Release Of Von Willebrand Factor Research Articles

Transient von Willebrand factor-mediated platelet influx stimulates liver regeneration after partial hepatectomy in mice.

In addition to their function in thrombosis and haemostasis, platelets play an important role in the stimulation of liver regeneration. It has been suggested that platelets deliver mitogenic cargo to the regenerating liver, and accumulation of platelets in the regenerating liver has been demonstrated. We studied kinetics of platelet influx in the regenerating liver and investigated the signal that initiates platelet influx. We visualized platelets in the liver remnant after partial hepatectomy in mice using intravital microscopy and assessed liver regeneration by examination of liver/body weight ratio and the number of proliferating hepatocytes examined by immunohistochemistry. We demonstrated rapid but transient platelet influx into the liver remnant after a partial liver resection. Liver regeneration in thrombocytopenic mice was substantially impaired as evidenced by a reduced liver-to-body weight ratio and decreased numbers of proliferating hepatocytes at day 3 compared to mice with normal platelet counts. In contrast, liver regeneration was only mildly impaired when thrombocytopaenia was induced 2hours after partial liver resection. Platelet influx into the liver remnant was virtually absent in the presence of an antibody to von Willebrand factor (VWF) suggesting that VWF release from liver sinusoidal endothelial cells mediates platelet influx. Additionally, liver regeneration in mice deficient in VWF was markedly impaired. A rapid but transient VWF-dependent platelet influx into the liver remnant drives platelet-mediated liver regeneration.

Zyxin regulates endothelial von Willebrand factor secretion by reorganizing actin filaments around exocytic granules

Endothelial exocytosis of Weibel–Palade body (WPB) is one of the first lines of defence against vascular injury. However, the mechanisms that control WPB exocytosis in the final stages (including the docking, priming and fusion of granules) are poorly understood. Here we show that the focal adhesion protein zyxin is crucial in this process. Zyxin downregulation inhibits the secretion of von Willebrand factor (VWF), the most abundant cargo in WPBs, from human primary endothelial cells (ECs) induced by cAMP agonists. Zyxin-deficient mice exhibit impaired epinephrine-stimulated VWF release, prolonged bleeding time and thrombosis, largely due to defective endothelial secretion of VWF. Using live-cell super-resolution microscopy, we visualize previously unappreciated reorganization of pre-existing actin filaments around WPBs before fusion, dependent on zyxin and an interaction with the actin crosslinker α-actinin. Our findings identify zyxin as a physiological regulator of endothelial exocytosis through reorganizing local actin network in the final stage of exocytosis.

How do we approach thrombocytopenia in critically ill patients?

A low platelet count is a frequently encountered haematological abnormality in patients treated in intensive treatment units (ITUs). Although severe thrombocytopenia (platelet count <20×109 /l) can be associated with bleeding, even moderate-degree thrombocytopenia is associated with organ failure and adverse prognosis. The aetiology for thrombocytopenia in ITU is often multifactorial and correcting one aetiology may not normalise the low platelet count. The classical view for thrombocytopenia in this setting is consumption associated with thrombin-mediated platelet activation, but other concepts, including platelet adhesion to endothelial cells and leucocytes, platelet aggregation by increased von Willebrand factor release, red cell damage and histone release, and platelet destruction by the complement system, have recently been described. The management of severe thrombocytopenia is platelet transfusion in the presence of active bleeding or invasive procedure, but the risk-benefit of prophylactic platelet transfusions in this setting is uncertain. In this review, the incidence and mechanisms of thrombocytopenia in patients with ITU, its prognostic significance and the impact on organ function is discussed. A practical approach based on the authors' experience is described to guide management of a critically ill patient who develops thrombocytopenia.

Histones link inflammation and thrombosis through the induction of Weibel–Palade body exocytosis

Background Damage-associated molecular patterns (DAMPs), including molecules such as DNA and histones, are released into the blood following cell death. DAMPs promote a procoagulant phenotype through enhancement of thrombin generation and platelet activation, thereby contributing to immunothrombosis. Weibel-Palade bodies (WPBs) are dynamic endothelial cell organelles that contain procoagulant and proinflammatory mediators, such as von Willebrand factor (VWF), and are released in response to cell stresses. VWF mediates platelet adhesion and aggregation, and has been implicated as a procoagulant component of the innate immune response. Objective To determine the influence of histones and DNA on WPB release, and characterize their association in models of inflammation. Methods We treated C57BL/6J mice and cultured endothelial cells with histones (unfractionated, lysine-rich or arginine-rich) and DNA, and measured WPB exocytosis. We used inhibitors to determine a mechanism of histone-induced WPB release invitro. We characterized the release of DAMPs and WPBs in response to acute and chronic inflammation in human and murine models. Results and conclusions Histones, but not DNA, induced the release of VWF (1.46-fold) from WBPs and caused thrombocytopenia (0.74-fold), which impaired arterial thrombus formation in mice. Histones induced WPB release from endothelial cells in a caspase-dependent, calcium-dependent and charge-dependent manner, and promoted platelet capture in a flow chamber model of VWF-platelet string formation. The levels of DAMPs and WPB-released proteins were elevated during inflammation, and were positively correlated in chronic inflammation. These studies showed that DAMPs can regulate the function and level of VWF by inducing its release from endothelial WPBs. This DAMP-WPB axis may propagate immunothrombosis associated with inflammation.

BLOC-2 subunit HPS6 deficiency affects the tubulation and secretion of von Willebrand factor from mouse endothelial cells

Hermansky-Pudlak syndrome (HPS) is a recessive disorder with bleeding diathesis, which has been linked to platelet granule defects. Both platelet granules and endothelial Weibel-Palade bodies (WPBs) are members of lysosome-related organelles (LROs) whose formation is regulated by HPS protein associated complexes such as BLOC (biogenesis of lysosome-related organelles complex) -1, -2, -3, AP-3 (adaptor protein complex-3) and HOPS (homotypic fusion and protein sorting complex). Von Willebrand factor (VWF) is critical to hemostasis, which is stored in a highly-multimerized form as tubules in the WPBs. In this study, we found the defective, but varying, release of VWF into plasma after desmopressin (DDAVP) stimulation in HPS1 (BLOC-3 subunit), HPS6 (BLOC-2 subunit), and HPS9 (BLOC-1 subunit) deficient mice. In particular, VWF tubulation, a critical step in VWF maturation, was impaired in HPS6 deficient WPBs. This likely reflects a defective endothelium, contributing to the bleeding tendency in HPS mice or patients. The differentially defective regulated release of VWF in these HPS mouse models suggests the need for precise HPS genotyping before DDAVP administration to HPS patients.

Heparins that block VEGF-A-mediated von Willebrand factor fiber generation are potent inhibitors of hematogenous but not lymphatic metastasis

Von Willebrand factor (VWF) serves as a nidus for platelet aggregation and thrombosis. We hypothesize that VWF fibers contribute to the development of venous thromboembolism (VTE) and to metastasis formation. Here, we show that vascular and lymphatic endothelial cells (ECs) express VWF in vitro and release VWF fibers after activation by tumor cell supernatants. In contrast, an ex vivo analysis of primary mouse tumors revealed the presence of VWF fibers in the blood microvasculature but not in lymphatic vessels. Unlike the anticoagulant Fondaparinux, an inhibitor of thrombin generation, the low-molecular-weight heparin (LMWH) Tinzaparin inhibited VWF fiber formation and vessel occlusion in tumor vessels by blocking thrombin-induced EC activation and vascular endothelial growth factor-A (VEGF-A)-mediated VWF release. Intradermal tumor cell inoculation in VWF- and ADAMTS13-deficient mice did not alter lymph node metastases compared with wild type animals. Interestingly, multiple tumor-free distal organs exhibited hallmarks of malignancy-related VTE, including luminal VWF fibers, platelet-rich thrombi and vessel occlusions. Furthermore, ADAMTS13 deficiency, characterized by prolonged intraluminal VWF network lifetimes, resulted in a severely increased number of metastatic foci in an experimental model of hematogenous lung seeding. Treatment with Tinzaparin inhibited tumor-induced release of VWF multimers, impeded platelet aggregation and decreased lung metastasis. Thus, our data strongly suggest a critical role of luminal VWF fibers in determining the occurrence of thrombosis and cancer metastasis. Moreover, the findings highlight LMWHs as therapeutic strategy to treat thrombotic complications while executing anti-metastatic activities.

Annexins A2 and A8 in endothelial cell exocytosis and the control of vascular homeostasis.

Blood vessel homeostasis is controlled by a variety of regulatory circuits that involve both the vessel-lining endothelial cells as well as the circulating blood cells and products thereof. One important feature is the control exerted by endothelial cells through regulated exocytosis of factors affecting blood coagulation and local inflammatory processes. These factors include two important adhesion proteins: the leukocyte receptor P-selectin and the pro-coagulant von Willebrand factor (VWF) that binds platelets and is involved in the formation of a platelet plug at sites of blood vessel injury. Failure to correctly produce and secrete P-selectin and VWF leads to pathologies such as von Willebrand disease, the most common inherited bleeding disorder. P-selectin and VWF are stored in unique secretory granules, the Weibel-Palade bodies (WPB), that undergo a complex maturation process and are acutely secreted following endothelial stimulation, e.g. in the course of inflammation or following blood vessel injury. Two annexins have been shown to be involved in different aspects of WPB biology: annexin A8 is required for proper WPB maturation and annexin A2 participates in late steps of WPB exocytosis. Thus, by affecting the stimulated release of P-selectin and VWF from endothelial cells, annexins fulfil important functions in the control of vascular homeostasis and could be considered as targets for influencing P-selectin- and VWF-dependent processes/pathologies.

Von Willebrand factor multimerization and the polarity of secretory pathways in endothelial cells

AbstractThe von Willebrand factor (VWF) synthesized and secreted by endothelial cells is central to hemostasis and thrombosis, providing a multifunctional adhesive platform that brings together components needed for these processes. VWF secretion can occur from both apical and basolateral sides of endothelial cells, and from constitutive, basal, and regulated secretory pathways, the latter two via Weibel-Palade bodies (WPB). Although the amount and structure of VWF is crucial to its function, the extent of VWF release, multimerization, and polarity of the 3 secretory pathways have only been addressed separately, and with conflicting results. We set out to clarify these relationships using polarized human umbilical vein endothelial cells (HUVECs) grown on Transwell membranes. We found that regulated secretion of ultra–large (UL)-molecular-weight VWF predominantly occurred apically, consistent with a role in localized platelet capture in the vessel lumen. We found that constitutive secretion of low-molecular-weight (LMW) VWF is targeted basolaterally, toward the subendothelial matrix, using the adaptor protein complex 1 (AP-1), where it may provide the bulk of collagen-bound subendothelial VWF. We also found that basally-secreted VWF is composed of UL-VWF, released continuously from WPBs in the absence of stimuli, and occurs predominantly apically, suggesting this could be the main source of circulating plasma VWF. Together, we provide a unified dataset reporting the amount and multimeric state of VWF secreted from the constitutive, basal, and regulated pathways in polarized HUVECs, and have established a new role for AP-1 in the basolateral constitutive secretion of VWF.

Cytomegalovirus-Associated CD4(+) CD28(null) Cells in NKG2D-Dependent Glomerular Endothelial Injury and Kidney Allograft Dysfunction.

Emerging data suggest that expansion of a circulating population of atypical, cytotoxic CD4(+) T cells lacking costimulatory CD28 (CD4(+) CD28(null) cells) is associated with latent cytomegalovirus (CMV) infection. The purpose of the current study was to increase the understanding of the relevance of these cells in 100 unselected kidney transplant recipients followed prospectively for a median of 54months. Multicolor flow cytometry of peripheral blood mononuclear cells before transplantation and serially posttransplantation was undertaken. CD4(+) CD28(null) cells were found predominantly in CMV-seropositive patients and expanded in the posttransplantation period. These cells were predominantly effector-memory phenotype and expressed markers of endothelial homing (CX3CR1) and cytotoxicity (NKG2D and perforin). Isolated CD4(+) CD27(-) CD28(null) cells proliferated in response to peripheral blood mononuclear cells previously exposed to CMV-derived (but not HLA-derived) antigens and following such priming incubation with glomerular endothelium resulted in signs of endothelial damage and apoptosis (release of fractalkine and von Willebrand factor; increased caspase 3 expression). This effect was mitigated by NKG2D-blocking antibody. Increased CD4(+) CD28(null) cell frequencies were associated with delayed graft function and lower estimated glomerular filtration rate at end follow-up. This study suggests an important role for this atypical cytotoxic CD4(+) CD28(null) cell subset in kidney transplantation and points to strategies that may minimize the impact on clinical outcomes.

Shear-Resistant Binding to von Willebrand Factor Allows Staphylococcus lugdunensis to Adhere to the Cardiac Valves and Initiate Endocarditis.

Staphylococcus lugdunensis is an emerging cause of endocarditis. To cause endovascular infections, S. lugdunensis requires mechanisms to overcome shear stress. We investigated whether platelets and von Willebrand factor (VWF) mediate bacterial adhesion to the vessel wall and the cardiac valves under flow. S. lugdunensis binding to VWF, collagen, and endothelial cells was studied in a parallel flow chamber in the absence and presence of platelets. In vivo adhesion of S. lugdunensis was evaluated in a mouse microvasculature perfusion model and a new mouse model of endocarditis. Contrary to other coagulase-negative staphylococci, S. lugdunensis bound to VWF under flow, thus enabling its adhesion to endothelial cells and to the subendothelial matrix. In inflamed vessels of the mesenteric circulation, VWF recruited S. lugdunensis to the vessel wall. In a novel endocarditis mouse model, local inflammation and the resulting release of VWF enabled S. lugdunensis to bind and colonize the heart valves. S. lugdunensis binds directly to VWF, which proved to be vital for withstanding shear forces and for its adhesion to the vessel wall and cardiac valves. This mechanism explains why S. lugdunensis causes more-aggressive infections, including endocarditis, compared with other coagulase-negative staphylococci.

Relative Contributions of von Willebrand Factor and Platelets in High Shear Thrombosis

Platelets and von Willebrand factor (VWF) are recognized as important mediators of thrombosis at high shear rates, but their relative contributions are unclear. We employ a stenotic microfluidic test system to induce thrombus formation to occlusion on collagen at pathologic shear rates of 3500-6000 s-1. To obtain blood analogs with reduced platelet and VWF concentrations, human whole blood was diluted with saline by 90% or 99% and hematocrit restored by adding washed red blood cells. Platelets and VWF were selectively restored to normal levels. Blood from patients with known von Willebrand disease (VWD) was also investigated with and without the addition of VWF to investigate the contribution of platelet VWF. Normal whole blood led to channel occlusion in all tests (occlusion time, tocc=6.1 ± 2.2 min). 90% dilution with restored VWF occluded in 6/7 subjects even without restored platelets (tocc=16.6 ± 1.4 min). 90% dilution with restored platelets occluded in 2/5 subjects (tocc=27.2 ± 1.8 min), showing that added VWF is more effective for restoring occlusion than platelets. Addition of plasma VWF to VWD blood restored occlusion in only 1/4 severe subjects (VWF:RCo<15) and did not reduce normal occlusion time in the occluding subject, suggesting that VWF release from platelets plays an important role in high shear thrombosis. Logistic regression shows that VWF concentration and platelet count are strong predictors of thrombotic occlusion. Efforts to control high shear thrombosis may focus on VWF in addition to platelet function.

Tissue factor: A potent stimulator of Von Willebrand factor synthesis by human umbilical vein endothelial cells.

Inflammation and dysfunction of endothelial cells are thought to be triggers for the secretion of Von Willebrand factor. The aim of this study was to examine the effects of the inflammatory cytokines interleukin-6 (IL-6), interleukin-8 (IL-8) and tumour necrosis factor-alpha (TNF-α) and the coagulation factors, tissue factor and thrombin on the release and cleavage potential of ultra-large von Willebrand factor (ULVWF) and its cleavage protease by cultured human umbilical vein endothelial cells (HUVEC). HUVEC were treated with IL-6, IL-8, and TNF-α, tissue factor (TF) and thrombin, and combinations thereof for 24 hours under static conditions. The cells were then exposed to shear stress after which the VWF-propeptide levels and the VWF cleavage protease, ADAMTS13 content were measured. All treatments and their combinations, excluding IL-6, significantly stimulated the secretion of VWF from HUVEC. The VWF secretion from the HUVEC was stimulated most by the combination of TF with TNF-α. Slightly lower levels of ADAMTS13 secretion were found with all treatments. This may explain the thrombogenicity of patients with inflammation where extremely high VWF levels and slightly lower ADAMTS13 levels are present.

Concordance of DDAVP Response in Siblings with Von Willebrand Disease or Hemophilia a

Background: Von Willebrand disease (VWD) is the most common congenital bleeding disorder, affecting 1 in 5,000 individuals. Von Willebrand factor (VWF) is an important component in platelet binding to form a primary hemostatic plug. Individuals with VWD have difficulty forming a platelet plug and thus have symptoms such as spontaneous bleeding from mucous membranes, epistaxis, excessive bleeding from wounds, and menorrhagia.DDAVP is a synthetic derivative of the antidiuretic hormone, vasopressin, and is currently used as a mainstay of therapy in VWD. The mechanism of action is thought to be a release of VWF from endothelial cell Weibel-Palade bodies through DDAVP's agonist effect on vasopressin V2 receptors, which, in effect increases plasma concentrations of VWF. Not every patient will respond to treatment, so DDAVP challenge tests are recommended in children with newly diagnosed VWD to identify responders in whom DDAVP may be used for the prevention or treatment of bleeding.Von Willebrand disease is inherited in an autosomal dominant pattern, so multiple members are commonly affected within a family. Siblings are likely to carry the same mutation and may or may not have a similar response to treatment. Our study is aimed at addressing the question of whether siblings of responders or non-responders in DDAVP trials have concordant results and therefore do not need DDAVP trial.Methods: With Human Investigation Committee and IRB approval we performed a retrospective chart review of patients who have undergone DDAVP trial at Children's Hospital of Michigan. Levels of VWF were measured at baseline and then at one, two and four hours. A positive response was defined as an increase in VWF of twofold over baseline, to at least 50 IU/dL. Results were then compared among sibling pairs to see if there was a similar response.Results: We examined 57 patients and had 2 related pairs. Before administering DDAVP, the average baseline of VWF activity, VWF antigen, and Factor VIII were 62.71, 60.95, and 73.58 respectively. 55 patients showed full response to trial with DDAVP. In each of our 2 sibling pairs, both family members showed full response.Discussion: Performing DDAVP trials in every child with symptomatic bleeding is not only costly, but also time consuming. In addition, the IV line placements, infusions, and blood draws involved can be a stressful and painful experience for the child. In a study by Archer et al, later published in BJH (2015), they tested response to DDAVP trial in children with low VWF, and had a 95% response rate. They concluded that, given the high response rate, the use of DDAVP challenge tests in pediatric patients with low VWF should be reduced.Conclusion: In our study, we found that in both sets of siblings, each member showed full response to trial with DDAVP. If there is a genetic correlation to whether or not one responds, these siblings may have benefited from not having to each undergo testing. We would like to further our investigation with a larger data set, and plan to look at a large multicenter cohort. Additionally, although not a primary endpoint of our study our data confirm the findings from the study by Archer et. al.. All of our patients with low VWF activity (>30%<50%) responded to DDAVP and DDAVP trials may not be needed in this group. DisclosuresNo relevant conflicts of interest to declare.

Comparing the Desmopressin (DDAVP) Challenge Test to DDAVP Clinical Response in Pediatric Patients with Von Willebrand's Disease: A Single Center Experience

IntroductionVon Willebrand's disease (VWD) is the most common inherited bleeding disorder. It is characterized by both quantitative and qualitative defects of the von Willebrand Factor (VWF) and generally manifests as menorrhagia, epistaxis and easy bruising. Initial treatment involves the use of desmopressin (DDAVP) that induces the release of VWF from the endothelium. Patients undergo a DDAVP challenge test that involves intravenous DDAVP (IV-DDAVP) infusion with multiple blood draws to determine an increase in the VWD panel. Due to the DDAVP challenge test being a costly and lengthy process, we sought to evaluate the prognostic utility of the challenge test in patients who responded favorably.MethodsA retrospective cohort study was conducted in pediatric patients aged 18 years or younger with a laboratory diagnosis of VWD who underwent the DDAVP challenge test between 2005 and 2015 at our institution. VWD panel results pre and post DDAVP challenge test were obtained from clinical records. A complete response was defined as a 2-fold increase at 1-3 hours or at least 1.5-fold increase at 4-6 hours post-infusion. Patients' clinical records were reviewed for initial presentation and response to DDAVP. SPSS version 23.0.0 was used to analyze demographic and laboratory data, with frequencies and paired t-tests performed for comparison where appropriate.ResultsSeventy two pediatric patients were identified with a diagnosis of VWD: 34 (47%) were VW type 1, 51 (72%) were females and 50 (69%) were Caucasian. Challenge results were available for 48 patients (66%): Post-DDAVP levels were significantly higher than baseline (p = <0.001) in the VWD panel for either times (1-3 or 4-6 hours). Thirty-seven patients (51%) had both challenge results and clinical symptoms reported with 78% (29/37) considered responders and 22% (8/37) considered non-responders to IV-DDAVP. 86% (25/29) of responders were initially placed on intranasal DDAVP (IN-DDAVP) alone while the others received combined therapy with addition of a plasma derived product (PDP). All non-responders initially received DDAVP therapy with some improvement in their clinical symptoms. Eleven (38% of responders) and four (50% of non-responders) were switched to a PDP, mostly for better symptom control. Six responders (16%) had side-effects with DDAVP.ConclusionA high proportion of our patients with VWD responded to the DDAVP challenge test, but subsequently required therapy with a PDP; and half of non-responders were able to use DDAVP. In our pediatric patient population, an initial IV-DDAVP challenge test did not identify those that would respond clinically to IN-DDAVP therapy long-term. Thus, more studies addressing the utility of the challenge test in VWD would be needed before drawing further conclusions on its prognostic value. DisclosuresNo relevant conflicts of interest to declare.

Abstract 15673: CRISPR-Cas9 Gene Editing in Mice Reveals Novel Thrombotic Function of a Human SNP in STXBP5

Introduction: Endothelial cells responds to vascular injury by exocytosis, releasing von Willebrand factor (vWF) into the blood stream. However, the regulation of endothelial vWF release remains poorly understood. Recent genome-wide association studies (GWAS) have identified syntaxin-binding protein 5 (STXBP5) as a candidate gene linked to changes in vWF plasma levels. One top nonsynonymous single nucleotide polymorphism (SNP), rs1039084 (hg19 chr6:g.147635413A&gt;G), encodes p. Asn436Ser substitution (STXBP5-N436S), and is associated with lower plasma vWF, higher bleeding score, and decreased venous thrombosis in humans. We recently discovered that STXBP5 inhibits endothelial vWF exocytosis and regulates thrombosis. However, the role of the STXBP5 genetic variants linked to vWF levels are not completely understood. Hypothesis: We hypothesized that STXBP5-N436S further inhibits endothelial exocytosis than wild type (STXBP5-WT). Methods: We overexpressed STXBP5-WT and STXBP5-N436S in cultured human endothelial cells and measured VWF release changes. Using CRISPR-Cas9 technique, we generated mice carrying the human rs1039084 SNP in Stxbp5 locus (Stxbp5-N437S mice). We conducted phenotypic analyses including endothelial exocytosis, hemostasis, and thrombosis in wild-type and Stxbp5-N437S mice. Results: In human endothelial cells, overexpression of STXBP5-N436S inhibits vWF exocytosis more potently than STXBP5-WT. Germline CRISPR-Cas9 gene editing efficiently and precisely knocked-in the human rs1039084 SNP in murine Stxbp5 locus, without causing detectable off-target genome cleavage. The baseline plasma vWF levels of Stxbp5-N437S mice are similar to WT mice, but Stxbp5-N437S mice showed impaired vWF exocytosis in response to epinephrine challenge. Moreover, Stxbp5-N437S mice have severe hemostasis defects displayed as prolonged bleeding time. Finally, Stxbp5-N437S mice have impaired mesenteric vessel thrombosis and carotid artery thrombosis. We are now studying the effects of the SNP upon STXBP5 structure and function. Conclusions: Our study validates the functional relevance of a candidate SNP identified by GWAS, and suggests that genetic variations within STXBP5 is a risk factor for thromboembolic disease.

Mechanism and functional impact of CD40 ligand-induced von Willebrand factor release from endothelial cells.

Co-stimulation via CD154 binding to CD40, pivotal for both innate and adaptive immunity, may also link haemostasis to vascular remodelling. Here we demonstrate that human platelet-bound or recombinant soluble CD154 (sCD154) elicit the release from and tethering of ultra-large (UL) von Willebrand factor (vWF) multimers to the surface of human cultured endothelial cells (ECs) exposed to shear stress. This CD40-mediated ULVWF multimer release from the Weibel-Palade bodies was triggered by consecutive activation of TRAF6, the tyrosine kinase c-Src and phospholipase Cγ1 followed by inositol-1,4,5 trisphosphate-mediated calcium mobilisation. Subsequent exposure to human washed platelets caused ULVWF multimer-platelet string formation on the EC surface in a shear stress-dependent manner. Platelets tethered to these ULVWF multimers exhibited P-selectin on their surface and captured labelled monocytes from the superfusate. When exposed to shear stress and sCD154, native ECs from wild-type but not CD40 or vWF-deficient mice revealed a comparable release of ULVWF multimers to which murine washed platelets rapidly adhered, turning P-selectin-positive and subsequently capturing monocytes from the perfusate. This novel CD154-provoked ULVWF multimer-platelet string formation at normal to fast flow may contribute to vascular remodelling processes requiring the perivascular or intravascular accumulation of pro-inflammatory macrophages such as arteriogenesis or atherosclerosis.

Mediators and molecular pathways involved in the regulation of neutrophil extracellular trap formation mediated by activated platelets.

In addition to being key elements in hemostasis and thrombosis, platelets amplify neutrophil function. We aimed to gain further insight into the stimuli, mediators, molecular pathways, and regulation of neutrophil extracellular trap formation mediated by human platelets. Platelets stimulated by lipopolysaccharide, a wall component of gram-negative bacteria, Pam3-cysteine-serine-lysine 4, a mimetic of lipopeptide from gram-positive bacteria, Escherichia coli, Staphylococcus aureus, or physiologic platelet agonists promoting neutrophil extracellular trap formation and myeloperoxidase-associated DNA activity under static and flow conditions. Although P-selectin or glycoprotein IIb/IIIa were not involved, platelet glycoprotein Ib, neutrophil cluster of differentiation 18, and the release of von Willebrand factor and platelet factor 4 seemed to be critical for the formation of neutrophil extracellular traps. The secretion of these molecules depended on thromboxane A(2) production triggered by lipopolysaccharide or Pam3-cysteine-serine-lysine 4 but not on high concentrations of thrombin. Accordingly, aspirin selectively inhibited platelet-mediated neutrophil extracellular trap generation. Signaling through extracellular signal-regulated kinase, phosphatidylinositol 3-kinase, and Src kinases, but not p38 or reduced nicotinamide adenine dinucleotide phosphate oxidase, was involved in platelet-triggered neutrophil extracellular trap release. Platelet-mediated neutrophil extracellular trap formation was inhibited by prostacyclin. Our results support a role for stimulated platelets in promoting neutrophil extracellular trap formation, reveal that an endothelium-derived molecule contributes to limiting neutrophil extracellular trap formation, and highlight platelet inhibition as a potential target for controlling neutrophil extracellular trap cell death.

All-trans retinoic acid modulates the balance of ADAMTS13 and VWF in human microvascular endothelial cells

PurposeTo better understand the antithrombotic property of All-trans retinoic acid (ATRA), we investigated whether ATRA may affect the balance between ADAMTS13 and von Willebrand factor (VWF) in human microvascular endothelial cell. MethodsCompared to tumor necrosis factor-alpha (TNF-α), we observed the effects of ATRA on the expression of ADAMTS13 and VWF. ADAMTS13mRNA in human microvascular endothelial cell (HMEC-1 cell line) were detected by real-time polymerase chain reaction amplification (RT-PCR). The levels of ADAMTS13 and VWF antigen were detected by western blot or enzyme-linked immunosorbent assay (ELISA), and the proteolytic activity of ADAMTS13 was also determined by using GST-VWF73-His peptide as a specific substrate. ResultsATRA significantly upregulated the expression of ADAMTS13mRNA in HMEC-1, while TNF-α inhibited ADAMTS13mRNA expression. ATRA could reverse the inhibition expression of ADAMTS13 by TNF-α. The results were confirmed from the levels of ADAMTS13 protein and its activity, while ATRA had no significant affection on triggering release of VWF. ConclusionsThis study provides the evidence that ATRA modulates the balance of ADAMTS13 and VWF in human microvascular endothelial cell, which might be a very relevant compartment for the antithrombotic property of ATRA.

Sequelae of radiotherapy-induced coronary artery disease in breast reconstruction

Sir, Approximately 37% of breast cancer patients who undergo breast conserving surgery or are found to have positive lymph nodes receive adjuvant radiotherapy [1]. Known side-effects of radiotherapy include wound breakdown, scarring, and radiation-induced angiosarcoma [2]. Less commonly cardiovascular complications can occur secondary to unwanted indirect irradiation of the heart, including coronary artery disease, pericarditis, and cardiomyopathy [3]. Chemotherapy for breast cancer can also induce cardiotoxicity, mainly secondary to anthracycline chemotherapy, which can lead to irreversible heart failure [4]. Cardiotoxicity has also been described with fluorouracil (5-FU) as angina-like chest pain, with incidences ranging from 1 to 68 % in the literature; cardiac events occur generally within 5 days after first administration [4]. We present an interesting case of a 45-year-old lady who underwent left mastectomy for breast cancer. This was followed by non-anthracycline chemotherapy and adjuvant radiotherapy receiving a total of 40 Gy over a course of 3 weeks. Two years later, she underwent delayed left breast reconstruction with a pedicled extended latissimus dorsi (ELD) musculocutaneous flap with a 220 cc round textured implant. She completed 5 years of tamoxifen and breast follow-up without any complications and remained diseasefree. Seven years after completion of radiotherapy, she presented to the emergency department with syncopal episodes and a four week history of exertional angina. Although she thought this pain was implant-related, her blood results showed a positive troponin (peak of 75 ng/L) and ECG’s showed T wave inversion in all the anterior leads, indicating an acute myocardial infarction. Coronary angiogram revealed critical coronary disease involving the left anterior descending, left circumflex and right coronary arteries (i.e. triple vessel disease). Her general risk factors were that she was an ex-smoker (2 pack years) with a family history of coronary heart disease. The cardiologists believed that her left-sided breast radiotherapy was the main predisposing factor for such unique triple vessel disease in a young patient. She was referred for urgent coronary artery bypass graft (CABG). Though the preoperative CT showed that the medial margin of the left breast implant was near the mid-sternal line (Fig. 1), this was probably positional, as clinically it appeared aesthetically acceptable (Fig. 2a). The patient underwent an uncomplicated triple CABG, and the left breast implant was preserved as it had not encroached the median sternotomy incision. Her postoperative breast aesthetic appearance remained unchanged (Fig. 2b). This letter highlights an interesting case of radiotherapyinduced triple vessel coronary artery disease and subsequent acute myocardial infarction in a young woman, following left sided irradiation for breast cancer. Microscopically, endothelial injury may occur secondary to the formation of reactive oxygen species and the activation of lysosomal enzymes within intima and media. Radiation promotes the adhesion of endothelial cells and the release of VonWillebrand factor inducing a prothrombotic state. Overall, atherosclerosis is * Hazim Sadideen hazim.sadideen@doctors.org.uk

Von Willebrand factor fibers promote cancer-associated platelet aggregation in malignant melanoma of mice and humans

AbstractTumor-mediated procoagulatory activity leads to venous thromboembolism and supports metastasis in cancer patients. A prerequisite for metastasis formation is the interaction of cancer cells with endothelial cells (ECs) followed by their extravasation. Although it is known that activation of ECs and the release of the procoagulatory protein von Willebrand factor (VWF) is essential for malignancy, the underlying mechanisms remain poorly understood. We hypothesized that VWF fibers in tumor vessels promote tumor-associated thromboembolism and metastasis. Using in vitro settings, mouse models, and human tumor samples, we showed that melanoma cells activate ECs followed by the luminal release of VWF fibers and platelet aggregation in tumor microvessels. Analysis of human blood samples and tumor tissue revealed that a promoted VWF release combined with a local inhibition of proteolytic activity and protein expression of ADAMTS13 (a disintegrin-like and metalloproteinase with thrombospondin type I repeats 13) accounts for this procoagulatory milieu. Blocking endothelial cell activation by the low-molecular-weight heparin tinzaparin was accompanied by a lack of VWF networks and inhibited tumor progression in a transgenic mouse model. Our findings implicate a mechanism wherein tumor-derived vascular endothelial growth factor-A (VEGF-A) promotes tumor progression and angiogenesis. Thus, targeting EC activation envisions new therapeutic strategies attenuating tumor-related angiogenesis and coagulation.