Platelet vWF Research Articles

Toll-like receptor expression and functional behavior in platelets from patients with systemic lupus erythematosus

BackgroundMultiple blood cell abnormalities participate in the development of inflammation in systemic lupus erythematosus (SLE). Although platelets have been suggested as one of these contributors through the release of their content during activation, there are limited specific data about their role as immune players in SLE. Materials and MethodsThirteen SLE patients were included. Flow cytometry was used to measure Toll-like receptors (TLR) 2, 4, and 9 in resting platelets, platelet-activation markers (PAC-1 binding, P-selectin, CD63, and CD40 ligand -L) and platelet-leukocyte aggregates before and after specific TLR stimulation. Soluble CD40L and von Willebrand factor (vWf) release from stimulated platelets was measured using ELISA. ResultsIn resting conditions, SLE platelets showed normal expression levels of TLR 2, 4 and 9. Platelet surface activation markers, soluble CD40L, and vWf release were normal at baseline and after TLR stimulation. Platelet-monocyte aggregates were elevated in resting conditions in SLE samples and showed only a marginal increase after TLR stimulation, while baseline and stimulated platelet-neutrophil and platelet-lymphocyte aggregates were normal. C-reactive protein levels positively correlated with platelet-monocyte aggregates both at baseline and after stimulation with the TLR-2 agonist PAM3CSK4, suggesting these complexes could reflect the inflammatory activity in SLE. In our cohort, 12 of 13 patients received treatment with hydroxychloroquine (HCQ), a known inhibitor of endosomal activity and a potential inhibitor of platelet activation. The fact that SLE platelets showed an adequate response to TLR agonists suggests that, despite this treatment, they retain the ability to respond to the increased levels of damage-associated molecular patterns (DAMPs), which represent known TLR ligands, present in the circulation of SLE patients. Interestingly, elevated plasma levels of high mobility group box 1 (HMGB1), a classical DAMP, correlated with vWf release from TLR-stimulated platelets, suggesting that HMGB1 may also be released by platelets, thereby creating a positive feedback loop for platelet activation that contributes to inflammation. ConclusionOur study demonstrates normal platelet TLR expression and function together with increased circulating platelet-monocyte aggregates. In addition, a direct correlation was observed between plasma HMGB1 levels and platelet vWf release following TLR2 stimulation. This platelet behavior in a group of patients undergoing HCQ treatment suggests that platelets could play a role in the inflammatory state of SLE.

Pervasive Platelet Secretion Defects in Patients with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)

Critically ill COVID-19 patients suffer from thromboembolic as well as bleeding events. Endothelial dysfunction, spiking of von Willebrand factor (vWF), and excessive cytokine signaling result in coagulopathy associated with substantial activation of plasmatic clotting factors. Thrombocytopenia secondary to extensive platelet activation is a frequent finding, but abnormal platelet dysfunction may also exist in patients with normal platelet counts. In this study, we performed analyses of platelet function and of von Willebrand factor in critically ill COVID-19 patients (n = 13). Platelet aggregometry was performed using ADP, collagen, epinephrin, and ristocetin. VWF and fibrinogen binding of platelets and CD62 and CD63 expression after thrombin stimulation were analyzed via flow cytometry. In addition, VWF antigen (VWF:Ag), collagen binding capacity (VWF:CB), and multimer analysis were performed next to routine coagulation parameters. All patients exhibited reduced platelet aggregation and decreased CD62 and CD63 expression. VWF binding of platelets was reduced in 12/13 patients. VWF:CB/VWF:Ag ratios were pathologically decreased in 2/13 patients and elevated in 2/13 patients. Critically ill COVID-19 patients exhibit platelet secretion defects independent of thrombocytopenia. Platelet exhaustion and VWF dysfunction may result in impaired primary hemostasis and should be considered when treating coagulopathy in these patients.

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.

Preferential uptake of SARS-CoV-2 by pericytes potentiates vascular damage and permeability in an organoid model of the microvasculature.

AimsThrombotic complications and vasculopathy have been extensively associated with severe COVID-19 infection, however the mechanisms inducing endotheliitis and the disruption of endothelial integrity in the microcirculation are poorly understood. We hypothesized that within the vessel wall, pericytes preferentially take up viral particles and mediate the subsequent loss of vascular integrity.Methods and ResultsImmunofluorescence of post-mortem patient sections were used to assess pathophysiological aspects of COVID19 infection. The effects of COVID-19 on the microvasculature were assessed using a vascular organoid model exposed to live viral particles or recombinant viral antigens. We find increased expression of the viral entry receptor ACE2 on pericytes when compared to vascular endothelium, and a reduction in the expression of the junctional protein CD144, as well as increased cell death, upon treatment with both live virus and/or viral antigens. We observe a dysregulation of genes implicated in vascular permeability including NOTCH3, angiopoietin-2 and TEK. Activation of vascular organoids with IL-1β did not have an additive effect on vascular permeability. Spike antigen was detected in some patients’ lung pericytes, which was associated with a decrease in CD144 expression and increased platelet recruitment and VWF deposition in the capillaries of these patients, with thrombi in large vessels rich in VWF and fibrin.ConclusionsTogether our data indicates that direct viral exposure to the microvasculature modelled by organoid infection and viral antigen treatment result in pericyte infection, detachment, damage and cell death, disrupting pericyte-endothelial cell crosstalk and increasing microvascular endothelial permeability, which can promote thrombotic and bleeding complications in the microcirculation.Translational PerspectiveEndotheliitis is a serious complication of severe COVID-19 patients which remains poorly understood. We identify a pericyte mediated mechanism by which the vasculature becomes compromised, contributing to thrombotic complications, highlighting important avenues for the development of therapies.

Carriers of ADAMTS13 Rare Variants Are at High Risk of Life-Threatening COVID-19.

Thrombosis of small and large vessels is reported as a key player in COVID-19 severity. However, host genetic determinants of this susceptibility are still unclear. Congenital Thrombotic Thrombocytopenic Purpura is a severe autosomal recessive disorder characterized by uncleaved ultra-large vWF and thrombotic microangiopathy, frequently triggered by infections. Carriers are reported to be asymptomatic. Exome analysis of about 3000 SARS-CoV-2 infected subjects of different severities, belonging to the GEN-COVID cohort, revealed the specific role of vWF cleaving enzyme ADAMTS13 (A disintegrin-like and metalloprotease with thrombospondin type 1 motif, 13). We report here that ultra-rare variants in a heterozygous state lead to a rare form of COVID-19 characterized by hyper-inflammation signs, which segregates in families as an autosomal dominant disorder conditioned by SARS-CoV-2 infection, sex, and age. This has clinical relevance due to the availability of drugs such as Caplacizumab, which inhibits vWF–platelet interaction, and Crizanlizumab, which, by inhibiting P-selectin binding to its ligands, prevents leukocyte recruitment and platelet aggregation at the site of vascular damage.

Characterization of the Pathogenicity of Novel Sequence Variants Identified in Type 1 VWD Subjects in the Zimmerman Program for the Molecular and Clinical Biology Study

Von Willebrand Disease (VWD) is the most prevalent inherited bleeding disorder. Von Willebrand factor's (VWF) complex intracellular processing may result in variable VWF expression which requires exploring the underlying mechanism related to individual variants. The mechanisms underlying type 1 VWD are not fully understood, although reduced secretion and increased clearance have been implicated. We aimed to characterize novel sequence variants (SV) identified in the VWF gene in type 1 VWD patients recruited through the Zimmerman Program in order to (1) define the underlying mechanism, (2) explore if mutations in a particular domain are mechanistically similar, and (3) correlate expression study data to patient phenotype.Homozygous and heterozygous expression in mammalian cells was used to study the effect of SV on VWF secretion, intracellular retention, function, and multimerization. Novel SV identified throughout the entire VWF protein were introduced into VWF-mycHis plasmid vector: V86M, W199X, C524Y, M947V, R960P, G994D, C996W, R1204W, Q1353X, E1660X, R1763Q, C2199Y, Q2256H, T2282I, P2524L, A2569E, C2693F, C2701Y, and C2754Y. Variant VWF cDNA was transfected homozygously and cotransfected heterozygously 1:1 with wild type (WT) VWF pCIneo into Human Embryonic Kidney cells (HEK293T). Supernatants (sup) and cell lysates from 3 separate transfections were collected and analyzed by ELISA for VWF:Ag, VWF binding to platelet glycoprotein 1b (VWF:GPIbM), VWF binding to collagen type III (VWF:CB). Typically VWF:GP1bM/VWF:Ag > 0.7 indicates normal platelet binding and VWF:CB/VWF:Ag > 0.7 indicates normal multimer structure. VWF multimer structure is analyzed by SDS-agarose electrophoresis and western blotting with differential staining of the VWF secreted from variant and WT alleles.V86M, M947V, R1204W, R1763Q, Q2256H, T2282I, P2524L, and A2569E demonstrated secretion of VWF in sup comparable to that of WT-VWF both when transfected homozygously and hetereozygously, normal VWF:GPIbM/VWF:Ag, VWF:CB/VWF:Ag and normal multimer structure. Two of the stop codon SV W199X and E1660X were not expressed homozgyously (as expected) but demonstrated normal secretion, function, and multimerization when cotransfected 1:1 with WT. Homozygous expression of R960P and C2693F demonstrated reduced VWF secretion, VWF:GPIbM/VWF:Ag, VWF:CB/VWF:Ag and abnormal multimerization. Heterozygous transfection with WT improved VWF secretion, but not multimer defects,reflected by a reduced VWF:CB/VWF:Ag. Homozygous expression of C524Y, G994D, C996W, Q1353X, C2199Y, C2701Y, and C2754Y resulted in absent or greatly reduced secretion with increased intracellular retention. Heterozygous expression resulted in reduced VWF secretion (<50% of WT) for all 7 variants. Q1353X, C2199Y, C2701Y, and C2754Y had normal VWF:CB/VWF:Ag, VWF:GPIbM/VWF:Ag, and multimer structure, but consisted primarily of secreted WT-VWF. G994D had VWF:CB/VWF:Ag ˂ 0.7 and reduced high molecular weight (HMW) multimers. Co-expression of C524Y and C996W with WT-VWF demonstrated nearly absent VWF secretion, indicating a strong dominant negative effect. The very low VWF:Ag levels precluded assessment of VWF function and multimerization.The VWF:CB/VWF:Ag ratio < 0.7 proved a reliable indicator of multimer structure. No clear domain-specific correlation of VWF secretion or multimer abnormality was observed. Co-expression results of Q1353X, C2199Y, C2701Y, C2754Y, C524Y, G994D, and C996W are consistent with the patient's phenotype, except that patients with C2701Y, and C524Y had slight defect in multimers. In contrast, the in-vitro G994D showed low VWF:CB/VWF:Ag and abnormal multimers but were normal in the patient. C2693F showed consistently abnormal multimer structure in both in vitro and in the patient while R960P had abnormal multimer structure in vitro only.In sum, 52.6% of the 19 SV theoretically are not causative of the VWD phenotype observed in the patients. These subjects had normal VWF propeptide/VWF:Ag suggesting reduced plasma survival is unlikely to be the causative mechanism. The VWD mechanism in these subjects remains undefined. Variants R960P, C2693F, and G994D (15.7%) behaved like Type 2 VWD SV with loss of HMW multimers. We identified 31.6% of the 19 VWF SV as likely to cause the type 1 VWD phenotype with reduced secretion. DisclosuresNo relevant conflicts of interest to declare.

Quantification of von Willebrand factor and ADAMTS-13 after traumatic injury: a pilot study

BackgroundVon Willebrand factor (VWF) is an acute phase reactant synthesized in the megakaryocytes and endothelial cells. VWF forms ultra-large multimers (ULVWF) which are cleaved by the metalloprotease ADAMTS-13, preventing spontaneous...

Platelet α-granules are required for occlusive high-shear-rate thrombosis

Abstractvon Willebrand factor (VWF) is essential for the induction of arterial thrombosis. In this study, we investigated the critical role of platelet VWF in occlusive thrombosis formation at high shear in mice that do not express platelet VWF (Nbeal2−/−). Using in silico modeling, in vitro high-shear microfluidics, and an in vivo Folts model of arterial thrombosis we reproduced the platelet dynamics that occur under pathological flow in a stenosed vessel. Computational fluid dynamics (CFDs) simulated local hemodynamics in a stenosis based on arterial geometries. The model predicted shear rates, time course of platelet adhesion, and time to occlusion. These predictions were validated in vitro and in vivo. Occlusive thrombosis developed in wild-type control mice that had normal levels of plasma VWF and platelet VWF in vitro and in vivo. Occlusive thrombosis did not form in the Nbeal2−/− mice that had normal plasma VWF and an absence of platelet VWF. Occlusive thrombosis was corrected in Nbeal2−/− microfluidic assays by the addition of exogenous normal platelets with VWF. Combining model and experimental data, we demonstrated the necessary requirement of platelet VWF in α-granules in forming an occlusive thrombus under high shear. These results could inspire new pharmacological targets specific to pathological conditions and prevent arterial thrombosis.

Meet the First Authors.

Meet the First Authors.

The impact of rivaroxaban on primary hemostasis in patients with venous thrombosis

Factor Xa inhibitors are safe and effective alternatives to warfarin, but several studies indicate that rivaroxaban may cause a different risk profile for bleeding. For instance, while the risk of major bleeding in general may be lower with rivaroxaban than for warfarin, the risk of gastrointestinal bleeding or abnormal uterine bleeding may be higher. The underlying mechanisms for these differences are not known, and the effect of rivaroxaban on primary hemostasis is poorly understood. The aim of this study was to investigate the effect of rivaroxaban on platelet function, P-selectin and von Willebrand factor (VWF) antigen and activity. Patients with venous thrombosis assigned to 3 months of treatment due to temporary risk factors were included. Blood was collected both during (on-treatment) and 4–6 weeks after end of treatment (without treatment). The platelet reactivity was assessed by light transmission aggregometry. P-selectin was measured by an enzyme-linked immunosorbent assay and vWF antigen and activity by latex immunoagglutination assays. Platelet reactivity during on-treatment (trough- and peak concentration) was similar to values without treatment. There was a trend toward a reduction of P-selectin during rivaroxaban treatment (peak concentration) compared to value without treatment (p = 0.06). There were no differences in vWF antigen and activity between the different time-points. We found no difference in platelet reactivity or vWF antigen/activity during rivaroxaban treatment compared with values without treatment. Apart from possibly causing a reduction of P-selectin, rivaroxaban seems not to influence primary hemostasis.

Prospective Diagnosis of VWD in a Large Cohort of Patients with Bleeding Symptoms through the Zimmerman Program

Prospective Diagnosis of VWD in a Large Cohort of Patients with Bleeding Symptoms through the Zimmerman Program

The elusive and heterogeneous pattern of type 2M von Willebrand disease: A diagnostic challenge.

To investigate type 2M heterogeneity and to establish the most appropriate diagnostic flowchart. Hemostatic and genetic VWF analyses were performed in 14 type 2M VWD patients carrying the p.G1324S, p.R1374H, p.R1374C, p.A1344_A1350del, or p.F1293L mutations. PFA-100 was always significantly prolonged, and ristocetin-induced platelet aggregation (RIPA) and VWF ristocetin cofactor (VWF:RCo) greatly reduced or absent. Plasma VWF antigen (VWF:Ag) was reduced except in the p.G1324S patient, while platelet VWF:Ag was normal or near normal except in the p.R1374C patients. The ratio of collagen binding (VWF:CB) to VWF:Ag was normal or near normal except in patients carrying the p.R1374H and p.A1344_A1350del mutations, whose large VWF multimers were slightly reduced. Multimer patterns were normal or lacking in large oligomers, or with larger than normal VWF components. Only PFA100, RIPA and VWF:RCo were always abnormal. We thus propose a minimal diagnostic test battery: RIPA (demonstrating the defective VWF-platelet interaction), VWF:Ag (exploring VWF synthesis), and VWF:CB and its ratio (to explore multimer patterns). Other tests would only serve for confirmation, if necessary.

Defective RAB1B-related megakaryocytic ER-to-Golgi transport in RUNX1 haplodeficiency: impact on von Willebrand factor

AbstractPatients with RUNX1 haplodeficiency have thrombocytopenia, platelet dysfunction, and deficiencies of α-granules and dense granules. Platelet expression profiling of a patient with a heterozygous RUNX1 mutation (c.969-323G>T) revealed decreased RAB1B, which encodes a small G protein. RAB GTPases regulate vesicle trafficking, and RAB1B is implicated in endoplasmic reticulum (ER)-to-Golgi transport in nonhematopoietic cells, but its role in megakaryocytes (MK) is unknown. We addressed the hypothesis that RAB1B is a transcriptional target of RUNX1 and that RAB1B regulates ER-to-Golgi transport in MK cells. Chromatin immunoprecipitation studies and electrophoretic mobility shift assay using phorbol 12-myristate 13-acetate (PMA)–treated human erythroleukemia cells revealed RUNX1 binding to RAB1B promoter region RUNX1 consensus sites, and their mutation reduced the promoter activity. RAB1B promoter activity and protein expression were inhibited by RUNX1 siRNA and enhanced by RUNX1 overexpression. These indicate that RAB1B is a direct RUNX1 target, providing a mechanism for decreased RAB1B in patient platelets. Vesicle trafficking from ER to Golgi in PMA-treated human erythroleukemia cells was impaired along with Golgi disruption on siRNA downregulation of RUNX1 or RAB1B. The effects of RUNX1 knockdown were reversed by RAB1B reconstitution. Trafficking of von Willebrand factor (vWF), an α-granule MK synthesized protein, was impaired with RUNX1 or RAB1B downregulation and reconstituted by ectopic RAB1B expression. Platelet vWF was decreased in patients with RUNX1 mutations. Thus, ER-to-Golgi transport, an early critical step in protein trafficking to granules, is impaired in megakaryocytic cells on RUNX1 downregulation, secondary to decreased RAB1B expression. Impaired RAB1B mediated ER-to-Golgi transport contributes to platelet α-granule defects in RUNX1 haplodeficiency.

Platelet Count, ADAMTS13 Activity, von Willebrand Factor Level and Survival in Patients with Colorectal Cancer: 5-Year Follow-up Study.

Distant metastasis is a major cause of colorectal cancer-related death, but the mechanism of tumour progression is not fully understood. There is growing evidence of an interaction between tumour cells and platelets which may influence tumour progression and metastasis formation. Quality and quantity of von Willebrand factor may regulate the interaction between tumour cells and platelets. Our aim was to measure the platelet count, von Willebrand factor antigen (VWF:Ag) levels and ADAMTS13 activity in a large (n = 232) cohort of colorectal cancer patients and to examine their relationships with the stage of the disease and 5-year survival without thrombotic complications using multivariable models. Significantly higher platelet counts (p = 0.005), VWF:Ag levels (p = 0.008) and decreased ADAMTS13 activity (p = 0.006) were observed in patients with metastatic disease. Results of the Kaplan-Meier analysis showed that lower platelet counts (p < 0.0001), lower VWF:Ag (p = 0.0008) levels and higher ADAMTS13 activity (p < 0.0001) were associated with better event-free survival. Finally, to investigate the association between overall event-free survival and the three study variables, multivariate Cox proportional hazard models were generated. All models were adjusted for age, gender and disease stage. Platelet count, ADAMTS13 activity or VWF:Ag level were incorporated and all of these variables turned out to be age-, gender- and stage-independent predictors of mortality (all hazard ratio >1.7, p < 0.05). In summary, this is the first observational study reporting association between higher mortality or thrombotic complications and increased platelet count, increased VWF:Ag levels and decreased ADAMTS13 activity in colorectal cancer.

Profile of von Willebrand factor antigen and von Willebrand factor propeptide in an overall TIA and ischaemic stroke population and amongst subtypes

IntroductionVon Willebrand factor propeptide (VWF:Ag II) is proposed to be a more sensitive marker of acute endothelial activation than von Willebrand factor antigen (VWF:Ag). Simultaneous data on VWF:Ag and VWF:Ag II profiles are very limited following TIA and ischaemic stroke. MethodsIn this prospective, observational, case-control study, plasma VWF:Ag and VWF:Ag II levels were quantified in 164 patients≤4weeks of TIA or ischaemic stroke (baseline), and then ≥14days (14d) and ≥90days (90d) later, and compared with those from 27 healthy controls. TIA and stroke subtyping was performed according to the TOAST classification. The relationship between VWF:Ag and VWF:Ag II levels and platelet activation status was assessed. Results‘Unadjusted’ VWF:Ag and VWF:Ag II levels were higher in patients at baseline, 14d and 90d than in controls (p≤0.03). VWF:Ag levels remained higher in patients than controls at baseline (p≤0.03), but not at 14d or 90d after controlling for differences in age or hypertension, and were higher in patients at baseline and 90d after controlling for smoking status (p≤0.04). ‘Adjusted’ VWF:Ag II levels were not higher in patients than controls after controlling for age, hypertension or smoking (p≥0.1). Patients with symptomatic carotid stenosis (N=46) had higher VWF:Ag and VWF:Ag II levels than controls at all time-points (p≤0.002). There was no significant correlation between platelet activation status and VWF:Ag or VWF:Ag II levels. ConclusionsVWF:Ag and VWF:Ag II levels are increased in an overall TIA and ischaemic stroke population, especially in patients with recently symptomatic carotid stenosis. VWF:Ag II was not superior to VWF:Ag at detecting acute endothelial activation in this cohort and might reflect timing of blood sampling in our study.

A GWAS of 170,000 Individuals Identifies Thousands of Alleles Perturbing Blood Cell Traits, Many of Which Are in Super Enhancers Setting Cell Identity

Adults release ~1011 blood cells into circulation every day. These cells derive, by clonal expansion, from a minuscule population of hematopoietic stem cells (HSCs), which differentiate into mature cells through progressively fate-restricted progenitor cells. In order to identify the genetic loci controlling these processes, we performed a hypothesis-free genome-wide association study (GWAS) of complete blood count data measured on 170,000 subjects in the UK Biobank and INTERVAL cohorts. This identified almost 3,000 causally distinct variants (hits) modulating hematopoiesis or blood cell clearance, including several hundred low frequency variants (minor allele frequencies <5%) with very large effect sizes.Over three quarters of the GWAS hits have no coding consequence and analysis of the 700 platelet hits showed a striking enrichment in the DNAse-I hypersensitivity sites of HSC-derived megakaryocytes (MKs) compared to those of HSC-derived erythroblasts (EBs), suggesting that a substantial proportions of blood trait heritability can be explained by variants modulating cell type specific genetic regulators.To understand the nature of gene regulation in the erythroid-megakaryocytic lineage we firstly used long-range chromosomal interaction data (from genome-wide promoter-capture chromosome conformation assays) and found that the platelet trait hits are enriched in regulatory elements that interact with a gene promoter in MKs. We then scanned the genome for super-enhancers (SEs) (regions characterized by a high density of enhancer elements and a strong H3K27ac histone modification). We identified 1067 SEs in MKs and 1294 SEs in EBs, with an overlap count of 400. Platelet GWAS hits were strongly enriched in MK SEs over MK typical enhancers (TEs) relative to red cell hits and were frequently localised in SEs specific to MKs. Finally, we found that SE (but not TE) MK-interacting genes are significantly enriched for gene-ontology (GO) terms for megakaryopoiesis, platelet function and hemostasis, supporting the notion that SEs play a major role in the determination of cell identity.To corroborate this, we focused on the second (SE2) of three SEs for the CD9-VWF gene cluster, which contained the GWAS hit rs2363877. We recalled 100 individuals by genotype at rs2363877 and found that the minor allele was associated with decreased expression of CD9 and increased expression of VWF in platelets. By flowing blood over collagen and a VWF-binding collagen-mimetic we showed this allele has a significant effect on the extent of ex-vivo thrombus formation. Finally, using CRISPR/Cas9 we eliminated SE2 from IPSC derived MKs and showed an increase in VWF expression.In summary, recently generated epigenetic and long-range chromosomal interaction data suggest that a significant fraction of platelet GWAS hits perturb super-enhancer activity in MKs with downstream effect on networks of genes. In the light of this the maxim that 'a GWAS hit acts through a single gene' cannot be sustained. DisclosuresNo relevant conflicts of interest to declare.

Platelet-derived VWF is not essential for normal thrombosis and hemostasis but fosters ischemic stroke injury in mice

AbstractVon Willebrand factor (VWF) is a key hemostatic protein synthesized in both endothelial cells and megakaryocytes. Megakaryocyte-derived VWF is stored in α-granules of platelets and is enriched in hyperactive “ultra-large” VWF multimers. To elucidate the specific contribution of platelet VWF in hemostasis and thrombosis, we performed crossed bone marrow transplantations between C57BL/6J and Vwf−/− mice to generate chimeric mice. Chimeric mice specifically lacking platelet VWF showed normal tail bleeding and carotid artery thrombosis, similar to wild-type mice. Chimeric mice with VWF present only in platelets were not able to support normal thrombosis and hemostasis. However, using a mouse model of transient middle cerebral artery occlusion, we observed that cerebral infarct sizes and fibrin(ogen) deposition in chimeric mice with only platelet VWF were significantly increased compared with Vwf−/− mice (P < .01). Blocking of the platelet VWF-glycoprotein (GP)Ib interaction abrogated this platelet VWF-mediated injury. These data suggest that whereas platelet-derived VWF does not play a crucial role in hemostasis and arterial thrombosis, it aggravates thrombo-inflammatory diseases such as stroke via a GPIb-dependent mechanism.

Drospirenone enhances GPIb‐IX‐V‐mediated platelet activation

Epidemiologic studies recently revealed that using drospirenone (DRSP)-containing contraceptives is associated with an increased risk of thrombosis in women. However, the underlying causality is unclear. To study the effects of DRSP on coagulation invitro and the probable mechanisms involved. First, the effects of DRSP on the activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT) and fibrinogen (FIB) were measured. Then, the effects of DRSP on platelet activation were investigated in response to low levels of collagen, adenosine 5'-diphosphate (ADP), thrombin, U46619, adrenaline and botrocetin/von Willebrand factor (VWF). DRSP has no direct effect on APTT, PT, TT, FIB and platelet aggregation induced by low levels of collagen, ADP, thrombin, U46619 or adrenaline. However, DRSP enhances botrocetin/VWF-induced platelet aggregation and VWF receptor glycoprotein Ib-IX-V (GPIb-IX-V)-mediated signaling. This enhancement can be blocked by the progesterone receptor membrane component 1 (PGRMC1) inhibitor AG205, or by the ADP scavenger apyrase and the cyclooxygenase inhibitor indomethacin. Although DRSP did not directly induce platelet activation, it obviously facilitated VWF receptor GPIb-IX-V-mediated platelet activation. The potential DRSP-binding protein PGRMC1 may play a role in this process. Our study also suggested that the inhibition of thromboxane A2 production and the activation of ADP receptors might prevent the side-effects of DRSP.

Platelets and physics: How platelets “feel” and respond to their mechanical microenvironment

During clot formation, platelets are subjected to various different signals and cues as they dynamically interact with extracellular matrix proteins such as von Willebrand factor (vWF), fibrin(ogen) and collagen. While the downstream signaling of platelet–ligand interactions is well-characterized, biophysical cues, such as hydrodynamic forces and mechanical stiffness of the underlying substrate, also mediate these interactions and affect the binding kinetics of platelets to these proteins. Recent studies have observed that, similar to nucleated cells, platelets mechanosense their microenvironment and exhibit dynamic physiologic responses to biophysical cues. This review discusses how platelet mechanosensing is affected by the hydrodynamic forces that dictate vWF–platelet interactions and fibrin polymerization and network formation. The similarities and differences in mechanosensing between platelets and nucleated cells and integrin-mediated platelet mechanosensing on both fibrin(ogen) and collagen are then reviewed. Further studies investigating how platelets interact with the mechanical microenvironment will improve our overall understanding of the hemostatic process.

Impact of Exercise on Hemostasis in Boys with Hemophilia a (HA) and B (HB): Principal Findings of the Sickkids Hemophilia Exercise Study

Introduction. We undertook a study to assess the impact of exercise on hemostatic parameters [FVIII, FIX, VWF, platelet count and function, thrombin generation time (TGT) and thromboelastography (TEG)] in boys with HA and HB. We did so to explore the hypothesis that subjects with non-severe HA and all subjects with HB would show an increase in platelet count, VWF and FVIII with exercise leading to an overall improvement in hemostasis.Methods.The study was approved by the institutional ethics board. Subjects (ages 5-18 years) meeting inclusion criteria were invited to participate. Baseline blood-work was drawn and subjects were then exercised on a stationary cycle ergometer using a progressive incremental Godfrey-protocol, aiming to attain 3-minutes of cycling at 85% of predicted maximum heart rate. Blood-work was repeated 5-minutes and 1-hour after exercise completion. Descriptive statistics were used to summarize parameters (mean ± SD). Statistical analyses were performed using the SAS version 9.2 software package. All calculated p-values were two-sided and p-values ≤ 0.05 were considered significant.Results. Thirty subjects [19 HA (7 mild; 6 moderate; 6 severe); 11 HB (2 mild; 6 moderate; 3 severe)] were enrolled. Mean age at participation was 12.8 (±3.8) years. Mean duration of exercise was 13.9 (±2.6) minutes. Baseline characteristics were similar for HA and HB subjects, except BMI, which was significantly higher in the HA cohort (p=0.01). For the entire study cohort, we found that platelet count, FVIII, VWF:Ag and VWF:RCo levels rose significantly by 5-minutes post-exercise (p values of <0.0001, 0.004, <0.0001 and 0.01 respectively), though only VWF:Ag and VWF:RCo levels remained significantly elevated at 1-hour (p=0.003 and 0.02 respectively). PFA-100 closure time with the collagen/epinephrine cartridge shortened significantly at 5 minutes (n=26; p=0.02), but not with the collagen/ADP cartridge (n=26; p=0.6). Subjects with severe HA showed no significant change in FVIII, VWF:Ag or VWF:RCo levels with exercise, but did show a rise in platelet counts (p=0.03). Post-pubertal boys (>12 years) had a significantly greater increase in FVIII, VWF:Ag and VWF:RCo levels as compared to pre-pubertal boys at both 5-minutes and 1-hour, despite having exercised for a shorter duration of time (13.2 minutes versus 15.1 minutes; p=0.04) (data for non-severe HA and all HB patients is elaborated in Fig. 1). Complete TEG and TGT data were available for 19 subjects (13 HA and 6 HB). No consistent improvement in TEG parameters was noted. TGT studies did show a significant improvement in peak thrombin generation (using 1 pM tissue factor) at 5-minutes post exercise (p=0.01), though this improvement was lost by 1-hour (p=0.9).Conclusion. Children with hemophilia (excluding severe HA) showed improvements in specific hemostatic parameters with exercise. These improvements were more robust in post-pubertal males. The change in coagulation profile could be discerned using PFA-100 and TGT but not TEG. We conclude that exercise may potentially improve hemostatic parameters (at least for a short time) in subjects with non-severe HA and all subjects with HB thus reducing the risk of bleeding. [Display omitted] Figure subscript. Impact of exercise on FVIII activity in non-severe HA subjects (1a) and HB subjects (1b); on VWF:RCo activity in non-severe HA subjects (1c) and HB subjects (1d); on VWF:Ag levels in non-severe HA subjects (1e) and HB subjects (1f) DisclosuresCarcao:Pfizer: This study was an investigator initiated study which was funded by Pfizer Other.