Natural purines like ATP, ADP and adenosine have crucial roles in platelet physiology. This knowledge has been significant in drug development and today ADP receptor antagonists are widely used for prevention of thrombotic events following myocardial infarction and ischaemic stroke. Recent studies have shown that a purine analogue bearing nitrate ester group (denoted MK128) has anti-inflammatory effects probably due to its ability to donate nitric oxide (NO). However, other pharmacological mechanisms may contribute to the observed effect. The aim of the present study was to establish the anti-platelet activity and elucidate the underlying molecular mechanism(s) of the purine analogue MK128. We found that MK128 reduced aggregation and secretion induced by the thrombin receptor agonist SFLLRN and nearly abolished aggregation and secretion induced by thromboxane A2 (TxA2) and collagen receptor agonists. The inhibition took place despite blockage of the NO/cGMP signalling system. Furthermore, interaction between MK128 and platelet purinergic receptors did not explain the observed inhibition. Instead, we found that MK128 concentration-dependently inhibited Rho-associated kinase (ROCK), which led to decreased ROCK-dependent myosin phosphatase target subunit (MYPT)-1 phosphorylation and suppression of platelet functional responses.