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...

We sought to determine the mechanisms for hyperactivity and abnormal platelet Ca(2+) homeostasis in diabetes. The glycosylated Hb (HbA(1c)) level was used as an index of glycemic control. Human platelets were loaded with Ca- green-fura red, and cytosolic Ca(2+) ([Ca(2+)](i)) and aggregation were simultaneously measured. In the first series of experiments, the platelets from diabetic and normal subjects were compared for the ability to release Ca(2+) or to promote Ca(2+) influx. A potent and relatively specific inhibitor of Na(+)/Ca(2+) exchange, 5-(4-chlorobenzyl)-2',4'-dimethylbenzamil (CB-DMB), increased the second phase of thrombin-induced Ca(2+) response, suggesting that the Na(+)/Ca(2+) exchanger works in the forward mode to mediate Ca(2+) efflux. In contrast, in the platelets from diabetics, CB-DMB decreased the Ca(2+) response, indicating that the Na(+)/Ca(2+) exchanger works in the reverse mode to mediate Ca(2+) influx. In the second series of experiments we evaluated the direct effect of hyperglycemia on platelets in vitro. We found that thrombin- and collagen-induced increases in [Ca(2+)](i) and aggregation were not acutely affected by high glucose concentrations of 45 mM. However, when the platelet-rich plasma was incubated with a high glucose concentration at 37 degrees C for 24 h, the second phase after thrombin activation was inhibited by CB-DMB. In addition, collagen-stimulated [Ca(2+)](i) response and aggregation were also increased. Thus in diabetes the direction and activity of the Na(+)/Ca(2+) exchanger is changed, which may be one of the mechanisms for the increased platelet [Ca(2+)](i) and hyperactivity. Prolonged hyperglycemia in vitro can induce similar changes, suggesting hyperglycemia per se may be the factor responsible for the platelet hyperactivity in diabetes.

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In septic shock, systemic vasodilation and myocardial depression contribute to the systemic hypotension observed. Both components can be attributed to the effects of mediators that are released as part of the inflammatory response. We previously found that lysozyme (Lzm-S), released from leukocytes, contributed to the myocardial depression that develops in a canine model of septic shock. Lzm-S binds to the endocardial endothelium, resulting in the production of nitric oxide (NO), which, in turn, activates the myocardial soluble guanylate cyclase (sGC) pathway. In the present study, we determined whether Lzm-S might also play a role in the systemic vasodilation that occurs in septic shock. In a phenylephrine-contracted canine carotid artery ring preparation, we found that both canine and human Lzm-S, at concentrations similar to those found in sepsis, produced vasorelaxation. This decrease in force could not be prevented by inhibitors of NO synthase, prostaglandin synthesis, or potassium channel inhibitors and was not dependent on the presence of the vascular endothelium. However, inhibitors of the sGC pathway prevented the vasodilatory activity of Lzm-S. In addition, Aspergillus niger catalase, which breaks down H(2)O(2), as well as hydroxyl radical scavengers, which included hydroquinone and mannitol, prevented the effect of Lzm-S. Electrochemical sensors corroborated that Lzm-S caused H(2)O(2) release from the carotid artery preparation. In conclusion, these results support the notion that when Lzm-S interacts with the arterial vasculature, this interaction results in the formation of H(2)O(2), which, in turn, activates the sGC pathway to cause relaxation. Lzm-S may contribute to the vasodilation that occurs in septic shock.

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