YC‐1 inhibited human platelet aggregation through NO‐independent activation of soluble guanylate cyclase

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Abstract
Our previous study demonstrated that YC‐1, a derivative of benzylindazole, is a novel activator of soluble guanylate cyclase (sGC) in rabbit platelets. This work investigated whether the antiplatelet effect of YC‐1 was mediated by a nitric oxide (NO)/sGC/cyclic GMP pathway in human platelets. In human washed platelets, YC‐1 inhibited platelet aggregation and ATP released induced by U46619 (2 μm), collagen (10 μg ml−1) and thrombin (0.1 u ml−1) in a concentration‐dependent manner with IC50 values of (μm) 2.1 ± 0.3, 11.7 ± 2.1 and 59.3 ± 7.1, respectively. In a 30,000 g supernatant fraction from human platelet homogenate, YC‐1 (5–100 μm) increased sGC activity in a concentration‐dependent manner. At the same concentration‐range, YC‐1 elevated cyclic GMP levels markedly, but only slightly elevated cyclic AMP levels in the intact platelets. MY‐5445, a selective inhibitor of cyclic GMP phosphodiesterase, potentiated the increases in cyclic GMP caused by YC‐1, and shifted the concentration‐anti‐aggregation curve of YC‐1 to the left. In contrast, HL‐725, a selective inhibitor of cyclic AMP phosphodiesterase, did not affect either the increases in cyclic nucleotides or the anti‐aggregatory effect caused by YC‐1. Methylene blue, an inhibitor of sGC, blocked the increases of cyclic GMP caused by YC‐1, and attenuated markedly the anti‐aggregatory effect of YC‐1. The adenylate cyclase inhibitor, 2′,5′‐dideoxyadenosine (DDA) did not affect YC‐1‐induced inhibition of platelet aggregation. Haemoglobin, which binds NO, prevented the activation of sGC and anti‐aggregatory effect caused by sodium nitroprusside, but did not affect YC‐1 responses. These results would suggest that YC‐1 activates sGC of human platelets by a NO‐independent mechanism, and exerts its antiplatelet effects through the sGC/cyclic GMP pathway.