Current treatments to prevent thrombosis, namely anticoagulants and platelets antagonists, remain complicated by the persistent risk of bleeding. Improved therapeutic strategies that diminish this risk would have a huge clinical impact. Antithrombotic agents that neutralize and inhibit polyphosphate (polyP) can be a powerful approach towards such a goal. Here, we report a design concept towards polyP inhibition, termed macromolecular polyanion inhibitors (MPI), with high binding affinity and specificity. Lead antithrombotic candidates are identified through a library screening of molecules which possess low charge density at physiological pH but which increase their charge upon binding to polyP, providing a smart way to enhance their activity and selectivity. The lead MPI candidates demonstrates antithrombotic activity in mouse models of thrombosis, does not give rise to bleeding, and is well tolerated in mice even at very high doses. The developed inhibitor is anticipated to open avenues in thrombosis prevention without bleeding risk, a challenge not addressed by current therapies.

Von Willebrand factor (VWF) mediates primary hemostasis and thrombosis in response to hydrodynamic forces. We previously showed that high shear promoted self-association of VWF into hyperadhesive strands, which can be attenuated by high density lipoprotein (HDL) and apolipoprotein(apo) A-I. Here, we show that low density lipoprotein (LDL) binds VWF under shear and enhances self-association. Vortexing VWF in tubes resulted in its loss from the solution and deposition onto tube surfaces, which was prevented by HDL. At a stabilizing HDL concentration of 1.2 mg/ml, increasing concentrations of LDL progressively increased VWF loss, the effect correlating with the LDL/HDL ratio and not the absolute concentration of the lipoproteins. Similarly, HDL diminished deposition of VWF in a post-in-channel microfluidic device, whereas LDL increased both the rate and extent of strand deposition, with both purified VWF and plasma. Hypercholesterolemic human plasma also displayed accelerated VWF accumulation in the microfluidic device. The initial rate of accumulation correlated linearly with the LDL/HDL ratio. In Adamts13-/- and Adamts13-/-LDLR-/-mice, high LDL levels enhanced VWF and platelet adhesion to the myocardial microvasculature, reducing cardiac perfusion, impairing systolic function, and producing early signs of cardiomyopathy. In wild-type mice, high plasma LDL concentrations also increased the size and persistence of VWF-platelet thrombi in ionophore-treated mesenteric microvessels, exceeding even that seen in similarly treated ADAMTS13-deficient mice that did not receive LDL infusion. We propose that targeting the interaction of VWF with itself and with LDL may improve the course of thrombotic microangiopathies, atherosclerosis, and other disorders with defective microvascular circulation.

Coronavirus-associated coagulopathy (CAC) is a morbid and lethal sequela of SARS-CoV-2 infection. CAC results from a perturbed balance between coagulation and fibrinolysis and occurs in conjunction with exaggerated activation of monocytes/macrophages (MO/Mφs) and the mechanisms that collectively govern this phenotype seen in CAC remain unclear. In this study, using experimental models that employ the murine betacoronavirus MHVA59, a well-established model of SARS-CoV-2 infection, we identify that the histone methyltransferase Mixed Lineage Leukemia 1 (MLL1/KMT2A) is an important regulator of MO/Mφ expression of procoagulant and profibrinolytic factors such as tissue factor (F3; TF), urokinase (PLAU), and urokinase receptor (PLAUR) (herein "coagulopathy-related factors") in non-infected and infected cells. We show that MLL1 concurrently promotes the expression of the proinflammatory cytokines while suppressing the expression of interferon a (IFNa), a well-known inducer of TF and PLAUR. Using in vitro models, we identify MLL1-dependent NFκB/RelA-mediated transcription of these coagulation-related factors and identify a context dependent MLL1-independent role for RelA in the expression of these factors in vivo. As functional correlates for these findings, we demonstrate that the inflammatory, procoagulant and profibrinolytic phenotypes seen in vivo after coronavirus infection were MLL1-dependent despite blunted Ifna induction in MO/Mφs. Finally, in an analysis of SARS-CoV-2 positive human samples, we identify differential upregulation of MLL1 and coagulopathy-related factor expression and activity in CD14+ MO/Mφs relative to non-infected and healthy controls. We also observed elevated plasma urokinase and TF activity in COVID-positive samples. Collectively, these findings highlight an important role for MO/Mφ MLL1 in promoting coronavirus-associated coagulopathy and inflammation.

No abstract

The antiplatelet effect of polyunsaturated fatty acids is primarily attributed to its metabolism to bioactive metabolites by oxygenases, such as lipoxygenases (LOX). Platelets have demonstrated the ability to generate 15‐LOX‐derived metabolites (15‐oxylipins); however, whether 15‐LOX is in the platelet or is required for the formation of 15‐oxylipins remains unclear. This study seeks to elucidate whether 15‐LOX is required for the formation of 15‐oxylipins in the platelet and determine their mechanistic effects on platelet reactivity. In this study, 15‐HETrE, 15‐HETE, and 15‐HEPE attenuated collagen‐induced platelet aggregation, and 15‐HETrE inhibited platelet aggregation induced by different agonists. The observed anti‐aggregatory effect was due to the inhibition of intracellular signaling including αIIbβ3 and protein kinase C activities, calcium mobilization, and granule secretion. While 15‐HETrE inhibited platelets partially through activation of peroxisome proliferator‐activated receptor β (PPARβ), 15‐HETE also inhibited platelets partially through activation of PPARα. 15‐HETrE, 15‐HETE, or 15‐HEPE inhibited 12‐LOX in vitro, with arachidonic acid as the substrate. Additionally, a 15‐oxylipin‐dependent attenuation of 12‐HETE level was observed in platelets following ex vivo treatment with 15‐HETrE, 15‐HETE, or 15‐HEPE. Platelets treated with DGLA formed 15‐HETrE and collagen‐induced platelet aggregation was attenuated only in the presence of ML355 or aspirin, but not in the presence of 15‐LOX‐1 or 15‐LOX‐2 inhibitors. Expression of 15‐LOX‐1, but not 15‐LOX‐2, was decreased in leukocyte‐depleted platelets compared to non‐depleted platelets. Taken together, these findings suggest that 15‐oxylipins regulate platelet reactivity; however, platelet expression of 15‐LOX‐1 is low, suggesting that 15‐oxylipins may be formed in the platelet through a 15‐LOX‐independent pathway.