A Mechanism of Impaired Platelet Response to ADP Stimulation in the CLIC1 Knockout Mice

Abstract number: P2137

Jiang¹ L, Kuffner¹ T, Qiu¹ MR, Joseph² J, Low² J, Matthaei³ K, M.-Smith⁴ M, Curmi¹ PMG, Breit¹ SN

¹¹Centre for Immunology, St. Vincent's Hospital and the University of New South Wales, Sydney, Australia ¹¹Centre for Immunology, St. Vincent's Hospital and the University of New South Wales, Sydney, Australia ²²Department of Haematology, St. Vincent's Hospital, Sydney, Australia ³³Gene Targeting Laboratory, John Curtin School of Medical Research, Australian National University, Canberra, Australia ⁴⁴Department of Physiology, University of Cambridge, Cambridge, UK

CLIC1 is a chloride ion channel protein of both cytoplasmic and subcellular membranes that is highly expressed in platelets. It has been known that platelet function is regulated by calcium hemostasis. As a counter-ion of Ca²⁺, Cl^- is implicated in the regulation of platelet function. However, its role is not yet fully understood. To determine the effect of CLIC1 on platelet function, we examined the activation and aggregation of platelets from CLIC1 knockout (KO) mice generated by our group. CLIC1 KO and wild type (WT) 129SvJ mice were bled by tail nicking or cardiac puncture. Platelet counts were determined using a haemocytometer. Platelet activation was measured by flow cytometry of surface GPIIbIIIa on diluted whole blood or washed platelets using PE-labelled antibody against the active form of GPIIbIIIa. Platelet aggregation was examined on platelet-rich plasma using an aggregometer. Our results show that compared to WT, the CLIC1 KO mice have an about 15% higher platelet count (KO: 835 ± 109 × 10³/µl, WT: 715 ± 110 × 10³/µl, P= 0.02). However, they are less responsive to ADP stimulation (0.5–10 µM), although they respond equally to thrombin and U46619, an analog of thromboxane A₂. Further studies using the antagonists of the ADP receptors show that MRS2179, the P2Y₁ antagonist, inhibited the ADP-induced platelet activation with greater potency in the WT compared to the CLIC1 KO, whereas the P2Y₁₂ antagonist inhibited the ADP response with equal potency in the WT and KO. These results indicate that the Gq-coupled Ca²⁺-mobilizing P2Y₁ receptor may be the main player responsible for the attenuated ADP response in the KO platelets. The lack of CLIC1 chloride channel in KO platelets may result in impaired Ca²⁺ regulation which is necessary for normal platelet activation.