Von Willebrand Factor Receptor Research Articles

Single-cell mass cytometry analysis unveils prothrombotic expression and heterogeneity in essential thrombocythemia

Abstract Background/Introduction Essential thrombocythemia (ET) is recognized for its hallmark elevated platelet count and a consequent increased risk of thromboembolic events including coronary events. Despite its clinical significance, the pathophysiological mechanisms predisposing ET patients to thrombosis remain largely undefined. Purpose The aim of this pioneering study was to shed light on the increased thrombotic risk in ET by conducting a detailed phenotypical characterization of platelet expression using single-cell mass cytometry, comparing ET patients to age- and sex-matched healthy controls. Methods Platelet samples from six patients diagnosed with ET and six healthy individuals were analyzed. This analysis utilized an extensive panel of 18 transmembrane regulators pivotal for platelet function and activation. Measurements were taken both at baseline and after ex-vivo stimulation with thrombin receptor-activating peptide (TRAP). Advanced clustering analysis via the FlowSOM algorithm helped identify specific subclusters of platelets with a prothrombotic expression profile. Results Our investigation revealed a significant overexpression of the activation marker CD62P (P-Selectin, p=0.049) and of the collagen receptor GPVI (p=0.044) in non-stimulated ET platelets (Figure 1A-C). Conversely, a notable decrease in expression was observed for the fibrinogen receptor GPIIb/IIIa units CD41 (p=0.036) and CD61 (p=0.044), as well as for the von Willebrand factor receptor CD42b (p=0.044). The FlowSOM analysis distinguished two prothrombotic subclusters of ET platelets, particularly highlighting one cluster, which was significantly overrepresented in ET (22.13% of total ET platelets versus 2.94% in controls, p=0.035, Figure 1D-E). Conclusion This study presents compelling evidence of a distinct prothrombotic phenotype in ET, marked by significant alterations in platelet expression profiles. The overexpression of CD62P and GPVI, alongside the underexpression of CD41, CD61, and CD42b, underscores the complex pathophysiological landscape of ET. Importantly, we identified a specific subcluster, particularly overrepresented in ET, with a specific prothrombic signature. Our findings, while only hypothesis generating, propose GPVI as a potential therapeutic target to mitigate thrombosis in ET patients, paving the way for targeted interventions in this high-risk population.

Platelet-Type von Willebrand Disease: Complex Pathophysiology and Insights on Novel Therapeutic and Diagnostic Strategies.

von Willebrand disease (VWD) is the most common well-studied genetic bleeding disorder worldwide. Much less is known about platelet-type VWD (PT-VWD), a rare platelet function defect, and a "nonidentical" twin bleeding phenotype to type 2B VWD (2B-VWD). Rather than a defect in the von Willebrand factor (VWF) gene, PT-VWD is caused by a platelet GP1BA mutation leading to a hyperaffinity of the glycoprotein Ibα (GPIbα) platelet surface receptor for VWF, and thus increased platelet clearing and high-molecular-weight VWF multimer elimination. Nine GP1BA gene mutations are known. It is historically believed that this enhanced binding was enabled by the β-switch region of GPIbα adopting an extended β-hairpin form. Recent evidence suggests the pathological conformation that destabilizes the compact triangular form of the R-loop-the GPIbα protein's region for VWF binding. PT-VWD is often misdiagnosed as 2B-VWD, even the though distinction between the two is crucial for proper treatment, as the former requires platelet transfusions, while the latter requires VWF/FVIII concentrate administration. Nevertheless, these PT-VWD treatments remain unsatisfactory, owing to their high cost, low availability, risk of alloimmunity, and the need to carefully balance platelet administration. Antibodies such as 6B4 remain undependable as an alternative therapy due to their questionable efficacy and high costs for this purpose. On the other hand, synthetic peptide therapeutics developed with In-Silico Protein Synthesizer to disrupt the association between GPIbα and VWF show preliminary promise as a therapy based on in vitro experiments. Such peptides could serve as an effective diagnostic technology for discriminating between 2B-VWD and PT-VWD, or potentially all forms of VWD, based on their high specificity. This field is rapidly growing and the current review sheds light on the complex pathology and some novel potential therapeutic and diagnostic strategies.

P151 Characterization of platelet activation profile in Inflammatory Bowel Disease

Abstract Background Platelet-related markers associated with a procoagulant state have been described for Immune-mediated Inflammatory Diseases (IMIDs), but a broader profiling of these abnormalities in Inflammatory Bowel Disease (IBD) is lacking. Methods Cross-sectional study to describe and compare platelet biomarkers of IBD outpatients, both Crohn’s Disease (CD) and Ulcerative Colitis (UC), vs healthy controls (HC). Patients over 18 years old with a diagnosis of CD or UC according to the European Crohn’s and Colitis Organization (ECCO) criteria were included. Fecal calproctectin (Fcal) >=150ug/g was used as a cutoff level for active disease. Exclusion criteria for IBD patients are listed under Table 1. Fibrinogen (FBG) receptor on platelet’s surface was determined with anti-CD41 and anti-CD61 monoclonal antibodies (mAbs); and von Willebrand factor receptor with anti-CD42a and anti-CD42b mAbs. Platelet activation markers [TRAP and ADP-induced activation of fibrinogen (FBG) receptor, and P-selectin (Psel) and CD63 exposure] were determined, respectively, through binding of PAC1, anti-P-selectin and anti-CD63 (mAbs). All variables were determined by flow cytometry. Results 55 patients (27 CD, 28 UC) and 75 HC have been included. 43 IBD patients had active disease (21 CD, 22 UC). Complete baseline characteristics of the IBD population are described in Table 1. Basal Psel (% of positive cells) was increased in CD vs UC (median [IQR]: 5.64 [4,4-10,4] vs 2,9 [1,8-6,8], p=0,02) and vs HC (3,5 [1,4-6,8], p=0,008). ADP-induced Psel was increased in CD vs HC (mean, SD: 49.92, SD 12.75 vs 41.943, SD13.15, p=0,007). Basal CD63 (% of positive cells) was significantly higher in active IBD vs HC (W = 1143, p = 0.02) and inactive IBD vs HC (W = 528, p= 0.006). CD42a (% of positive cells) was decreased in IBD vs HC (W = 1128.00, p=0.007) and CD vs HC (W = 777.00, p =0.04). CD42b (% of positive cells) was also decreased in IBD vs HC (mean, SD: 106.9 SD, 17.32 vs 115.7, SD 28.42, p=0,01) and CD vs HC (104.06 SD 14.49, p=0,03). No differences in basal or TRAP/ADP induced PAC binding, and in CD41 or CD61 expression were observed among groups. Multiple linear regression showed association of Fcal with basal Psel in IBD patients (p=0,02); and Spearman’s correlation showed negative moderate correlation between Fcal and basal Psel (r=0,3; p=0,003) Conclusion Features of platelets in IBD suggest increased platelet degranulation and reduced vWF receptor with regards to healthy controls, specially in CD, but no differences in FBG-related receptors. Inverse moderate correlation between Fcal and Psel in IBD patients suggests depletion of intra-platelet granules at higher levels of gut inflammation.

Deep proteomic analysis of obstetric antiphospholipid syndrome by DIA-MS of extracellular vesicle enriched fractions

Proteins in the plasma/serum mirror an individual’s physiology. Circulating extracellular vesicles (EVs) proteins constitute a large portion of the plasma/serum proteome. Thus, deep and unbiased proteomic analysis of circulating plasma/serum extracellular vesicles holds promise for discovering disease biomarkers as well as revealing disease mechanisms. We established a workflow for simple, deep, and reproducible proteome analysis of both serum large and small EVs enriched fractions by ultracentrifugation plus 4D-data-independent acquisition mass spectrometry (4D-DIA-MS). In our cohort study of obstetric antiphospholipid syndrome (OAPS), 4270 and 3328 proteins were identified from large and small EVs enriched fractions respectively. Both of them revealed known or new pathways related to OAPS. Increased levels of von Willebrand factor (VWF) and insulin receptor (INSR) were identified as candidate biomarkers, which shed light on hypercoagulability and abnormal insulin signaling in disease progression. Our workflow will significantly promote our understanding of plasma/serum-based disease mechanisms and generate new biomarkers.

PACSIN2 regulates platelet integrin β1 hemostatic function

BackgroundUpon vessel injury, platelets adhere to exposed matrix constituents via specific membrane receptors, including the von Willebrand factor receptor glycoprotein (GP)Ib-IX-V complex and integrins β1 and β3. In platelets, the Fes/CIP4-homology Bin-Amphiphysin-Rvs protein PACSIN2 associates with the cytoskeletal and scaffolding protein filamin A (FlnA), linking GPIbα and integrins to the cytoskeleton. ObjectivesHere we investigated the role of PACSIN2 in platelet function. MethodsPlatelet parameters were evaluated in mice lacking PACSIN2 and platelet integrin β1. ResultsPacsin2–/– mice displayed mild thrombocytopenia, prolonged bleeding time, and delayed thrombus formation in a ferric chloride-mediated carotid artery injury model, which was normalized by injection of control platelets. Pacsin2–/– platelets formed unstable thrombi that embolized abruptly in a laser-induced cremaster muscle injury model. Pacsin2–/– platelets had hyperactive integrin β1, as evidenced by increased spreading onto surfaces coated with the collagen receptor α2β1-specific peptide GFOGER and increased binding of the antibody 9EG7 directed against active integrin β1. By contrast, Pacsin2–/– platelets had normal integrin αIIbβ3 function and expressed P-selectin normally following stimulation through the collagen receptor GPVI or with thrombin. Deletion of platelet integrin β1 in Pacsin2–/– mice normalized platelet count, hemostasis, and thrombus formation. A PACSIN2 peptide mimicking the FlnA-binding site mediated the pull-down of a FlnA rod 2 construct by integrin β7, a model for integrin β-subunits. ConclusionsPacsin2–/– mice displayed severe thrombus formation defects due to hyperactive platelet integrin β1. The data suggest that PACSIN2 binding to FlnA negatively regulates platelet integrin β1 hemostatic function.

Lentiviral gene therapy reverts GPIX expression and phenotype in Bernard-Soulier syndrome type C

Bernard-Soulier syndrome (BSS) is a rare congenital disease characterized by macrothrombocytopenia and frequent bleeding. It is caused by pathogenic variants in three genes (GP1BA, GP1BB, or GP9) that encode for the GPIbα, GPIbβ, and GPIX subunits of the GPIb-V-IX complex, the main platelet surface receptor for von Willebrand factor, being essential for platelet adhesion and aggregation. According to the affected gene, we distinguish BSS type A1 (GP1BA), type B (GP1BB), or type C (GP9). Pathogenic variants in these genes cause absent, incomplete, or dysfunctional GPIb-V-IX receptor and, consequently, a hemorrhagic phenotype. Using gene-editing tools, we generated knockout (KO) human cellular models that helped us to better understand GPIb-V-IX complex assembly. Furthermore, we developed novel lentiviral vectors capable of correcting GPIX expression, localization, and functionality in human GP9-KO megakaryoblastic cell lines. Generated GP9-KO induced pluripotent stem cells produced platelets that recapitulated the BSS phenotype: absence of GPIX on the membrane surface and large size. Importantly, gene therapy tools reverted both characteristics. Finally, hematopoietic stem cells from two unrelated BSS type C patients were transduced with the gene therapy vectors and differentiated to produce GPIX-expressing megakaryocytes and platelets with a reduced size. These results demonstrate the potential of lentiviral-based gene therapy to rescue BSS type C.

Abstract P198: Associations Between Plasma Proteome and Leukocyte Mitochondrial DNA Copy Number: The Atherosclerosis Risk in Communities Study (ARIC)

Introduction: The number of copies of the mitochondrial genome, termed mitochondrial DNA copy number (MTCN), is a proxy for mitochondrial function and varies by cell. Critically, lower MTCN has been associated with aging and chronic diseases such as dementia and cancer. We conducted a large-scale proteomics analysis of leukocyte MTCN (L-MTCN) to elucidate pathways modulated by L-MTCN that may be involved in diseases of aging. Hypothesis: We hypothesize that L-MTCN is significantly associated to plasma proteins that have been implicated in biological pathways of L-MTCN and related diseases. Methods: Based on whole genome sequencing (WGS) data measured in samples collected in ARIC visits 1, 2, and 3 (i.e., baseline for the current study), L-MTCN was calculated as (2 * coverage of the mitochondrial genome) / coverage of the autosome, using the median coverage of 1000 base pair bins overlapping the mitochondrial genome (16 total) and the median coverage of ~142,000 higher quality autosomal bins. Only samples with read lengths of 150 or 151 were included in subsequent analyses, and L-MTCN estimates were inverse normalized within read length group and WGS group before being merged together. This normalized L-MTCN was then analyzed as a predictor for an association with 4,870 plasma proteins measured by SOMAscan v4 in ARIC visit 3 in race-specific generalized linear models, adjusting for the following covariates at baseline: WGS sample visit, age, gender, field center, smoking status, and a measure of kidney function. Bonferroni correction was applied to account for the number of proteins tested in 5,007 White participants (p≤1x10 -5 ). Associations were tested for replication in 1,178 Black participants and considered significant if protein estimates had the same direction of association as Whites at p-value ≤ 2.2x10 -4 . Results: Among White participants, 230 proteins were significantly associated with L-MTCN. Of these proteins S100A9, GP1Bα, EGFR, THBS2, and FCGR3B were significantly replicated among Black participants. Strongest associations with L-MTCN were seen with GP1Bα (Estimate: 0.121) and S100A9 (Estimate: -0.191). Biologically S100A9 is a calgranulin and together with S100A8 forms calprotectin, which have roles mediating inflammatory processes. GP1Bα helps mediate platelet activation and forms the glycoprotein Ib-IX-V complex which acts as a platelet surface receptor for von Willebrand factor facilitating platelet adhesion. Conclusion: This large proteomics analysis identified 230 plasma proteins significantly associated with L-MTCN. Some of the proteins have been connected to inflammatory diseases, cardiovascular disease, and cancer in the literature. These results provide insight into proteins involved in biological pathways related to L-MTCN.

ДИНАМИКА ФУНКЦИОНАЛЬНОЙ АКТИВНОСТИ ТРОМБОЦИТОВ У НЕДОНОШЕННЫХ НОВОРОЖДЕННЫХ ДЕТЕЙ С ИНФЕКЦИОННО-ВОСПАЛИТЕЛЬНЫМИ ЗАБОЛЕВАНИЯМИ В РАННЕМ НЕОНАТАЛЬНОМ ПЕРИОДЕ

Platelet involvement in inflammatory and immune responses and its functional activity in various pathological conditions are actively studied. Despite there are scientific publications characterizing platelets of newborns vs. adults the data on the features of the expression of activation markers on platelets of premature infants with a complicated course of the early neonatal period are not enough. The purpose of the research was to analyze the expression of glycoproteins and activation molecules on the surface of peripheral blood platelets in newborns with congenital infection depending on gestational age (GA). Materials and methods of research: a prospective single-center case-control study was conducted in the National Medical Research Center for Obstetrics, Gynecology and Perinatology named after V.I. Kulakov (Moscow, Russia) in March 2018 - September 2019. The expression of activation markers on resting and activated platelets on the 1st and 3rd days of life (DOL) in 158 newborns aged 24 to 38 weeks was analyzed using the flow cytometry. The patients were divided into 3 groups: group 1 (n=20) of newborns with GA 24 to 28 weeks; group 2 (n=52) of newborns with GA of 29 to 33 weeks; and group 3 (n=86) of newborns with GA of 34 weeks and older. Results: in severely preterm (G1) compared with infants of older GA on the 3rd DOL platelets had higher MPV: 11.3 (10.4-12.3) vs. 10.1 (9.6-10.3), p=0.011, and PDW: 13.1 (12.2-15.6) vs. 11.3 (10.6-12.3), p<0.001. In infectious diseases in children with GA of 29 to 33 weeks there is a lower expression of phosphatidylserine on activated platelets in the 1st DOL: 8.8 (7.5-11.1) vs. 11.2 (10.3-12.1), p=0.046, and lower the content of activated platelets expressing P-selectin on the 3rd DOL: 89.5 (87.1-92.7) vs. 93.1 (90.4-94.4), p=0.048. In newborns with GA of 34 weeks and older with congenital infection in the 1st DOL there was a higher expression of von Willebrand Factor receptor revealed: 13.9 (12.3-16.4) vs. 12.4 (10.9-14.1), p=0.028, and a higher content of dense granules in activated platelets: 0.73 (0.65-0.79) vs. 0.67 (0.60-0.71), p=0.008. By the 3rd DOL there was an increase in MPV and PDW in all groups of children (p<0.05) recorded. Conclusion: in premature infants with congenital infection the expression of structural and activation molecules on platelets undergoes greater changes during the first three days of life than in children of similar GA without infectious pathology.

Biomechanical activation of blood platelets via adhesion to von Willebrand factor studied with mesoscopic simulations.

Platelet adhesion and activation are essential initial processes of arterial and microvascular hemostasis, where high hydrodynamic forces from the bloodflow impede coagulation. The process relies on von Willebrand factor (VWF)-a linear multimeric protein of blood plasma plays a pivotal role in mechanochemical regulation of shear-induced platelet aggregation (SIPA). Adhesive interactions between VWF and glycoprotein receptors GPIb are crucial for platelet recruitment under high shear stress in fluid. Recent advances in experimental studies revealed that mechanical tension on the extracellular part of GPIb may trigger a cascade of biochemical reactions in platelets leading to activation of integrins [Formula: see text] (also known as GPIIb/IIIa) and strengthening of the adhesion. The present paper is aimed at investigation of this process by three-dimensional computer simulations of platelet adhesion to surface-grafted VWF multimers in pressure-driven flow of platelet-rich plasma. The simulations demonstrate that GPIb-mediated mechanotransduction is a feasible way of platelet activation and stabilization of platelet aggregates under high shear stress. Quantitative understanding of mechanochemical processes involved in SIPA would potentially promote the discovery of new anti-platelet medication and the development of multiscale numerical models of platelet thrombosis and hemostasis.

Histone-stimulated platelet adhesion to mouse cremaster venules in vivo is dependent on von Willebrand factor.

Extracellular histones are known mediators of platelet activation, inflammation, and thrombosis. Von Willebrand Factor (vWF) and Toll-like receptor 4 (TLR4) have been implicated in pro-inflammatory and prothrombotic histone responses. The objective of this study was to assess the role of vWF and TLR4 on histone-induced platelet adhesion in vivo. Intravital microscopy of the mouse cremaster microcirculation, in the presence of extracellular histones or saline control, was conducted in wild-type, vWF-deficient, and TLR4-deficient mice to assess histone-mediated platelet adhesion. Platelet counts following extracellular histone exposure were conducted. Platelets were isolated from vWF-deficient mice and littermates to assess the role of vWF on histone-induced platelet aggregation. Histones promoted platelet adhesion to cremaster venules in vivo in wild-type animals, as well as in TLR4-deficient mice to a comparable degree. Histones did not lead to increased platelet adhesion in vWF-deficient mice, in contrast to littermate controls. In all genotypes, histones resulted in thrombocytopenia. Histone-induced platelet aggregation ex vivo was similar in vWF-deficient mice and littermate controls. Histone-induced platelet adhesion to microvessels in vivo is vWF-dependent and TLR4-independent. Platelet-derived vWF was not necessary for histone-induced platelet aggregation ex vivo. These data are consistent with the notion that endothelial vWF, rather than platelet vWF, mediates histone-induced platelet adhesion in vivo.

Serum levels of laminin and von Willebrand factor in COVID-19 survivors 6 months after discharge

ObjectivesThe aim of this study was to evaluate the clinical characteristics, pulmonary diffusion function, chest computed tomography (CT), and serum lung cell damage indicators of coronavirus disease 2019 (COVID-19) survivors 6 months after discharge. MethodsData of COVID-19 survivors discharged from hospital between January 21, 2020 and January 11, 2021 and healthy controls were collected. Serum levels of surfactant protein D (SP-D), the receptor for advanced glycation end products (RAGE), laminin, and von Willebrand factor (vWF) were measured in the healthy controls and COVID-19 survivors 6 months after discharge. The relationships between serum lung cell damage indicator levels and various parameters were explored. ResultsFifty-two COVID-19 survivors (31 with non-severe disease and 21 with severe disease) and 30 controls were included. Serum levels of laminin in COVID-19 survivors 6 months after discharge were significantly higher than those in the controls. The increase was more significant in elderly and female patients. Serum levels of RAGE and vWF were not statistically different from those of the controls. However, 6 months after discharge, COVID-19 survivors with abnormal chest CT and those in the severe group had higher vWF levels. ConclusionsCOVID-19 patients had abnormal lung injury indicators 6 months after discharge. The recovery time after infection is currently unknown, and long-term observation is required.

S2907 Portal Vein Thrombosis in the COVID-19 Era: A Rare Presentation

Introduction: An increased incidence of venous thromboembolism (VTE) has been reported with SARS-COV2 (severe acute respiratory syndrome coronavirus 2) infections. VTE in unusual sites like the portal vein is limited to case reports. This report adds to the growing body of literature about thrombotic complications with SARS-COV2 infections. Case Description/Methods: A female aged 39 with a medical history of diabetes, hypertension and cholecystectomy presented with three days of right upper quadrant (RUQ) pain. She denied substance use, cirrhosis, autoimmune disease or previous VTE. On presentation, vitals were stable. She had RUQ tenderness. Blood work revealed elevated alkaline phosphatase and transaminases. White cell count, bilirubin and coagulation were normal. Imaging revealed occlusive thrombus of the right portal vein proximal to the bifurcation with peripheral extension (images 1 and 2). Extensive hypercoagulable and infectious workup was negative. She was immediately started on enoxaparin. A chest x ray was suggestive of SARS-COV2 infection and confirmatory testing was positive. Her symptoms improved and lab abnormalities resolved. She was discharged home with oral anticoagulants and instructions to self-isolate. Discussion: Most reported cases of Portal vein thrombosis (PVT) with SARS-COV2 are incidental. Our patient presented with RUQ abdominal pain, highlighting an unusual presentation. PVT is rare and occurs with cirrhosis, connective tissue disease, malignancies, pancreatitis and thrombophilia. These were absent in our patient and other causes of hypercoagulability such as antiphospholipid syndrome, proteins C, S and antithrombin deficiencies, Factor V mutations were ruled out. SARS-COV2 infected patients experience a hypercoagulable state with the incidence of thrombotic complications as high as 31%. Some have suggested that systemic inflammation leads to endothelial dysfunction, causing Von-Willebrand Factor and toll-like receptor activation, leading to platelet aggregation and coagulation cascade initiation. Others have speculated that inflammatory cytokines bind to endothelial surface angiotensin converting enzyme receptor leading to lymphocytic endotheliitis and prothrombotic gene overexpression. Anticoagulation should be used in PVT to reduce complications of intestinal infarction and portal hypertension. Recanalization occurs in 60% of cases with anticoagulation in the first week. We used therapeutic enoxaparin and switched to an oral anticoagulant for a 6-month course.Figure 1.: Image 1 a and b: CT scan showing right portal vein thrombosis, just at its bifurcation (blue arrows) c: chest x ray showing bilateral patchy opacities.

Candida albicans elicits protective allergic responses via platelet mediated T helper 2 and T helper 17 cell polarization

Fungal airway infection (airway mycosis) is an important cause of allergic airway diseases such as asthma, but the mechanisms by which fungi trigger asthmatic reactions are poorly understood. Here, we leverage wild-type and mutant Candida albicans to determine how this common fungus elicits characteristic Th2 and Th17 cell-dependent allergic airway disease in mice. We demonstrate that rather than proteinases that are essential virulence factors for molds, C.albicans instead promoted allergic airway disease through the peptide toxin candidalysin. Candidalysin activated platelets through the Von Willebrand factor (VWF) receptor GP1bα to release the Wnt antagonist Dickkopf-1 (Dkk-1) to drive Th2 and Th17 cell responses that correlated with reduced lung fungal burdens. Platelets simultaneously precluded lethal pulmonary hemorrhage resulting from fungal lung invasion. Thus, in addition to hemostasis, platelets promoted protection against C.albicans airway mycosis through an antifungal pathway involving candidalysin, GP1bα, and Dkk-1 that promotes Th2 and Th17 responses.

Comparison of Different Phenotypes of Bernard-Soulier Syndrome

Introduction. Bernard-Soulier syndrome (BSS) is one of the rare inherited platelet disorder. It is caused by deficiency or defect of the glycoprotein (GP) Ib-IX-V complex on the surface of platelets - the main receptor for von Willebrand factor (vWF). Dissociation of hemostasis in BSS leads to microcirculatory and mixed types of bleeding episodes - mucocutaneous bleedings, spontaneous and posttraumatic hematomas and severe hemorrhagic episodes during surgery.Materials and methods. We diagnosed 2 Caucasian women of age 32 and 29 years (patient 1 and patient 2 with different phenotypes of BSS) using total blood count, clotting time tests, fibrinogen, platelet aggregation with ristomicin, ADP, collagen, arachnid acid, adrenaline, platelet morphology analysis and flow cytometry assay. Flow cytometry assay was performed on isolated from 3 mL of citrated blood platelets. Platelets were loaded with mepacrine (1 mM) and then stimulated with thrombin at 88 nM. Samples from 10 healthy donors were used as controls. We evaluated CD61, PAC1, CD42b, mepacrine, CD62p, annexin V on active and non-active platelets.Results. Both patients had similar results in standard coagulation test: normal or shortened APTT (26-29 sec), normal fibrinogen (1.8-2.5 g/l), increased activity of vWF (209-275%) and FVIII (140-287%). Data of light aggregometry was classic for BSS: decrease of platelet aggregation with ristomicin and normal with other parameters. The peripheral blood smear showed large platelets and absence of neutrophil inclusions.Despite similarity of primary laboratory analysis patient 1 and patient 2 have very different phenotypes of BSS. Patient 1 had first clinical manifestation at age of 2 - large posttraumatic hematomas and then was treated from ITP without any results. At age of 13 menorrhagia had started and was slightly managed by prescribing hormonal therapy. At 16 yrs she had hemorrhage into the abdominal cavity. A therapeutic laparotomy was performed, complicated by the development of bleeding in the early postoperative period. At age of 31, the patient was hospitalized with a diagnosis of acute cerebrovascular accidents of hemorrhagic type with the formation of subdural hematoma with a repeated spontaneous brain hemorrhage in 1 month.Patient 2 was diagnosed with BSS at age of 6 yrs. Clinical pictures is characterized by menorrhagia from age of 14 yrs, that normalized at age of 20. Although she was hospitalized for many times due to severe anemia (Hb 40-50 g/l) of unknown reason. At age of 29 patient 2 was diagnosed with angiectasia of jejunum that appeared to be hidden source of blood loss. After treating it with eptacog alfa (activated) and proscribing gastrointestinal therapy anemia syndrome was managed.We compared the results of flow cytometry of patient 1 and patient 2. СD42b was 0% in both patients. Patient 1 also had a deficiency of dense granules of platelets and elevated levels of CD 61 and CD62. Patient 2 had no other platelet deficiencies and highly elevated parameters of CD61, PAC1, mepacrine, CD62p (up to 3 times).Conclusions. The compensatory high activation of other components of platelets in patient 2 can be evaluated as a result of a mild phenotype. Combined defect of BSS and deficiency of dense granules also can be a result of severe hemorrhage in patient 1. It is important to include flow cytometry of platelets in standard algorithm of diagnostic to evaluate more correctly the phenotype of platelet disorders. DisclosuresNo relevant conflicts of interest to declare.

A Factor VIII Fusion with an Inactivated FIX Generates a Biologically Active Molecule with Delayed Clearance in a FVIII/VWF Knockout Mouse Model

Background: Developing a delayed clearance factor VIII for the treatment of haemophilia A is of importance due to the cost and inconvenience associated with current FVIII products which require several intravenous injections each week to maintain therapeutic FVIII levels. While there are a number of extended half-life rFVIII products in development, they only have a ~1.8-fold extension of half-life due to the dominant influence of von Willebrand factor (VWF).Objective: To generate a long-acting rFVIII through fusion to inactive FIX allowing intermolecular binding of FVIII and FIX as well as a dynamic equilibrium with FVIII being free to bind endogenous FIXa. We hypothesized that the fusion to inactive FIX will protect FVIII from proteolytic inactivation, create steric hindrance for binding to VWF and FVIII clearance receptors and thereby delay the in vivo clearance of FVIII.Methods: We cloned a novel rFVIII fusion molecule linked through its C-terminus to an inactivated factor IX designated 13A1FVIII. 13A1FVIII was expressed in mammalian cells (CHO) and protein expression confirmed by western blotting. Biological activity was tested using a chromogenic assay and thrombin generation test and clearance examined in VWF/FVIII double knock out mice.Results: 13A1FVIII protein purification was achieved by affinity based chromatography with >95% purity as judged by silver staining. Purified 13A1FVIII bioactivity was confirmed in vitro using a chromogenic FVIII assay and an activated partial thromboplastin time (aPTT) test. In a PK study using VWF/FVIII double knock out mice, 13A1FVIII exhibited a reduced plasma clearance over Advate rFVIII with terminal half-life of 32hrs vs 12hrs, respectively. The observed 2.7-fold increase in half-life was achieved independent of VWF.Conclusions: A fusion of FVIII to inactive FIX retains biological activity in two in vitro tests of haemostasis. The fusion protein also demonstrates delayed plasma clearance and an increase in circulating half-life in a haemophiliac mouse model. The results suggest it is possible to delay FVIII clearance independent of VWF using this fusion protein approach. DisclosuresNo relevant conflicts of interest to declare.

Exome Sequencing Studies Identify Mutations in STAB2 As a Genetic Risk for Venous Thromboembolic Disease

Deep vein thrombosis and pulmonary embolism, collectively referred to as venous thromboembolism (VTE), are the third leading cause of cardiovascular death in the United States. Genetic factors account for 50-60% of VTE risk and a recent meta-analysis of genome-wide association studies confirmed that common variants in F5, ABO, F2 and seven other very low effect size loci are associated with VTE. Rare mutations in the anticoagulant genes PROC (protein C), PROS1 (protein S) and SERPINC1 (antithrombin III) have been linked to highly penetrant VTE risk in family studies but no genome-wide study of rare variants has been reported to date.We performed whole exome sequencing (WES) in 373 unrelated individuals of European ancestry with well characterized unprovoked VTE and compared results to a previously genome sequenced control cohort of 5784 unrelated Europeans. We then identified single nucleotide variants, small insertion or deletions that were present at allele frequencies less than 0.005% in ExAC and had equal sequencing coverage between groups. Using a dominant model and a collapsing association analysis, we looked for genes that were more likely to harbor “knockout” (frame shift, nonsense, splice site and damaging missense) mutations in cases versus controls. Strikingly, ranked by p-value, among 17,395 genes tested, the top 4 genes were PROS1 (P= 2.01E-09, odds ratio: 11.8), STAB2 (P=2.70E-7, odds ratio 3.37), PROC (P=3.24E-05, odds ratio 11.0) and SERPINC1 (P=1.10E-04, odds ratio 8.5), Figure. These findings suggest that heterozygous deficiency for the STAB2 encoded protein, stabilin-2, confers VTE susceptibility similar to the previously well described phenotypes of heterozygous protein C, protein S and antithrombin III deficiency.Stabilin-2 is a single transmembrane glycoprotein scavenger receptor expressed on venous endothelium of liver sinusoids and spleen. We detected damaging STAB2 mutations in 29 (7%) VTE cases versus 141 (2%) of controls. Common and uncommon variants in STAB2 have been associated with elevated plasma von Willebrand factor (VWF) and coagulation factor VIII levels, which are known risk factors for VTE. Stabilin-2 is implicated as a clearance receptor for VWF, so that haploinsufficiency of stabilin-2 may increase plasma VWF half-life. To test this hypothesis, we examined 1162 individuals in the Twins UK Study (VTE status unknown) with available whole genome sequencing data and measured their plasma VWF levels by AlphaLISA. VWF levels were adjusted by log transformation and ABO blood type tagging SNPs. We identified 38 individuals (3.2%, all heterozygous) with rare damaging variants in STAB2. VWF levels were significantly higher in individuals (N=30) with rare STAB2 variants compared to those without (N=1016) (Mann-Whitney, P= 0.0046).To further examine stabilin-2 function in vivo, we compared Stab2 deficient mice (Stab2 -/-) to wild type (Stab2 +/+) controls. Stab2 -/- mice had elevated plasma levels of factor VIII:C (118%, p=0.0006), but no differences in VWF (p=0.58). Next, we examined thrombosis in a mouse model where venous blood clotting was induced by inferior vena cava stenosis. Thrombi were weighed and longitudinal sections were analyzed by quantitative immunohistochemistry 24 hours post-stenosis. Stab2 -/- mice developed significantly larger thrombi than Stab2 +/+ mice (194%, n=19, p=0.003) but no difference in DVT incidence (64% vs 61%, P= 0.85). Despite equivalent hematocrit (P= 0.91) and white type thrombus (P> 0.05), thrombi from Stab2 -/- mice were comprised of more red type thrombus (185%, P= 0.004).Through an unbiased analysis of whole exome sequencing in 6,157 individuals, this studyconfirms the role of haploinsufficiency of PROC, PROS1 and SERPINC1 for VTE susceptibility and identifies STAB2 deficiency as a potential new risk factor. Analysis of an independent human cohort of 1162 Europeans demonstrates that STAB2 haploinsufficiency is associated with elevated VWF levels, a known risk factor for VTE. Finally, a mouse model of Stab2 deficiency demonstrates enhanced thrombosis, though without an association with elevated VWF levels. Taken together, these results suggest that rare genetic variants in STAB2 collectively play a role in individuals with unprovoked VTE by prolonging the plasma half-life of procoagulant factors such as VWF, adding an important new gene to the short list of known risk factors for this common disease. [Display omitted] DisclosuresReitsma:Leiden University Medical Center; VarmX B.V.: Equity Ownership, Patents & Royalties: co-inventor on a patent application related to this abstract.

Identification of the Flavobacterium johnsoniae cysteate-fatty acyl transferase required for capnine synthesis and for efficient gliding motility.

Sulfonolipids (SLs) are bacterial lipids that are structurally related to sphingolipids. Synthesis of this group of lipids seems to be mainly restricted to Flavobacterium, Cytophaga and other members of the phylum Bacteroidetes. These lipids have a wide range of biological activities: they can induce multicellularity in choanoflagellates, act as von Willebrand factor receptor antagonists, inhibit DNA polymerase, or function as tumour suppressing agents. In Flavobacterium johnsoniae, their presence seems to be required for efficient gliding motility. Until now, no genes/enzymes involved in SL synthesis have been identified, which has been limiting for the study of some of the biological effects these lipids have. Here, we describe the identification of the cysteate-fatty acyl transferase Fjoh_2419 required for synthesis of the SL precursor capnine in F. johnsoniae. This enzyme belongs to the α-oxoamine synthase family similar to serine palmitoyl transferases, 2-amino-3-oxobutyrate coenzyme A ligase and 8-amino-7-oxononanoate synthases. Expression of the gene fjoh_2419 in Escherichia coli caused the formation of a capnine-derived molecule. Flavobacterium johnsoniae mutants deficient in fjoh_2419 lacked SLs and were more sensitive to many antibiotics. Mutant growth was not affected in liquid medium but the cells exhibited defects in gliding motility.

Impaired adhesion of neutrophils expressing Slc44a2/HNA-3b to VWF protects against NETosis under venous shear rates

AbstractGenome-wide association studies linked expression of the human neutrophil antigen 3b (HNA-3b) epitope on the Slc44a2 protein with a 30% decreased risk of venous thrombosis (VT) in humans. Slc44a2 is a ubiquitous transmembrane protein identified as a receptor for von Willebrand factor (VWF). To explain the link between Slc44a2 and VT, we wanted to determine how Slc44a2 expressing either HNA-3a or HNA-3b on neutrophils could modulate their adhesion and activation on VWF under flow. Transfected HEK293T cells or neutrophils homozygous for the HNA-3a– or HNA-3b–coding allele were purified from healthy donors and perfused in flow chambers coated with VWF at venous shear rates (100 s−1). HNA-3a expression was required for Slc44a2-mediated neutrophil adhesion to VWF at 100 s−1. This adhesion could occur independently of β2 integrin and was enhanced when neutrophils were preactivated with lipopolysaccharide. Moreover, specific shear conditions with high neutrophil concentration could act as a “second hit,” inducing the formation of neutrophil extracellular traps. Neutrophil mobilization was also measured by intravital microscopy in venules from SLC44A2-knockout and wild-type mice after histamine-induced endothelial degranulation. Mice lacking Slc44a2 showed a massive reduction in neutrophil recruitment in inflamed mesenteric venules. Our results show that Slc44a2/HNA-3a is important for the adhesion and activation of neutrophils in veins under inflammation and when submitted to specific shears. The fact that neutrophils expressing Slc44a2/HNA-3b have a different response on VWF in the conditions tested could thus explain the association between HNA-3b and a reduced risk for VT in humans.

Pretreatment with melatonin improves ovarian tissue cryopreservation for transplantation

BackgroudMelatonin has anti-inflammatory and antioxidative actions at the mitochondrial level. This indole-containing molecule may protect ovarian grafts during the process of cryopreservation. Therefore, we aimed to determine whether melatonin pretreatment improves rat ovarian graft quality.MethodsTwenty-six female rats were allocated to two study groups of thirteen animals each: 1) control group: ovaries cryopreserved using the standard protocol; and 2) melatonin group: ovaries cryopreserved in a medium with melatonin. Ten rats of each group were submitted to 24-h freezing, and whole ovaries autologous and avascular transplantation with retroperitoneal placement. After postoperative (PO) day 15, daily vaginal smears were obtained for estrous cycle characterization. Between PO days 30 and 35, the animals were euthanized and ovarian grafts were recovered for histological and immunohistochemical (Ki-67, cleaved caspase-3, TUNEL, von Willebrand factor, estrogen, and progesterone receptors) analyses. The ovaries of the three remaining rats from each group were studied immediately after thawing to assess the effects of cryopreservation. ANOVA and Tukey’s tests were used and the rejection level of the null hypothesis was set at 0.05 or 5% (p < 0.05).ResultsMelatonin promoted faster restart of the estrous cycle and increased the expression of mature follicles, collagen type I, von Willebrand factor, Ki-67, and cleaved caspase-3 on corpora lutea and estrogen receptors in the ovaries as compared to control. There was a reduction in apoptosis by TUNEL on follicles, corpora lutea, and collagen type III.ConclusionBased on the evaluated parameters, melatonin may promote the quality of ovarian grafts. Reproductive function enhancement should be further studied.

Comprehensive Description of Monoallelic GP1BA Variants Associated with Thrombocytopenia

GP1BA encodes the glycoprotein (GP) 1bα subunit of the GP1b/IX/V complex, the platelet receptor for von Willebrand factor (VWF) and a mediator of outside-in signalling that contributes to platelet activation. Biallelic variants in GPIBA that prevent surface expression of GPIB/IX/V cause the recessive disorder Bernard Soulier Syndrome (BSS; OMIM #23100) in which there is often severe bleeding caused by thrombocytopenia and platelet dysfunction. Thrombocytopenia has also been associated with monoallelic missense variants in the GP1bα R loop (residues 227-242) which enhance VWF binding (platelet-type von Willebrand disease (pVWD); OMIM #177820). Other monoallelic GPIBA missense variants such as the Bolzano variant (p.Ala172Val; OMIM #153670) have been associated with dominant thrombocytopenia without altered VWF levels. In order to better describe the relationship between monoallelic GPIBA variants and thrombocytopenia we evaluated data from the NIHR BioResource for Rare Diseases, (NBR-RD) and the Inherited Platelet Disorders programmes at the Universities of Birmingham (UK), Murcia (Spain) and Toronto (Canada) in which patients with unexplained platelet disorders underwent diagnostic next generation sequencing. Using the BeviMed method for genetic association we compared the genotypes at rare nonsynoymous variants of 105 unrelated thrombocytopenia cases (platelet count (PLT) &amp;lt;130x109/L or the human phenotype ontology term 'HP 001873 thrombocytopenia') in the NBR-BR with those of 10,152 unrelated participants without thrombocytopenia. There was a strong statistical association (posterior probability 0.97) between dominantly inherited GPIBA variants and thrombocytopenia. Considering the five variants within the NBR-BR that drove this association alongside genotype data from the other case collections, we identified a total of 21 different monoallelic GPIBA coding variants that were rare (gnomAD allele frequency &amp;lt;1/1000) and had a CADD pathogenicity score &amp;gt;15. These were present in 29 index cases (18 females, median age 37 years) with unexplained thrombocytopenia and included the variant predicting the p.Asn150Ser substitution which was identified in seven independent index cases. The variants comprised 18 missense, two inframe insertions (one with an additional missense change) and one insertion resulting in a frame shift. Seven had been previously associated with thrombocytopenia. Sixteen of the variants predicted amino acid substitutions or single residue insertions within the GP1bα leucine rich repeat region (LRR; residues 48-200) or in adjacent regions of the LRR N- and C-terminal caps. Cases with variants in the LRR region typically displayed mild mucocutaneous bleeding, abnormal bleeding after minor trauma or surgery and heavy menstrual bleeding. The platelet count was typically 60-100 x109/L and mean platelet volume greater than 11 fL. There were two missense variants in the GP1bα R loop that have been previously associated with pVWD and two further previously unreported missense variants in the cytoplasmic domain. The final variant was a monoallelic single nucleotide insertion predicted to cause a frame shift and absent expression of GP1bα and has been previously reported as a biallelic variant associated with BSS. These data indicate that in a multicentre cross-sectional cohort of cases with otherwise unexplained thrombocytopenia, monoallelic missense GPIBA variants affecting the LRR or adjacent regions were more prevalent than R loop variants associated with pVWD or variants causing absent GP1bα expression associated with biallelic BSS. The LRR region contains the GP1bα-VWF binding site which is predicted to be disrupted by the missense variants observed in this study. The LRR region is also the location of the GP1bα Bolzano variant and six other monoallelic variants previously associated with thrombocytopenia in previous single case reports. These observations extend the repertoire of GPIBA variants associated with thrombocytopenia and although requiring experimental confirmation of pathogenicity, suggest a common molecular pathogenesis for dominant GPIBA-associated thrombocytopenia in which reduced circulating platelet numbers arises from defective GP1bα-VWF interactions. Figure Disclosures Turro: Tachyon Ventures: Other: Partner &amp; Scientific Director.