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Background: Thrombus formation in vitro under flow conditions is one of the most widely used methods to study haemostasis and to evaluate the activity of potential antithrombotic compounds. Assessment of the results of these experiments is often based on a quantification of microscopic images of thrombi. In a majority of reported analysis all thrombi visualised in an image are quantified as one homogenous class. In some protocols, qualitative assessment of thrombi morphology based on a visual comparison of evaluated images with representative images of predefined classes of thrombi are performed by experienced analysts. In presented paper we show how the quantitative analysis can be improved by classification of thrombi on the basis of defined morphological features prior to quantification and we suggest that machine learning-based approach can improve this way of analysis. Methods: We tested the applicability of machine learning-based segmentation and classification of thrombi images to improve the outcome of quantification of the results of flow chamber assays. For this, we used the public domain machine learning software Ilastik for bioimage analysis developed at the European Molecular Biology Laboratory. A model was trained to distinguish two classes of thrombi based on certain morphological features which apparently correspond to the stage of thrombus development. Thrombi formed in the presence of a model antiplatelet compound—abciximab or in control conditions were quantified with the use of this model and the results were compared to quantification where all thrombi were quantified as a homogenous class. Results: Machine learning-based analysis was capable of effective distinguishing of two classes of morphologically distinct platelet aggregates. The use of the model which segmented and quantified only the objects recognized as compacted structures provided results which better mirrored the actual effect of an antiplatelet treatment than quantification based on all structures. Conclusions: Classification of thrombi enabled by machine learning increases the relevance of quantitative information and allows better evaluation of the results of in vitro thrombosis assays. [ABSTRACT FROM AUTHOR]

Numerous in vitro experiments have confirmed that a dysfunctional endothelium is characterized by, inter alia, a higher affinity for binding of platelets and leukocytes. However, there is still no direct evidence for greater interaction between platelets and intact endothelium in in vivo animal models of diabetes. Therefore, the present study examines the pro-adhesive properties of endothelium change in vivo as an effect of streptozotocin (STZ)-induced diabetes and the role of two key platelet receptors: GPIb-IX-V and GPIIb/IIIa. Mice of C57BL strain with streptozotocin-induced diabetes were used in the study. Flow cytometry was used to assess basal activation and reactivity of platelets. Adhesion of platelets to the vascular wall was visualized with the use of intravital microscopy in mesentery. The contribution of GPIIb/IIIa and GPIb-IX-V was evaluated by the injection of Fab fragments of respective antibodies. The integrity of the endothelium and vWf expression were evaluated histochemically. Basal activation and reactivity of platelets in streptozotocin-diabetic mice were elevated. Blood platelets adhered more often to the vascular wall of diabetic mice than nondiabetic animals: 11.9 (6.4; 32.8) plt/min/mm2 (median [IQR]) vs 2.7 (1.3; 6.4) plt/min/mm2. The injection of anti-GPIbα antibodies decreased the number of adhering platelets from 89.5 (34.0; 113.1) plt/min/mm2 (median [IQR]) in mice treated with isotype antibodies to 3.1 (1.7; 5.6) plt/min/mm2 in mice treated with blocking antibodies. The effect of GPIIb/IIIa blockage was not significant. Immunohistochemistry revealed a higher expression of vWF in the endothelium of STZ mice, but no substantial changes in endothelial morphology were detected. To conclude, the study shows that the platelets interact more frequently with the mesenteric vascular bed in mice with 1-month STZ-induced diabetes than in healthy mice. These interactions are mediated via platelet GPIb-IX-V and are driven by increased expression of vWF in endothelial cells. [ABSTRACT FROM AUTHOR]

Abstract: The role of cyclooxygenase-2 (COX-2) in restoring the functions of impaired endothelium is attracting considerable attention, notably the function of COX-2-derived vasodilatory prostaglandins is disputed in the context of the regulation function in the impaired vascular beds. We have examined the hypothesis that COX-2 activity contributes more to vasodilation in hyperglycemic animals than in healthy counterparts, and that COX-2 derived vasodilatory prostaglandins (PGI2 and PGE2) are responsible for this effect. Using the Langendorff heart perfusion system, the effects of COX-2 inhibition were monitored on both basal and bradykinin-induced coronary flows in Sprague–Dawley rats given 8-week streptozotocin-induced diabetes and in age-matched controls (n=15). Secretions of PGI2 and PGE2, both total and the COX-2 dependent pools, have also been compared. The selective COX-2 inhibitor, N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide (NS-398), had no effect on coronary flow in the diabetic group of animals. Thus, the compensatory role of COX-2 in regulation of vascular tone in experimental diabetes found in other experimental models was not confirmed. However, COX-2 activity significantly contributed to PGI2 synthesis in healthy rats, with prostacyclin secretion being two-fold decreased by NS-398. Contrary to our hypothesis, neither prostacyclin nor PGE2 production differed between the experimental groups under the basal conditions. Bradykinin had no effect on the secretion of PGI2 in either group, but increased PGE2 synthesis in healthy animals, although not in the streptozotocin group. PGE2 production in response to bradykinin was COX-2-dependent in control animals. We conclude that, in rats with 8-week streptozotocin-induced diabetes, the activity of COX-2 in coronary vasculature is not significantly enhanced. [Copyright &y& Elsevier]

Large inter-individual variation in platelet response to endogenous agonists and pharmacological agents, including resistance to antiplatelet therapy, prompts a search for novel platelet inhibitors and development new antithrombotic strategies. The present in vitro study evaluates the beneficial effects of three adenosine receptor (AR) agonists (regadenoson, LUF 5835 and NECA), different in terms of their selectivity for platelet adenosine receptors, when used alone and in combination with P2Y12 inhibitors, such as cangrelor or prasugrel metabolite. The anti-platelet effects of AR agonists were evaluated in healthy subjects (in the whole group and after stratification of individuals into high- and low-responders to P2Y12 inhibitors), using whole blood techniques, under flow (thrombus formation) and static conditions (study of platelet activation and aggregation). Compared to P2Y12 antagonists, AR agonists were much less or not effective under static conditions, but demonstrated similar antiplatelet activity in flow. In most cases, AR agonists significantly enhanced the anti-platelet effect of P2Y12 antagonists, despite possessing different selectivity profiles and antiplatelet activities. Importantly, their inhibitory effects in combination with P2Y12 antagonists were similar in high- and low-responders to P2Y12 inhibitors. In conclusion, a combination of anti-platelet agents acting via the P1 and P2 purinergic receptors represents a promising alternative to existing antithrombotic therapy. [ABSTRACT FROM AUTHOR]

Large inter-individual variation in platelet response to endogenous agonists and pharmacological agents, including resistance to antiplatelet therapy, prompts a search for novel platelet inhibitors and development new antithrombotic strategies. The present in vitro study evaluates the beneficial effects of three adenosine receptor (AR) agonists (regadenoson, LUF 5835 and NECA), different in terms of their selectivity for platelet adenosine receptors, when used alone and in combination with P2Y 12 inhibitors, such as cangrelor or prasugrel metabolite. The anti-platelet effects of AR agonists were evaluated in healthy subjects (in the whole group and after stratification of individuals into high- and low-responders to P2Y 12 inhibitors), using whole blood techniques, under flow (thrombus formation) and static conditions (study of platelet activation and aggregation). Compared to P2Y 12 antagonists, AR agonists were much less or not effective under static conditions, but demonstrated similar antiplatelet activity in flow. In most cases, AR agonists significantly enhanced the anti-platelet effect of P2Y 12 antagonists, despite possessing different selectivity profiles and antiplatelet activities. Importantly, their inhibitory effects in combination with P2Y 12 antagonists were similar in high- and low-responders to P2Y 12 inhibitors. In conclusion, a combination of anti-platelet agents acting via the P1 and P2 purinergic receptors represents a promising alternative to existing antithrombotic therapy.