In HUVECs, plasma-induced transcriptional changes are mediated by thrombin-induced PAR-1 activation. Rivaroxaban downregulated the expression of pro-inflammatory markers and tissue factor to a similar extent to dabigatran.

Objective Coagulation factor XI (FXI) has been shown to contribute to thrombus formation on collagen or tissue factor (TF)-coated surfaces in vitro and in vivo by enhancing thrombin generation. Whether the role of the intrinsic pathway of coagulation is restricted to the local site of thrombus formation is unknown. This study was aimed to determine whether FXI could promote both proximal and distal platelet activation and aggregate formation in the bloodstream. Approach and Results Pharmacological blockade of FXI activation or thrombin activity in blood did not affect local platelet adhesion, yet reduced local platelet aggregation, thrombin localization and fibrin formation on immobilized collagen and TF under shear flow, ex vivo. Downstream of the thrombus formed on immobilized collagen or collagen and 10 pM TF, platelet CD62P expression and microaggregate formation and progressive platelet consumption were significantly reduced in the presence of FXI-function blocking antibodies or a thrombin inhibitor in a shear rate- and time-dependent manner. In a non-human primate model of thrombus formation, we found that inhibition of FXI reduced single platelet consumption in the bloodstream distal to a site of thrombus formation. Conclusions This study demonstrates that the FXI-thrombin axis contributes to distal platelet activation and procoagulant microaggregate formation in the blood flow downstream of the site of thrombus formation. Our data highlights FXI as a novel therapeutic target for inhibiting distal thrombus formation without affecting proximal platelet adhesion.

Background: Activated coagulation factor XI (FXIa) contributes to the development and propagation of thrombosis but plays only a minor role in hemostasis; therefore, it is an attractive antithrombotic target. Objectives:To evaluate the pharmacology of asundexian (BAY 2433334), a small molecule inhibitor targeting FXIa, in vitro and in various rabbit models. Methods:The effects of asundexian on FXIa activity, selectivity versus other proteases, plasma thrombin generation, and clotting assays were evaluated. Antithrombotic effects were determined in FeCl 2 -and arterio-venous (AV) shunt models. Asundexian was administered intravenously or orally, before or during thrombus formation, and with or without antiplatelet drugs (aspirin and ticagrelor). Potential effects of asundexian on bleeding were evaluated in ear-, gum-, and liver injury models.Results: Asundexian inhibited human FXIa with high potency and selectivity. It reduced FXIa activity, thrombin generation triggered by contact activation or low concentrations of tissue factor, and prolonged activated partial thromboplastin time in human, rabbit, and various other species, but not in rodents. In the FeCl 2 -injury models, asundexian reduced thrombus weight versus control, and in the arterial model when added to aspirin and ticagrelor. In the AV shunt model, asundexian reduced thrombus weight when administered before or during thrombus formation. Asundexian alone or in combination with antiplatelet drugs did not increase bleeding times or blood loss in any of the models studied. Conclusions:Asundexian is a potent oral FXIa inhibitor with antithrombotic efficacy in arterial and venous thrombosis models in prevention and intervention settings, without increasing bleeding.

Aggregation and spreading of α-Synuclein (αSyn) are hallmarks of several neurodegenerative diseases, thus monitoring human αSyn (hαSyn) in animal models or cell cultures is vital for the field. However, the detection of native hαSyn in such systems is challenging. We show that the nanobody NbSyn87, previously-described to bind hαSyn, also shows cross-reactivity for the proteasomal subunit Rpn10. As such, when the NbSyn87 is expressed in the absence of hαSyn, it is continuously degraded by the proteasome, while it is stabilized when it binds to hαSyn. Here, we exploit this feature to design a new Fluorescent Reporter for hαSyn (FluoReSyn) by fusing NbSyn87 to fluorescent proteins, which results in fluorescence signal fluctuations depending on the presence and amounts of intracellular hαSyn. We characterize this biosensor in cells and tissues to finally reveal the presence of transmittable αSyn in human cerebrospinal fluid, demonstrating the potential of FluoReSyn for clinical research and diagnostics.