTS13 (rADAMTS13) into Adamts13-/- mice resulted in similar changes in plasma VWF multimer distribution (Fig. 1) and antigen levels (not shown). Importantly, all mice tolerated well to the high dose of rFVIII infusion without major thrombotic events during weeks of follow-up. The restoration of plasma FVIII to 100-200% of normal levels in the fviii-/- mice with AAV8-mediated gene therapy also led to a profound degradation of plasma VWF multimers and significantly reduced plasma levels of VWF antigen (not shown). Finally, a similar pattern of dramatically reduced plasma VWF multimers was observed in 3 out of 4 patients with hereditary TTP following treatment with plasma-derived FVIII concentrates (not shown). Conclusions: Our results demonstrate that supplementation of FVIII at physiological or super-physiological concentrations dramatically accelerates the degradation of VWF multimers in mice and human. These findings further support our hypothesis that FVIII is a physiological cofactor of ADAMTS13 in regulating VWF homeostasis. Our findings may also offer a scientific explanation how FVIII concentrates or perhaps rFVIII might work for the treatment of hereditary TTP and other arterial thrombosis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Endothelial VWF is critical for the pathogenesis of vaso-occlusive episode in a mouse model of sickle cell disease

Vaso-occlusive episode (VOE) is a common and critical complication of sickle cell disease (SCD). Its pathogenesis is incompletely understood. von Willebrand factor (VWF), a multimeric plasma hemostatic protein synthesized and secreted by endothelial cells and platelets, is increased during a VOE. However, whether and how VWF contributes to the pathogenesis of VOE is not fully understood. In this study, we found increased VWF levels during tumor necrosis factor (TNF)-induced VOE in a humanized mouse model of SCD. Deletion of endothelial VWF decreased hemolysis, vascular occlusion, and organ damage caused by TNF-induced VOE in SCD mice. Moreover, administering ADAMTS13, the VWF-cleaving plasma protease, reduced plasma VWF levels, decreased inflammation and vaso-occlusion, and alleviated organ damage during VOE. These data suggest that promoting VWF cleavage via ADAMTS13 may be an effective treatment for reducing hemolysis, inflammation, and vaso-occlusion during VOE.

Contribution of Select ISTH-BAT Subscores to the Total Score in Type 1 VWD

Background:von Willebrand Disease (VWD) is the most common inherited bleeding disorder with partial quantitative deficiency in von Willebrand Factor (VWF); Type 1 comprises 80% of VWD. Clinical manifestations include mucocutaneous bleeding, prolonged bleeding after surgery or injury and excessive menorrhagia in females which have also been reported in normal subjects. A diagnosis of Type 1 VWD is based upon reduced plasma VWF, abnormal mucocutaneous bleeding symptoms and usually a family history of bleeding and VWD. Evaluating abnormal bleeding symptoms involves a detailed bleeding history, which can be quite subjective and time consuming. To address these challenges, the Vicenza-based Bleeding Assessment Tool (BAT) was developed as a validated score and has evolved into the current International Society on Thrombosis and Haemostasis-Bleeding Assessment Tool (ISTH-BAT). A large multi-center NIH supported study (The Zimmerman Program for the Molecular and Clinical Biology of VWD), began in 2005 to correlate clinical laboratory testing, bleeding severity determined by the ISTH-BAT and genetic variability in VWD. In this current study, we investigated which bleeding symptoms were most sensitive in applying the ISTH-BAT to the diagnosis of Type I VWD.Methods:Subjects were enrolled in the Zimmerman Program . Each symptom in the validated ISTH-BAT questionnaire was scored from 0-4 (no symptoms to symptom intervention) and included epistaxis, bruising, oral bleeding, hematuria, gastrointestinal (GI) bleeding, head bleeding, hemarthrosis, hematoma and prolonged bleeding with minor wounds, tooth extraction, surgery, menstruation and child birth. Other scored bleeding symptoms included bleeding at circumcision, excessive umbilical stump bleeding, venipuncture bleeding, vacuum extraction delivery bleeding, ovulatory bleeding and cephalohematoma. The scores of each symptom were analyzed in both healthy controls and subjects diagnosed with Type 1 VWD. Only subjects with VWF antigen (VWF:Ag) and/or VWF ristocetin cofactor activity (VWF:RCo) <30 IU/dL were considered in the comparative analysis. VWF levels were performed by a central laboratory, including VWF:Ag, VWF:RCo, VWF propeptide (VWFpp) and VWF collagen binding with type III collagen (VWF:CB). Statistical analysis included Mann-Whitney test, Chi-square or a Fisher's exact test, Pearson correlations and Spearman correlations. Results were considered significant for a two-sided p-value <0.05. A regression analysis was performed to examine which subscores contributed the most to the total score of the ISTH-BAT.Results:Analysis included 383 subjects (Female=262, Male=121) enrolled in the Zimmerman Program . Of these subjects, 244 were healthy controls (mean age of 38.93 [range 10-73 years of age]) and 139 had Type 1 VWD (mean age of 19.46 [range 1-76 years of age]). Within the Type 1 subjects, there were more females >12 years of age (no gender difference < 12 years of age) and greater number of Caucasians and younger subjects. Type 1 VWD subjects scored significantly higher than the healthy control group in both ISTH-BAT total scores (p<0.0001) as well as all subscores (p<0.001). In comparing bleeding symptoms from healthy controls with Type 1 VWD subjects: epistaxis 4% vs 46%, bruising 16% vs 64%, minor wound bleeding 1% vs 42%, oral bleeding 3% vs 35%, tooth extraction bleeding 3% vs 18%, GI bleeding 3% vs 11%, hematuria 0% vs 11%, surgical bleeding 4% vs 23%, menstrual bleeding 60% vs 85%, bleeding after delivery 2% vs 12%, other bleeding 0% vs 11% (47% were circumcision bleeding), head bleeding 0% vs 4%, hemarthrosis 0% vs 11%, and hematoma 0% vs 7%. Regression analysis revealed that epistaxis, bruising, excessive bleeding with minor wounds, oral bleeding, and menstrual bleeding subscores contributed the most to the ISTH-BAT total score (p<0.016).Conclusion:The ISTH-BAT currently is strongly predictive of bleeding symptoms in the diagnosis of Type 1 VWD. Though all ISTH-BAT subscores correlated with a diagnosis of Type 1 VWD, some subscores contributed more significantly to the total score than others and were more prevalent than in normal subjects. Accordingly, the sensitivity and specificity of ISTH-BAT subscores are critical to the assessment of bleeding in the diagnosis of Type 1 VWD. DisclosuresFlood:Shire: Consultancy; CSL Behring: Consultancy. Gill:Bayer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Shire: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Behring: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. James:CSL Behring: Research Funding; Shire: Research Funding; Bayer: Research Funding.

Toward Personalized Treatment for Patients with Low von Willebrand Factor and Quantitative von Willebrand Disease.

The biological mechanisms involved in the pathogenesis of type 2 and type 3 von Willebrand disease (VWD) have been studied extensively. In contrast, although accounting for the majority of VWD cases, the pathobiology underlying partial quantitative VWD has remained somewhat elusive. However, important insights have been attained following several recent cohort studies that have investigated mechanisms in patients with type 1 VWD and low von Willebrand factor (VWF), respectively. These studies have demonstrated that reduced plasma VWF levels may result from either (1) decreased VWF biosynthesis and/or secretion in endothelial cells and (2) pathological increased VWF clearance. In addition, it has become clear that some patients with only mild to moderate reductions in plasma VWF levels in the 30 to 50 IU/dL range may have significant bleeding phenotypes. Importantly in these low VWF patients, bleeding risk fails to correlate with plasma VWF levels and inheritance is typically independent of the VWF gene. Although plasma VWF levels may increase to > 50 IU/dL with progressive aging or pregnancy in these subjects, emerging data suggest that this apparent normalization in VWF levels does not necessarily equate to a complete correction in bleeding phenotype in patients with partial quantitative VWD. In this review, these recent advances in our understanding of quantitative VWD pathogenesis are discussed. Furthermore, the translational implications of these emerging findings are considered, particularly with respect to designing personalized treatment plans for VWD patients undergoing elective procedures.

The physical spacing between the von Willebrand factor D'D3 and A1 domains regulates platelet adhesion in vitro and in vivo

Background Previous exvivo studies using truncated VWF (von Willebrand factor) suggest that domain-level molecular architecture may control platelet-GpIbα binding function. Objective We determined if this is the case with multimeric VWF invivo. Methods Full-length human VWF ('hV') was modified with a 22-amino acid mucinous stretch at either the N-terminus of VWF-A1 to create 'hNV' or C-terminus to yield 'hCV'. This extends the physical distance between VWF-A1 and the adjacent domains by ~6nm. Similar mucin inserts were also introduced into a human-murine chimera ('h[mA1]V') where murine-A1 replaced human-A1 in hV. This yielded 'h[mA1]NV' and 'h[mA1]CV', with N- and C-terminal inserts. The constructs were tested exvivo and invivo. Results Mucin insertion at the N-terminus, but not C-terminus, in both types of constructs resulted in >50-fold increase in binding to immobilized GpIbα. N-terminal insertion also resulted in greater shear-induced platelet activation, more thrombus formation on collagen, enhanced platelet accumulation and slower platelet translocation on immobilized VWF in microfluidics assays. Hydrodynamic injection-based expression of h[mA1]NV, but not h[mA1]V or h[mA1]CV, in VWF-/- mice caused profound thrombocytopenia, reduced plasma VWF concentrations, lower multimer distribution, and incessant tail bleeding that is reminiscent of von Willebrand disease type 2B. Platelet plugs were noted in the portal veins and hepatic arteries. An anti-D'D3 mAb DD3.3 that displays enhanced binding to VWF containing the N-terminal mucin insert also exhibited increased binding to wild-type VWF under shear and upon ristocetin addition. Conclusion Conformation changes at the VWF D'D3-A1 interface may be a key regulator of thrombosis invivo. Structural features at the A1-A2 interface are likely of less significance.

Novel insights into the clinical phenotype and pathophysiology underlying low VWF levels

AbstractCritical clinical questions remain unanswered regarding diagnosis and management of patients with low von Willebrand factor (VWF) levels (30-50 IU/dL). To address these questions, the Low VWF Ireland Cohort (LoVIC) study investigated 126 patients registered with low VWF levels. Despite marginally reduced plasma VWF levels, International Society of Thrombosis and Haemostasis Bleeding Assessment Tool (ISTH BAT) confirmed significant bleeding phenotypes in the majority of LoVIC patients. Importantly, bleeding tendency did not correlate with plasma VWF levels within the 30 to 50 IU/dL range. Furthermore, bleeding phenotypes could not be explained by concurrent hemostatic defects. Plasma factor VIII to VWF antigen (VWF:Ag) ratios were significantly increased in LoVIC patients compared with controls (P < .0001). In contrast, VWF propeptide to VWF:Ag ratios >3 were observed in only 6% of the LoVIC cohort. Furthermore, platelet-VWF collagen binding activity levels were both significantly reduced compared with controls (P < .05). In response to 1-desamino-8-D-arginine vasopressin (DDAVP), peak VWF:Ag levels exceeded 100 IU/dL in 88% of patients and was sustained >100 IU/dL after 4 hours in 72% of subjects. In conclusion, our novel data suggest that low VWF levels can be associated with significant bleeding and are predominantly due to reductions in VWF synthesis and/or constitutive secretion. Although enhanced VWF clearance may contribute to the pathophysiology in some individuals, the absolute reduction in VWF plasma half-life is usually mild and not sufficient to significantly impact upon the duration of DDAVP-induced VWF response. This trial was registered at www.clinicaltrials.gov as #NCT03167320.

Novel Insights into the Clinical Phenotype and Pathophysiology Underlying Low VWF Levels: The Low Von Willebrand Factor in Ireland Cohort (LoVIC) Study

▪Cohort studies have identified VWF gene mutations in ~65% of patients with type 1 Von Willebrand Disease (VWD). However, in patients with mild reductions in Von Willebrand Factor (VWF) levels, VWF mutations are less common and linkage studies suggest that the reduced plasma VWF levels are often independent of the VWFgene. Important clinical questions therefore remain unanswered regarding diagnosis and management of patients with Low VWF (levels 30-50 IU/dL). Evidence-based diagnostic criteria are needed for establishing the diagnosis of Low VWF, and the relationship between Low VWF levels and bleeding phenotype needs to be defined. Furthermore, the molecular mechanisms responsible for the reduced plasma VWF levels remain unclear. To address these questions, we established the Low VWF Ireland Cohort (LoVIC) study. In contrast to previous Type 1 VWD studies, this prospective longitudinal cohort study recruited only patients with Low VWF levels, (defined by bleeding history and lowest VWF levels 30-50 IU/dL on two occasions, three months apart) and recruited 140 adult Irish patients.Although Bleeding Assessment Tools (BATs) have been used in type 1 VWD, their utility has not been studied in patients with Low VWF. As determined using either the ISTH BAT or the Condensed MCMDM-1 VWD score, we observed significant bleeding histories in the majority of LoVIC patients. For example, among female patients (n=112), 77% had positive ISTH BAT scores (≥6) and 72% had positive MCMDM-1 scores (≥4). Importantly, bleeding tendency did not correlate with plasma VWF levels within the 30-50 IU/dL range. To further investigate this bleeding phenotype, hemostatic studies (including platelet aggregation and coagulation factor assays) were performed. Additional mild coagulation defects were identified in only 10 subjects. Furthermore, abnormal multimer patterns were identified in only 3 patients. To investigate the pathophysiology underlying Low VWF levels, plasma FVIII:C and VWF propeptide (VWF:pp) levels were defined. Interestingly, plasma FVIII:C/VWF:Ag ratios were significantly increased in LoVIC patients compared to normal controls (mean 1.3 versus 1.07; p<0.001). In contrast, increased plasma VWF:pp/VWF:Ag ratios > 3 were observed in only 6% of the total cohort. Taken together, these data demonstrate that the reduced plasma VWF levels in patients with Low VWF are predominantly attributable to decreased VWF synthesis and/or secretion rather than enhanced VWF clearance. To determine whether quantitative and/or qualitative abnormalities in platelet (plt)-VWF may influence bleeding phenotype in patients with Low VWF, we studied plt-VWF:Ag and VWF:CB in 50 consecutive LoVIC patients. In keeping with the hypothesis that reduced VWF synthesis plays a key role in the pathogenesis underlying Low VWF levels, plt-VWF:Ag and plt-VWF:CB levels were both significantly reduced in LoVIC patients compared to controls (mean plt-VWF:Ag 0.16 versus 0.21 IU/109/L, p<0.05; mean plt-VWF:CB 0.18 versus 0.34 IU/109/L, p<0.001).To further study the molecular mechanisms underlying Low VWF levels, plasma VWF:Ag and VWF:RCo responses following desmopressin (DDAVP) administration were examined. In 88% of patients, the post-DDAVP peak levels exceeded 100 IU/dL. Importantly, the response to DDAVP was also sustained with both VWF:Ag and VWF:RCo above 100 IU/dL after 4 hours in 72% subjects. These DDAVP responses demonstrate that Weibel Palade body stores of VWF are maintained in patients with Low VWF, and that the regulated pathway of VWF secretion is intact. Furthermore, the sustained plasma VWF response observed following DDAVP supports the hypothesis that enhanced VWF clearance does not play a major role in the pathogenesis of Low VWF levels.In conclusion, we demonstrate that Low VWF levels are associated with a significant bleeding phenotype. In addition, we further show that in the majority of patients with Low VWF levels, the bleeding phenotype cannot be explained by the presence of concomitant bleeding disorders. Finally, our novel data demonstrate that Low VWF levels are due in large part to reductions in VWF synthesis and/or constitutive secretion. Although enhanced VWF clearance may contribute to the pathophysiology in a minority of individuals with Low VWF, the absolute reduction in VWF plasma half-life is usually mild and not sufficient to significantly impact upon the duration of DDAVP-induced VWF response. DisclosuresLavin:Baxalta: Consultancy, Research Funding. O'Sullivan:Pzifer: Research Funding. O'Connell:Baxalta: Consultancy, Research Funding. James:Octapharma: Research Funding; Bayer: Research Funding; CSL Behring: Research Funding; Biogen: Consultancy; Basalt: Consultancy. Di Paola:CSL BEhring: Consultancy; Biogen: Consultancy. O'Donnell:Bayer: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Baxalta: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novo Nordisk: Research Funding, Speakers Bureau; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Leo Pharma: Speakers Bureau; CSL Behring: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees.

Pharmacological and Genetic Inhibition of Autophagy Decreases the Secretion of High Molecular Weight Von Willebrand Factor from Endothelial Cells in Vitro and in Vivo

The regulation of von Willebrand factor (VWF) processing, packaging, and secretion is important for primary hemostasis. Recently, autophagy has been implicated in modulating VWF maturation and Weibel-Palade body (WPB) morphology. Additionally, treatment of mice and humans with chloroquine (CQ), an anti-malarial agent and pharmacological inhibitor of autophagy, is shown to increase bleeding time. Therefore, we hypothesize that targeting autophagic flux may be therapeutic for arterial thrombotic disorders such as thrombotic thrombocytopenic purpura (TTP). To test this hypothesis, we first treated human umbilical vein endothelial cells (HUVECs) with CQ in culture and showed that CQ dose-dependently decreased VWF antigen levels and multimer sizes in the conditioned medium of histamine-stimulated cells (not shown). Knockdown of an autophagy-related protein (Atg7) with shRNA in HUVECs had similar effect to CQ treatment in VWF secretion and multimer distribution (not shown). More interestingly, daily injections (i.p.) of CQ at 60 mg/kg into Adamts13-/- mice (CAST/Ei strain) for 7 days reduced plasma VWF concentration by more than 60% compared to vehicle control (Fig. 1). Interestingly, VWF secreted from CQ-treated mice remained functional as collagen binding activity/antigen ratio was comparable to vehicle control mice. However, multimer analysis demonstrated the selective lack of ultra large VWF in plasma of Adamts13-/- mice treated with CQ as compared with those treated with vehicle alone (Fig. 2). These results indicate that targeting autophagy pathway with a pharmacological agent, such as CQ, may modulate VWF secretion and, thereby, arterial thrombosis. Our ongoing experiments are to determine the therapeutic efficacy of CQ and other autophagy inhibitors in murine models of arterial thrombosis and TTP. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Storage and secretion of naturally occurring von Willebrand factor A domain variants.

Von Willebrand disease (VWD) is a bleeding disorder characterized by reduced plasma von Willebrand factor (VWF) levels or functionally abnormal VWF. Low VWF plasma levels in VWD patients are the result of mutations in the VWF gene that lead to decreased synthesis, impaired secretion, increased clearance or a combination thereof. However, expression studies of variants located in the A domains of VWF are limited. We therefore characterized the biosynthesis of VWF mutations, located in the VWF A1-A3 domains, that were found in families diagnosed with VWD. Human Embryonic Kidney 293 (HEK293) cells were transiently transfected with plasmids encoding full-length wild-type VWF or mutant VWF. Six mutations in the A1-A3 domains were expressed. We found that all mutants, except one, showed impaired formation of elongated pseudo-Weibel-Palade bodies (WPB). In addition, two mutations also showed reduced numbers of pseudo-WPB, even in the heterozygous state, and increased endoplasmic reticulum retention, which is in accordance with the impaired regulated secretion seen in patients. Regulated secretion upon stimulation of transfected cells reproduced the in vivo situation, indicating that HEK293 cells expressing VWF variants found in patients with VWD can be used to properly assess defects in regulated secretion.

VWF Binding to Types I, III or VI Collagen In the ZPMCB-VWD with Identification of Subjects with Selective Reduced Plasma VWF:CB

Abstract 235 Von Willebrand Disease (VWD) is a common bleeding disorder caused by quantitative (types 1 and 3) and qualitative (type 2) abnormalities in von Willebrand factor (VWF). Defective VWF binding to collagen (VWF:CB) has been identified in VWD patients, but the type(s) and amount of collagen vary between recommended assays. We measured VWF:CB using separate assays for types I, III, and VI collagen in plasmas from 233 healthy controls and 315 VWD index cases (261 type 1, 37 type 2 and 17 type 3) recruited into the Zimmerman Program for the Molecular and Clinical Biology of VWD (ZPMCB-VWD). Additional studies included VWF antigen (Ag), VWF ristocetin-cofactor (RCo), VWF multimer, and VWF gene sequence analysis. VWF:CB was tested using ELISA assays with type I human placental collagen (5 ug/ml), type III human placental collagen (1 ug/ml), and by a commercial human placental type VI VWF:CB assay (Technoclone, Austria). Normal ranges were established for VWF:CB and VWF:CB/Ag ratios with the ZPMCB-VWD healthy control population. Three subjects (2 healthy controls and 1 type 1 VWD subject) were identified with reduced VWF:CB and reduced VWF:CB/Ag ratios to type VI collagen, despite normal results with type I and type III collagen. The two control subjects had VWF:CB/Ag ratios of 0.43 and 0.51 for type VI collagen. The patient with type 1 VWD had absent type VI VWF:CB and a VWF:Ag of 54 IU/dL with a history of clinical epistaxis and an EU Bleeding Score of 8 (normal less than or equal to 3). VWF sequencing demonstrated that these three individuals had an A1 loop polymorphism, R1399H, which has been previously reported as a VWF polymorphism. No other individuals in our study had this sequence change. Of particular note was that the VWF:CB/Ag ratios for types I and III collagen were each normal – suggesting a selective abnormality. As anticipated, collagen binding with all collagens was undetectable in the type 3 VWD subjects. Type 2A and 2B VWD subjects demonstrated reduced VWF:CB and VWF:CB/Ag ratios to all three types of collagen. Type 2M and 2N VWD subjects exhibited normal VWF:CB/Ag ratios to all three types of collagen, but one type 2M patient with an I1425F A1 loop mutation had a reduced VWF:CB/Ag ratio of 0.58. In the VWF A1 domain crystal structure, amino acid 1425 is in close proximity to amino acid 1399, suggesting the conformation of that region may be critical to type VI collagen binding to VWF. Since the frequency of the R1399H polymorphism is estimated to be 2% of the population (Sadler and Ginsburg, 1993), defective binding to type VI collagen may be an important contributor to the variability in the bleeding phenotype of VWD. Disclosures: Lentz: Novo Nordisk: Consultancy. Montgomery:GTI Diagnostics, Inc: Consultancy.

The Prevalence of Reduced VWF Survival Phenotype (Type 1C VWD) in Type 1 Index Cases Recruited into the TS Zimmerman Program for the Molecular and Clinical Biology of VWD (ZPMCB - VWD).

The decreased survival of von Willebrand factor (VWF) in plasma has recently been identified as a novel mechanism causing type 1 VWD. Patients with this phenotype are characterized by a substantially reduced plasma VWF:Ag (< 30 IU/dL), a robust response to DDAVP, and a reduced half-life of VWF:Ag after DDAVP administration. Although a normal distribution of plasma VWF multimers appears to be present, the multimer satellite bands are decreased in intensity. A few mutations have been identified in type 1C VWD patients including C1130F/G/R, W1144G, R1205H, and S2179F. Identification of type 1 VWD patients with this phenotype is clinically important because DDAVP may not be the treatment of choice in this subset of patients due to the reduced half-life of the VWF released into plasma. Although previous studies have facilitated the characterization of this phenotype, the prevalence of the reduced survival, type 1C VWD has not yet been determined. We have previously reported that assay of plasma VWF:Ag and VWF propeptide (VWFpp) can be used to identify type 1C VWD patients having reduced VWF survival. These individuals have a substantially increased ratio of steady-state plasma VWFpp/VWF:Ag in contrast to normal individuals where the ratio is equal to one. We subsequently established blood group-specific normal ranges for VWFpp/VWF:Ag by assay of 279 healthy blood donors. Here we report the assay of VWF:Ag and VWFpp in 192 normal controls and 61 type 1 VWD index cases recruited from eight US centers within the TS Zimmerman Program for the Molecular and Clinical Biology of VWD (ZPMCB - VWD) study. VWD type 1C individuals previously reported from our center were excluded from the current study. Using VWF:Ag and VWFpp levels determined for the 192 normal controls, we established a 2-standard deviation normal range for VWFpp/VWF:Ag ratio of 0.5 to 1.6, consistent with our previous studies. Of the 61 type 1 VWD index cases, three individuals were identified with a substantially increased VWFpp/VWF:Ag ratio (mean = 5.0). One individual was found to have the well-characterized R1205H VWF gene mutation that has been associated with reduced VWF survival. These individuals demonstrated laboratory parameters consistent with a reduced VWF survival phenotype including moderate to severely decreased plasma VWF:Ag (mean = 16.6 IU/dL), a diminished intensity of multimer satellite bands amongst an essentially normal distribution of plasma multimers, and an elevated bleeding score (mean = 4.7). Together these data indicate a reduced survival phenotype (type 1C) in 4.9% of type 1 VWD index cases in the ZPMCB - VWD study. Identification of type 1 VWD individuals with a reduced plasma VWF survival phenotype is clinically essential as these individuals may require alternative treatment strategies.

Deletion of Pro1648-Leu1650 in VWF73, the Conservative Substrate for ADAMTS13, Causes von Willebrand Disease.

The multimer distribution of Von Willebrand factor (VWF) in plasma is regulated by the specific VWF cleaving protease ADAMTS13, which cleaves at the Y1605-M1606 bond in the A2 domain of VWF under the shear stress, plays paramount roles in mediating platelet adhesion to the subendothelium during vascular damage. Quantitative deficiency or qualitative abnormity in VWF caused by the mutations in the VWF gene leads to von Willebrand disease (vWD). There exist three types of vWD. Type 1 vWD is characterized by the partial quantitative deficiency of VWF and normal multimers. Type 3 refers to complete deficiency of VWF. Type 2 vWD refers to the qualitative deficiency of VWF and is subdivided into types of 2A, 2B, 2M, 2N. Meanwhile, the subtype of 2A vWD is also subdivided into two groups regarding ADAMTS13-dependent proteolysis of VWF. Group I includes the mutations G1505R, S1506L, L1540P, V1607D, which hinder the multimer assembly and diminish the secretion of VWF while group II includes R1597W, R1597Q, G1505E, I1628T, E1628K, which make VWF more susceptible to ADAMTS13 -dependent proteolysis. All these published point mutations cluster in the A2 domain of VWF and the corresponding mutation mechanism upon VWF has been elucidated. We have identified a patient with bleeding symptoms and reduced plasma VWF antigen, factor VIII and ristocetin cofactor activity, compatible with clinical von Willebrand disease. Analysis of proband's plasma VWF multimers in low resolution agarose gels demonstrated similar results compared to the healthy. The patient carried a heterozygous deletion mutation from position 1648 to 1650 resulting in loss of three consecutive amino acids (ProIleLeu) in the pre-pro-VWF. It has been demonstrated that the minimal substrate for ADAMTS13 is intact VWF73, a region from Asp1596 to Arg1668 of von Willebrand factor. The novel deletion mutation in this patient occurred in the intact VWF73 and its mutated effect upon cleavage by ADAMTS13 could be clarified by further experiments such as in vitro recombinant expression of mutated VWF and might strengthen our understanding of the interaction between VWF and ADAMTS13.

Reversal of Acquired von Willebrand Disease after Allogeneic Hematopoietic Stem Cell Transplantation in a Patient with High Risk Chronic Lymphocytic Leukemia.

Acquired von Willebrand (AvWD) disease is a relatively rare bleeding disorder and since the initial description, fewer than 300 cases of AvWD have been reported. The most frequently observed association is with dysproteinemias such as monoclonal gammopathy of undetermined significance (MGUS) and plasma cell proliferative disorders such as Waldenstroem macroglobulinemia and multiple myeloma. Other hematological diseases associated with AvWD have been reported including lymphoproliferative and myeloproliferative disorders. We report the case of a 59 year old male patient with an AvWD associated with a chronic lymphocytic leukemia.In November 2000 this patient was diagnosed with a B cell chronic lymphocytic leukemia (CLL). Because of an initial Binet-Stage A no specific therapy was initiated. In October 2003 the CLL progressed to Binet-Stage B and molecular examinations revealed an unfavorable risk profile with a 11q22.3-q23.1 deletion and an unmutated IgVH gene status. Because of the presence of an HLA-identical brother the patient was enrolled into a pilot study of allogeneic stem cell transplantation following conditioning with fludarabine and cyclophosphamid in patients with high-risk chronic lymphocytic leukemia. Four cycles of fludarabine and cyclophosphamide were given prior to allogeneic stem cell transplantation and induced a partial remission.Since 2000 the patient reported a new clinical bleeding tendency, presenting mainly as frequent epistaxis. Further laboratory investigations showed a prolonged activated partial thromboplastin time (50 sec.) and a reduced plasma von Willebrand factor (vWF) antigen and vWF activity of 6% and 2%, respectively. This was accompanied by a reduced Factor VIII activity of 14% (factor IX-, XI-, XII- activity > 100%, respectively; lupus anticoagulant assay negative; immunofixation negativ). A vWF multimer analysis showed selective decrease in the high-molecular-weight multimers as seen in type II congenital vWD.The reduced intensity conditioning (RIC) regimens consisted the following combination: Fludarabine 30mg/m² /d (=150mg/m² total dose) and Cyclophosphamide 500mg/m² /d (=2500mg/m² total dose) (day -7 through day -3), GVHD prophylaxis with Cyclosporin A (CsA) and mycophenolate mofetil (MMF).A therapeutic trial of desmopressin (DDAVP) was insufficient and therefore a continuous infusion of the factor VIII/vWF concentrate (Haemate® P; Aventis Behring) was administered from the start of conditioning until platelet recovery. Under this regimen no bleeding complication was observed, neither spontaneously nor during intensive procedures. In accordance with previous reports of replacement therapy in AvWD we observed a shortened half-life of the vWF.On day 30 after allogenic hematopoietic stem cell transplantation we observed for the first time a normalization of the activated partial thromboplastin time followed by a gradual increase of the vWF antigen and activity. On day 60 we were able to document the normalization of both the vWF antigen and activity and the vWF multimer analysis. This nomalisation was coincident with the establishment of a complete donor chimerism for the first time.We therefore believe that this case shows a form of AvWD associated with chronic lymphocytic leukemia and its reversal by treatment of the underlying disease by allogeneic hematopoietic stem cell transplantation. [Display omitted]

Long-term angiotensin-converting enzyme inhibition reduces plasma asymmetric dimethylarginine and improves endothelial nitric oxide bioavailability and coronary microvascular function in patients with syndrome X

Angiotensin-converting enzyme (ACE) inhibition has been shown to improve clinical myocardial ischemia in patients with syndrome X (angina pectoris, positive treadmill exercise test, normal coronary angiograms, and no evidence of coronary spasm). This study was conducted to investigate the effects of long-term ACE inhibitors on endothelial nitric oxide (NO) metabolism and coronary microvascular function in patients with syndrome X. After a 2-week washout period, 20 patients with syndrome X were randomized to receive either enalapril, an ACE inhibitor, 5 mg twice daily (n = 10) or placebo (n = 10) in a double-blind design for 8 weeks. Another 6 age- and gender-matched subjects with negative treadmill exercise tests were also studied as controls. Compared with control subjects, patients with syndrome X had significantly reduced coronary flow reserve, reduced plasma levels of nitrate and nitrite (NOx), and a reduced plasma l-arginine to asymmetric dimethylarginine (ADMA) ratio (an index of systemic NO metabolism), as well as reduced endothelial function. These patients also had increased plasma levels of ADMA, which is an endogenous inhibitor of NO synthase and of von Willebrand factor, a marker of endothelial injury. Baseline characteristics including exercise performance and coronary flow reserve were similar between enalapril and placebo groups. After an 8-week treatment period, exercise duration (p = 0.001) and coronary flow reserve (p = 0.001) significantly improved with enalapril but not with placebo. Enalapril treatment, but not placebo, reduced plasma von Willebrand factor (p = 0.03) and ADMA levels (p = 0.01) and increased NOx levels (p = 0.01) and the ratio of l-arginine to ADMA (p <0.01). In patients with syndrome X, the plasma NOx level was positively and ADMA level inversely correlated with coronary flow reserve before and after the treatment. In conclusion, long-term ACE inhibitor treatment with enalapril improved coronary microvascular function as well as myocardial ischemia in patients with syndrome X. This may be related to the improvement of endothelial NO bioavailability with the reduction of plasma ADMA levels.

Comparative Mapping of Distal Murine Chromosome 11 and Human 17q21.3 in a Region Containing a Modifying Locus for Murine Plasma von Willebrand Factor Level

Type 1 von Willebrand disease (VWD) is a common inherited disorder characterized by mild to moderate bleeding and reduced levels of von Willebrand factor (VWF). An animal model for human type 1 VWD, the RIIIS/J mouse strain, exhibits a prolonged bleeding time and reduced plasma VWF levels. We have previously mapped the defect in RIIIS/J to distal mouse Chr 11, distinct from theVwflocus on Chr 6. This locus,Mvwf,was localized to an ∼0.5-cM interval, tightly linked toGip,distal toNgfr,and proximal toHoxb.We have now used these genetic markers to construct a contig of yeast and bacterial artificial chromosomes and bacteriophage P1 clones spanning the ∼300-kbMvwfnonrecombinant interval. In a comparative mapping approach, mouse homologues of mapped human expressed sequence tags (ESTs) were localized relative to the candidate interval. Twenty-one sequence-tagged sites and ESTs from the corresponding human syntenic region 17q21.3 were ordered using the high-resolution Stanford TNG3 radiation hybrid panel. Based on the resulting radiation hybrid map and our mouse genetic and physical maps, the order of human and mouse genes in a >0.7-cM region appears to be conserved. Six genes localized to theMvwfnonrecombinant interval by comparative mapping included orthologs ofGNGT2, ATP6N1,and a nuclear domain protein. Seven other genes or ESTs were excluded from the candidate interval, including orthologs ofPHB, PDK2,a speckle-type protein, and a UDP-galactose transporter. Using exon trapping, 10 additional putative expressed sequences were identified within theMvwfnonrecombinant interval, including a previously cloned murine glycosyltransferase as well as exons showing sequence similarity to genes forCaenorhabditis elegansandSaccharomyces cerevisiaepredicted proteins, anArabidopsis thalianaubiquitin-conjugating enzyme, and aGallus gallusmRNA zipcode-binding protein. Further characterization of these putative genes could identify the dominant mutation responsible for low plasma VWF levels in RIIIS/J mice. These data may also aid in the localization of other disease loci mapped to this region, including the gene for tricho-dento-osseous syndrome and a murine locus for susceptibility to ozone-induced acute lung injury.

Family Study on Type I von Willebrand Disease with Aberrant 200kDa Fragment of Reduced Plasma von Willebrand Factor

Family Study on Type I von Willebrand Disease with Aberrant 200kDa Fragment of Reduced Plasma von Willebrand Factor

Reduced Plasma von Willebrand Factor Fragments in Patients with Various Types of von Willebrand Disease

We previously reported a new analytical immunoblotting method to detect reduced plasma von Willebrand factor (vWF) fragments using polyclonal anti-reduced vWF antibody and demonstrated that the reduced normal plasma vWF consists of at least five or six fragments, 240kDa for the main fragment and 216 (220, 210), 194, 164 and 144kDa for minor fragments. In this study, we describe the reduced fragments in patients with various types of von Willebrand disease (vWD) (type I: 7 patients, type IIA: 4 patients and type IIB: 3 patients) using this method. Of seven patients with type I vWD, one patient with an equally low level of ristocetin cofactor activity (vWF: RCo) and vWF antigen (vWF: Ag) showed normal relative proportions of each fragment. However, three of four patients with a low level of vWF: RCo compared to vWF: Ag showed a relatively increased proportion of the 164kDa fragment. The remaining two patients demonstrated a new 200kDa reduced fragment in addition to those of normal plasma vWF. In type II A vWD, all patients showed a relatively increased proportion of the 164kDa fragment. In type IIB vWD, two of three patients also showed a relatively increased proportion of the 164kDa fragment. These results suggest that plasma vWF with a low vWF: RCo compared with the vWF: Ag ratio tends to show an increased proportion of the 164kDa reduced fragment, even in type I vWD with a normal multimeric structure. Fragment analysis of reduced vWF in patients with vWD is useful to understand the abnormality at the molecular level, especially in type I vWD.