<div><h4>Altered whole blood thrombin generation and hyperresponsive platelets in patients with pancreatic cancer.</h4><i>Willems RAL, Konings J, Huskens D, Middelveld H, ... de Laat B, Roest M</i><br /><b>Background</b><br />Thromboembolic disease is a major complication in patients with pancreatic ductal adenocarcinoma (PDAC). Patients with PDAC often have altered blood cell counts, which are associated with venous thromboembolism (VTE) development. The high thrombotic risk in patients with PDAC may be partially caused by procoagulant blood cells.<br /><b>Objectives</b><br />The aim of this study was to compare blood cell-dependent coagulation between patients with PDAC (n = 18) and healthy controls matched for age and sex (n = 18).<br /><b>Methods</b><br />Thrombin generation (TG) was measured in whole blood (WB) and plasma. The capacity of platelets to release granules (PGRCs) was measured in WB. We explored the occurrence of thromboembolic events in patients with PDAC during a 6-month follow-up.<br /><b>Results</b><br />Patients showed an increased endogenous thrombin potential in WB compared with controls. This difference was not observed in plasma, indicating a procoagulant effect of blood cells. Both in WB and plasma, the lag time was prolonged in patients compared with controls. Patients had hyperresponsive platelets, with a shorter time to peak granule release. Of the 18 patients with PDAC, 4 developed a venous thromboembolism (22%) and 1 developed an arterial thrombosis (6%). A shorter lag time in WB, but not in plasma, and an increased PGRC were associated with thromboembolic events.<br /><b>Conclusion</b><br />Patients with PDAC have an increased and delayed WB TG coagulation profile compared with controls. A shorter lag time in WB TG and increased PGRC are associated with the incidence of thromboembolic events. Platelets appear to be key players in thrombosis development. Measuring hemostasis in WB could improve thrombosis risk estimation in patients with PDAC.<br /><br />Copyright © 2024 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.<br /><br /><small>J Thromb Haemost: 01 Apr 2024; 22:1132-1144</small></div>

<div><h4>Density-based lipoprotein depletion improves extracellular vesicle isolation and functional analysis.</h4><i>Merij LB, da Silva LR, Palhinha L, Gomes MT, ... de Oliveira Trugilho MR, Hottz ED</i><br /><b>Background</b><br />Blood plasma is the main source of extracellular vesicles (EVs) in clinical studies aiming to identify biomarkers and to investigate pathophysiological processes, especially regarding EV roles in inflammation and thrombosis. However, EV isolation from plasma has faced the fundamental issue of lipoprotein contamination, representing an important bias since lipoproteins are highly abundant and modulate cell signaling, metabolism, and thromboinflammation.<br /><b>Objectives</b><br />Here, we aimed to isolate plasma EVs after depleting lipoproteins, thereby improving sample purity and EV thromboinflammatory analysis.<br /><b>Methods</b><br />Density-based gradient ultracentrifugation (G-UC) was used for lipoprotein depletion before EV isolation from plasma through size-exclusion chromatography (SEC) or serial centrifugation (SC). Recovered EVs were analyzed by size, concentration, cellular source, ultrastructure, and bottom-up proteomics.<br /><b>Results</b><br />G-UC efficiently separated lipoproteins from the plasma, allowing subsequent EV isolation through SEC or SC. Combined analysis from EV proteomics, cholesterol quantification, and apoB-100 detection confirmed the significant reduction in lipoproteins from isolated EVs. Proteomic analysis identified similar gene ontology and cellular components in EVs, regardless of lipoprotein depletion, which was consistent with similar EV cellular sources, size, and ultrastructure by flow cytometry and transmission electron microscopy. Importantly, lipoprotein depletion increased the detection of less abundant proteins in EV proteome and enhanced thromboinflammatory responses of platelets and monocytes stimulated in vitro with EV isolates.<br /><b>Conclusion</b><br />Combination of G-UC+SEC significantly reduced EV lipoprotein contamination without interfering in EV cellular source, gene ontology, and ultrastructure, allowing the recovery of highly pure EVs with potential implications for functional assays and proteomic and lipidomic analyses.<br /><br />Copyright © 2024 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.<br /><br /><small>J Thromb Haemost: 01 May 2024; 22:1372-1388</small></div>

<div><h4>A new look at an old body: molecular determinants of Weibel-Palade body composition and von Willebrand factor exocytosis.</h4><i>Hordijk S, Carter T, Bierings R</i><br /><AbstractText>Endothelial cells, forming a monolayer along blood vessels, intricately regulate vascular hemostasis, inflammatory responses, and angiogenesis. A key determinant of these functions is the controlled secretion of Weibel-Palade bodies (WPBs), which are specialized endothelial storage organelles housing a presynthesized pool of the hemostatic protein von Willebrand factor and various other hemostatic, inflammatory, angiogenic, and vasoactive mediators. This review delves into recent mechanistic insights into WPB biology, including the biogenesis that results in their unique morphology, the acquisition of intraluminal vesicles and other cargo, and the contribution of proton pumps to organelle acidification. Additionally, in light of a number of proteomic approaches to unravel the regulatory networks that control WPB formation and secretion, we provide a comprehensive overview of the WPB exocytotic machinery, including their molecular and cellular mechanisms.</AbstractText><br /><br />Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.<br /><br /><small>J Thromb Haemost: 01 May 2024; 22:1290-1303</small></div>

<div><h4>Long-term simulated microgravity fosters carotid aging-like changes via Piezo1.</h4><i>Zhang J, Wang X, Fu Z, Xing C, ... Li J, Gao F</i><br /><b>Aims</b><br />Elucidating the impacts of long-term spaceflight on cardiovascular health is urgently needed in face of the rapid development of human space exploration. Recent reports including the NASA Twins Study on vascular deconditioning and aging of astronauts in spaceflight are controversial. The aims of this study were to elucidate whether long-term microgravity promotes vascular aging and the underlying mechanisms.<br /><b>Methods and results</b><br />Hindlimb unloading (HU) by tail suspension was used to simulate microgravity in rats and mice. The dynamic changes of carotid stiffness in rats during 8 weeks of HU were determined. Simulated microgravity led to carotid artery aging-like changes as evidenced by increased stiffness, thickness, fibrosis, and elevated senescence biomarkers in the HU rats. Specific deletion of the mechanotransducer Piezo1 in vascular smooth muscles significantly blunted these aging-like changes in mice. Mechanistically, mechanical stretch-induced activation of Piezo1 elevated microRNA-582-5p in vascular smooth muscle cells, with resultant enhanced synthetic cell phenotype and increased collagen deposition via PTEN/PI3K/Akt signalling. Importantly, inhibition of miRNA-582-5p alleviated carotid fibrosis and stiffness not only in HU rats but also in aged rats.<br /><b>Conclusions</b><br />Long-term simulated microgravity induces carotid aging-like changes via the mechanotransducer Piezo1-initiated and miRNA-mediated mechanism.<br /><br />© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.<br /><br /><small>Cardiovasc Res: 30 Apr 2024; 120:548-559</small></div>

<div><h4>Histone content, and thus DNA content, is associated with differential in vitro lysis of acute ischemic stroke clots.</h4><i>Akkipeddi SMK, Rahmani R, Ellens NR, Kohli GS, ... Morrell CN, Bender MT</i><br /><b>Background</b><br />Fibrin, von Willebrand factor, and extracellular DNA from neutrophil extracellular traps all contribute to acute ischemic stroke thrombus integrity.<br /><b>Objectives</b><br />In this study, we explored how the proteomic composition of retrieved thromboemboli relates to susceptibility to lysis with distinct thrombolytics.<br /><b>Methods</b><br />Twenty-six retrieved stroke thromboemboli were portioned into 4 segments, with each subjected to 1 hour of in vitro lysis at 37 °C in 1 of 4 solutions: tissue plasminogen activator (tPA), tPA + von Willebrand factor-cleaving ADAMTS-13, tPA + DNA-cleaving deoxyribonuclease (DNase) I, and all 3 enzymes. Lysis, characterized by the percent change in prelysis and postlysis weight, was compared across the solutions and related to the corresponding abundance of proteins identified on mass spectrometry for each of the thromboemboli used in lysis.<br /><b>Results</b><br />Solutions containing DNase resulted in approximately 3-fold greater thrombolysis than that with the standard-of-care tPA solution (post hoc Tukey, P < .01 for all). DNA content was directly related to lysis in solutions containing DNase (Spearman\'s ρ > 0.39 and P < .05 for all significant histones) and inversely related to lysis in solutions without DNase (Spearman\'s ρ < -0.40 and P < .05 for all significant histones). Functional analysis suggests distinct pathways associated with susceptibility to thrombolysis with tPA (platelet-mediated) or DNase (innate immune system-mediated).<br /><b>Conclusion</b><br />This study demonstrates synergy of DNase and tPA in thrombolysis of stroke emboli and points to DNase as a potential adjunct to our currently limited selection of thrombolytics in treating acute ischemic stroke.<br /><br />Copyright © 2024 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.<br /><br /><small>J Thromb Haemost: 01 May 2024; 22:1410-1420</small></div>

<div><h4>Prothrombin conversion and thrombin decay in patients with cirrhosis-role of prothrombin and antithrombin deficiencies.</h4><i>Sinegre T, Abergel A, Lecompte T, Lebreton A</i><br /><b>Background</b><br />Thrombin generation (TG) in the presence of thrombomodulin (TG-TM) in the plasma of patients with cirrhosis (PWC) is tilted toward a hypercoagulable phenotype. Low protein C and elevated factor VIII levels play a role, but other determinants, such as the prothrombin/antithrombin pair, must also be studied.<br /><b>Objectives</b><br />The objectives were (i) to quantitatively assess the subprocesses (prothrombin conversion and thrombin decay) and (ii) to understand the underlying mechanism by studying TG dynamics after prothrombin and antithrombin plasma level correction in PWC.<br /><b>Methods</b><br />We studied TG-TM in plasma samples of 36 healthy controls (HCs) and 41 PWC with prothrombin and antithrombin levels of <70% and after their correction. We initiated coagulation with an intermediate picomolar concentration of tissue factor. We determined the overall thrombin potential, prothrombin conversion, and thrombin decay.<br /><b>Results</b><br />TG-TM was increased in PWC compared with HC due to impaired thrombin inhibition. Indeed, thrombin decay capacity (min<sup>-1</sup>) decreased from 0.37 (0.35-0.40) in HC to 0.33 (0.30-0.37) in the Child-Turcotte-Pugh A (CTP-A; P = .09), 0.27 (0.26-0.30) in the CTP-B (P < .001), and 0.20 (0.19-0.20) in the CTP-C (P < .001) group. Concomitant correction of prothrombin and antithrombin increased endogenous thrombin potential with prothrombin conversion surpassing thrombin decay. By contrast, when we corrected only antithrombin, TG-TM was normalized and even consistent with a hypocoagulable phenotype in the CTP-C group.<br /><b>Conclusion</b><br />Our results highlight that in PWC, hypercoagulability (evidenced in the presence of TM) is due to impaired thrombin decay, whereas low prothrombin levels do not translate into decreased prothrombin conversion, likely due to altered TM-activated protein C negative feedback.<br /><br />Copyright © 2024 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.<br /><br /><small>J Thromb Haemost: 01 May 2024; 22:1347-1357</small></div>

<div><h4>Histone demethylase KDM5 regulates cardiomyocyte maturation by promoting fatty acid oxidation, oxidative phosphorylation, and myofibrillar organization.</h4><i>Deogharia M, Venegas-Zamora L, Agrawal A, Shi M, ... Marian AJ, Gurha P</i><br /><b>Aims</b><br />Human pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) provide a platform to identify and characterize factors that regulate the maturation of CMs. The transition from an immature foetal to an adult CM state entails coordinated regulation of the expression of genes involved in myofibril formation and oxidative phosphorylation (OXPHOS) among others. Lysine demethylase 5 (KDM5) specifically demethylates H3K4me1/2/3 and has emerged as potential regulators of expression of genes involved in cardiac development and mitochondrial function. The purpose of this study is to determine the role of KDM5 in iPSC-CM maturation.<br /><b>Methods and results</b><br />KDM5A, B, and C proteins were mainly expressed in the early post-natal stages, and their expressions were progressively downregulated in the post-natal CMs and were absent in adult hearts and CMs. In contrast, KDM5 proteins were persistently expressed in the iPSC-CMs up to 60 days after the induction of myogenic differentiation, consistent with the immaturity of these cells. Inhibition of KDM5 by KDM5-C70 -a pan-KDM5 inhibitor, induced differential expression of 2372 genes, including upregulation of genes involved in fatty acid oxidation (FAO), OXPHOS, and myogenesis in the iPSC-CMs. Likewise, genome-wide profiling of H3K4me3 binding sites by the cleavage under targets and release using nuclease assay showed enriched of the H3K4me3 peaks at the promoter regions of genes encoding FAO, OXPHOS, and sarcomere proteins. Consistent with the chromatin and gene expression data, KDM5 inhibition increased the expression of multiple sarcomere proteins and enhanced myofibrillar organization. Furthermore, inhibition of KDM5 increased H3K4me3 deposits at the promoter region of the ESRRA gene and increased its RNA and protein levels. Knockdown of ESRRA in KDM5-C70-treated iPSC-CM suppressed expression of a subset of the KDM5 targets. In conjunction with changes in gene expression, KDM5 inhibition increased oxygen consumption rate and contractility in iPSC-CMs.<br /><b>Conclusion</b><br />KDM5 inhibition enhances maturation of iPSC-CMs by epigenetically upregulating the expressions of OXPHOS, FAO, and sarcomere genes and enhancing myofibril organization and mitochondrial function.<br /><br />© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.<br /><br /><small>Cardiovasc Res: 07 May 2024; 120:630-643</small></div>

<div><h4>A dispensable role of oligodendrocyte-derived laminin-α5 in brain homeostasis and intracerebral hemorrhage.</h4><i>Kang M, Nirwane A, Ruan J, Adithan A, ... Xu L, Yao Y</i><br /><AbstractText>Laminin, a major component of the basal lamina in the CNS, is also expressed in oligodendrocytes (OLs). However, the function of OL-derived laminin remains largely unknown. Here, we performed loss-of-function studies using two OL-specific laminin-α5 conditional knockout mouse lines. Both mutants were grossly normal and displayed intact blood-brain barrier (BBB) integrity. In a mouse model of intracerebral hemorrhage (ICH), control mice and both mutants exhibited comparable hematoma size and neurological dysfunction. In addition, similar levels of hemoglobin and IgG leakage were detected in the mutant brains compared to the controls, indicating comparable BBB damage. Consistent with this finding, subsequent studies revealed no differences in tight junction protein (TJP) and caveolin-1 expression among control and knockout mice, suggesting that neither paracellular nor transcellular mechanism was affected in the mutants. Furthermore, compared to the controls, both mutant lines showed comparable oligodendrocyte number, oligodendrocyte proliferation rate, MBP/MAG levels, and SMI-32 expression, highlighting a minimal role of OL-derived laminin-α5 in OL biology. Together, these findings highlight a dispensable role of OL-derived laminin-α5 in both brain homeostasis and ICH pathogenesis.</AbstractText><br /><br /><br /><br /><small>J Cereb Blood Flow Metab: 01 Apr 2024; 44:611-623</small></div>