Measurement and Manipulation of Intracellular Ca2+ in Single Platelets and Megakaryocytes

Intracellular free Ca²⁺ is a key second messenger in virtually all cells. In unstimulated platelets and megakaryocytes, [Ca²⁺ ]_i is approx 100 nM and can be rapidly elevated by a variety of different agonists that activate phospholipase-C via heterotrimeric G proteins or receptor kinases (1 –4 ). Phospholipase-C (PLC) generates cytosolic IP₃ , which releases Ca²⁺ by opening cation channels on the intracellular stores. Agonists also evoke Ca²⁺ influx across the plasma membrane, although the exact nature of these pathways and their mechanism of activation and contribution under physiological conditions remain poorly understood and, in fact, highly controversial. Release of Ca²⁺ from the stores activates a Ca²⁺ influx pathway in both the platelet and megakaryocyte (5 –7 ); electrophysiological measurements have shown that ICRAC (calcium-release activated calcium current), one of the main candidates for the current underlying store-dependent Ca²⁺ influx, is present in the precursor cell (3 ,8 ). Non-store-dependent Ca²⁺ influx also seems to exist in the platelet (9 ) and TRPC6, a nonselective cation channel known to be activated by the PLC product diacylglycerol (DAG), has recently been detected in platelets and megakaryocytic cell lines (10 ). Finally, platelets possess at least one type of receptor-operated Ca²⁺ -permeable channel, the ATP-gated P2X₁ receptor, which allows rapid Ca²⁺ entry independently of store release (11 ,12 ).