Thrombomodulin (TM) is a vascular endothelial cell (EC) receptor that is a cofactor for thrombin-mediated activation of the anticoagulant protein C. The extracellular NH2-terminal domain of TM has homology to C-type lectins that are involved in immune regulation. Using transgenic mice that lack this structure (TMLeD/LeD), we show that the lectin-like domain of TM interferes with polymorphonuclear leukocyte (PMN) adhesion to ECs by intercellular adhesion molecule 1–dependent and –independent pathways through the suppression of extracellular signal–regulated kinase (ERK)1/2 activation. TMLeD/LeD mice have reduced survival after endotoxin exposure, accumulate more PMNs in their lungs, and develop larger infarcts after myocardial ischemia/reperfusion. The recombinant lectin-like domain of TM suppresses PMN adhesion to ECs, diminishes cytokine-induced increase in nuclear factor κB and activation of ERK1/2, and rescues ECs from serum starvation, findings that may explain why plasma levels of soluble TM are inversely correlated with cardiovascular disease. These data suggest that TM has antiinflammatory properties in addition to its role in coagulation and fibrinolysis.

Acute renal failure (ARF) remains a major clinical challenge, especially in the intensive care setting. Mortality of ARF combined with acute lung injury (ALI) is even higher and may reach 80%. Recent studies have suggested a remote effect of ARF on pulmonary homeostasis. However, it is unknown whether and to what extent ARF clinically affects pulmonary function, in particular oxygenation. For elucidation of the impact of ARF on aseptic ALI, a murine two-hit model that consists of acute uremia (AU) and subsequent ALI was developed. AU was induced by renal ischemia-reperfusion (inflammatory AU) or bilateral nephrectomy (noninflammatory AU). ALI was initiated by intratracheal HCl instillation and characterized by severe, PMN-dependent decrease in arterial partial pressure of O 2 (>70%) in nonuremic mice. Uremic mice, by contrast, showed a significant protection from ALI (decrease in arterial partial pressure of O 2 <40%); this was independent of the type of AU. Reconstitution experiments, in which uremic neutrophils were injected into nonuremic mice and vice versa, identified uremic neutrophils as the primary mediators. Between normal and uremic neutrophils, there were no differences in apoptosis or superoxide production. Pulmonary recruitment of uremic neutrophils, however, was significantly attenuated compared with that of normal neutrophils. This defect was associated with altered surface expression of L-selectin; sialyl Lewis x , an L-selectin counterreceptor, previously was proved to be critical in aseptic ALI. In conclusion, it is shown that AU but not renal inflammation attenuates aseptic, neutrophil-dependent ALI and exerts an anti-inflammatory effect by attenuating pulmonary neutrophil recruitment.

Many patients with chronic kidney disease (CKD) receive anticoagulation or antiplatelet therapy due to atrial fibrillation, coronary artery disease, thromboembolic disease, or peripheral artery disease. The treatment usually includes vitamin K antagonists (VKAs) and/or platelet aggregation inhibitors. The direct oral anticoagulants (DOAC) inhibiting factor Xa or thrombin represent an alternative for VKAs. In patients with acute and chronic kidney disease, caution is warranted, as DOACs can accumulate as they are partly eliminated by the kidneys. Thus, they can potentially increase the bleeding risk in patients with CKD. In patients with an estimated glomerular filtration rate (eGFR) above 60 mL/min, DOACs can be used safely with greater efficacy and safety as compared to VKAs. In patients with CKD 3, DOACs are as effective as VKAs with a lower bleeding rate. The more the renal function declines, the lower is the advantage of DOACs over VKAs. Thus, use of DOACs should be avoided in patients with an eGFR below 30 mL/min, particularly, the compounds with a high renal elimination. Available data suggest that DOACs can also be used safely in older patients. In this review, use of DOACs in comparison with VKAs, heparins, and heparinoids, together with special considerations in patients with impaired renal function will be discussed.

Rationale: Chronic thromboembolic pulmonary hypertension (CTEPH) is characterized by defective thrombus resolution, pulmonary artery obstruction, and vasculopathy. TGFβ (transforming growth factor-β) signaling mutations have been implicated in pulmonary arterial hypertension, whereas the role of TGFβ in the pathophysiology of CTEPH is unknown. Objective: To determine whether defective TGFβ signaling in endothelial cells contributes to thrombus nonresolution and fibrosis. Methods and Results: Venous thrombosis was induced by inferior vena cava ligation in mice with genetic deletion of TGFβ1 in platelets (Plt.TGFβ-KO) or TGFβ type II receptors in endothelial cells (End.TGFβRII-KO). Pulmonary endarterectomy specimens from CTEPH patients were analyzed using immunohistochemistry. Primary human and mouse endothelial cells were studied using confocal microscopy, quantitative polymerase chain reaction, and Western blot. Absence of TGFβ1 in platelets did not alter platelet number or function but was associated with faster venous thrombus resolution, whereas endothelial TGFβRII deletion resulted in larger, more fibrotic and higher vascularized venous thrombi. Increased circulating active TGFβ1 levels, endothelial TGFβRI/ALK1 (activin receptor-like kinase), and TGFβRI/ALK5 expression were detected in End.TGFβRII-KO mice, and activated TGFβ signaling was present in vessel-rich areas of CTEPH specimens. CTEPH-endothelial cells and murine endothelial cells lacking TGFβRII simultaneously expressed endothelial and mesenchymal markers and transcription factors regulating endothelial-to-mesenchymal transition, similar to TGFβ1-stimulated endothelial cells. Mechanistically, increased endothelin-1 levels were detected in TGFβRII-KO endothelial cells, murine venous thrombi, or endarterectomy specimens and plasma of CTEPH patients, and endothelin-1 overexpression was prevented by inhibition of ALK5, and to a lesser extent of ALK1. ALK5 inhibition and endothelin receptor antagonization inhibited mesenchymal lineage conversion in TGFβ1-exposed human and murine endothelial cells and improved venous thrombus resolution and pulmonary vaso-occlusions in End.TGFβRII-KO mice. Conclusions: Endothelial TGFβ1 signaling via type I receptors and endothelin-1 contribute to mesenchymal lineage transition and thrombofibrosis, which were prevented by blocking endothelin receptors. Our findings may have relevant implications for the prevention and management of CTEPH.

Oxidative stress and inflammation of the vessel wall contribute to prothrombotic states. The antioxidative protein paraoxonase-2 (PON2) shows reduced expression in human atherosclerotic plaques and endothelial cells in particular. Supporting a direct role for PON2 in cardiovascular diseases, deficiency in mice promotes atherogenesis through incompletely understood mechanisms. Here, we show that deregulated redox regulation in deficiency causes vascular inflammation and abnormalities in blood coagulation. In unchallenged mice, we find increased oxidative stress and endothelial dysfunction. Bone marrow transplantation experiments and studies with endothelial cells provide evidence that increased inflammation, indicated by circulating interleukin-6 levels, originates from deficiency in the vasculature. Isolated endothelial cells from mice display increased tissue factor (TF) activity in vitro. Coagulation times were shortened and platelet procoagulant activity increased in mice relative to wild-type controls. Coagulation abnormalities of mice were normalized by anti-TF treatment, demonstrating directly that TF increases coagulation. PON2 reexpression in endothelial cells by conditional reversal of the knockout cassette, restoration in the vessel wall using bone marrow chimeras, or treatment with the antioxidant-acetylcysteine normalized the procoagulant state. These experiments delineate a PON2 redox-dependent mechanism that regulates endothelial cell TF activity and prevents systemic coagulation activation and inflammation.

Vascular calcification is a severe consequence of several pathological processes with a lack of effective therapy. Recent studies suggest that circulating and resident mesenchymal stem cells (MSC) contribute to the osteogenic program of vascular calcification. Molecular mechanisms underlying MSC osteogenic potential and differentiation remain, however, sparsely explored. We investigated a role for the complement receptor C5aR in these processes. We found that expression of C5aR was upregulated upon differentiation of human MSC to osteoblasts. C5aR inhibition by silencing and specific antagonist impaired osteogenic differentiation. We demonstrate that C5aR expression upon MSC differentiation was regulated by the multifunctional urokinase receptor (uPAR). uPAR targeting by siRNA resulted in complete abrogation of C5aR expression and consequently in the inhibition of MSC-osteoblast differentiation. We elucidated the NFkB pathway as the mechanism utilized by the uPAR-C5aR axis. MSC treatment with the NFkB inhibitor completely blocked the differentiation process. Nuclear translocation of the p65 RelA component of the NFkB complex was induced under osteogenic conditions and impaired by the inhibition of uPAR or C5aR. Dual-luciferase reporter assays demonstrated enhanced NFkB signaling upon MSC differentiation, whereas uPAR and C5aR downregulation lead to inhibition of the NFkB activity. We show involvement of the Erk1/2 kinase in this cascade. In vivo studies in a uPAR/LDLR double knockout mouse model of diet-induced atherosclerosis revealed impaired C5aR expression and calcification in aortic sinus plaques in uPAR -/ -/LDLR -/ -versus uPAR + / + /LDLR -/ -control animals. These results suggest that uPAR-C5aR axis via the underlying NFkB transcriptional program controls osteogenic differentiation with functional impact on vascular calcification in vivo.

Patients diagnosed with pseudohypoparathyroidism type Ia (PHP Ia) suffer from hormonal resistance and abnormal postural features, in a condition classified as Albright hereditary osteodystrophy (AHO) syndrome. this syndrome is linked to a maternally inherited mutation in the GNAS complex locus, encoding for the GTPase subunit Gsα. Here, we investigated how platelet phenotype and omics analysis can assist in the often difficult diagnosis. By coupling to the IP receptor, Gsα induces platelet inhibition via adenylyl cyclase and cAMP-dependent protein kinase A (PKA). In platelets from seven patients with suspected AHO, one of the largest cohorts examined, we studied the PKA-induced phenotypic changes. Five patients with a confirmed GNAS mutation, displayed impairments in Gsαdependent VASP phosphorylation, aggregation, and microfluidic thrombus formation. Analysis of the platelet phosphoproteome revealed 2,516 phosphorylation sites, of which 453 were regulated by Gsα-PKA. Common changes in the patients were: (1) a joint panel of upregulated and downregulated phosphopeptides; (2) overall PKA dependency of the upregulated phosphopeptides; (3) links to key platelet function pathways. In one patient with GNAS mutation, diagnosed as non-AHo, the changes in platelet phosphoproteome were reversed. this combined approach thus revealed multiple phenotypic and molecular biomarkers to assist in the diagnosis of suspected PHP Ia. Pseudohypoparathyroidism (PHP) characterises a heterogeneous group of disorders, of which the common feature is an end-organ resistance to parathyroid hormone. Patients diagnosed with pseudohypoparathyroidism

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