EXAMINING VARIABILITY IN THE DIAGNOSIS AND MANAGEMENT OF PEOPLE WITH BLEEDING DISORDERS OF UNKNOWN CAUSE: COMMUNICATION FROM THE ISTH SSC SUBCOMMITTEE ON VON WILLEBRAND FACTOR.

BACKGROUND: Bleeding disorder of unknown cause (BDUC) is characterised by a bleeding phenotype in the setting of normal haemostatic testing. No standardised diagnostic criteria or treatment algorithms exist for people with BDUC. To address the unmet need, the International Society on Thrombosis and Haemostasis von Willebrand Factor Scientific Subcommittee (ISTH VWF SCC) performed a real-world survey, aimed at addressing knowledge gaps, developing consensus pathways and ultimately improving care. OBJECTIVES AND METHODS: We sought to determine current international clinical practices in the investigation, registration, and treatment of people with BDUC through an online structured survey of health care providers (HCPs) who managed patient with bleeding disorders. RESULTS: Two hundred and sixteen respondents from 39 countries were included in the final analysis. The clinical assessment of those with a possible bleeding disorder varied, with only 55% excluding hypermobility but high levels (80%) of bleeding assessment tools (BAT) usage. In haemostatic testing only the prothrombin time (PT) and activated partial thromboplastin time (APTT) tests gained universal support. Tranexamic acid (TXA) was favoured for prophylaxis for minor (71%)/major (59%) surgeries and pregnancy (58%) but advice on the treatment advised if bleeding occurred was heterogeneous. The management of heavy menstrual bleeding (HMB) in women despite combined oral contraceptive pill (COCP) use also proved challenging with HCPs selecting multiple alternative strategies. CONCLUSION: Significant variation exists in the recognition, registration and management of people with BDUC worldwide. This survey emphasises the need for consensus pathways to diagnose and treat BDUC to standardise and improve care for patients internationally.

Novel functions for Von Willebrand factor.

For many years, it has been known that VWF interacts with FVIII, 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, more than 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 impact the functional properties for some of its ligands. In keeping with those observations, exciting new roles for VWF in regulating a series of non-hemostatic biological functions has also emerged. These include inflammation, wound healing, angiogenesis, and bone metabolism. Finally, recent evidence supports the hypothesis that the non-hemostatic 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 impact cellular biology. In addition, we consider the evidence that non-hemostatic VWF functions may contribute to the pathogenesis of human diseases beyond thrombosis and bleeding.

O-glycan determinants regulate VWF trafficking to Weibel-Palade bodies.

ABSTRACT: von Willebrand factor (VWF) undergoes complex posttranslational modification within endothelial cells (ECs) before secretion. This includes significant N- and O-linked glycosylation. Previous studies have demonstrated that changes in N-linked glycan structures significantly influence VWF biosynthesis. In contrast, although abnormalities in VWF O-linked glycans (OLGs) have been associated with enhanced VWF clearance, their effect on VWF biosynthesis remains poorly explored. Herein, we report a novel role for OLG determinants in regulating VWF biosynthesis and trafficking within ECs. We demonstrate that alterations in OLGs (notably reduced terminal sialylation) lead to activation of the A1 domain of VWF within EC. In the presence of altered OLG, VWF multimerization is reduced and Weibel-Palade body (WPB) formation significantly impaired. Consistently, the amount of VWF secreted from WPB after EC activation was significantly reduced in the context of O-glycosylation inhibition. Finally, altered OLG on VWF not only reduced the amount of VWF secreted after EC activation but also affected its hemostatic efficacy. Notably, VWF secreted after WPB exocytosis consisted predominantly of low molecular weight multimers, and the length of tethered VWF string formation on the surface of activated ECs was significantly reduced. In conclusion, our data therefore support the hypothesis that alterations in O-glycosylation pathways directly affect VWF trafficking within human EC. These findings are interesting given that previous studies have reported altered OLG on plasma VWF (notably increased T-antigen expression) in patients with von Willebrand disease.

Standardization of definition and management for bleeding disorder of unknown cause: communication from the SSC of the ISTH.

In many patients referred with significant bleeding phenotype, laboratory testing fails to define any hemostatic abnormalities. Clinical practice with respect to diagnosis and management of this patient cohort poses significant clinical challenges. We recommend that bleeding history in these patients should be objectively assessed using the International Society on Thrombosis and Haemostasis (ISTH) bleeding assessment tool. Patients with increased bleeding assessment tool scores should progress to hemostasis laboratory testing. To diagnose bleeding disorder of unknown cause (BDUC), normal complete blood count, prothrombin time, activated partial thromboplastin time, thrombin time, von Willebrand factor antigen, von Willebrand factor function, coagulation factors VIII, IX, and XI, and platelet light transmission aggregometry should be the minimum laboratory assessment. In some laboratories, additional specialized hemostasis testing may be performed to identify other rare causes of bleeding. We recommend that patients with a significant bleeding phenotype but normal laboratory investigations should be registered with a diagnosis of BDUC in preference to other terminology. Global hemostatic tests and markers of fibrinolysis demonstrate variable abnormalities, and their clinical significance remains uncertain. Targeted genomic sequencing examining candidate hemostatic genes has a low diagnostic yield. Underlying BDUC should be considered in patients with heavy menstrual bleeding since delays in diagnosis often extend to many years and negatively impact quality of life. Treatment options for BDUC patients include tranexamic acid, desmopressin, and platelet transfusions.

Glycolytic reprogramming fuels myeloid cell-driven hypercoagulability.

BACKGROUND: Myeloid cell metabolic reprogramming is a hallmark of inflammatory disease; however, its role in inflammation-induced hypercoagulability is poorly understood. OBJECTIVES: We aimed to evaluate the role of inflammation-associated metabolic reprogramming in regulating blood coagulation. METHODS: We used novel myeloid cell-based global hemostasis assays and murine models of immunometabolic disease. RESULTS: Glycolysis was essential for enhanced activated myeloid cell tissue factor expression and decryption, driving increased cell-dependent thrombin generation in response to inflammatory challenge. Similarly, inhibition of glycolysis enhanced activated macrophage fibrinolytic activity through reduced plasminogen activator inhibitor 1 activity. Macrophage polarization or activation markedly increased endothelial protein C receptor (EPCR) expression on monocytes and macrophages, leading to increased myeloid cell-dependent protein C activation. Importantly, inflammation-dependent EPCR expression on tissue-resident macrophages was also observed in vivo. Adipose tissue macrophages from obese mice fed a high-fat diet exhibited significantly enhanced EPCR expression and activated protein C generation compared with macrophages isolated from the adipose tissue of healthy mice. Similarly, the induction of colitis in mice prompted infiltration of EPCR+ innate myeloid cells within inflamed colonic tissue that were absent from the intestinal tissue of healthy mice. CONCLUSION: Collectively, this study identifies immunometabolic regulation of myeloid cell hypercoagulability, opening new therapeutic possibilities for targeted mitigation of thromboinflammatory disease.

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 Nijmegen ultra-sensitive Bethesda Assay detects very low-titer factor VIII inhibitors in patients with congenital and acquired hemophilia A.

BACKGROUND: An inhibitor can develop in congenital hemophilia A (HA) patients against exogenous infused factor (F)VIII, whereas in acquired HA (AHA) inhibitors initially develop against endogenous FVIII. Inhibitors can be detected with the Nijmegen Bethesda Assay (NBA), which has an international cut-off level of 0.60 Nijmegen Bethesda Units/mL (NBU/mL). Thereby, very low-titer inhibitors may remain undetected. AIM: To describe the design and validation of the Nijmegen ultra-sensitive Bethesda Assay (NusBA) for the detection of very low-titer inhibitors. METHODS: The NusBA is a modification of the NBA in which the ratio of patient plasma to normal pooled plasma is changed from 1:1 to 9:1. Analytical validation was performed according to the CLSI EP10 guideline in order to determine trueness and reproducibility. Clinical validation was performed in two cohorts of congenital HA patients (82 adults) with pharmacokinetic data and four AHA patients. The limit of quantitation (LOQ) was determined by measuring plasma samples spiked with inhibitor levels in the low range (0.05-0.80 NBU/mL). RESULTS: The LOQ for the NusBA was 0.10 NusBU/mL, with a coefficient of variation of 24.2 %. Seven (8.5 %) congenital HA patients had a positive NusBA result, of which only one was detected with the NBA. There was no correlation between NusBA and FVIII half-life. In three of the AHA patients the NusBA remained positive, when the NBA became negative. DISCUSSION: The NusBA is able to detect very low-titer FVIII inhibitors of ≥0.10 NBU/mL. Thereby, it may have added value in early inhibitor detection and therapy adjustments in patients with congenital HA and AHA.

VWF-ADAMTS13 axis dysfunction in children with sickle cell disease treated with hydroxycarbamide vs blood transfusion.

Previous studies have reported elevated von Willebrand factor (VWF) levels in patients with sickle cell disease (SCD) and demonstrated a key role for the VWF-ADAMTS13 axis in the pathobiology of SCD vaso-occlusion. Although blood transfusion is the gold standard for stroke prevention in SCD, the biological mechanisms underpinning its improved efficacy compared with hydroxycarbamide are not fully understood. We hypothesized that the improved efficacy of blood transfusion might relate to differences in VWF-ADAMTS13 axis dysfunction. In total, 180 children with a confirmed diagnosis of SCD (hemoglobin SS) on hydroxycarbamide (n = 96) or blood transfusion (n = 84) were included. Despite disease-modifying treatment, plasma VWF and VWF propeptide were elevated in a significant proportion of children with SCD (33% and 47%, respectively). Crucially, all VWF parameters were significantly higher in the hydroxycarbamide compared with the blood transfusion cohort (P < .05). Additionally, increased levels of other Weibel-Palade body-stored proteins, including factor VIII (FVIII), angiopoietin-2, and osteoprotegerin were observed, indicated ongoing endothelial cell activation. Children treated with hydroxycarbamide also had higher FVIII activity and enhanced thrombin generation compared with those in the blood transfusion cohort (P < .001). Finally, hemolysis markers strongly correlated with VWF levels (P < .001) and were significantly reduced in the blood transfusion cohort (P < .001). Cumulatively, to our knowledge, our findings demonstrate for the first time that despite treatment, ongoing dysfunction of the VWF-ADAMTS13 axis is present in a significant subgroup of pediatric patients with SCD, especially those treated with hydroxycarbamide.

Unraveling coagulation factor-mediated cellular signaling.

Blood coagulation is initiated in response to blood vessel injury or proinflammatory stimuli, which activate coagulation factors to coordinate complex biochemical and cellular responses necessary for clot formation. In addition to these critical physiologic functions, plasma protein factors activated during coagulation mediate a spectrum of signaling responses via receptor-binding interactions on different cell types. In this review, we describe examples and mechanisms of coagulation factor signaling. We detail the molecular basis for cell signaling mediated by coagulation factor proteases via the protease-activated receptor family, considering new insights into the role of protease-specific cleavage sites, cofactor and coreceptor interactions, and distinct signaling intermediate interactions in shaping protease-activated receptor signaling diversity. Moreover, we discuss examples of how injury-dependent conformational activation of other coagulation proteins, such as fibrin(ogen) and von Willebrand factor, decrypts their signaling potential, unlocking their capacity to contribute to aberrant proinflammatory signaling. Finally, we consider the role of coagulation factor signaling in disease development and the status of pharmacologic approaches to either attenuate or enhance coagulation factor signaling for therapeutic benefit, emphasizing new approaches to inhibit deleterious coagulation factor signaling without impacting hemostatic activity.

A cross-sectional follow-up study of physical activity in adults with moderate and severe haemophilia.

AIM: To conduct a cross-sectional follow-up assessment of physical activity (PA) in people with moderate and severe haemophilia (PwMSH) from the Irish Personalised Approach to the Treatment of Haemophilia (iPATH) study. METHODS: Between June-December 2021, participants' PA was measured over one week using accelerometery, and was compared with their previously measured data from the original iPATH assessment. Self-awareness of PA and the impact of the Covid-19 pandemic on PA, pain, mobility and function were retrospectively examined using a survey. RESULTS: Of 30 participants who returned surveys [n = 19, severe (FVIII, <.01 IU/mL); n = 4, moderate (FVIII, .01-.05 IU/mL); n = 7, severe (FIX, <.01 IU/mL); age: 47 (36, 55) years], 28 completed accelerometery (follow-up time: 3 years). There were no significant differences in accelerometer PA (all p > .05), but achievement of World Health Organisation guidelines increased (67.9%-75.0%; p = .646). Increased self-awareness of PA was reported by 76.7%, and 66.7% reported desires to become more physically active. Compared to normal, most reported either no differences or lower levels of PA during lockdown restrictions. Self-reported PA increased for most when restrictions eased from April 2021 onwards. Beyond the pandemic, concerns included pain and access to exercise resources. CONCLUSION: Self-reported PA throughout the pandemic was variable, whilst there were no significant differences in objectively measured PA between assessment periods, despite reports of increased self-awareness and desires to be physically active at follow-up. Further qualitative research is needed to design personalised PA and health interventions, capturing perspectives of patients, their families, and multi-disciplinary haemophilia healthcare providers.

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.

Heterogeneity in the half-life of factor VIII concentrate in patients with hemophilia A is due to variability in the clearance of endogenous von Willebrand factor.

BACKGROUND: Previous studies have reported marked interindividual variation in factor VIII (FVIII) clearance in patients with hemophilia (PWH) and proposed a number of factors that influence this heterogeneity. OBJECTIVES: To investigate the importance of the clearance rates of endogenous von Willebrand factor (VWF) compared with those of other FVIII half-life modifiers in adult PWH. METHODS: The half-life of recombinant FVIII was determined in a cohort of 61 adult PWH. A range of reported modifiers of FVIII clearance was assessed (including plasma VWF:antigen and VWF propeptide levels; VWF-FVIII binding capacity; ABO blood group; and nonneutralizing anti-FVIII antibodies). The FVIII-binding region of the VWF gene was sequenced. Finally, the effects of variation in FVIII half-life on clinical phenotype were investigated. RESULTS: We demonstrated that heterogeneity in the clearance of endogenous plasma VWF is a key determinant of variable FVIII half-life in PWH. Both ABO blood group and age significantly impact FVIII clearance. The effect of ABO blood group on FVIII half-life in PWH is modulated entirely through its effect on the clearance rates of endogenous VWF. In contrast, the age-related effect on FVIII clearance is, at least in part, VWF independent. In contrast to previous studies, no major effects of variation in VWF-FVIII binding affinity on FVIII clearance were observed. Although high-titer immunoglobulin G antibodies (≥1:80) were observed in 26% of PWH, these did not impact FVIII half-life. Importantly, the annual FVIII usage (IU/kg/y) was significantly (p = .0035) increased in patients with an FVIII half-life of <12 hours. CONCLUSION: Our data demonstrate that heterogeneity in the half-life of FVIII concentrates in patients with hemophilia A is primarily attributable to variability in the clearance of endogenous VWF.

Low von Willebrand Disease: A Bleeding Disorder of Unknown Cause?

von Willebrand disease (VWD) represents the most common inherited bleeding disorder. The majority of VWD cases are characterized by partial quantitative reductions in plasma von Willebrand factor (VWF) levels. Management of patients with mild to moderate VWF reductions in the range of 30 to 50 IU/dL poses a common clinical challenge. Some of these low VWF patients present with significant bleeding problems. In particular, heavy menstrual bleeding and postpartum hemorrhage can cause significant morbidity. Conversely, however, many individuals with mild plasma VWF:Ag reductions do not have any bleeding sequelae. In contrast to type 1 VWD, most patients with low VWF do not have detectable pathogenic VWF sequence variants, and bleeding phenotype correlates poorly with residual VWF levels. These observations suggest that low VWF is a complex disorder caused by variants in other genes beyond VWF. With respect to low VWF pathobiology, recent studies have shown that reduced VWF biosynthesis within endothelial cells likely plays a key role. However, pathological enhanced VWF clearance from plasma has also been described in approximately 20% of low VWF cases. For low VWF patients who require hemostatic treatment prior to elective procedures, tranexamic acid and desmopressin have both been shown to be efficacious. In this article, we review the current state of the art regarding low VWF. In addition, we consider how low VWF represents an entity that appears to fall between type 1 VWD on the one hand and bleeding disorders of unknown cause on the other.

Endothelial cell activation, Weibel-Palade body secretion, and enhanced angiogenesis in severe COVID-19.

Background: Severe COVID-19 is associated with marked endothelial cell (EC) activation that plays a key role in immunothrombosis and pulmonary microvascular occlusion. However, the biological mechanisms through which SARS-CoV-2 causes EC activation and damage remain poorly defined. Objectives: We investigated EC activation in patients with acute COVID-19, and specifically focused on how proteins stored within Weibel-Palade bodies may impact key aspects of disease pathogenesis. Methods: Thirty-nine patients with confirmed COVID-19 were recruited. Weibel-Palade body biomarkers (von Willebrand factor [VWF], angiopoietin-2 [Angpt-2], and osteoprotegerin) and soluble thrombomodulin (sTM) levels were determined. In addition, EC activation and angiogenesis were assessed in the presence or absence of COVID-19 plasma incubation. Results: Markedly elevated plasma VWF antigen, Angpt-2, osteoprotegerin, and sTM levels were observed in patients with acute COVID-19. The increased levels of both sTM and Weibel-Palade body components (VWF, osteoprotegerin, and Angpt-2) correlated with COVID-19 severity. Incubation of COVID-19 plasma with ECs triggered enhanced VWF secretion and increased Angpt-2 expression, as well as significantly enhanced in vitro EC tube formation and angiogenesis. Conclusion: We propose that acute SARS-CoV-2 infection leads to a complex and multifactorial EC activation, progressive loss of thrombomodulin, and increased Angpt-2 expression, which collectively serve to promote a local proangiogenic state.

Synovitis and joint health in patients with haemophilia: Statements from a European e-Delphi consensus study.

INTRODUCTION: Synovitis, a common feature in haemophilia, is triggered by the presence of blood in joints, and represents the first step towards the development of chronic arthropathy. Synovitis may be detected early by means of ultrasound or magnetic resonance imaging scan; clinical joint scores are less sensitive in this setting. Regular long-term prophylaxis with clotting factor concentrates, as primary prophylaxis and tailored to individual needs, has high efficacy in preventing synovitis. In general, higher factor levels lower bleeding risk, but no direct correlation between factor levels and synovitis incidence has been demonstrated. AIM: This study aimed to develop an expert consensus relating to the definition, pathophysiology, diagnosis, prevention, follow-up and treatment of synovitis, recognising its relevance for joint health and taking into account existing knowledge gaps. METHODS: A Delphi consensus study was designed and performed. An expert group prepared 22 statements based on existing literature; a wider expert panel subsequently voted on these. RESULTS: Retention of panellists was high. Four statements required amending and consensus on all statements was achieved after three rounds of voting. CONCLUSION: This e-Delphi consensus study addressed the importance of synovitis in joint health of people with haemophilia and highlighted knowledge gaps in this field. Studies on the natural course of synovitis are lacking and the biological mechanisms underlying this process are not yet fully elucidated. While basic and clinical research proceeds in this field, expert consensus can help guide clinicians in their routine clinical practice, and Delphi methodology is often used to produce best-practice guidelines.