The P2Y12 Receptor Antagonist Selatogrel Dissolves Preformed Platelet Thrombi In Vivo

Abstract

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.

Keywords: P2Y12 receptor; haemostasis; platelets; thrombosis; thrombus dissolution.

Figure 1

Selatogrel inhibits fibrinogen binding after subcutaneous applications in mice. Binding of FITC–fibrinogen to ADP-activated mouse platelets. Panel (A): Fibrinogen binding to platelets, expressed in arbitrary units as the mean fluorescence intensity (MFI) (average ± standard deviation). Asterisks indicate p < 0.001 in a Student’s t-test; ns, not significant. Panel (B): Time course of the inhibition of FITC–fibrinogen. Selatogrel (0.2 μg/kg) was injected subcutaneously in the lower abdomen of mice and blood samples were taken before injection (0) and 10, 20, 40, and 60 min after injection. Blood samples were processed as described in the Materials and Methods section. Maximal binding of FITC–fibrinogen in the absence of a P2Y12 receptor antagonist was defined as a 100% value. All other data are expressed relative to the 100% control. Fibrinogen binding (%) (y-axis) is presented relative to time (minutes) (x-axis). Error bars indicate the standard deviation (n = 3).

Figure 2

Modified Folts model in guinea pigs. Blood flow velocity (volts) (y-axis) is shown relative to time (minutes) (x-axis). Mechanical injury of the carotid artery is indicated by a white arrow. The black tracing indicates CFVs in the absence of antagonists. Selatogrel injection (30 μg/kg s.c.) is indicated by a black arrow. Subsequent CFVs are indicated by grey tracings. The green arrow indicates the initiation of antithrombotic activity, and the red arrows indicate the increase in blood flow velocity, suggestive of dissolution of the existing platelet thrombus after selatogrel application.

Figure 3

Intravital microscopy in mice. Laser-induced thrombus formation was monitored by real-time intravital microscopy. Panel (A): Increases in integrated fluorescence intensity (y-axis) are indicative of platelet incorporation into the growing thrombus and were monitored over time (x-axis). Black tracing represents the incorporation of platelets into a growing platelet-rich thrombus induced in the absence of an antagonist. The grey tracing represents the incorporation of platelets where selatogrel was injected 62 s after laser injury (1 μg/kg) (grey arrow). Panel (B): The incorporation of fibrin into growing platelet thrombi was quantified using a fluorescence-labelled anti-fibrin monoclonal antibody and expressed as the ratio of the fibrin signal relative to the observed platelet signal (y-axis). The fibrin/platelet signal ratio is presented at timepoints of 100, 200, and 300 s after laser injury. Black bars represent the absence of an antagonist, and grey bars represent the condition in the presence of selatogrel infusion (1 μg/kg). Brackets indicate a statistical comparison; ns, not significant, *** = p < 0.05.

Figure 4

Dissolution of consolidated thrombi. Platelet thrombus formation was induced by laser injury. The stability of the platelet thrombi was monitored for up to 65 min. Panel (A): Platelet thrombus size in the absence of an antagonist. Representative images of platelet thrombi (red) at T0 (maximal size of the initial thrombus) and at 30 min (T30), 35 min (T35), and 65 min (T65). Panel (B): Area under the curve of the integrated fluorescence intensity normalised to the initial thrombus for T30, T35, and T65 (y-axis); ns, not significant. Panel (C): Representative images of platelet thrombi (red) at T0 (maximal size) and at 30 min (T30), 35 min (T35), and 65 min (T65). Selatogrel was infused at T30, and the thrombus sizes at T35 and T65 are shown. Panel (D): Area under the curve of the integrated fluorescence intensity normalised to the initial thrombus (T0) (y-axis). In the presence of selatogrel, the differences between integrated fluorescence intensity at T35 and T65 relative to T30 were statistically significant (* = p < 0.05) (ns, not significant).

Figure 5

Dissolution of human platelet aggregates. Light transmission aggregation with human platelet-rich plasma containing physiological calcium concentrations. Light transmission is expressed as the percentage aggregation (y-axis, right hand side). Prior to the initiation of the experiment, the aggregometer was calibrated with platelet-poor plasma (100% aggregation) and platelet-rich plasma (0% aggregation). Aggregation was induced by the addition of ADP (10 μM), open arrows. Vehicle (Control) or selatogrel were injected at the peak aggregation (filled arrows, black = control, red = selatogrel 1 μM).