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.

von Willebrand factor propeptide variants lead to impaired storage and ER retention in patient-derived endothelial colony-forming cells.

BACKGROUND: von Willebrand factor (VWF) is synthesized by vascular endothelial cells and megakaryocytes. The VWF propeptide is critical for multimerization and acts as an intra-molecular chaperone for mature VWF in sorting to its storage organelles, Weibel-Palade bodies (WPBs). In the Canadian Type 3 VWD study, almost half of the identified variants were in the VWF propeptide and these were associated with an increased bleeding phenotype. OBJECTIVE: To investigate VWF propeptide variants that cause quantitative von Willebrand disease (VWD) by utilizing patient-derived endothelial colony-forming cells (ECFCs). PATIENTS/METHODS: Endothelial colony-forming cells were isolated from five Type 3 VWD patients from four families with the following variants: (1) homozygous p.Asp75_Gly178del (deletion of exons 4 and 5 deletion; Ex4-5del), (2) homozygous p.Cys633Arg, (3) homozygous p.Arg273Trp, and (4) p.Pro293Glnfs*164 and p.Gln419* inherited in the compound heterozygous state. Additionally, ECFCs were isolated from six family members (two Type 1 VWD, four unaffected). RESULTS: Endothelial colony-forming cells from the Type 3 patient with the compound heterozygous genotype exhibited a true null VWF cellular phenotype, with negligible VWF detected. In contrast, the other three propeptide variants presented a similar expression pattern in homozygous ECFCs where VWF was synthesized but not packaged in WPBs, and variant VWF had an increased association with the endoplasmic reticulum (ER) marker, protein disulfide-isomerase (PDI), indicating an ER-retention phenotype. The biosynthetic phenotype was similar but to a lesser degree in heterozygous ECFCs expressing the non-null variants. CONCLUSION: This study further elucidates the importance of the VWF propeptide in the VWD phenotype using patient-derived cells.

Novel cysteine substitution p.(Cys1084Tyr) causes variable expressivity of qualitative and quantitative VWF defects.

von Willebrand factor (VWF) is an extremely cysteine-rich multimeric protein that is essential for maintaining normal hemostasis. The cysteine residues of VWF monomers form intra- and intermolecular disulfide bonds that regulate its structural conformation, multimer distribution, and ultimately its hemostatic activity. In this study, we investigated and characterized the molecular and pathogenic mechanisms through which a novel cysteine variant p.(Cys1084Tyr) causes an unusual, mixed phenotype form of von Willebrand disease (VWD). Phenotypic data including bleeding scores, laboratory values, VWF multimer distribution, and desmopressin response kinetics were investigated in 5 members (2 parents and 3 daughters) of a consanguineous family. VWF synthesis and secretion were also assessed in a heterologous expression system and in a transient transgenic mouse model. Heterozygosity for p.(Cys1084Tyr) was associated with variable expressivity of qualitative VWF defects. Heterozygous individuals had reduced VWF:GPIbM (<0.40 IU/mL) and VWF:CB (<0.35 IU/mL), as well as relative reductions in high-molecular-weight multimers, consistent with type 2A VWD. In addition to these qualitative defects, homozygous individuals also displayed reduced factor VIII (FVIII):C/VWF:Ag, leading to very low FVIII levels (0.03-0.1 IU/mL) and reduced VWF:Ag (<0.40 IU/mL) and VWF:GPIbM (<0.30 IU/mL). Accelerated VWF clearance and impaired VWF secretion contributed to the fully expressed homozygous phenotype with impaired secretion arising because of disordered disulfide connectivity.

Multifaceted pathomolecular mechanism of a VWF large deletion involved in the pathogenesis of severe VWD.

An in-frame heterozygous large deletion of exons 4 through 34 of the von Willebrand factor (VWF) gene was identified in a type 3 von Willebrand disease (VWD) index patient (IP), as the only VWF variant. The IP exhibited severe bleeding episodes despite prophylaxis treatment, with a short VWF half-life after infusion of VWF/factor VIII concentrates. Transcript analysis confirmed transcription of normal VWF messenger RNA besides an aberrant deleted transcript. The IP endothelial colony-forming cells (ECFCs) exhibited a defect in the VWF multimers and Weibel-Palade bodies (WPBs) biogenesis, although demonstrating normal VWF secretion compared with healthy cells. Immunostaining of IP-ECFCs revealed subcellular mislocalization of WPBs pro-inflammatory cargos angiopoietin-2 (Ang2, nuclear accumulation) and P-selectin. Besides, the RNA-sequencing (RNA-seq) analysis showed upregulation of pro-inflammatory and proangiogenic genes, P-selectin, interleukin 8 (IL-8), IL-6, and GROα, copackaged with VWF into WPBs. Further, whole-transcriptome RNA-seq and subsequent gene ontology (GO) enrichment analysis indicated the most enriched GO-biological process terms among the differentially expressed genes in IP-ECFCs were regulation of cell differentiation, cell adhesion, leukocyte adhesion to vascular endothelial, blood vessel morphogenesis, and angiogenesis, which resemble downstream signaling pathways associated with inflammatory stimuli and Ang2 priming. Accordingly, our functional experiments exhibited an increased endothelial cell adhesiveness and interruption in endothelial cell-cell junctions of the IP-ECFCs. In conclusion, the deleted VWF has a dominant-negative impact on multimer assembly and the biogenesis of WPBs, leading to altered trafficking of their pro-inflammatory cargos uniquely, which, in turn, causes changes in cellular signaling pathways, phenotype, and function of the endothelial cells.

Stabilin-2 deficiency increases thrombotic burden and alters the composition of venous thrombi in a mouse model.

BACKGROUND: Stabilin-2 is an endocytic scavenger receptor that mediates the clearance of glycosaminoglycans, phosphatidylserine-expressing cells, and the von Willebrand factor-factor VIII (FVIII) complex. In a genome-wide screening study, pathogenic loss-of-function variants in the human STAB2 gene associated with an increased incidence of unprovoked venous thromboembolism (VTE). However, the specific mechanism(s) by which stabilin-2 deficiency influences the pathogenesis of VTE is unknown. OBJECTIVES: The aim of this study was to assess the influence of stabilin-2 on deep vein thrombosis (DVT) and to characterize the underlying prothrombotic phenotype of stabilin-2 deficiency in a mouse model. METHODS: DVT was induced using the inferior vena cava (IVC) stenosis model in two independent cohorts (littermates and non-littermates) of wild-type (Stab2+/+ ) and stabilin-2 (Stab2-/- )-deficient mice. Thrombus structure and contents were quantified by immunohistochemistry. Plasma procoagulant activity was assessed and complete blood counts were performed. RESULTS: Incidence of thrombus formation was not altered between Stab2+/+ and Stab2-/- mice. When thrombi were formed, Stab2-/- mice developed significantly larger thrombi than Stab2+/+ controls. Thrombi from Stab2-/- mice contained significantly more leukocytes and citrullinated histone H3 than Stab2+/+ thrombi. Stab2-/- mice had increased FVIII activity. Circulating levels of monocytes and granulocytes were significantly elevated in Stab2-/- mice, and Stab2-/- mice had elevated plasma cell-free DNA 24 hours post-IVC stenosis compared to their Stab2+/+ counterparts. CONCLUSIONS: These data suggest that stabilin-2 deficiency associates with a prothrombotic phenotype involving elevated levels of neutrophil extracellular trap-releasing leukocytes coupled with endogenous procoagulant activity, resulting in larger and qualitatively distinct venous thrombi.

Functional Roles of the von Willebrand Factor Propeptide.

The primary polypeptide sequence of von Willebrand factor (VWF) includes an N-terminal 741-amino acid VWF propeptide (VWFpp). In cells expressing VWF, the VWFpp performs two critical functions. In the Golgi, VWFpp mediates the intermolecular disulfide linkages that generate high-molecular-weight VWF multimers. Subsequently, the VWFpp, which is proteolytically cleaved from mature VWF by furin, functions to generate the endothelial storage organelles (Weibel-Palade bodies) in which VWF and a distinct collection of proteins are stored, and from where they undergo regulated secretion from the endothelium. The VWFpp is secreted from endothelial cells as dimers and circulates in plasma with at least some of the dimers associating with a noncovalent manner with the D'D3 domain of mature VWF. The VWFpp has a half-life of 2 to 3 hours in plasma, but to date no extracellular function has been determined for the molecule. Nevertheless, its large size and several biologically interesting structural features (two sets of vicinal cysteines and an RGD sequence) suggest that there may be roles that the VWFpp plays in hemostasis or associated physiological processes such as angiogenesis or wound repair.

Genetic determinants of VWF clearance and FVIII binding modify FVIII pharmacokinetics in pediatric hemophilia A patients.

Factor VIII (FVIII) pharmacokinetic (PK) properties show high interpatient variability in hemophilia A patients. Although previous studies have determined that age, body mass index, von Willebrand factor antigen (VWF:Ag) levels, and ABO blood group status can influence FVIII PK, they do not account for all observed variability. In this study, we aim to describe the genetic determinants that modify the FVIII PK profile in a population of 43 pediatric hemophilia A patients. We observed that VWF:Ag and VWF propeptide (VWFpp)/VWF:Ag, but not VWFpp, were associated with FVIII half-life. VWFpp/VWF:Ag negatively correlated with FVIII half-life in patients with non-O blood type, but no correlation was observed for type O patients, suggesting that von Willebrand factor (VWF) half-life, as modified by the ABO blood group, is a strong regulator of FVIII PK. The FVIII-binding activity of VWF positively correlated with FVIII half-life, and the rare or low-frequency nonsynonymous VWF variants p.(Arg826Lys) and p.(Arg852Glu) were identified in patients with reduced VWF:FVIIIB but not VWF:Ag. Common variants at the VWF, CLEC4M, and STAB2 loci, which have been previously associated with plasma levels of VWF and FVIII, were associated with the FVIII PK profile. Together, these studies characterize the mechanistic basis by which VWF clearance and ABO glycosylation modify FVIII PK in a pediatric population. Moreover, this study is the first to identify non-VWF and non-ABO variants that modify FVIII PK in pediatric hemophilia A patients.

The scavenger receptor SCARA5 is an endocytic receptor for von Willebrand factor expressed by littoral cells in the human spleen.

BACKGROUND: Scavenger receptors play a significant role in clearing aged proteins from the plasma, including the large glycoprotein coagulation factors von Willebrand factor (VWF) and factor VIII (FVIII). A large genome-wide association study meta-analysis has identified genetic variants in the gene SCARA5, which encodes the class A scavenger receptor SCARA5, as being associated with plasma levels of VWF and FVIII. OBJECTIVES: The ability of SCARA5 to regulate the clearance of VWF-FVIII was characterized. METHODS: VWF-FVIII interactions with SCARA5 were evaluated by solid phase binding assays and in vitro cell based assays. The influence of SCARA5 deficiency on VWF:Ag and half-life was assessed in a murine model. The expression pattern of SCARA5 and its colocalization with VWF was evaluated in human tissues. RESULTS: VWF and the VWF-FVIII complex bound to human recombinant SCARA5 in a dose- and calcium-dependent manner. SCARA5 expressing HEK 293T cells bound and internalized VWF and the VWF-FVIII complex into early endosomes. In vivo, SCARA5 deficiency had a modest influence on the half-life of human VWF. mRNA analysis and immunohistochemistry determined that human SCARA5 is expressed in kidney podocytes and the red pulp, white pulp, and marginal zone of the spleen. VWF was found to colocalize with SCARA5 expressed by littoral cells lining the red pulp of the human spleen. CONCLUSIONS: SCARA5 is an adhesive and endocytic receptor for VWF. In human tissues, SCARA5 is expressed by kidney podocytes and splenic littoral endothelial cells. SCARA5 may have a modest influence on VWF clearance in humans.

Galectin-1 and Galectin-3 Constitute Novel-Binding Partners for Factor VIII.

OBJECTIVE: Recent studies have demonstrated that galectin-1 (Gal-1) and galectin-3 (Gal-3) can bind von Willebrand factor and directly modulate von Willebrand factor-dependent early thrombus formation in vivo. Because the glycans expressed on human factor VIII (FVIII) are similar to those of von Willebrand factor, we investigated whether galectins might also bind and modulate the activity of FVIII. APPROACH AND RESULTS: Immunosorbant assays and surface plasmon resonance analysis confirmed that Gal-1 and Gal-3 bound purified FVIII with high affinity. Exoglycosidase removal of FVIII N-linked glycans significantly reduced binding to both Gal-1 and Gal-3. Moreover, combined removal of both the N- and O-glycans of FVIII further attenuated Gal-3 binding. Notably, specific digestion of FVIII high-mannose glycans at N239 and N2118 significantly impaired FVIII affinity for Gal-1. Importantly Gal-1, but not Gal-3, bound to free FVIII in the plasma milieu, and significantly inhibited FVIII functional activity. Interestingly, commercial recombinant FVIII (rFVIII) concentrates are manufactured in different cell lines and differ in their glycosylation profiles. Although the biological mechanism has not been defined, recent studies in previously untreated patients with severe hemophilia A reported significant differences in inhibitor development associated with different rFVIII products. Interestingly, Gal-1 and Gal-3 both displayed enhanced affinity for BHK-rFVIII compared with CHO-rFVIII. Furthermore, binding of Gal-1 and Gal-3 to BDD-FVIII was markedly reduced compared with full-length rFVIII. CONCLUSIONS: We have identified Gal-1 and Gal-3 as novel-binding partners for human FVIII and demonstrated that Gal-1 binding can influence the procoagulant activity of FVIII.

Altered glycosylation of platelet-derived von Willebrand factor confers resistance to ADAMTS13 proteolysis.

Platelet-von Willebrand factor (VWF) is stored within α-granules and accounts for ∼20% of total VWF in platelet-rich plasma. This platelet-VWF pool is distinct from plasma-VWF and is enriched in high molecular weight multimers (HMWM). Previous studies have described significant functional discrepancies between platelet-VWF and plasma-VWF; however, the molecular basis of these differences is not well understood. We have characterized terminal glycan expression on platelet-VWF. Our findings demonstrate that platelet-VWF exists as a distinct natural glycoform. In particular, N-linked sialylation is markedly reduced (>50%) compared with plasma-VWF. Moreover, unlike plasma-VWF, platelet-VWF does not express AB blood group determinants, although precursor H antigen expression is similar to that on plasma-VWF. Because of this differential glycosylation, platelet-VWF exhibits specific resistance to ADAMTS13 proteolysis. Thus platelet activation at sites of vascular injury results in the release of high local concentrations of HMWM platelet-VWF that is more resistant to ADAMTS13, thereby facilitating platelet-plug formation.

Elucidating the role of carbohydrate determinants in regulating hemostasis: insights and opportunities.

Recent improvement in modern analytical technologies has stimulated an explosive growth in the study of glycobiology. In turn, this has lead to a richer understanding of the crucial role of N- and O-linked carbohydrates in dictating the properties of the proteins to which they are attached and, in particular, their centrality in the control of protein synthesis, longevity, and activity. Given their importance, it is unsurprising that both gross and subtle defects in glycosylation often contribute to human disease pathology. In this review, we discuss the accumulating evidence for the significance of glycosylation in mediating the functions of the plasma glycoproteins involved in hemostasis and thrombosis. In particular, the role of naturally occurring coagulation protein glycoforms and inherited defects in carbohydrate attachment in modulating coagulation is considered. Finally, we describe the therapeutic opportunities presented by new insights into the role of attached carbohydrates in shaping coagulation protein function and the promise of carbohydrate modification in the delivery of novel therapeutic biologics with enhanced functional properties for the treatment of hemostatic disorders.

Elevated factor VIII levels and risk of venous thrombosis.

Modern thrombophilia testing fails to identify any underlying prothrombotic tendency in a significant number of patients presenting with objectively confirmed venous thromboemboembolism (VTE). This observation has led to a search for other novel inherited or acquired human thrombophilias. Although a number of putative mechanisms have been described, the evidence behind many of these candidates remains weak. In contrast, an increasing body of work supports the hypothesis that increased plasma factor VIII (FVIII) levels may be important in this context. An association between elevated plasma FVIII levels and VTE was first described in the Leiden Thrombophilia Study (LETS). Subsequently, these conclusions have been supported by an increasing number of independent case-control studies. Cumulatively, these studies have clearly demonstrated that high FVIII levels constitute a prevalent, dose-dependent risk factor for VTE. Furthermore, more recent studies have shown that the risk of recurrent venous thrombosis is also significantly increased in patients with high FVIII levels. In this review, we present the evidence supporting the hypothesis that elevated FVIII levels constitute a clinically important thrombophilia. In addition, we examine the biological mechanisms that may underlie persistently elevated FVIII levels, and the pathways through which high FVIII may serve to increase thrombotic risk.