James S. O Donnell

Markedly increased risk of thrombosis has been described as a key clinical feature of the recently described VEXAS (vacuoles, E1 enzyme, X linked, autoinflammatory, somatic) syndrome. Critically however, the pathological mechanisms underlying VEXAS-associated hypercoagulability remain poorly understood. We addressed this question in a cohort of 40 VEXAS patients referred to the National Institutes of Health with defined pathological UBA1 sequence variants. In keeping with previous reports, twenty-one (52.5%) VEXAS patients in our cohort experienced thrombotic events. Plasma VWF and FVIII activity levels were both increased in VEXAS compared to age-matched controls. Consistent with endothelial cell (EC) activation and Weibel Palade body exocytosis, plasma VWF propeptide and angiopoietin II levels were also significantly elevated in VEXAS subjects. Furthermore, an accumulation of high molecular weight VWF multimers was seen in VEXAS patients. In keeping with ongoing endotheliopathy, plasma levels of soluble Vascular Cell adhesion Molecule-1 (VCAM-1) and soluble thrombomodulin (sTM) were significantly higher in VEXAS patients compared to controls. Finally, significantly increased thrombin generation and activated protein C resistance (APCR) were also observed in plasma from VEXAS patients. In conclusion, our novel findings demonstrate that VEXAS syndrome is associated with significant EC activation and endotheliopathy. This results in both quantitative and qualitative alterations in plasma VWF levels, increased FVIII levels, enhanced thrombin generation and an APCR phenotype. Further studies will be required to determine whether therapeutic interventions targeted at EC and/or the VWF-ADAMTS13 axis may be useful in VEXAS management.

Despite significant insights into factor VIII (FVIII) biosynthesis, the mechanisms involved in FVIII clearance remain poorly defined. To investigate genetic modifiers of FVIII clearance, we studied 106 SNPs across 43 different loci previously reported to influence plasma FVIII and/or von Willebrand (VWF) levels and reviewed their association with FVIII pharmacokinetic (PK) parameters in 52 patients with hemophilia A. Marked inter-individual variability in FVIII clearance was observed. Plasma VWF levels, ABO blood group and age all influenced FVIII PK parameters. Collectively however, linear regression analysis demonstrated that these explained less than 40% of the observed variance in FVIII clearance. Consistent with previous reports, SNPs mapped to the FVIII/VWF clearance receptors STAB2, ASGR2, and CLEC4M were associated with FVIII clearance. Importantly however, we identified four additional genetic signals marked by SNPs rs4904820 (TC2N), rs3124768 (ADAMTS13), rs12979891 (RASIP1/MAMSTR/FUT2) and rs6494314 (C2CD4B) that were also significantly associated with FVIII PK parameters. Of particular interest, the FUT2 gene on chromosome 19q13 determines Secretor blood group status and thus determines secretion of ABO(H) glycan determinants into plasma. In addition to the association between the rs12979891 SNP and FVIII clearance, we further observed an effect of Secretor genotype on FVIII PK parameters, with prolonged FVIII half-life in non-secretor individuals compared to homozygous secretors. Furthermore, plasma FVIII levels were significantly increased in Fut2 knockout mice. Cumulatively, these data identify novel modifiers of FVIII clearance in vivo. Given that plasma FVIII levels constitute a dose-dependent risk factor for cardiovascular disease, these findings are of direct clinical importance.

Since the first description of a patient with von Willebrand disease (VWD) back in 1926, significant advances have been made in understanding the biology of von Willebrand factor (VWF). Under normal conditions, in vivo biosynthesis of VWF is restricted to endothelial cells and megakaryocytes only. This biosynthesis involves complex post-translational modifications (including glycosylation and multimerization) which play a key role in enabling the hemostatic functions of VWF. As a result, VWF circulates in normal plasma as a series of heterogeneous multimers that can modulate tethering of platelets and primary hemostasis at sites of vascular injury. In addition, VWF also influences secondary hemostasis by serving as a chaperone molecule and protecting factor VIII from proteolysis and premature clearance. The molecular mechanisms underlying the pro-hemostatic functions of VWF have been comprehensively characterized. These insights serve to underpin the current classification of different VWD subtypes. Interestingly, accumulating evidence over the past decade has identified an array of new ligands that are able to bind to VWF. Consistent with these data, recent studies have further suggested a series of novel and non-hemostatic biological functions for VWF. These include potential roles for VWF in regulating inflammation, wound healing, angiogenesis and tumor cell metastasis. Further research in the coming years will be required to determine the clinical significance of these non-hemostatic roles of VWF. Defining the molecular mechanisms involved may offer exciting opportunities to develop novel anti-VWF targeted treatment approaches for important unmet clinical needs.

An essential component of low von Willebrand factor (VWF)/type 1 von Willebrand disease (VWD) diagnosis is to identify patients with an increased bleeding phenotype, as most individuals with VWF levels in the 30 to 50 IU/dL range do not bleed. The International Society on Thrombosis and Haemostasis-bleeding assessment tool (ISTH-BAT), widely used for assessing bleeding severity, has recently been shown to be age dependent. Although age may also influence ISTH-BAT scores in individuals with VWF levels between 30 and 50 IU/dL, and subsequently affect low VWF diagnosis, this relationship has not been investigated. Therefore, we analyzed 325 participants from the Zimmerman Program, of whom 220 (67.7%) had abnormal ISTH-BAT scores, whereas 105 (32.3%) had normal scores. Our analysis demonstrates that age critically influences the likelihood of attaining an abnormal ISTH-BAT score and, consequently, being registered with a formal diagnosis of low VWF/type 1 VWD. For example, children first assessed at ≥10 years, are twice as likely to have an abnormal ISTH-BAT compared with those first investigated at <10 years (P < .001). In addition, the prevalence of abnormal ISTH-BAT scores was significantly higher in women aged ≥44 years (91.8%) compared with women aged 18 to 28 years (66.7%; P = .004). Finally, we demonstrate that the change in abnormal ISTH-BAT threshold at the age of 18 years critically affects low VWF diagnosis, owing to lower rates of abnormal scores in young adults (P = .006). In conclusion, we demonstrate that the likelihood of a low VWF/type 1 VWD diagnosis is influenced by the age at which ISTH-BAT is first assessed in individuals with mild-to-moderately reduced VWF levels.

Gold nanoparticles decorated with a sialic acid-displaying glycopeptide exhibit reduced cellular uptake and liver accumulation, offering a promising strategy to enhance the safety and therapeutic performance of nanomaterials.

The half-life of recombinant human von Willebrand factor (rVWF) expressed in CHO cells (CHO-rVWF; Vonicog alfa; and Vonvendi/Veyvondi) is significantly longer than that of plasma-derived VWF (pdVWF). This finding is intriguing because CHO cells do not generate α2-6 sialylation, which constitutes the majority of human pdVWF sialylation. We hypothesized that glycan differences might regulate the longer half-life of CHO-rVWF. In lectin plate-binding assays and liquid chromatography–mass spectrometry analysis, we confirmed that CHO-rVWF lacked α2-6–linked sialylation. Conversely, however, α2-3–linked sialylation was significantly increased on CHO-rVWF, which also had reduced exposed β-galactose (β-Gal) compared to pdVWF. Consistent with human data, CHO-rVWF clearance was significantly (P < .001) reduced in VWF–/– mice compared to pdVWF. However, clearance of asialo-pdVWF and asialo–CHO-rVWF were identical. In keeping with the in vivo half-life prolongation, CHO-rVWF binding to murine macrophages (P = .012) and HepG2 cells (P = .001) was significantly decreased compared to pdVWF. Furthermore, CHO-rVWF binding to purified macrophage-galactose-type lectin (MGL) receptor and asialoglycoprotein receptor (ASGPR) was also significantly reduced. In contrast to pdVWF, in vivo studies in MGL1–/– mice and Asgr1–/– mice demonstrated that neither MGL nor ASGPR plays significant roles in regulating CHO-rVWF clearance. Together, our findings demonstrate that enhanced α2-3–linked sialylation on CHO-rVWF is responsible for its extended half-life.

Previous reports have highlighted that some low VWF patients with significant bleeding were diagnosed based upon an isolated but persistent reduction in plasma VWF activity levels in the 30-50 IU/dL range. These patients had plasma VWF:Ag levels > 50 IU/dL and thus had 'qualitative' rather than 'quantitative' low VWF. Although the clinical importance of functional VWF defects in type 2 VWD is well recognized, the translational implications of mild functional defects in patients with qualitative low VWF (low VWF-QL) have not been defined. To address this clinically important question, we combined low VWF datasets from the low VWF in Ireland cohort and the low VWF in Erasmus MC studies. Overall, we observed that low VWF-QL was common and accounted for approximately 50% of our combined low VWF cohort. Importantly, our findings demonstrate that many of these patients with mild isolated functional VWF defects in the 30-50 IU/dl range had significant bleeding phenotypes, even though their plasma VWF:Ag levels were within the normal range. In addition, we further show that low VWF-QL is a distinct clinic-pathological entity compared to type 2 VWD. Finally, our studies highlight that low VWF-QL is predominantly due to abnormalities in VWF biosynthesis within endothelial cells that are occurring largely independent of identifiable pathological VWF sequence variants. Cumulatively, these novel observations have important clinical implications for the diagnosis and management of patients with mild functional VWF defects.

Rondaptivon pegol (previously BT200) is a pegylated RNA aptamer that binds to the A1 domain of von Willebrand factor (VWF). Recent clinical trials demonstrated that BT200 significantly increased plasma VWF–factor VIII levels by attenuating VWF clearance. The biological mechanism(s) through which BT200 attenuates in vivo clearance of VWF has not been defined. We hypothesized that BT200 interaction with the VWF-A1 domain may increase plasma VWF levels by attenuating macrophage-mediated clearance. We observed that full-length and VWF-A1A2A3 binding to macrophages and VWF-A1 domain binding to lipoprotein receptor–related protein 1 (LRP1) cluster II and cluster IV were concentration-dependently inhibited by BT200. Additionally, full-length VWF binding to LRP1 expressed on HEK293T (HEK-LRP1) cells was also inhibited by BT200. Importantly, BT200 interacts with the VWF-A1 domain in proximity to a conserved cluster of 4 lysine residues (K1405, K1406, K1407, and K1408). Alanine mutagenesis of this K1405-K1408 cluster (VWF-4A) significantly (P < .001) attenuated binding of VWF to both LRP1 clusters II and IV. Furthermore, in vivo clearance of VWF-4A was significantly (P < .001) reduced than that of wild-type VWF. BT200 did not significantly inhibit binding of VWF-4A to LRP1 cluster IV or HEK-LRP1 cells. Finally, BT200 interaction with the VWF-A1 domain also inhibited binding to macrophage galactose lectin and the SR-AI scavenger receptor. Collectively, our findings demonstrate that BT200 prolongs VWF half-life by attenuating macrophage-mediated clearance and specifically the interaction of K1405-K1408 in the VWF-A1 domain with macrophage LRP1. These data support the concept that targeted inhibition of VWF clearance pathways represents a novel therapeutic approach for von Willebrand disease and hemophilia A.

For many years, it has been known that von Willebrand factor (VWF) interacts with factor VIII, collagen, and platelets. In addition, the key roles played by VWF in regulating normal hemostasis have been well defined. However, accumulating recent evidence has shown that VWF can interact with a diverse array of other novel ligands. To date, over 60 different binding partners have been described, with interactions mapped to specific VWF domains in some cases. Although the biological significance of these VWF-binding interactions has not been fully elucidated, recent studies have identified some of these novel ligands as regulators of various aspects of VWF biology, including biosynthesis, proteolysis, and clearance. Conversely, VWF binding has been shown to directly affect the functional properties for some of its ligands. In keeping with those observations, exciting new roles for VWF in regulating a series of nonhemostatic biological functions have also emerged. These include inflammation, wound healing, angiogenesis, and bone metabolism. Finally, recent evidence supports the hypothesis that the nonhemostatic functions of VWF directly contribute to pathogenic mechanisms in a variety of diverse diseases including sepsis, malaria, sickle cell disease, and liver disease. In this manuscript, we review the accumulating data regarding novel ligand interactions for VWF and critically assess how these interactions may affect cellular biology. In addition, we consider the evidence that nonhemostatic VWF functions may contribute to the pathogenesis of human diseases beyond thrombosis and bleeding.