Macrophage Galactose Lectin Contributes to the Regulation of FVIII (Factor VIII) Clearance in Mice-Brief Report.

BACKGROUND: Although most plasma FVIII (Factor VIII) circulates in complex with VWF (von Willebrand factor), a minority (3%-5%) circulates as free-FVIII, which is rapidly cleared. Consequently, 20% of total FVIII may be cleared as free-FVIII. Critically, the mechanisms of free-FVIII clearance remain poorly understood. However, recent studies have implicated the MGL (macrophage galactose lectin) in modulating VWF clearance. METHODS: Since VWF and FVIII share similar glycosylation, we investigated the role of MGL in FVIII clearance. FVIII binding to MGL was assessed in immunosorbent and cell-based assays. In vivo, FVIII clearance was assessed in MGL1-/- and VWF-/-/FVIII-/- mice. RESULTS: In vitro-binding studies identified MGL as a novel macrophage receptor that binds free-FVIII in a glycan-dependent manner. MGL1-/- and MGL1-/- mice who received an anti-MGL1/2 blocking antibody both showed significantly increased endogenous FVIII activity compared with wild-type mice (P=0.036 and P<0.0001, respectively). MGL inhibition also prolonged the half-life of infused FVIII in FVIII-/- mice. To assess whether MGL plays a role in the clearance of free FVIII in a VWF-independent manner, in vivo clearance experiments were repeated in dual VWF-/-/FVIII-/- mice. Importantly, the rapid clearance of free FVIII in VWF-/-/FVIII-/- mice was significantly (P=0.012) prolonged in the presence of anti-MGL1/2 antibodies. Finally, endogenous plasma FVIII levels in VWF-/- mice were significantly increased following MGL inhibition (P=0.016). CONCLUSIONS: Cumulatively, these findings demonstrate that MGL plays an important role in regulating macrophage-mediated clearance of both VWF-bound FVIII and free-FVIII in vivo. We propose that this novel FVIII clearance pathway may be of particular clinical importance in patients with type 2N or type 3 Von Willebrand disease.

Von Willebrand Disease: Gaining a global perspective.

INTRODUCTION: Recent guidelines for von Willebrand Disease (VWD) highlighted the challenges in diagnosis and management. Identifying the number of persons with VWD (PwVWD) internationally will help target support to aid diagnosis of PwVWD. AIM: To examine international registration rates of PwVWD, the influence of income status, geographical region and the age and sex profile. Cumulatively, these data will be used to inform future strategy from the World Federation of Haemophilia (WFH) to address unmet clinical and research needs. METHODS: Data from the 2018/2019 WFH Annual Global Survey (AGS) were analysed, providing a global perspective on VWD registration. RESULTS: Registration rates are lowest in South Asia (0.6/million population) and highest in Europe/Central Asia (50.9/million population, 0.005%), but below the expected prevalence rate (0.1%). National economic status impacted VWD registration rates, reflecting variation in access to optimal healthcare infrastructure. Females represented the majority of PwVWD globally, however, in low-income countries (LIC) males predominated. Age profile varied, with markedly higher rates of paediatric registrations in North America, Middle East and North Africa and South Asia. Rates of type 3 VWD registrations were significantly influenced by economic status (81% of VWD diagnoses in LIC), suggesting only the most severe VWD types are diagnosed in resource limited settings. CONCLUSION: Significant variation in registration rates of PwVWD exist internationally and is influenced by income status and the presence of HTC networks. Improved understanding of registration rates will enable targeting of advocacy to improve awareness, diagnosis and support for PwVWD internationally. KEY POINTS: Registration rates of People with Von Willebrand Disease (PwVWD) vary internationally and are influenced by national income status Although females represent the majority of PwVWD globally, in low income countries (LIC) males predominated, possibly related to stigma surrounding gynaecological bleeding. Rates of type 3 VWD registration were significantly influenced by economic status (81% of VWD diagnoses in LIC), suggesting only the most severe VWD types are diagnosed in resource limited settings.

The relationship between ABO blood group, von Willebrand factor, and primary hemostasis.

Numerous studies have reported significant associations between ABO blood group and risk of cardiovascular disease. These studies have consistently demonstrated that thrombotic risk is significantly reduced in individuals in blood group O. Nevertheless, the biological mechanisms through which ABO influences hemostasis have remained poorly understood. Exciting recent data have provided novel insights into how these ABO effects are modulated and have highlighted that ABO group significantly influences platelet plug formation at sites of vascular injury (primary hemostasis). In particular, ABO affects multiple aspects of von Willebrand factor (VWF) biology. In keeping with their reduced thrombotic risk, plasma VWF levels are ∼25% lower in healthy group O compared with healthy group non-O individuals. In addition, blood group O VWF demonstrates enhanced susceptibility to ADAMTS13 proteolysis. Finally, preliminary findings suggest that the interaction of group O VWF with platelets may also be reduced. Although the molecular mechanisms underlying these ABO effects on VWF have not been fully elucidated, it seems likely that they are mediated in large part by the ABO(H) carbohydrate structures that are carried on both the N- and O-linked glycans of VWF. Interestingly, ABO(H) determinants are also expressed on several different platelet surface glycoprotein receptors. Recent studies support the hypothesis that ABO group not only exerts major quantitative and qualitative effects on VWF, but also affect specific aspects of platelet function. Given the severe morbidity and the mortality associated with thrombotic disorders, defining the mechanisms underlying these ABO effects is not only of scientific interest, but also of direct clinical importance.

Sialylation on O-linked glycans protects von Willebrand factor from macrophage galactose lectin-mediated clearance.

Terminal sialylation determines the plasma half-life of von Willebrand factor (VWF). A role for macrophage galactose lectin (MGL) in regulating hyposialylated VWF clearance has recently been proposed. In this study, we showed that MGL influences physiological plasma VWF clearance. MGL inhibition was associated with a significantly extended mean residence time and 3-fold increase in endogenous plasma VWF antigen levels (P<0.05). Using a series of VWF truncations, we further demonstrated that the A1 domain of VWF is predominantly responsible for enabling the MGL interaction. Binding of both full-length and VWF-A1-A2-A3 to MGL was significantly enhanced in the presence of ristocetin (P<0.05), suggesting that the MGL-binding site in A1 is not fully accessible in globular VWF. Additional studies using different VWF glycoforms demonstrated that VWF O-linked glycans, clustered at either end of the A1 domain, play a key role in protecting VWF against MGLmediated clearance. Reduced sialylation has been associated with pathological, increased clearance of VWF in patients with von Willebrand disease. Herein, we demonstrate that specific loss of α2-3 linked sialylation from O-glycans results in markedly increased MGL-binding in vitro, and markedly enhanced MGL-mediated clearance of VWF in vivo. Our data further show that the asialoglycoprotein receptor (ASGPR) does not have a significant role in mediating the increased clearance of VWF following loss of O-sialylation. Conversely however, we observed that loss of N-linked sialylation from VWF drives enhanced circulatory clearance predominantly via the ASGPR. Collectively, our data support the hypothesis that in addition to regulating physiological VWF clearance, the MGL receptor works in tandem with ASGPR to modulate enhanced clearance of aberrantly sialylated VWF in the pathogenesis of von Willebrand disease.

Von Willebrand factor propeptide in severe coronavirus disease 2019 (COVID-19): evidence of acute and sustained endothelial cell activation.

Endothelial cell (EC) activation plays a key role in the pathogenesis of pulmonary microvascular occlusion, which is a hallmark of severe coronavirus disease 2019 (COVID-19). Consistent with EC activation, increased plasma von Willebrand factor antigen (VWF:Ag) levels have been reported in COVID-19. Importantly however, studies in other microangiopathies have shown that plasma VWF propeptide (VWFpp) is a more sensitive and specific measure of acute EC activation. In the present study, we further investigated the nature of EC activation in severe COVID-19. Markedly increased plasma VWF:Ag [median (interquatile range, IQR) 608·8 (531-830)iu/dl] and pro-coagulant factor VIII (FVIII) levels [median (IQR) 261·9 (170-315) iu/dl] were seen in patients with severe severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Sequential testing showed that these elevated VWF-FVIII complex levels remained high for up to 3 weeks. Similarly, plasma VWFpp levels were also markedly elevated [median (IQR) 324·6 (267-524) iu/dl]. Interestingly however, the VWFpp/VWF:Ag ratio was reduced, demonstrating that decreased VWF clearance contributes to the elevated plasma VWF:Ag levels in severe COVID-19. Importantly, plasma VWFpp levels also correlated with clinical severity indices including the Sequential Organ Failure Assessment (SOFA) score, Sepsis-Induced Coagulopathy (SIC) score and the ratio of arterial oxygen partial pressure to fractional inspired oxygen (P/F ratio). Collectively, these findings support the hypothesis that sustained fulminant EC activation is occurring in severe COVID-19, and further suggest that VWFpp may have a role as a biomarker in this setting.

The Biological Significance of von Willebrand Factor O-Linked Glycosylation.

Glycosylation is a key posttranslational modification, known to occur on more than half of all secreted proteins in man. As such, the role of N- and O-linked glycan structures in modulating various aspects of protein biology is an area of much research. Given their prevalence, it is perhaps unsurprising that variations in glycan structures have been demonstrated to play critical roles in modulating protein function and have been implicated in the pathophysiology of human diseases. von Willebrand factor (VWF), a plasma glycoprotein that is essential for normal hemostasis, is heavily glycosylated, containing 13 N-linked and 10 O-linked glycans. Together, these carbohydrate chains account for 20% of VWF monomeric mass, and have been shown to modulate VWF structure, function, and half-life. In this review, we focus on the specific role played by O-linked glycans in modulating VWF biology. Specifically, VWF O-linked glycans have been shown to modulate tertiary protein structure, susceptibility to ADAMTS13 proteolysis, platelet tethering, and VWF circulatory half-life.

Advances in the Management of Cancer-Associated Thrombosis.

The association between cancer and venous thromboembolism (VTE) has been established for more than 150 years. Nevertheless, cancer-associated thrombosis still remains a major clinical challenge and is associated with significant morbidity and mortality for patients with cancer. The clinical presentation of cancer-associated thrombosis can be distinct from that of a patient without an underlying malignancy. Moreover, specific cancer types, including pancreatic cancer and hematological malignancies, as well as advanced stage disease can confer a significant thrombotic risk. This risk is further augmented by specific anticancer treatment modalities. The pathophysiology of cancer-associated thrombosis is complex and multifactorial. However, understanding the biological mechanisms underpinning VTE risk may provide insight into novel targeted prophylaxis in cancer patients. Over the last decade, low-molecular-weight heparin has been the preferred anticoagulant agent for patients with cancer-associated thrombosis due to improved efficacy compared with vitamin K antagonists. However, the advent of direct oral anticoagulants (DOACs) has added to the repertoire of ammunition now at the disposal of clinicians to aid in the management of cancer-associated thrombosis. Several randomized controlled trials have now been published, demonstrating DOAC as a noninferior alternative for both the treatment and prevention of cancer-associated thrombosis. Notwithstanding this, limitations for their widespread use remain, with the potential for increased bleeding risk, drug interactions, and poor DOAC metabolism. This review discusses the evidence base for the incidence and risk factors associated with VTE in cancer, development, and refinement of risk prediction models and novel advances in the therapeutic management of cancer-associated thrombosis.

Blood group alters platelet binding kinetics to von Willebrand factor and consequently platelet function.

Blood type O is associated with a lower risk of myocardial infarction. Platelets play a critical role in myocardial infarction. It is not known whether the expression of blood group antigens on platelet proteins alters platelet function; we hypothesized that platelet function would be different between donors with blood type O and those with non-O. To address this hypothesis, we perfused blood from healthy type O donors (n = 33) or non-O donors (n = 54) over pooled plasma derived von Willebrand factor (VWF) protein and purified blood type-specific VWF at arterial shear and measured platelet translocation dynamics. We demonstrate for the first time that type O platelets travel farther at greater speeds before forming stable bonds with VWF. To further characterize these findings, we used a novel analytical model of platelet interaction. Modeling revealed that the kinetics for GPIb/VWF binding rate are significantly lower for type O compared with non-O platelets. Our results demonstrate that platelets from type O donors interact less with VWF at arterial shear than non-O platelets. Our results suggest a potential mechanism for the reduced risk of myocardial infarction associated with blood type O.

Increased galactose expression and enhanced clearance in patients with low von Willebrand factor.

Glycan determinants on von Willebrand factor (VWF) play critical roles in regulating its susceptibility to proteolysis and clearance. Abnormal glycosylation has been shown to cause von Willebrand disease (VWD) in a number of different mouse models. However, because of the significant technical challenges associated with accurate assessment of VWF glycan composition, the importance of carbohydrates in human VWD pathogenesis remains largely unexplored. To address this, we developed a novel lectin-binding panel to enable human VWF glycan characterization. This methodology was then used to study glycan expression in a cohort of 110 patients with low VWF compared with O blood group-matched healthy controls. Interestingly, significant interindividual heterogeneity in VWF glycan expression was seen in the healthy control population. This variation included terminal sialylation and ABO(H) blood group expression on VWF. Importantly, we also observed evidence of aberrant glycosylation in a subgroup of patients with low VWF. In particular, terminal α(2-6)-linked sialylation was reduced in patients with low VWF, with a secondary increase in galactose (Gal) exposure. Furthermore, an inverse correlation between Gal exposure and estimated VWF half-life was observed in those patients with enhanced VWF clearance. Together, these findings support the hypothesis that loss of terminal sialylation contributes to the pathophysiology underpinning low VWF in at least a subgroup of patients by promoting enhanced clearance. In addition, alterations in VWF carbohydrate expression are likely to contribute to quantitative and qualitative variations in VWF levels in the normal population. This trial was registered at www.clinicaltrials.gov as #NCT03167320.

COVID19 coagulopathy in Caucasian patients.

Although the pathophysiology underlying severe COVID19 remains poorly understood, accumulating data suggest that a lung-centric coagulopathy may play an important role. Elevated D-dimer levels which correlated inversely with overall survival were recently reported in Chinese cohort studies. Critically however, ethnicity has major effects on thrombotic risk, with a 3-4-fold lower risk in Chinese compared to Caucasians and a significantly higher risk in African-Americans. In this study, we investigated COVID19 coagulopathy in Caucasian patients. Our findings confirm that severe COVID19 infection is associated with a significant coagulopathy that correlates with disease severity. Importantly however, Caucasian COVID19 patients on low molecular weight heparin thromboprophylaxis rarely develop overt disseminated intravascular coagulation (DIC). In rare COVID19 cases where DIC does develop, it tends to be restricted to late-stage disease. Collectively, these data suggest that the diffuse bilateral pulmonary inflammation observed in COVID19 is associated with a novel pulmonary-specific vasculopathy termed pulmonary intravascular coagulopathy (PIC) as distinct to DIC. Given that thrombotic risk is significantly impacted by race, coupled with the accumulating evidence that coagulopathy is important in COVID19 pathogenesis, our findings raise the intriguing possibility that pulmonary vasculopathy may contribute to the unexplained differences that are beginning to emerge highlighting racial susceptibility to COVID19 mortality.

A novel role for the macrophage galactose-type lectin receptor in mediating von Willebrand factor clearance.

Previous studies have shown that loss of terminal sialic acid causes enhanced von Willebrand factor (VWF) clearance through the Ashwell-Morrell receptor (AMR). In this study, we investigated (1) the specific importance of N- vs O-linked sialic acid in protecting against VWF clearance and (2) whether additional receptors contribute to the reduced half-life of hyposialylated VWF. α2-3-linked sialic acid accounts for <20% of total sialic acid and is predominantly expressed on VWF O-glycans. Nevertheless, specific digestion with α2-3 neuraminidase (α2-3Neu-VWF) was sufficient to cause markedly enhanced VWF clearance. Interestingly, in vivo clearance experiments in dual VWF-/-/Asgr1-/- mice demonstrated enhanced clearance of α2-3Neu-VWF even in the absence of the AMR. The macrophage galactose-type lectin (MGL) is a C-type lectin that binds to glycoproteins expressing terminal N-acetylgalactosamine or galactose residues. Importantly, the markedly enhanced clearance of hyposialylated VWF in VWF-/-/Asgr1-/- mice was significantly attenuated in the presence of an anti-MGL inhibitory antibody. Furthermore, dose-dependent binding of human VWF to purified recombinant human MGL was confirmed using surface plasmon resonance. Additionally, plasma VWF:Ag levels were significantly elevated in MGL1-/- mice compared with controls. Collectively, these findings identify MGL as a novel macrophage receptor for VWF that significantly contributes to the clearance of both wild-type and hyposialylated VWF.

Advances in understanding the molecular mechanisms that maintain normal haemostasis.

Blood clot formation to stem bleeding from an injured blood vessel arises from a complex series of cellular and biochemical events, which, when dysregulated, predispose to an increased risk of thrombosis or bleeding. Similarly, haemostatic regulation of clot growth and size is exquisitely controlled by a series of anticoagulant 'checkpoints', that exert their inhibitory activity at distinct stages in the steps leading to clot formation. Although the major plasma protein constituents required for haemostasis have now been largely elucidated and the molecular events that lead to clot formation are well understood, defining a fuller appreciation of the importance, location and regulation of each haemostatic process remains a fertile area of ongoing research. In this review article, we first provide an overview of the original 'waterfall' or 'cascade' hypothesis of blood coagulation as it was defined in the 1960s. We subsequently discuss how this original model has been refined over time to incorporate accumulating data that has enabled a more nuanced consideration of the role of specific proteins, receptors and lipids in dictating the spatial and temporal development of a blood clot.

Advances in understanding the molecular mechanisms of venous thrombosis.

Venous thromboembolism (VTE) represents a leading cause of global mortality, however, the determinants that contribute to thrombus development remain incompletely understood. In this review, we discuss the role of inherited abnormalities of blood coagulation in VTE pathogenesis. In addition, we also consider recent emerging data suggesting other molecular and cellular determinants may also contribute. Specifically, we describe the role played by the inflamed endothelium, and the dysregulated responses to inflammatory stimuli that create a platform for pathological clot formation. We review the accumulating evidence that blood cells, contact pathway factors and protein disulphide isomerases all play key roles in VTE development. Finally, we discuss new insights into the role of metabolites arising from commensal gut bacteria and their potential role in facilitating VTE. This overview provides an update on these state-of-the-art developments and the opportunities they provide for new antithrombotic therapies with enhanced efficacy and improved safety profiles.

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

Critical 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.

von Willebrand factor clearance - biological mechanisms and clinical significance.

The mechanisms involved in regulating von Willebrand factor (VWF) clearance remain poorly understood. However recent studies have shown that macrophages play a critical role in regulating the half-life of VWF, and have identified specific lectin (including asialoglycoprotein, macrophage galactose-type lectin, Sigec-5 and C-type lectin domain family 4 member M) and scavenger receptors (including low-density lipoprotein receptor-related protein-1, scavenger receptor A1 and stabilin-2) that are involved in VWF clearance. Further studies will be required to determine the relative importance of these individual receptors with respect to physiological and pathological VWF clearance. Nevertheless, recent clinical data have highlighted the importance of enhanced VWF clearance in the pathogenesis of type 1 von Willebrand disease (VWD). Moreover, increased clearance also contributes to reduced VWF levels in many patients with type 2 and type 3 VWD. Improved understanding regarding VWF clearance is not only of direct biological relevance, but may also have important implications for the development of novel therapeutic agents with extended plasma half-lives for the treatment of both VWD and haemophilia A.

Emerging Roles for von Willebrand Factor in Cancer Cell Biology.

von Willebrand factor (VWF) is a complex multimeric plasma glycoprotein that plays critical roles in normal hemostasis. However, additional novel roles for VWF in modulating cancer cell biology, and in particular tumor metastasis, have recently been reported. Markedly elevated plasma VWF levels were associated with advanced tumor stage and metastatic disease. These observations have raised the question of whether VWF may be involved in regulating tumor progression. Interestingly, novel findings indicate that VWF is expressed by a variety of tumor cells of nonendothelial origin. Critically, tumor cells that exhibit de novo acquired VWF expression demonstrate enhanced binding to endothelial cells (EC) and platelets, and increased extravasation through EC barriers. Furthermore, in vitro studies have shown that VWF can bind a variety of different tumor cells mediated by specific receptors expressed on the tumor cell surface. The concept that VWF is important in modulating tumor metastasis is further supported by in vivo experiments demonstrating that antibody-mediated VWF inhibition significantly attenuated murine metastasis. Intriguingly, however, VWF binding to specific human tumor cell lines results in apoptosis. In this study, the authors provide an overview of recent advances supporting a role for VWF in regulating multiple aspects of cancer cell biology.

Emerging roles for hemostatic dysfunction in malaria pathogenesis.

Severe Plasmodium falciparum malaria remains a leading cause of mortality, particularly in sub-Saharan Africa where it accounts for up to 1 million deaths per annum. In spite of the significant mortality and morbidity associated with cerebral malaria (CM), the molecular mechanisms involved in the pathophysiology of severe malaria remain surprisingly poorly understood. Previous studies have demonstrated that sequestration of P falciparum-infected erythrocytes within the microvasculature of the brain plays a key role in the development of CM. In addition, there is convincing evidence that both endothelial cell activation and platelets play critical roles in the modulating the pathogenesis of severe P falciparum malaria. In this review, we provide an overview of recent studies that have identified novel roles through which hemostatic dysfunction may directly influence malaria pathogenesis. In particular, we focus on emerging data suggesting that von Willebrand factor, coagulation cascade activation, and dysfunction of the protein C pathway may be of specific importance in this context. These collective insights underscore a growing appreciation of the important, but poorly understood, role of hemostatic dysfunction in malaria progression and, importantly, illuminate potential approaches for novel therapeutic strategies. Given that the mortality rate associated with CM remains on the order of 20% despite the availability of effective antimalarial therapy, development of adjunctive therapies that can attenuate CM progression clearly represents a major unmet need. These emerging data are thus not only of basic scientific interest, but also of direct clinical significance.

N-linked glycans within the A2 domain of von Willebrand factor modulate macrophage-mediated clearance.

Enhanced von Willebrand factor (VWF) clearance is important in the etiology of von Willebrand disease. However, the molecular mechanisms underlying VWF clearance remain poorly understood. In this study, we investigated the role of VWF domains and specific glycan moieties in regulating in vivo clearance. Our findings demonstrate that the A1 domain of VWF contains a receptor-recognition site that plays a key role in regulating the interaction of VWF with macrophages. In A1-A2-A3 and full-length VWF, this macrophage-binding site is cryptic but becomes exposed following exposure to shear or ristocetin. Previous studies have demonstrated that the N-linked glycans within the A2 domain play an important role in modulating susceptibility to ADAMTS13 proteolysis. We further demonstrate that these glycans presented at N1515 and N1574 also play a critical role in protecting VWF against macrophage binding and clearance. Indeed, loss of the N-glycan at N1515 resulted in markedly enhanced VWF clearance that was significantly faster than that observed with any previously described VWF mutations. In addition, A1-A2-A3 fragments containing the N1515Q or N1574Q substitutions also demonstrated significantly enhanced clearance. Importantly, clodronate-induced macrophage depletion significantly attenuated the increased clearance observed with N1515Q and N1574Q in both full-length VWF and A1-A2-A3. Finally, we further demonstrate that loss of these N-linked glycans does not enhance clearance in VWF in the presence of a structurally constrained A2 domain. Collectively, these novel findings support the hypothesis that conformation of the VWF A domains plays a critical role in modulating macrophage-mediated clearance of VWF in vivo.

A novel role for von Willebrand factor in the pathogenesis of experimental cerebral malaria.

Plasmodium falciparum malaria infection is associated with an early marked increase in plasma von Willebrand factor (VWF) levels, together with a pathological accumulation of hyperreactive ultra-large VWF (UL-VWF) multimers. Given the established critical role of platelets in malaria pathogenesis, these increases in plasma VWF raise the intriguing possibility that VWF may play a direct role in modulating malaria pathogenesis. To address this hypothesis, we used an established murine model of experimental cerebral malaria (ECM), in which wild-type (WT) C57BL/6J mice were infected with Plasmodium berghei ANKA. In keeping with findings in children with P falciparum malaria, acute endothelial cell activation was an early and consistent feature in the murine model of cerebral malaria (CM), resulting in significantly increased plasma VWF levels. Despite the fact that murine plasma ADAMTS13 levels were not significantly reduced, pathological UL-VWF multimers were also observed in murine plasma following P berghei infection. To determine whether VWF plays a role in modulating the pathogenesis of CM in vivo, we further investigated P berghei infection in VWF(-/-) C57BL/6J mice. Clinical ECM progression was delayed, and overall survival was significantly prolonged in VWF(-/-) mice compared with WT controls. Despite this protection against ECM, no significant differences in platelet counts or blood parasitemia levels were observed between VWF(-/-) and WT mice. Interestingly, however, the degree of ECM-associated enhanced blood-brain barrier permeability was significantly attenuated in VWF(-/-) mice compared with WT controls. Given the significant morbidity and mortality associated with CM, these novel data may have direct translational significance.

Plasmin Cleaves Von Willebrand Factor at K1491-R1492 in the A1-A2 Linker Region in a Shear- and Glycan-Dependent Manner In Vitro.

OBJECTIVE: Previous studies have demonstrated a role for plasmin in regulating plasma von Willebrand factor (VWF) multimer composition. Moreover, emerging data have shown that plasmin-induced cleavage of VWF is of particular importance in specific pathological states. Interestingly, plasmin has been successfully used as an alternative to ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif) in a mouse model of thrombotic thrombocytopenic purpura. Consequently, elucidating the molecular mechanisms through which plasmin binds and cleaves VWF is not only of basic scientific interest but also of direct clinical importance. Our aim was to investigate factors that modulate the susceptibility of human VWF to proteolysis by plasmin. APPROACH AND RESULTS: We have adapted the VWF vortex proteolysis assay to allow for time-dependent shear exposure studies. We show that globular VWF is resistant to plasmin cleavage under static conditions, but is readily cleaved by plasmin under shear. Although both plasmin and ADAMTS13 cleave VWF in a shear-dependent manner, plasmin does not cleave at the Tyr1605-Met1606 ADAMTS13 proteolytic site in the A2 domain. Rather under shear stress conditions, or in the presence of denaturants, such as urea or ristocetin, plasmin cleaves the K1491-R1492 peptide bond within the VWF A1-A2 linker region. Finally, we demonstrate that VWF susceptibility to plasmin proteolysis at K1491-R1492 is modulated by local N-linked glycan expression within A1A2A3, and specifically inhibited by heparin binding to the A1 domain. CONCLUSIONS: Improved understanding of the plasmin-VWF interaction offers exciting opportunities to develop novel adjunctive therapies for the treatment of refractory thrombotic thrombocytopenic purpura.