Screening for von Willebrand disease does not impact posttonsillectomy bleeding in a low-risk population.

BACKGROUND: Bleeding is an important complication in children following tonsillectomy. Screening with coagulation tests prior to procedure is common to assess bleeding risk in the perioperative period, although ASH/ASPHO Choosing Wisely guidelines recommend against routine PT/PTT testing. Our aim was to compare von Willebrand factor antigen (VWF:Ag) and activity levels among patients with postoperative bleeding following tonsillectomy to evaluate for potential risk for bleeding. PROCEDURE: Eligible subjects were aged 0-18 without significant personal or family history of major bleeding. Postoperative bleeding diaries were collected and symptoms measured using a postoperative bleeding score. Plasma VWF levels were drawn at time of anesthesia administration. RESULTS: Postoperative bleeding occurred in 248 cases out of 1399 total subjects. Median VWF:Ag was 86 in patients with postoperative bleeding scores of 1-2, 86 for scores 3-4, 84 for scores 5-6, and 83 for scores >6, with no significant difference among groups (p = .98). Additionally, no difference was observed for subjects with multiple days of postoperative bleeding as compared to those with only 1 day of postoperative bleeding. Finally, no difference in VWF:Ag was observed for subjects whose first reported bleed occurred early in the postoperative course compared to those whose first reported bleed occurred later. VWF:Ag does not correlate with severity of bleeding, time of onset of first bleeding event, or recurrence of bleeding in healthy children with no personal or family history of bleeding who have postoperative bleeding following tonsillectomy. CONCLUSIONS: This data does not support routine von Willebrand disease screening prior to tonsillectomy.

The role of genetics in the pathogenesis and diagnosis of type 1 Von Willebrand disease.

PURPOSE OF REVIEW: Von Willebrand disease (VWD) is a common bleeding disorder, but diagnosis of VWD is challenging, particularly with type 1 VWD. Although most clinicians use specific tests of von Willebrand factor (VWF) activity to classify patients with VWD, genetic testing for VWF defects is another potential method of diagnosis. RECENT FINDINGS: Studies of patients with type 1 VWD report consistently that many, but not all, study participants have VWF gene defects. Certain populations, including those with VWF levels less than 30 IU/dl and those with clearance defects, are more likely to have a VWF sequence variant. In addition, a number of loci outside the VWF gene have been shown to affect VWF levels, including ABO, CLEC4M, STXBP5, and STAB2. SUMMARY: Genetic defects in VWF are common, but not all defects lead to disease. Type 1 VWD in particular does not always have an associated VWF sequence variant. New data stemming from genome-wide association studies on modifier genes suggest that the etiology of type 1 VWD is multifactorial.

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

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

von Willebrand factor propeptide: biology and clinical utility.

von Willebrand factor (VWF) is a large multimeric glycoprotein that mediates the attachment of platelets to damaged endothelium and also serves as the carrier protein for coagulation factor VIII (FVIII), protecting it from proteolytic degradation. Quantitative or qualitative defects in VWF result in von Willebrand disease (VWD), a common inherited bleeding disorder. VWF is synthesized with a very large propeptide (VWFpp) that is critical for intracellular processing of VWF. VWFpp actively participates in the process of VWF multimerization and is essential for trafficking of VWF to the regulated storage pathway. Mutations identified within VWFpp in VWD patients are associated with altered VWF structure and function. The assay of plasma VWFpp has clinical utility in assessing acute and chronic vascular perturbation associated with diseases such as thrombotic thrombocytopenic purpura, sepsis, and diabetes among others. VWFpp assay also has clear utility in the diagnosis of VWD subtypes, particularly in discriminating true type 3 subjects from type 1C (reduced plasma survival of VWF), which is clinically important and has implications for therapeutic treatment.

VWF propeptide in defining VWD subtypes.

In this issue of Blood, Sanders and coworkers define the pathophysiology of types 1, 2, and 3 von Willebrand disease (VWD) in the Willebrand in the Netherlands (WiN) study by using the ratios of von Willebrand factor propeptide (VWFpp) or factor VIII activity to VWF antigen.

Crucial role for the VWF A1 domain in binding to type IV collagen.

Von Willebrand factor (VWF) contains binding sites for platelets and for vascular collagens to facilitate clot formation at sites of injury. Although previous work has shown that VWF can bind type IV collagen (collagen 4), little characterization of this interaction has been performed. We examined the binding of VWF to collagen 4 in vitro and extended this characterization to a murine model of defective VWF-collagen 4 interactions. The interactions of VWF and collagen 4 were further studied using plasma samples from a large study of both healthy controls and subjects with different types of von Willebrand disease (VWD). Our results show that collagen 4 appears to bind VWF exclusively via the VWF A1 domain, and that specific sequence variations identified through VWF patient samples and through site-directed mutagenesis in the VWF A1 domain can decrease or abrogate this interaction. In addition, VWF-dependent platelet binding to collagen 4 under flow conditions requires an intact VWF A1 domain. We observed that decreased binding to collagen 4 was associated with select VWF A1 domain sequence variations in type 1 and type 2M VWD. This suggests an additional mechanism through which VWF variants may alter hemostasis.

Allosteric activation of ADAMTS13 by von Willebrand factor.

The metalloprotease ADAMTS13 cleaves von Willebrand factor (VWF) within endovascular platelet aggregates, and ADAMTS13 deficiency causes fatal microvascular thrombosis. The proximal metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), Cys-rich (C), and spacer (S) domains of ADAMTS13 recognize a cryptic site in VWF that is exposed by tensile force. Another seven T and two complement C1r/C1s, sea urchin epidermal growth factor, and bone morphogenetic protein (CUB) domains of uncertain function are C-terminal to the MDTCS domains. We find that the distal T8-CUB2 domains markedly inhibit substrate cleavage, and binding of VWF or monoclonal antibodies to distal ADAMTS13 domains relieves this autoinhibition. Small angle X-ray scattering data indicate that distal T-CUB domains interact with proximal MDTCS domains. Thus, ADAMTS13 is regulated by substrate-induced allosteric activation, which may optimize VWF cleavage under fluid shear stress in vivo. Distal domains of other ADAMTS proteases may have similar allosteric properties.

G protein-dependent basal and evoked endothelial cell vWF secretion.

von Willebrand factor (vWF) secretion by endothelial cells (ECs) is essential for hemostasis and thrombosis; however, the molecular mechanisms are poorly understood. Interestingly, we observed increased bleeding in EC-Gα13(-/-);Gα12(-/-) mice that could be normalized by infusion of human vWF. Blood from Gα12(-/-) mice exhibited significantly reduced vWF levels but normal vWF multimers and impaired laser-induced thrombus formation, indicating that Gα12 plays a prominent role in EC vWF secretion required for hemostasis and thrombosis. In isolated buffer-perfused mouse lungs, basal vWF levels were significantly reduced in Gα12(-/-), whereas thrombin-induced vWF secretion was defective in both EC-Gαq(-/-);Gα11(-/-) and Gα12(-/-) mice. Using siRNA in cultured human umbilical vein ECs and human pulmonary artery ECs, depletion of Gα12 and soluble N-ethylmaleimide-sensitive-fusion factor attachment protein α (α-SNAP), but not Gα13, inhibited both basal and thrombin-induced vWF secretion, whereas overexpression of activated Gα12 promoted vWF secretion. In Gαq, p115 RhoGEF, and RhoA-depleted human umbilical vein ECs, thrombin-induced vWF secretion was reduced by 40%, whereas basal secretion was unchanged. Finally, in vitro binding assays revealed that Gα12 N-terminal residues 10-15 mediated the binding of Gα12 to α-SNAP, and an engineered α-SNAP binding-domain minigene peptide blocked basal and evoked vWF secretion. Discovery of obligatory and complementary roles of Gα12 and Gαq/11 in basal vs evoked EC vWF secretion may provide promising new therapeutic strategies for treatment of thrombotic disease.

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

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

The B subunits of Shiga-like toxins induce regulated VWF secretion in a phospholipase D1-dependent manner.

Shiga toxin (Stx) causes diarrhea-associated hemolytic uremic syndrome by damaging renal microvascular endothelium. The pentameric B subunits of Stx types 1 and 2 (Stx1B and Stx2B) are sufficient to stimulate acute VWF secretion from endothelial cells, but Stx1B and Stx2B exert distinct effects on Ca(2+) and cAMP pathways. Therefore, we investigated other signaling components in StxB-induced VWF exocytosis. Incubation of HUVECs with StxB transiently increased phospholipase D (PLD) activity. Inhibition of PLD activity or shRNA-mediated PLD1 knockdown abolished StxB-induced VWF secretion. In addition, treatment with StxB triggered actin polymerization, enhanced endothelial monolayer permeability, and activated RhoA. PLD activation and VWF secretion induced by Stx1B were abolished on protein kinase Cα (PKCα) inhibition or gene silencing but were only moderately reduced by Rho or Rho kinase inhibitors. Conversely, PLD activation and VWF exocytosis induced by Stx2B were reduced by Rho/Rho kinase inhibitors and dominant-negative RhoA, whereas attenuation of PKCα did not affect either process. Another PLD1 activator, ADP-ribosylation factor 6, was involved in VWF secretion induced by Stx1B or Stx2B, but not histamine. These data indicate that Stx1B and Stx2B induce acute VWF secretion in a PLD1-dependent manner but do so by differentially modulating PKCα, RhoA, and ADP-ribosylation factor 6.

Intersection of mechanisms of type 2A VWD through defects in VWF multimerization, secretion, ADAMTS-13 susceptibility, and regulated storage.

Type 2A VWD is characterized by the absence of large VWF multimers and decreased platelet-binding function. Historically, type 2A variants are subdivided into group 1, which have impaired assembly and secretion of VWF multimers, or group 2, which have normal secretion of VWF multimers and increased ADAMTS13 proteolysis. Type 2A VWD patients recruited through the T. S. Zimmerman Program for the Molecular and Clinical Biology of VWD study were characterized phenotypically and potential mutations identified in the VWF D2, D3, A1, and A2 domains. We examined type 2A variants and their interaction with WT-VWF through expression studies. We assessed secretion/intracellular retention, multimerization, regulated storage, and ADAMTS13 proteolysis. Whereas some variants fit into the traditional group 1 or 2 categories, others did not fall clearly into either category. We determined that loss of Weibel-Palade body formation is associated with markedly reduced secretion. Mutations involving cysteines were likely to cause abnormalities in multimer structure but not necessarily secretion. When coexpressed with wild-type VWF, type 2A variants negatively affected one or more mechanisms important for normal VWF processing. Type 2A VWD appears to result from a complex intersection of mechanisms that include: (1) intracellular retention or degradation of VWF, (2) defective multimerization, (3) loss of regulated storage, and (4) increased proteolysis by ADAMTS13.

VWF mutations and new sequence variations identified in healthy controls are more frequent in the African-American population.

Diagnosis and classification of VWD is aided by molecular analysis of the VWF gene. Because VWF polymorphisms have not been fully characterized, we performed VWF laboratory testing and gene sequencing of 184 healthy controls with a negative bleeding history. The controls included 66 (35.9%) African Americans (AAs). We identified 21 new sequence variations, 13 (62%) of which occurred exclusively in AAs and 2 (G967D, T2666M) that were found in 10%-15% of the AA samples, suggesting they are polymorphisms. We identified 14 sequence variations reported previously as VWF mutations, the majority of which were type 1 mutations. These controls had VWF Ag levels within the normal range, suggesting that these sequence variations might not always reduce plasma VWF levels. Eleven mutations were found in AAs, and the frequency of M740I, H817Q, and R2185Q was 15%-18%. Ten AA controls had the 2N mutation H817Q; 1 was homozygous. The average factor VIII level in this group was 99 IU/dL, suggesting that this variation may confer little or no clinical symptoms. This study emphasizes the importance of sequencing healthy controls to understand ethnic-specific sequence variations so that asymptomatic sequence variations are not misidentified as mutations in other ethnic or racial groups.

The common VWF variant p.Y1584C: detailed pathogenic examination of an enigmatic sequence change.

BACKGROUND: As knowledge of the human genome has advanced, so too has the recognition that interpretation of the pathogenic nature of sequence variants can be challenging. The von Willebrand factor (VWF) gene exhibits a significant degree of sequence variability, and the first VWF variant associated with type 1 von Willebrand disease (VWD), c.4751 A>G, p.Y1584C, was described in 2003. However, since that time, the pathogenic nature of this variant has remained unclear, being assigned properties ranging from a risk factor to a pathogenic variant. OBJECTIVES: To provide additional evaluation on the interpretation of pathogenicity for this common VWF variant. METHODS: Fifty-eight subjects with only the p.Y1584C variant were recruited from 2 cohort studies (the Zimmerman Program and the Canadian type 1 VWD study). Clinical and laboratory phenotypes were assessed. RESULTS: The prevalence of the p.Y1584C variant in our cohorts was 23- to 27-fold higher than that in large normal population databases. Significantly more p.Y1584C subjects had an abnormal bleeding score when compared to Y1584 individuals. In comparison with a group of 35 subjects without the p.Y1584C variant, subjects with the variant had lower mean VWF:antigen and VWF:ristocetin cofactor values and significantly higher VWF propeptide/VWF:antigen ratios suggestive of enhanced clearance. CONCLUSION: Collectively, the results of this analysis suggest that p.Y1584C is likely pathogenic, however, due to influences such as incomplete penetrance, variable expressivity, and other genetic modifiers like ABO blood group, the straightforward assignment of pathogenicity to this variant is inevitably challenging.

Distinguishing ASH clinical practice guidelines from other forms of ASH clinical advice.

ABSTRACT: The American Society of Hematology (ASH) develops a variety of resources that provide guidance to clinicians on the diagnosis and management of blood diseases. These resources include clinical practice guidelines (CPGs) and other forms of clinical advice. Although both ASH CPGs and other forms of clinical advice provide recommendations, they differ with respect to the methods underpinning their development, the principal type of recommendations they offer, their transparency and concordance with published evidence, and the time and resources required for their development. It is crucial that end users be aware of the differences between CPGs and other forms of clinical advice and that producers and publishers of these resources use clear and unambiguous terminology to facilitate their distinction. The objective of this article is to highlight the similarities and differences between ASH CPGs and other forms of ASH clinical advice and discuss the implications of these differences for end users.

Collagen binding provides a sensitive screen for variant von Willebrand disease.

BACKGROUND: von Willebrand factor (VWF) is a multimeric protein that binds platelets and collagen, facilitating hemostasis at sites of vessel injury. Measurement of VWF multimer distribution is critical for diagnosis of variant von Willebrand disease (VWD), particularly types 2A and 2B, but the typical measurement by gel electrophoresis is technically difficult and time-consuming. A comparison of VWF collagen binding (VWF:CB) and VWF multimer distribution was performed to evaluate the utility of VWF:CB as a diagnostic test. METHODS: Participants were enrolled in the Zimmerman Program for the Molecular and Clinical Biology of VWD. VWF:CB was analyzed with type III collagen and multimer distribution by agarose gel electrophoresis. The study population included 146 healthy controls, 351 individuals with type 1 VWD, and 77 with type 2 VWD. Differences between individuals with multimer group results within (controls) and outside the reference intervals were assessed with Mann-Whitney tests. RESULTS: The mean VWF:CB/VWF antigen ratio was 1.10 for individuals with multimer distribution within the reference intervals and 0.51 for those with multimer distribution outside the reference intervals (P < 0.001). Sensitivity of VWF:CB for multimer abnormalities was 100% for healthy controls, 99% for patients with type 1, and 100% for patients with type 2A and type 2B VWD using a VWF:CB/VWF antigen cutoff ratio of 0.6, and decreased to 99% for all patients with a ratio of 0.7. With the exception of individuals with novel or unclassified mutations, the VWF:CB was able to correctly categorize participants with variant VWD. CONCLUSIONS: These findings suggest that VWF:CB may substitute for multimer distribution in initial VWD testing, although further studies are needed to validate the clinical utility of VWF:CB.

Gain-of-function GPIb ELISA assay for VWF activity in the Zimmerman Program for the Molecular and Clinical Biology of VWD.

von Willebrand disease (VWD) is a common bleeding disorder, but diagnosis is sometimes challenging because of issues with the current von Willebrand factor (VWF) assays, VWF antigen (VWF:Ag) and VWF ristocetin cofactor activity (VWF:RCo), used for diagnosis. We evaluated 113 healthy controls and 164 VWD subjects enrolled in the T.S. Zimmerman Program for the Molecular and Clinical Biology of VWD for VWF:Ag, VWF:RCo, and a new enzyme-linked immunosorbent assay (ELISA)-based assay of VWF-glycoprotein Ib (GPIb) interactions using a gain-of-function GPIb construct (tGPIbα(235Y;239V)) as a receptor to bind its ligand VWF in an assay independent of ristocetin (VWF:IbCo ELISA). Healthy controls, type 1, 2A, 2M, and 2N subjects had VWF:RCo/VWF:Ag ratios similar to the ratio obtained with VWF:IbCo ELISA/VWF:Ag. Type 2B VWD subjects, however, had elevated VWF:IbCo ELISA/VWF:Ag ratios. Type 3 VWD subjects had undetectable (< 1.6 U/dL) VWF:IbCo ELISA values. As previously reported, VWF:RCo/VWF:Ag ratio was decreased with a common A1 domain polymorphism, D1472H, as was direct binding to ristocetin for a 1472H A1 loop construct. The VWF:IbCo ELISA, however, was not affected by D1472H. The VWF:IbCo ELISA may be useful in testing VWF binding to GPIb, discrimination of type 2 variants, and in the diagnosis of VWD as it avoids some of the pitfalls of VWF:RCo assays.

Enhanced VWF clearance in low VWF pathogenesis: limitations of the VWFpp/VWF:Ag ratio and clinical significance.

Increased von Willebrand factor (VWF) clearance plays a key role in the pathogenesis of type 1 and type 2 von Willebrand disease (VWD). However, the pathological mechanisms involved in patients with mild to moderate reductions in plasma VWF:Ag (range, 30-50 IU/dL; low VWF) remain poorly understood. In this study, we investigated the hypothesis that enhanced VWF clearance may contribute to the pathobiology of low VWF. Patients with low VWF were recruited to the LoVIC study after ethics approval and receipt of informed consent. Desmopressin was administered IV in 75 patients, and blood samples were drawn at baseline and at the 1-hour and 4-hour time points. As defined by recent ASH/ISTH/NHF/WFH guidelines, 20% of our low-VWF cohort demonstrated significantly enhanced VWF clearance. Importantly, from a clinical perspective, this enhanced VWF clearance was seen after desmopressin infusion, but did not affect the steady-state VWF propeptide (VWFpp)-to-VWF antigen (VWF:Ag) ratio (VWFpp/VWF:Ag) in most cases. The discrepancy between the VWFpp/VWF:Ag ratio and desmopressin fall-off rates in patients with mild quantitative VWD may have reflected alteration in VWFpp clearance kinetics. Finally, bleeding scores were significantly lower in patients with low VWF with enhanced VWF clearance, compared with those in whom reduced VWF biosynthesis represented the principle pathogenic mechanism. This trial was registered at http://www.clinicaltrials.gov as #NCT03167320.

Characterization of copy-number variants in a large cohort of patients with von Willebrand disease reveals a relationship between disrupted regions and disease type.

Background: Genetic analysis for von Willebrand disease (VWD) commonly utilizes DNA sequencing to identify variants in the von Willebrand factor (VWF) gene; however, this technique cannot always detect copy-number variants (CNVs). Additional mapping of CNVs in patients with VWD is needed. Objectives: This study aimed to characterize CNVs in a large sample of VWF mutation-negative VWD patients. Methods: To determine the role of CNVs in VWD, a VWF high-resolution comparative genomic hybridization array was custom-designed to avoid multiple sequence variations, repeated sequences, and the VWF pseudogene. This was performed on 204 mutation-negative subjects for whom clinical variables were also available. Results: Among the 204 patients, 7 unique CNVs were found, with a total of 24 CNVs (12%). Of the 7 unique CNVs, 1 was novel, 1 was found in a VWF database, and 5 were previously reported. All patients with type 1C VWD and a CNV had the same exon 33 and 34 in-frame deletion. Certain clinical variables were also significantly different between those with and without CNVs. Conclusion: The in-frame deletion in patients with type 1C VWD exactly matches the D4N module of the D4 domain, a region where mutations and deletions are known to affect clearance. We observed significantly higher VWF-to-ristocetin cofactor levels in patients with type 1C VWD and a CNV than in patients without a CNV, suggesting a relationship between CNVs and the increased clearance observed in patients with type 1C VWD. Glycoprotein IbM activity was significantly lower in patients with type 1 VWD and a CNV than in patients without a CNV, suggesting that platelet binding is more affected by CNVs than single base pair mutations. This work elucidates some of the underlying genetic mechanisms of CNVs in these patients.

The mutation N528S in the von Willebrand factor (VWF) propeptide causes defective multimerization and storage of VWF.

We characterized a consanguineous Turkish family suffering from von Willebrand disease (VWD) with significant mucocutaneous and joint bleeding. The relative reduction of large plasma von Willebrand factor (VWF) multimers and the absent VWF triplet structure was consistent with type 2A (phenotype IIC) VWD. Surprisingly, platelet VWF was completely deficient of multimers beyond the VWF protomer, suggesting defective alpha-granular storage of larger multimers. Patients were nearly unresponsive to desmopressin acetate, consistent with a lack of regulated VWF release from endothelial cell Weibel-Palade bodies, suggesting defective storage also in endothelial cells. We identified an N528S homozygous mutation in the VWF propeptide D2 domain, predicting the introduction of an additional N-glycosylation site at amino acid 526 in close vicinity to a "CGLC" disulphide isomerase consensus sequence. Expression studies in mammalian cells demonstrated that N528S-VWF was neither normally multimerized nor trafficked to storage granules. However, propeptide containing the N528S mutation trafficked normally to storage granules. Our data indicate that the patients' phenotype is the result of defective multimerization, storage, and secretion. In addition, we have identified a potentially novel pathogenic mechanism of VWD, namely a transportation and storage defect of mature VWF due to defective interaction with its transporter, the mutant propeptide.

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

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