Antithrombotic Effects of Fostamatinib in Combination with Conventional Antiplatelet Drugs.

New antithrombotic medications with less effect on haemostasis are needed for the long-term treatment of acute coronary syndromes (ACS). The platelet receptor glycoprotein VI (GPVI) is critical in atherothrombosis, mediating platelet activation at atherosclerotic plaque. The inhibition of spleen tyrosine kinase (Syk) has been shown to block GPVI-mediated platelet function. The aim of our study was to investigate if the Syk inhibitor fostamatinib could be repurposed as an antiplatelet drug, either alone or in combination with conventional antiplatelet therapy. The effect of the active metabolite of fostamatinib (R406) was assessed on platelet activation and function induced by atherosclerotic plaque and a range of agonists in the presence and absence of the commonly used antiplatelet agents aspirin and ticagrelor. The effects were determined ex vivo using blood from healthy volunteers and aspirin- and ticagrelor-treated patients with ACS. Fostamatinib was also assessed in murine models of thrombosis. R406 mildly inhibited platelet responses induced by atherosclerotic plaque homogenate, likely due to GPVI inhibition. The anti-GPVI effects of R406 were amplified by the commonly-used antiplatelet medications aspirin and ticagrelor; however, the effects of R406 were concentration-dependent and diminished in the presence of plasma proteins, which may explain why fostamatinib did not significantly inhibit thrombosis in murine models. For the first time, we demonstrate that the Syk inhibitor R406 provides mild inhibition of platelet responses induced by atherosclerotic plaque and that this is mildly amplified by aspirin and ticagrelor.

Novel antiplatelet strategies targeting GPVI, CLEC-2 and tyrosine kinases.

Antiplatelet medications comprise the cornerstone of treatment for diseases that involve arterial thrombosis, including acute coronary syndromes (ACS), stroke and peripheral arterial disease. However, antiplatelet medications may cause bleeding and, furthermore, thrombotic events may still recur despite treatment. The interaction of collagen with GPVI receptors on the surface of platelets has been identified as one of the major players in the pathophysiology of arterial thrombosis that occurs following atherosclerotic plaque rupture. Promisingly, GPVI deficiency in humans appears to have a minimal impact on bleeding. These findings together suggest that targeting platelet GPVI may provide a novel treatment strategy that provides additional antithrombotic efficacy with minimal disruption of normal hemostasis compared to conventional antiplatelet medications. CLEC-2 is gaining interest as a therapeutic target for a variety of thrombo-inflammatory disorders including deep vein thrombosis (DVT) with treatment also predicted to cause minimal disruption to hemostasis. GPVI and CLEC-2 signal through Src, Syk and Tec family tyrosine kinases, providing additional strategies for inhibiting both receptors. In this review, we summarize the evidence regarding GPVI and CLEC-2 and strategies for inhibiting these receptors to inhibit platelet recruitment and activation in thrombotic diseases.

Amplified inhibition of atherosclerotic plaque-induced platelet activation by glenzocimab with dual antiplatelet therapy.

BACKGROUND: Aspirin and platelet P2Y12 inhibitors, such as ticagrelor, suboptimally inhibit microvascular thrombosis during ST-elevation myocardial infarction. Glycoprotein (GP) IIb/IIIa inhibitors may further inhibit this but cause excessive bleeding. OBJECTIVES: We investigated whether combination of glenzocimab, a GPVI inhibitor, with aspirin and ticagrelor provides additional antithrombotic effects, as GPVI has a critical role in atherothrombosis but minimal involvement in hemostasis. METHODS: We investigated the effects of glenzocimab (monoclonal antibody Fab fragment) using blood from healthy donors and patients with acute coronary syndrome treated with aspirin and ticagrelor. Platelets were stimulated with multiple agonists, including atherosclerotic plaque, from patients undergoing carotid endarterectomy. RESULTS: Aspirin and ticagrelor partially inhibited atherosclerotic plaque-induced platelet aggregation by 48% compared with control (34 ± 3 vs 65 ± 4 U; P < .001). Plaque-induced platelet aggregation, adhesion, secretion, and activation were critically dependent on GPVI activation. Glenzocimab alone reduced plaque-induced aggregation by 75% compared with control (16 ± 4 vs 65 ± 4 U; P < .001) and by >95% when combined with aspirin and ticagrelor (3 ± 1 vs 65 ± 4 U; P < .001). Glenzocimab reduced platelet aggregation, adhesion, and thrombin generation when added to blood of aspirin- and ticagrelor-treated patients with acute coronary syndrome. Glenzocimab shared several antithrombotic effects with the GPIIb/IIIa inhibitor eptifibatide with less effect on general hemostasis assessed by rotational thromboelastometry. In a murine intravital model of ST-elevation myocardial infarction, genetic depletion of GPVI reduced microvascular thrombosis. CONCLUSION: Addition of glenzocimab to aspirin and ticagrelor enhances platelet inhibition via multiple mechanisms of atherothrombosis. Compared with a GPIIb/IIIa inhibitor, glenzocimab shares multiple antithrombotic effects, with less inhibition of mechanisms involved in general hemostasis.

The Btk inhibitor AB-95-LH34 potently inhibits atherosclerotic plaque-induced thrombus formation and platelet procoagulant activity.

BACKGROUND: New antithrombotic therapies with less effect on bleeding are needed for coronary artery disease. The Btk inhibitor ibrutinib blocks atherosclerotic plaque-mediated thrombus formation. However, it is associated with increased bleeding, possibly due to non-Btk-mediated effects. Btk-deficient patients do not have bleeding issues, suggesting selective Btk inhibition as a promising antithrombotic strategy. OBJECTIVES: To compare the antithrombotic effects of the highly selective Btk inhibitor AB-95-LH34 (LH34) with ibrutinib. METHODS: Glycoprotein VI and G-protein coupled receptor-mediated platelet function and signaling were analyzed in healthy human donor platelets by lumi-aggregometry, flow adhesion, and western blot following 1 h treatment with inhibitors in vitro. RESULTS: LH34 showed similar inhibition of Btk-Y223 phosphorylation as ibrutinib, but had no off-target inhibition of Src-Y418 phosphorylation. Similar dose-dependent inhibition of aggregation to atherosclerotic plaque material was observed for both. However, in response to Horm collagen, which also binds integrin α2ß1, LH34 exhibited less marked inhibition than ibrutinib. Both LH34 and ibrutinib inhibited platelet adhesion and aggregation to plaque material at arterial shear. Ibrutinib demonstrated the most potent effect, with complete blockade at high concentrations. Platelet activation (P-selectin) and procoagulant activity (phosphatidylserine exposure) in thrombi were inhibited by LH34 and completely blocked by ibrutinib at high concentrations. Furthermore, plaque-induced thrombin generation was reduced by higher concentrations of LH34 and ibrutinib. CONCLUSIONS: LH34 potently inhibits atherosclerotic plaque-induced thrombus formation and procoagulant platelet activity in vitro, with less off-target inhibition of Src than ibrutinib, suggesting it is a promising antiplatelet therapy with the potential for reduced bleeding side effects.