Effect of Different Doses of Acetylsalicylic Acid on the Antithrombotic Activity of Clopidogrel in a Mouse Arterial Thrombosis Model.

Objective- Dual-antiplatelet therapy with acetylsalicylic acid and a P2Y12 antagonist, such as clopidogrel, is the standard of care for acute coronary syndromes. However, the drugs have divergent effects on the formation of cAMP, an inhibitory second messenger. Thus, by inhibiting the synthesis of prostacyclin, acetylsalicylic acid reduces cAMP formation, whereas clopidogrel potentiates it. Therefore, with higher doses of acetylsalicylic acid, the potentiation of cAMP production by clopidogrel may be attenuated, which could limit the antithrombotic potential of the drug combination. The purpose of this study was to examine this possibility in vivo. Approach and Results- Mice were given oral acetylsalicylic acid at varying doses, oral clopidogrel (5 mg/kg body weight), or both. At doses of 0.15 and 0.6 mg/kg, acetylsalicylic acid inhibited arachidonic acid-induced platelet aggregation, but only 0.6 mg/kg acetylsalicylic acid, or higher, decreased the plasma levels of 6-keto-prostaglandin-F1α, the stable metabolite of prostacyclin. When given with clopidogrel, laser injury-induced arterial thrombi were significantly larger with the 0.6 mg/kg dose of acetylsalicylic acid than with the 0.15 mg/kg dose. Thrombi in mice treated with clopidogrel and the 0.15 mg/kg dose of acetylsalicylic acid were smaller than in mice treated with clopidogrel alone, suggesting that acetylsalicylic acid can add to the antithrombotic effect of clopidogrel but that higher doses of acetylsalicylic acid blunt the antithrombotic effect of clopidogrel. Conclusions- These findings support the use of lower, prostacyclin-preserving, doses of acetylsalicylic acid in conjunction with clopidogrel.

Abnormal megakaryocyte development and platelet function in Nbeal2(-/-) mice.

Gray platelet syndrome (GPS) is an inherited bleeding disorder associated with macrothrombocytopenia and α-granule-deficient platelets. GPS has been linked to loss of function mutations in NEABL2 (neurobeachin-like 2), and we describe here a murine GPS model, the Nbeal2(-/-) mouse. As in GPS, Nbeal2(-/-) mice exhibit splenomegaly, macrothrombocytopenia, and a deficiency of platelet α-granules and their cargo, including von Willebrand factor (VWF), thrombospondin-1, and platelet factor 4. The platelet α-granule membrane protein P-selectin is expressed at 48% of wild-type levels and externalized upon platelet activation. The presence of P-selectin and normal levels of VPS33B and VPS16B in Nbeal2(-/-) platelets suggests that NBEAL2 acts independently of VPS33B/VPS16B at a later stage of α-granule biogenesis. Impaired Nbeal2(-/-) platelet function was shown by flow cytometry, platelet aggregometry, bleeding assays, and intravital imaging of laser-induced arterial thrombus formation. Microscopic analysis detected marked abnormalities in Nbeal2(-/-) bone marrow megakaryocytes, which when cultured showed delayed maturation, decreased survival, decreased ploidy, and developmental abnormalities, including abnormal extracellular distribution of VWF. Our results confirm that α-granule secretion plays a significant role in platelet function, and they also indicate that abnormal α-granule formation in Nbeal2(-/-) mice has deleterious effects on megakaryocyte survival, development, and platelet production.

Tissue factor microparticles and haemophilia.

Treatment choices for haemophilia patients with inhibitors are suboptimal. Tissue factor-bearing microparticles home to thrombi in a cell adhesion molecule-dependent fashion. Their potential utility as a procoagulant is discussed along with the challenges of evaluating this approach in mouse models of haemophilia.