The aptamer BT200 blocks interaction of K1405-K1408 in the VWF-A1 domain with macrophage LRP1.

ABSTRACT: 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.

Type 1 VWD classification revisited: novel insights from combined analysis of the LoVIC and WiN studies.

ABSTRACT: There is significant ongoing debate regarding type 1 von Willebrand disease (VWD) defintion. Previous guidelines recommended patients with von Willebrand factor (VWF) levels <30 IU/dL be diagnosed type 1 VWD, whereas patients with significant bleeding and VWF levels from 30 to 50 IU/dL be diagnosed with low VWF. To elucidate the relationship between type 1 VWD and low VWF in the context of age-induced increases in VWF levels, we combined data sets from 2 national cohort studies: 162 patients with low VWF from the Low VWF in Ireland Cohort (LoVIC) and 403 patients with type 1 VWD from the Willebrand in The Netherlands (WiN) studies. In 47% of type 1 VWD participants, VWF levels remained <30 IU/dL despite increasing age. Conversely, VWF levels increased to the low VWF range (30-50 IU/dL) in 30% and normalized (>50 IU/dL) in 23% of type 1 VWD cases. Crucially, absolute VWF antigen (VWF:Ag) levels and increase of VWF:Ag per year overlapped between low VWF and normalized type 1 VWD participants. Moreover, multiple regression analysis demonstrated that VWF:Ag levels in low VWF and normalized type 1 VWD patients would not have been different had they been diagnosed at the same age (ß = 0.00; 95% confidence interval, -0.03 to 0.04). Consistently, no difference was found in the prevalence of VWF sequence variants; factor VIII activity/VWF:Ag or VWF propeptide/VWF:Ag ratios; or desmopressin responses between low VWF and normalized type 1 VWD patients. In conclusion, our findings demonstrate that low VWF does not constitute a discrete clinical or pathological entity. Rather, it is part of an age-dependent type 1 VWD evolving phenotype. Collectively, these data have important implications for future VWD classification criteria.

The Role of Myeloid Cells in Thromboinflammatory Disease.

Inflammation contributes to the development of thrombosis, but the mechanistic basis for this association remains poorly understood. Innate immune responses and coagulation pathways are activated in parallel following infection or injury, and represent an important host defense mechanism to limit pathogen spread in the bloodstream. However, dysregulated proinflammatory activity is implicated in the progression of venous thromboembolism and arterial thrombosis. In this review, we focus on the role of myeloid cells in propagating thromboinflammation in acute inflammatory conditions, such as sepsis and coronavirus disease 2019 (COVID-19), and chronic inflammatory conditions, such as obesity, atherosclerosis, and inflammatory bowel disease. Myeloid cells are considered key drivers of thromboinflammation via upregulated tissue factor activity, formation of neutrophil extracellular traps (NETs), contact pathway activation, and aberrant coagulation factor-mediated protease-activated receptor (PAR) signaling. We discuss how strategies to target the intersection between myeloid cell-mediated inflammation and activation of blood coagulation represent an exciting new approach to combat immunothrombosis. Specifically, repurposed anti-inflammatory drugs, immunometabolic regulators, and NETosis inhibitors present opportunities that have the potential to dampen immunothrombotic activity without interfering with hemostasis. Such therapies could have far-reaching benefits for patient care across many thromboinflammatory conditions.

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.

Immunothrombosis and the molecular control of tissue factor by pyroptosis: prospects for new anticoagulants.

The interplay between innate immunity and coagulation after infection or injury, termed immunothrombosis, is the primary cause of disseminated intravascular coagulation (DIC), a condition that occurs in sepsis. Thrombosis associated with DIC is the leading cause of death worldwide. Interest in immunothrombosis has grown because of COVID-19, the respiratory disease caused by SARS-CoV-2, which has been termed a syndrome of dysregulated immunothrombosis. As the relatively new field of immunothrombosis expands at a rapid pace, the focus of academic and pharmacological research has shifted from generating treatments targeted at the traditional 'waterfall' model of coagulation to therapies better directed towards immune components that drive coagulopathies. Immunothrombosis can be initiated in macrophages by cleavage of the non-canonical inflammasome which contains caspase-11. This leads to release of tissue factor (TF), a membrane glycoprotein receptor that forms a high-affinity complex with coagulation factor VII/VIIa to proteolytically activate factors IX to IXa and X to Xa, generating thrombin and leading to fibrin formation and platelet activation. The mechanism involves the post-translational activation of TF, termed decryption, and release of decrypted TF via caspase-11-mediated pyroptosis. During aberrant immunothrombosis, decryption of TF leads to thromboinflammation, sepsis, and DIC. Therefore, developing therapies to target pyroptosis have emerged as an attractive concept to counteract dysregulated immunothrombosis. In this review, we detail the three mechanisms of TF control: concurrent induction of TF, caspase-11, and NLRP3 (signal 1); TF decryption, which increases its procoagulant activity (signal 2); and accelerated release of TF into the intravascular space via pyroptosis (signal 3). In this way, decryption of TF is analogous to the two signals of NLRP3 inflammasome activation, whereby induction of pro-IL-1ß and NLRP3 (signal 1) is followed by activation of NLRP3 (signal 2). We describe in detail TF decryption, which involves pathogen-induced alterations in the composition of the plasma membrane and modification of key cysteines on TF, particularly at the location of the critical, allosterically regulated disulfide bond of TF in its 219-residue extracellular domain. In addition, we speculate towards the importance of identifying new therapeutics to block immunothrombotic triggering of TF, which can involve inhibition of pyroptosis to limit TF release, or the direct targeting of TF decryption using cysteine-modifying therapeutics.

The protein C pathways.

PURPOSE OF REVIEW: To provide an overview of the state-of-the-art in protein C (PC) pathway research. RECENT FINDINGS: The PC pathway is crucial for maintaining hemostasis to prevent venous thromboembolism. This is evident from genetic mutations that result in impaired PC pathway activity and contribute to increased venous thromboembolism risk in affected individuals. In addition to its anticoagulant role, activated PC (APC) also mediates a complex, pleiotropic role in the maintenance of vascular cell health, which it achieves via anti-inflammatory and antiapoptotic cell signaling on endothelial cells. Emerging data have demonstrated that cell signaling by APC, mediated by multiple receptor interactions on different cell types, also confers cytoprotective and anti-inflammatory benefits. Defects in both arms of the PC pathway are associated with increased susceptibility to thrombo-inflammatory disease in various preclinical thrombotic, proinflammatory and neurological disease models. Moreover, recent studies have identified attenuation of anticoagulant PC pathway activity as an exciting therapeutic opportunity to promote hemostasis in patients with inherited or acquired bleeding disorders. SUMMARY: In this review, we provide an overview of some recent developments in our understanding of the PC pathways.

Dysregulated haemostasis in thrombo-inflammatory disease.

Inflammatory disease is often associated with an increased incidence of venous thromboembolism in affected patients, although in most instances, the mechanistic basis for this increased thrombogenicity remains poorly understood. Acute infection, as exemplified by sepsis, malaria and most recently, COVID-19, drives 'immunothrombosis', where the immune defence response to capture and neutralise invading pathogens causes concurrent activation of deleterious prothrombotic cellular and biological responses. Moreover, dysregulated innate and adaptive immune responses in patients with chronic inflammatory conditions, such as inflammatory bowel disease, allergies, and neurodegenerative disorders, are now recognised to occur in parallel with activation of coagulation. In this review, we describe the detailed cellular and biochemical mechanisms that cause inflammation-driven haemostatic dysregulation, including aberrant contact pathway activation, increased tissue factor activity and release, innate immune cell activation and programmed cell death, and T cell-mediated changes in thrombus resolution. In addition, we consider how lifestyle changes increasingly associated with modern life, such as circadian rhythm disruption, chronic stress and old age, are increasingly implicated in unbalancing haemostasis. Finally, we describe the emergence of potential therapies with broad-ranging immunothrombotic functions, and how drug development in this area is challenged by our nascent understanding of the key molecular and cellular parameters that control the shared nodes of proinflammatory and procoagulant pathways. Despite the increasing recognition and understanding of the prothrombotic nature of inflammatory disease, significant challenges remain in effectively managing affected patients, and new therapeutic approaches to curtail the key pathogenic steps in immune response-driven thrombosis are urgently required.

Heterogeneity in Bleeding Tendency and Arthropathy Development in Individuals with Hemophilia.

People with hemophilia (PWH) have an increased tendency to bleed, often into their joints, causing debilitating joint disease if left untreated. To reduce the incidence of bleeding events, PWH receive prophylactic replacement therapy with recombinant factor VIII (FVIII) or FIX. Bleeding events in PWH are typically proportional to their plasma FVIII or IX levels; however, in many PWH, bleeding tendency and the likelihood of developing arthropathy often varies independently of endogenous factor levels. Consequently, many PWH suffer repeated bleeding events before correct dosing of replacement factor can be established. Diagnostic approaches to define an individual's bleeding tendency remain limited. Multiple modulators of bleeding phenotype in PWH have been proposed, including the type of disease-causing variant, age of onset of bleeding episodes, plasma modifiers of blood coagulation or clot fibrinolysis pathway activity, interindividual differences in platelet reactivity, and endothelial anticoagulant activity. In this review, we summarize current knowledge of established factors modulating bleeding tendency and discuss emerging concepts of additional biological elements that may contribute to variable bleeding tendency in PWH. Finally, we consider how variance in responses to new gene therapies may also necessitate consideration of patient-specific tailoring of treatment. Cumulatively, these studies highlight the need to reconsider the current "one size fits all" approach to treatment regimens for PWH and consider therapies guided by the bleeding phenotype of each individual PWH at the onset of therapy. Further characterization of the biological bases of bleeding heterogeneity in PWH, combined with the development of novel diagnostic assays to identify those factors that modulate bleeding risk in PWH, will be required to meet these aspirations.

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.

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.

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.

The Immunoregulatory Activities of Activated Protein C in Inflammatory Disease.

Tissue injury prompts the initiation of host defense responses to limit blood loss, restrict pathogen entry, and promote repair. Biochemical and cellular pathways that lead to blood coagulation serve a fundamental role in generating a physical barrier at the wound site, but have also evolved to promote immune response to injury. Similarly, anticoagulant pathways that attenuate clot formation also regulate innate and adaptive immune responses. Of particular importance is activated protein C (APC) which serves as a principal regulator of thrombin generation, shapes the innate immune response to infection, and has been shown to contribute to the adaptive immune response in several preclinical models of autoimmune disease. APC controls blood coagulation by proteolytic degradation of procoagulant activated cofactors essential for fibrin clot development, but also cleaves multiple additional substrates and interacts with cell surface receptors to exert additional physiologically important roles. In this review, we focus on the molecular mechanisms utilized by APC to limit inflammation and, in particular, current understanding of the basis for APC anticoagulant and signaling activities. In particular, we provide an overview of established and emerging signaling pathways initiated by APC on endothelial cells, monocytes, neutrophils, dendritic cells, and T cells to control and regulate immune cell physiology. Finally, we consider the impact of APC activity in the context of both acute and chronic inflammatory disease, and the continuing efforts to harness the immunoregulatory properties of recombinant APC for therapeutic use.

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.

Activated protein C ß-glycoform promotes enhanced noncanonical PAR1 proteolysis and superior resistance to ischemic injury.

Activated protein C (APC) is an anticoagulant protease that initiates cell signaling via protease-activated receptor 1 (PAR1) to regulate vascular integrity and inflammatory response. In this study, a recombinant APC variant (APC(N329Q)) mimicking the naturally occurring APC-ß plasma glycoform was found to exhibit superior PAR1 proteolysis at a cleavage site that selectively mediates cytoprotective signaling. APC(N329Q) also enhanced integrin αMß2-dependent PAR1 proteolysis to exert significantly improved antiinflammatory activity on macrophages compared with wild-type APC. Recent therapeutic applications of recombinant APC in ischemic stroke models have used APC variants with limited anticoagulant activity to negate potential bleeding side effects. Using a mouse model of ischemic stroke and late t-PA intervention, the neuroprotective activity of a murine APC variant with limited anticoagulant activity (mAPC(PS)) was compared with an identical APC variant except for the absence of glycosylation at the APC-ß sequon (mAPC(PS/N329Q)). Remarkably, mAPC(PS/N329Q) limited cerebral ischemic injury and reduced brain lesion volume significantly more effectively than mAPC(PS). Collectively, this study reveals the importance of APC glycosylation in controlling the efficacy of PAR1 proteolysis by APC and demonstrates the potential of novel APC variants with superior cytoprotective signaling function as enhanced therapeutic agents for the treatment of ischemic stroke.