The Signaling Molecule CalDAG-GEFI Represents a Novel Target for Antithrombotic Therapy.

The signaling molecule Ca²⁺ and diacylglycerol regulated guanine nucleotide exchange factor I (CalDAG-GEFI) plays a crucial role in the immediate, Ca²⁺ dependent activation of Rap1 and intergrin αaIIbβ3 in stimulated platelets. Our previous studies demonstrated that signaling by protein kinase C and the Gi-coupled receptor for ADP, P2Y12, provides an alternative pathway that facilitates the delayed but prolonged activation of Rap1. P2Y12 inhibitors such as clopidogrel bisulfate (Plavix) are currently considered the gold standard for the prevention and treatment of thrombotic complications. The aims of the study were to: 1) evaluate CalDAG-GEFI signaling as a potential new target for antithrombotic therapy and 2) to compare the contribution of CalDAG-GEFI and P2Y12 signaling to thrombus formation in pre-clinical models of models of thrombotic disease.

Thrombus formation was compared in: wildtype (WT), CalDAG-GEFI^-/-, WT treated with clopidogrel (clopidogrel was administered orally 24h and 3h prior to the experiment at a dosage of 0,075mg/g bodyweight) and CalDAG-GEFI^-/- mice treated with clopidogrel. Thrombosis was studied in three different models: 1) laser-induced thrombosis in the microcirculation of the cremasteric muscle (thrombin-dependent, localized), tissue factor-induced pulmonary thromboembolism (thrombin-dependent, systemic) and FeCl3-induced thrombosis in the mesentery (collagen/thrombin-dependent, localized). As shown previously, clopidogrel treatment significantly reduced but did not abolish laser-induced thrombus formation in WT mice. Laser induced thrombosis was completely inhibited in arterioles of CalDAG-GEFI ^-/- mice, while significant thrombus formation was observed in venules. Clopidogrel treatment of CalDAG-GEFI-/- mice further reduced venous thrombus formation, providing in vivo confirmation that platelet aggregation in the absence of CalDAG-GEFI requires P2Y12 signaling. In line with these findings, we observed that CalDAG-GEF-/- mice were partially protected from tissue factor-induced pulmonary thromboembolism when compared to clopidogrel-treated CalDAG-GEF-/- mice. In the model of FeCl3-induced thrombosis, CalDAG-GEFI-/- platelets adhered to the damaged vascular wall but failed to form thrombi in both venules and arterioles. In contrast clopidogrel-treated WT mice were protected from FeCl3-induced vessel occlusion but continuously formed embolizing thrombi of considerable size that contained activated platelets. This phenomenon was detectable in both arterioles and venules and might be of clinical relevance.

Our studies identify CalDAG-GEFI as a promising new target for antiplatelet therapy. CalDAG-GEFI inhibition will have a strong antithrombotic effect as it is a critical component of the near-immediate platelet response to exposed extracellular matrix and/or soluble agonists. However, backup by PKC/P2Y12 signaling should allow for the formation of small but stable mural thrombi, especially under low flow conditions as found in veins and in venules, thus limiting the risk of bleeding complications.

Poncz:Diagnostica Stago: Patents & Royalties.