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Aims: P-selectin is a key surface adhesion molecule for the interaction of platelets with leukocytes. We have shown previously that the N-terminal domain of Staphylococcus aureus extracellular fibrinogen-binding protein (Efb) binds to P-selectin and interferes with platelet-leukocyte aggregate formation. Here, we aimed to identify the minimal Efb motif required for binding platelets and to characterize its ability to interfering with the formation of platelet-leukocyte aggregates. Methods and Results: Using a library of synthetic peptides, we mapped the platelet-binding site to a continuous 20 amino acid stretch. The peptide Efb68-87 was able to bind to resting and, to a greater extent, thrombin-stimulated platelets in the absence of fibrinogen. Dot blots, pull-down assays and P-selectin glycoprotein ligand-1 (PSGL-1) competitive binding experiments identified P-selectin as the cellular docking site mediating Efb68-87 platelet binding. Accordingly, Efb68-87 did not bind to other blood cells and captured platelets from human whole blood under low shear stress conditions. Efb68-87 did not affect platelet activation as tested by aggregometry, flow cytometry and immunoblotting, but inhibited the formation of platelet-leukocyte aggregates (PLAs). Efb68-87 also interfered with the platelet-dependent stimulation of neutrophil extracellular traps (NETs) formation in vitro. Conclusions: We have identified Efb68-87 as a novel selective platelet-binding peptide. Efb68-87 binds directly to P-selectin and inhibits interactions of platelets with leukocytes that lead to PLA and NET formation. As PLAs and NETs play a key role in thromboinflammation, Efb68-87 is an exciting candidate for the development of novel selective inhibitors of the proinflammatory activity of platelets.

Aims: P-selectin is a key surface adhesion molecule for the interaction of platelets with leukocytes. We have shown previously that the N-terminal domain of Staphylococcus aureus extracellular fibrinogen-binding protein (Efb) binds to P-selectin and interferes with platelet-leukocyte aggregate formation. Here, we aimed to identify the minimal Efb motif required for binding platelets and to characterize its ability to interfering with the formation of platelet-leukocyte aggregates. Methods and Results: Using a library of synthetic peptides, we mapped the platelet-binding site to a continuous 20 amino acid stretch. The peptide Efb68-87 was able to bind to resting and, to a greater extent, thrombin-stimulated platelets in the absence of fibrinogen. Dot blots, pull-down assays and P-selectin glycoprotein ligand-1 (PSGL-1) competitive binding experiments identified P-selectin as the cellular docking site mediating Efb68-87 platelet binding. Accordingly, Efb68-87 did not bind to other blood cells and captured platelets from human whole blood under low shear stress conditions. Efb68-87 did not affect platelet activation as tested by aggregometry, flow cytometry and immunoblotting, but inhibited the formation of platelet-leukocyte aggregates (PLAs). Efb68-87 also interfered with the platelet-dependent stimulation of neutrophil extracellular traps (NETs) formation in vitro. Conclusions: We have identified Efb68-87 as a novel selective platelet-binding peptide. Efb68-87 binds directly to P-selectin and inhibits interactions of platelets with leukocytes that lead to PLA and NET formation. As PLAs and NETs play a key role in thromboinflammation, Efb68-87 is an exciting candidate for the development of novel selective inhibitors of the proinflammatory activity of platelets.

Background Persistent symptoms including breathlessness, fatigue, and decreased exercise tolerance have been reported in patients after acute SARS-CoV-2 infection. The biological mechanisms underlying this “long COVID” syndrome remain unknown. However, autopsy studies have highlighted the key roles played by pulmonary endotheliopathy and microvascular immunothrombosis in acute COVID-19. Objectives To assess whether endothelial cell activation may be sustained in convalescent COVID-19 patients and contribute to long COVID pathogenesis. Patients and Methods Fifty patients were reviewed at a median of 68 days following SARS-CoV-2 infection. In addition to clinical workup, acute phase markers, endothelial cell (EC) activation and NETosis parameters and thrombin generation were assessed. Results Thrombin generation assays revealed significantly shorter lag times (p < .0001, 95% CI −2.57 to −1.02 min), increased endogenous thrombin potential (p = .04, 95% CI 15–416 nM/min), and peak thrombin (p < .0001, 95% CI 39–93 nM) in convalescent COVID-19 patients. These prothrombotic changes were independent of ongoing acute phase response or active NETosis. Importantly, EC biomarkers including von Willebrand factor antigen (VWF:Ag), VWF propeptide (VWFpp), and factor VIII were significantly elevated in convalescent COVID-19 compared with controls (p = .004, 95% CI 0.09–0.57 IU/ml; p = .009, 95% CI 0.06–0.5 IU/ml; p = .04, 95% CI 0.03–0.44 IU/ml, respectively). In addition, plasma soluble thrombomodulin levels were significantly elevated in convalescent COVID-19 (p = .02, 95% CI 0.01–2.7 ng/ml). Sustained endotheliopathy was more frequent in older, comorbid patients, and those requiring hospitalization. Finally, both plasma VWF:Ag and VWFpp levels correlated inversely with 6-min walk tests. Conclusions Collectively, our findings demonstrate that sustained endotheliopathy is common in convalescent COVID-19 and raise the intriguing possibility that this may contribute to long COVID p

Background Persistent symptoms including breathlessness, fatigue, and decreased exercise tolerance have been reported in patients after acute SARS-CoV-2 infection. The biological mechanisms underlying this “long COVID” syndrome remain unknown. However, autopsy studies have highlighted the key roles played by pulmonary endotheliopathy and microvascular immunothrombosis in acute COVID-19. Objectives To assess whether endothelial cell activation may be sustained in convalescent COVID-19 patients and contribute to long COVID pathogenesis. Patients and Methods Fifty patients were reviewed at a median of 68 days following SARS-CoV-2 infection. In addition to clinical workup, acute phase markers, endothelial cell (EC) activation and NETosis parameters and thrombin generation were assessed. Results Thrombin generation assays revealed significantly shorter lag times (p < .0001, 95% CI −2.57 to −1.02 min), increased endogenous thrombin potential (p = .04, 95% CI 15–416 nM/min), and peak thrombin (p < .0001, 95% CI 39–93 nM) in convalescent COVID-19 patients. These prothrombotic changes were independent of ongoing acute phase response or active NETosis. Importantly, EC biomarkers including von Willebrand factor antigen (VWF:Ag), VWF propeptide (VWFpp), and factor VIII were significantly elevated in convalescent COVID-19 compared with controls (p = .004, 95% CI 0.09–0.57 IU/ml; p = .009, 95% CI 0.06–0.5 IU/ml; p = .04, 95% CI 0.03–0.44 IU/ml, respectively). In addition, plasma soluble thrombomodulin levels were significantly elevated in convalescent COVID-19 (p = .02, 95% CI 0.01–2.7 ng/ml). Sustained endotheliopathy was more frequent in older, comorbid patients, and those requiring hospitalization. Finally, both plasma VWF:Ag and VWFpp levels correlated inversely with 6-min walk tests. Conclusions Collectively, our findings demonstrate that sustained endotheliopathy is common in convalescent COVID-19 and raise the intriguing possibility that this may contribute to long COVID p

Background Laboratory analyses of blood samples are essential for diagnostics and therapy monitoring of patients with bleeding and thromboembolic diseases. Following publication of the core curriculum for clinical thrombosis and hemostasis, the International Society on Thrombosis and Haemostasis (ISTH) recognized that thrombosis and hemostasis laboratory specialists require distinct competencies that differ from medical doctors working clinically with patients. To address this gap the ISTH formed a working group of international hemostasis and thrombosis laboratory specialists to develop an evidence‐based core curriculum for laboratory specialists. Objective This research sought consensus from the international community on core competencies required for laboratory specialists in thrombosis and hemostasis. Methods A draft list of 64 competencies was developed and an online stakeholder survey was circulated electronically to 15 302 ISTH members and contacts in the wider international community. The results were analyzed and used to develop the final approved core curriculum. Results Three hundred and thirty responses contained meaningful data, with broad international representation of specialists. No draft competencies were excluded, and 58 were rated as “does” or “shows how.” The Leik measure of consensus for most competences was “moderate” (n = 30) or “fair” (n = 32). Conclusions The development of an international core curriculum for laboratory specialists provides a foundation for the development and enhancement of education and quality management of the laboratory. Although there is no formal designation for laboratory specialists, international governing bodies and regulatory organizations are encouraged to consider the diagnostic core curriculum for development and accreditation of more standardized educational programs and formal assessment across jurisdictions.

Background Laboratory analyses of blood samples are essential for diagnostics and therapy monitoring of patients with bleeding and thromboembolic diseases. Following publication of the core curriculum for clinical thrombosis and hemostasis, the International Society on Thrombosis and Haemostasis (ISTH) recognized that thrombosis and hemostasis laboratory specialists require distinct competencies that differ from medical doctors working clinically with patients. To address this gap the ISTH formed a working group of international hemostasis and thrombosis laboratory specialists to develop an evidence‐based core curriculum for laboratory specialists. Objective This research sought consensus from the international community on core competencies required for laboratory specialists in thrombosis and hemostasis. Methods A draft list of 64 competencies was developed and an online stakeholder survey was circulated electronically to 15 302 ISTH members and contacts in the wider international community. The results were analyzed and used to develop the final approved core curriculum. Results Three hundred and thirty responses contained meaningful data, with broad international representation of specialists. No draft competencies were excluded, and 58 were rated as “does” or “shows how.” The Leik measure of consensus for most competences was “moderate” (n = 30) or “fair” (n = 32). Conclusions The development of an international core curriculum for laboratory specialists provides a foundation for the development and enhancement of education and quality management of the laboratory. Although there is no formal designation for laboratory specialists, international governing bodies and regulatory organizations are encouraged to consider the diagnostic core curriculum for development and accreditation of more standardized educational programs and formal assessment across jurisdictions.

The intermediate filament protein nestin is expressed during embryonic development, but considered largely restricted to areas of regeneration in the adult. Here, we perform a body-wide transcriptome and protein-profiling analysis to reveal that nestin is constitutively, and highly-selectively, expressed in adult human endothelial cells (EC), independent of proliferative status. Correspondingly, we demonstrate that it is not a marker for tumour EC in multiple malignancy types. Imaging of EC from different vascular beds reveals nestin subcellular distribution is shear-modulated. siRNA inhibition of nestin increases EC proliferation, and nestin expression is reduced in atherosclerotic plaque neovessels. eQTL analysis reveals an association between SNPs linked to cardiovascular disease and reduced aortic EC nestin mRNA expression. Our study challenges the dogma that nestin is a marker of proliferation, and provides insight into its regulation and function in EC. Furthermore, our systems-based approach can be applied to investigate body-wide expression profiles of any candidate protein., Export Date: 22 October 2018; Article; Correspondence Address: Butler, L.M.; Science for Life Laboratory, School of Biotechnology, Kungliga Tekniska Högskolan (KTH) Royal Institute of TechnologySweden; email: Lynn.Butler@ki.se. QC 20181127

Endothelial cells line blood vessels and regulate hemostasis, inflammation, and blood pressure. Proteins critical for these specialized functions tend to be predominantly expressed in endothelial cells across vascular beds. Here, we present a systems approach to identify a panel of human endothelial-enriched genes using global, body-wide transcriptomics data from 124 tissue samples from 32 organs. We identified known and unknown endothelial-enriched gene transcripts and used antibody-based profiling to confirm expression across vascular beds. The majority of identified transcripts could be detected in cultured endothelial cells from various vascular beds, and we observed maintenance of relative expression in early passage cells. In summary, we describe a widely applicable method to determine cell-type-specific transcriptome profiles in a whole-organism context, based on differential abundance across tissues. We identify potential vascular drug targets or endothelial biomarkers and highlight candidates for functional studies to increase understanding of the endothelium in health and disease., QC 20170310

Objective: Proliferation of smooth muscle cells (SMCs) can stabilize atherosclerotic lesions but the molecular mechanisms that regulate this process in humans are largely unknown. We have previously shown that heparan sulfate proteoglycans (HSPGs), such as perlecan, regulate SMC growth in animal models by modulating heparin-binding mitogens. Since perlecan is expressed at low levels in human atherosclerosis, we speculated that the effect of heparan sulfate (HS) in human disease was rather influenced by HS degradation and investigated the expression of heparanase (HPSE) in human carotid endarterectomies. Methods and results: Gene expression analysis from 127 endarterectomies in the BiKE database revealed increased expression of HPSE in carotid plaques compared with normal arteries, and a further elevation in symptomatic lesions. Increased HPSE protein expression in symptomatic plaque tissue was verified by tissue microarrays. HPSE mRNA levels correlated positively with expression of inflammatory markers IL-18, RANTES and IL-1β, and also T-cell co-stimulatory molecules, such as B7.2, CD28, LFA-1 and 4-1BB. Previously reported single nucleotide polymorphisms within HPSE were associated with differential mRNA expression in plaques. Immunohistochemistry revealed that inflammatory cells were major producers of HPSE in plaque tissue. HPSE immunoreactivity was also observed in SMCs adjacent to the necrotic core and was co-localized to deposits of fibrin. Conclusions: This study demonstrates increased expression of HPSE in human atherosclerosis associated with inflammation, coagulation and plaque instability. Since HS can regulate SMC proliferation and influence plaque stability, the findings suggest that HPSE degradation of HS take part in the regulation of SMC function in human atherosclerosis.

BACKGROUND: Particulate matter (PM) is a key component of ambient air pollution and has been associated with an increased risk of thrombotic events and mortality. The underlying mechanisms remain unclear. OBJECTIVES: To study the mechanisms of PM-driven procoagulant activity in human plasma and to investigate mainly, the coagulation driven by ultrafine particles (UFPs; < 0.1 μm) in genetically modified mice. METHODS: Thrombin generation in response to PM of different sizes was assessed in normal human platelet-poor plasma, as well as in plasmas deficient in the intrinsic pathway proteases factors XII (FXII) or XI (FXI). In addition, UFPs were intratracheally instilled in wild-type (WT) and FXII-deficient (FXII(-/-) ) mice and plasma thrombin generation was analyzed in plasma from treated mice at 4 and 20 h post-exposure. RESULTS: In normal human plasma, thrombin generation was enhanced in the presence of PM, whereas PM-driven thrombin formation was completely abolished in FXII- and FXI-deficient plasma. UFPs induced a transient increase in tissue factor (TF)-driven thrombin formation at 4 h post-instillation in WT mice compared with saline instillation. Intratracheal instillation of UFPs resulted in a procoagulant response in WT mice plasma at 20 h, whereas it was entirely suppressed in FXII(-/-) mice. CONCLUSIONS: Overall, the data suggest that PM promotes its early procoagulant actions mostly through the TF-driven extrinsic pathway of coagulation, whereas PM-driven long lasting thrombogenic effects are predominantly mediated via formation of activated FXII. Hence, FXII-driven thrombin formation may be relevant to an enhanced thrombotic susceptibility upon chronic exposure to PM in humans.