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Background Previous studies have demonstrated that the A1A2A3 domains of von Willebrand factor (VWF) play a key role in regulating macrophage-mediated clearance in vivo. In particular, the A1-domain has been shown to modulate interaction with macrophage low-density lipoprotein receptor-related protein-1 (LRP1) clearance receptor. Furthermore, N-linked glycans within the A2-domain have been shown to protect VWF against premature LRP1-mediated clearance. Importantly, however, the specific regions within A1A2A3 that enable macrophage binding have not been defined. Objective and methods To address this, we utilized site-directed PEGylation and introduced novel targeted N-linked glycosylation within A1A2A3-VWF and subsequently examined VWF clearance. Results Conjugation with a 40-kDa polyethylene glycol (PEG) moiety significantly extended the half-life of A1A2A3-VWF in VWF-/- mice in a site-specific manner. For example, PEGylation at specific sites within the A1-domain (S1286) and A3-domain (V1803, S1807) attenuated VWF clearance in vivo, compared to wild-type A1A2A3-VWF. Furthermore, PEGylation at these specific sites ablated binding to differentiated THP-1 macrophages and LRP1 cluster II and cluster IV in-vitro. Conversely, PEGylation at other positions (Q1353-A1-domain and M1545-A2-domain) had limited effects on VWF clearance or binding to LRP1.Novel N-linked glycan chains were introduced at N1803 and N1807 in the A3-domain. In contrast to PEGylation at these sites, no significant extension in half-life was observed with these N-glycan variants. Conclusions These novel data demonstrate that site specific PEGylation but not site specific N-glycosylation modifies LRP1-dependent uptake of the A1A2A3-VWF by macrophages. This suggests that PEGylation, within the A1- and A3-domains in particular, may be used to attenuate LRP1-mediated clearance of VWF.

The factor VIII–von Willebrand factor (VWF) complexFVIII is a plasma sialoglycoprotein that plays a critical role in maintaining normal hemostasis. Patients with severe hemophilia A have markedly reduced plasma FVIII levels, and thus typically show a significant bleeding phenotype. Accumulating data suggest that plasma FVIII is predominantly derived from biosynthesis within sinusoidal cells and endothelial cells (ECs), particularly in the liver and lung 1. FVIII is initially synthesized as an inactive 2332 amino acid polypeptide composed of three distinct domain types: A, B, and C (domain structure A1–a1–A2–a2–B–a3–A3–C1–C2). Prior to secretion, this single‐chain FVIII undergoes complex post‐translational modification that includes significant glycosylation, sulfation, and limited intracellular proteolytic processing. Consequently, plasma FVIII circulates as a heterodimeric protein consisting of a heavy chain (A1–a1–A2–a2–B) and a light chain (a3–A3–C1–C2), held together through a metal ion‐dependent interaction.

In this issue of the Journal of Thrombosis and Haemostasis, Peyvandi et al present further insights from the Survey of Inhibitors in Plasma-Product Exposed Toddlers (SIPPET) study regarding the relative immunogenicity of recombinant- (rFVIII) versus plasma-derived FVIII (pdFVIII) in patients with haemophilia A [1]. In a post-hoc analysis of this prospective randomised controlled trial, the timing and severity of inhibitor development for previously untreated patients (PUPs) treated with rFVIII was compared with that in PUPs treated with pdFVIII. In keeping with previous studies, the period of greatest risk for inhibitor development in PUPs was observed within the first 15 exposure days (EDs). This article is protected by copyright. All rights reserved.

Background Activated protein C (APC) cell signaling is largely reliant upon its ability to mediate protease-activated receptor 1 (PAR1) proteolysis when bound to the endothelial cell protein C receptor (EPCR). Furthermore, EPCR-bound PC modulates PAR1 signaling by thrombin to induce APC-like endothelial cell (EC) cytoprotection. Objective The molecular determinants of EPCR-dependent cytoprotective PAR1 signaling remain poorly defined. To address this, a PC-factor VII (FVII) chimera (PCFVII−82) possessing FVII N-terminal domains and conserved EPCR binding was characterized. Methods APCFVII−82 anticoagulant activity was measured by calibrated automated thrombography and FVa degradation assays. APCFVII−82 signaling activity was characterised using reporter assays of PAR1 proteolysis and EC barrier integrity. APCFVII−82 anti-inflammatory activity was assessed by its inhibition of NF-κB activation and cytokine secretion from monocytes. Results PCFVII−82 was activated normally by thrombin on ECs, but was unable to inhibit plasma thrombin generation. Surprisingly, APCFVII−82 did not mediate EPCR-dependent PAR1 proteolysis, confer PAR1-dependent protection of thrombin-induced EC barrier disruption, or limit PAR1-dependent attenuation of IL-6 release from LPS-stimulated macrophages. Interestingly, EPCR occupation by active-site blocked APCFVII−82 was, like FVII, unable to mimic EC barrier stabilization induced by PC upon PAR1 proteolysis by thrombin. APCFVII−82 did, however, diminish LPS-induced NF-κB activation and TNF-α release from monocytes in an apolipopotein E receptor 2 (ApoER2)-dependent manner, with similar efficacy as wild type APC. Conclusions These data identify a novel role for APC light chain amino acid residues outwith the EPCR binding site in enabling cytoprotective PAR1 signaling. This article is protected by copyright. All rights reserved.

Essentials von Willebrands factor (VWF) glycosylation plays a key role in modulating in vivo clearance. VWF glycoforms were used to examine the role of specific glycan moieties in regulating clearance. Reduction in sialylation resulted in enhanced VWF clearance through asialoglycoprotein receptor. Progressive VWF N-linked glycan trimming resulted in increased macrophage-mediated clearance. Click to hear Dr Denis discuss clearance of von Willebrand factor in a free presentation from the ISTH Academy SUMMARY: Background Enhanced von Willebrand factor (VWF) clearance is important in the etiology of both type 1 and type 2 von Willebrand disease (VWD). In addition, previous studies have demonstrated that VWF glycans play a key role in regulating in vivo clearance. However, the molecular mechanisms underlying VWF clearance remain poorly understood. Objective To define the molecular mechanisms through which VWF N-linked glycan structures influence in vivo clearance. Methods By use of a series of exoglycosidases, different plasma-derived VWF (pd-VWF) glycoforms were generated. In vivo clearance of these glycoforms was then assessed in VWF-/- mice in the presence or absence of inhibitors of asialoglycoprotein receptor (ASGPR), or following clodronate-induced macrophage depletion. Results Reduced amounts of N-linked and O-linked sialylation resulted in enhanced pd-VWF clearance modulated via ASGPR. In addition to this role of terminal sialylation, we further observed that progressive N-linked glycan trimming also resulted in markedly enhanced VWF clearance. Furthermore, these additional N-linked glycan effects on clearance were ASGPR-independent, and instead involved enhanced macrophage clearance that was mediated, at least in part, through LDL receptor-related protein 1. Conclusion The carbohydrate determinants expressed on VWF regulate susceptibility to proteolysis by ADAMTS-13. In addition, our findings now further demonstrate that non-sialic acid carbohydrate determinants expressed on VWF also play an unexpectedly important role in modulating in vivo clearance through both hepatic ASGPR-dependent and macrophage-dependent pathways. In addition, these data further support the hypothesis that variation in VWF glycosylation may be important in the pathophysiology underlying type 1C VWD.

Essentials Treatment options are limited for refractory bleeding in acquired von Willebrand Syndrome (AVWS). Lenalidomide therapy was studied in two patients with AVWS due to monoclonal gammopathy (MG). Lenalidomide increased von Willebrand factor (VWF), lowered VWF clearance and resolved bleeding. Lenalidomide is a potential treatment option for refractory bleeding in AVWS secondary to MG. SummaryBackground Acquired von Willebrand syndrome (AVWS) is associated with lymphoproliferative disorders, including monoclonal gammopathy (MG) of undetermined significance (MGUS) and multiple myeloma. Patients commonly present with significant bleeding complications that are difficult to manage, owing to a markedly reduced von Willebrand factor (VWF) half-life. Objectives To investigate the use of the immunomodulatory drug lenalidomide in two patients with severe refractory bleeding caused by AVWS associated with MGs. Results In both patients, lenalidomide treatment resulted in significant clinical improvement, and marked increases in plasma VWF antigen (VWF:Ag) and VWF ristocetin cofactor levels. This normalization in plasma VWF levels was sustained for > 2 years in both patients. Furthermore, in one patient, plasma VWF levels remain normal for at least 14 months following discontinuation of lenalidomide treatment. To investigate the molecular mechanisms underlying these observations, VWF propeptide (VWFpp)/VWF:Ag ratios were analyzed to assess VWF clearance. At enrolment, plasma VWFpp/VWF:Ag ratios were significantly elevated in both patients. Importantly, lenalidomide treatment resulted in normalization of VWFpp/VWF:Ag ratios in both patients. These novel data suggest that lenalidomide functions to attenuate enhanced VWF clearance in AVWS. Interestingly, in a patient with MGUS, lenalidomide treatment was associated with a significant increase in plasma VWF levels, despite no major change in paraprotein level. Conclusions Collectively, our findings suggest that lenalidomide constitutes a novel therapeutic option for the management of AVWS associated with MG. The biological mechanism(s) through which lenalidomide causes a sustained increase in plasma VWF levels in AVWS independently of paraprotein level requires further study, but is in part modulated through inhibition of enhanced VWF clearance.