The mechanism of collagen-induced human platelet activation was examined using Ca 2؉ , Na ؉ , and the pHsensitive fluorescent dyes calcium green/fura red, sodium-binding benzofuran isophthalate, and 2 ,7 -bis(2-car- Collagen is the most thrombogenic component of the subendothelium (1). Following vascular damage, collagen is exposed to circulating platelets and both acts as a substrate for the adhesion of platelets (2-4) and induces platelet activation (4). The prevailing evidence proposes that two receptors are involved in the platelet response to collagen; integrin ␣ 2  1 acts to adhere platelets to collagen, allowing platelets to interact with the lower affinity receptor glycoprotein VI, which is mainly responsible for platelet activation (3,5).Many of the platelet responses to collagen progress simultaneously when platelets adhere to collagen. At high concentrations, collagen activation of platelets has been shown to proceed through activation of phospholipase C␥2 and subsequent cleavage of phosphatidylinositol 4,5-bisphosphate into inositol 1,4,5-trisphosphate and 1,2-diacylglycerol (6, 7). Inositol 1,4,5-trisphosphate induces the release of calcium from the dense tubular system (8, 9), whereas 1,2-diacylglycerol activates protein kinase C (10). The collagen-induced inositol 1,4,5-trisphosphate-mediated increase in [Ca 2ϩ ] i is accompanied by an influx of calcium from the extracellular milieu (11, 12). 1,2-Diacylglycerol and calcium mediate the characteristic platelet activation responses such as shape change, granule secretion, and aggregation.At lower concentrations, many of the effects of collagen are enhanced by its production of thromboxane A 2 (TXA) 1 (6, 13-15). The collagen-induced increase in [Ca 2ϩ ] i can be decreased by inhibiting the production of TXA via the pretreatment of platelets with cyclooxygenase inhibitors such as aspirin (11,16,17).Calcium is an important second messenger in the platelet activation cascade. At rest, a [Ca 2ϩ ] i of ϳ100 nM is maintained by a balance between the leak of Ca 2ϩ into the platelet and the concurrent efflux of free Ca 2ϩ across the plasma membrane of the platelet and accumulation in intracellular stores (18,19). Ca 2ϩ is moved out across the plasma membrane through the actions of the plasma membrane Ca 2ϩ -ATPase and the Na ϩ / Ca 2ϩ exchanger (NCX). Plasma membrane Ca 2ϩ -ATPases are membrane-inserted enzymes that use the energy of ATP hydrolysis to move Ca 2ϩ against its gradient and across the membrane. The NCX is capable of moving Ca 2ϩ into or out of the platelet cytosol in exchange for Na ϩ (20, 21). In the resting state, the NCX removes Ca 2ϩ from the platelet cytosol. Internally, Ca 2ϩ is transported into the dense tubular system by the sarco/endoplasmic reticulum Ca 2ϩ -ATPases 2b and 3 (22,23).In response to a moderate dose of collagen (10 g/ml), ϳ70% of the increase in [Ca 2ϩ ] i is due to the influx of Ca 2ϩ from the extracellular milieu, with the remainder as a function of Ca 2ϩ release from the dense tubular system (12). Because voltagega...