The P2Y12 Receptor Antagonist Selatogrel Dissolves Preformed Platelet Thrombi In Vivo

Selatogrel, a potent and reversible antagonist of the P2Y12 receptor, inhibited FeCl3-induced thrombosis in rats. Here, we report the anti-thrombotic effect of selatogrel after subcutaneous applications in guinea pigs and mice. Selatogrel inhibited platelet function only 10 min after subcutaneous application in mice. In addition, in a modified Folts thrombosis model in guinea pigs, selatogrel prevented a decrease in blood-flow, indicative of the inhibition of ongoing thrombosis, approximately 10 min after subcutaneous injection. Selatogrel fully normalised blood flow; therefore, we speculate that it may not only prevent, but also dissolve, platelet thrombi. Thrombus dissolution was investigated using real-time intravital microscopy in mice. The infusion of selatogrel during ongoing platelet thrombus formation stopped growth and induced the dissolution of the preformed platelet thrombus. In addition, platelet-rich thrombi were given 30 min to consolidate in vivo. The infusion of selatogrel dissolved the preformed and consolidated platelet thrombi. Dissolution was limited to the disintegration of the occluding part of the platelet thrombi, leaving small mural platelet aggregates to seal the blood vessel. Therefore, our experiments uncovered a novel advantage of selatogrel: the dissolution of pre-formed thrombi without the disintegration of haemostatic seals, suggesting a bipartite benefit of the early application of selatogrel in patients with acute thrombosis.

Fibrinolysis in Platelet Thrombi

The extent and duration of occlusive thrombus formation following an arterial atherothrombotic plaque disruption may be determined by the effectiveness of endogenous fibrinolysis. The determinants of endogenous fibrinolysis are the subject of much research, and it is now broadly accepted that clot composition as well as the environment in which the thrombus was formed play a significant role. Thrombi with a high platelet content demonstrate significant resistance to fibrinolysis, and this may be attributable to an augmented ability for thrombin generation and the release of fibrinolysis inhibitors, resulting in a fibrin-dense, stable thrombus. Additional platelet activators may augment thrombin generation further, and in the case of coronary stenosis, high shear has been shown to strengthen the attachment of the thrombus to the vessel wall. Neutrophil extracellular traps contribute to fibrinolysis resistance. Additionally, platelet-mediated clot retraction, release of Factor XIII and resultant crosslinking with fibrinolysis inhibitors impart structural stability to the thrombus against dislodgment by flow. Further work is needed in this rapidly evolving field, and efforts to mimic the pathophysiological environment in vitro are essential to further elucidate the mechanism of fibrinolysis resistance and in providing models to assess the effects of pharmacotherapy.

Nonbacterial thrombotic endocarditis as an initial presentation of advanced cholangiocarcinoma in a young patient: A case report

Nonbacterial thrombotic endocarditis, a form of noninfectious thrombotic endocarditis, is mainly characterized by deposition of sterile platelet thrombi on heart valves. Usually, it is observed in advanced malignancy. Herein, we report a case of a previously healthy male with recent unprovoked deep vein thrombosis presented with acute ischemic stroke. The echocardiogram revealed aortic and mitral valve masses. Eventually, he was discovered to have advanced cholangiocarcinoma. The present case, apart from being the youngest reported case, is among the few reported cases which manifest the association between cholangiocarcinoma and nonbacterial thrombotic endocarditis.

Novel Stenotic Microchannels to Study Thrombus Formation in Shear Gradients: Influence of Shear Forces and Human Platelet-Related Factors

Thrombus formation in hemostasis or thrombotic disease is initiated by the rapid adhesion, activation, and aggregation of circulating platelets in flowing blood. At arterial or pathological shear rates, for example due to vascular stenosis or circulatory support devices, platelets may be exposed to highly pulsatile blood flow, while even under constant flow platelets are exposed to pulsation due to thrombus growth or changes in vessel geometry. The aim of this study is to investigate platelet thrombus formation dynamics within flow conditions consisting of either constant or variable shear. Human platelets in anticoagulated whole blood were exposed ex vivo to collagen type I-coated microchannels subjected to constant shear in straight channels or variable shear gradients using different stenosis geometries (50%, 70%, and 90% by area). Base wall shears between 1800 and 6600 s−1, and peak wall shears of 3700 to 29,000 s−1 within stenoses were investigated, representing arterial-pathological shear conditions. Computational flow-field simulations and stenosis platelet thrombi total volume, average volume, and surface coverage were analysed. Interestingly, shear gradients dramatically changed platelet thrombi formation compared to constant base shear alone. Such shear gradients extended the range of shear at which thrombi were formed, that is, platelets became hyperthrombotic within shear gradients. Furthermore, individual healthy donors displayed quantifiable differences in extent/formation of thrombi within shear gradients, with implications for future development and testing of antiplatelet agents. In conclusion, here, we demonstrate a specific contribution of blood flow shear gradients to thrombus formation, and provide a novel platform for platelet functional testing under shear conditions.

Binding of Coagulation Factor XIII Zymogen to Activated Platelet Subpopulations: Roles of Integrin αIIbβ3 and Fibrinogen

Factor XIIIa (fXIIIa) is a transglutaminase that plays a crucial role in fibrin clot stabilization and regulation of fibrinolysis. It is known to bind to procoagulant platelets. In contrast, the zymogen fXIII interaction with platelets is not well characterized. We investigated the interaction of zymogen fXIII with activated platelet subpopulations. Confocal microscopy and flow cytometry using fluorescently labelled factors and antibodies. Phosphatidylserine (PS)-positive activated platelets bound 700 to 800 molecules/cell of fXIII at 100 nM, while both PS-negative activated platelets and resting platelets bound 200 to 400 molecules/cell. The binding was reversible, calcium-independent and linear within the fXIII concentration range of up to 1,000 nM. fXIII predominantly bound to the caps of procoagulant platelets and co-localized with fibrinogen. Exogenous fibrinogen promoted fXIII binding by activated PS-negative platelets; this effect was abolished by the integrin αIIbβ3 antagonist monafram. The fXIII binding was 1.5- to 3-fold decreased for platelets from four patients with grey platelet syndrome, and was variable for platelets from six patients with Glanzmann's thrombasthenia. Strong platelet stimulation, fibrinogen and αIIbβ3 play essential roles in fXIII binding, without any of them fXIII does not bind to platelets. The preferential binding in the cap-like structures might be important for increasing local fXIII concentration in platelet thrombi.

A new method for accurate platelet thrombi volume measurement using a confocal microscope

<p class="Abstract">The accuracy of quantitative measurements represents an essential pre-requisite to the characterization and definition of the complex dynamic phenomena occurring in the field of cell biology. In research projects that involve the induction of blood coagulation under flow in microfluidic artificial channels, thrombus volume is an important quantity for estimation as a significant index related to the individual thrombotic risk profile. Concerning its importance in the early diagnosis of cardiovascular diseases, the estimated thrombus volume should reflect and represent reality. In 3D confocal microscopy, systematic errors can arise from distortions of the axial distance, whose accurate calibration remains a challenge. As a result, the 3D reconstructions show a noticeable axial elongation, and the volume measurements are thus overestimated. In this paper, a 400-600 % volume overestimation is demonstrated, and a new easy to use and automatic calibration procedure is outlined for this specific microfluidic and optical context. The adaptive algorithm proposed leads to the automatic compensation of the elongation error and to the accurate thrombus volume measurement. The method has been calibrated using fluorescent beads of known volume, validated with groups of several distinct platelets and finally applied on platelet thrombi.</p>

Thrombotic Thrombocytopenic Purpura after Ischemic Stroke

Background and purpose: Thrombotic thrombocytopenic purpura (TTP) is a rare disorder, the exact cause of which is unknown. In addition to thrombocytopenia, it is characterized by hemolytic anemia, changing neurological symptoms, fever, and renal abnormalities. Most of these findings are caused by the formation of platelet thrombi in capillaries and arterioles throughout the body. This article reports a case of TTP after Ischemic Stroke and its purpose was to describe the clinical and pathophysiologic features of TTP. We describe a 52-year-old woman who presented three onset of ischemic stroke. A third episode of ischemic stroke was complicated by TTP. Hemolytic anemia and thrombocytopenia were identified.

Inflammation, von Willebrand factor, and ADAMTS13

Increasing evidence indicates that inflammation can cause thrombosis by a von Willebrand factor (VWF)-mediated mechanism that includes endothelial activation, secretion of VWF, assembly of hyperadhesive VWF strings and fibers, cleavage by ADAMTS13, and adhesion and deposition of VWF-platelet thrombi in the vasculature. This mechanism appears to contribute to thrombosis not only in small vessels, but also in large vessels. Inflammation and VWF contribute to atherogenesis and may contribute to arterial and venous thrombosis as well as stroke. Elucidation of the mechanism will hopefully identify new targets and suggest new approaches for prevention and intervention.