Insulin inhibits platelet aggregation through nitric oxide synthesis by stimulating platelet insulin activated nitric oxide synthase. Impaired platelet insulin activated nitric oxide synthase in acute myocardial infarction (AMI) patients had been reported and thus our aim was to identify and isolate the factors impairing insulin activated nitric oxide in acute myocardial infarction patients' plasma and study its effect on platelets aggregation in vitro. The insulin activated nitric oxide synthase inhibitor was identified as a protein and was purified from the plasma of AMI subjects using DEAE cellulose and Sephadex G-50 column, molecular weight determined by SDS-PAGE, nitric oxide quantified by methaemoglobin method, inhibitor protein quantified in plasma by immunoblot and ELISA, platelet aggregation studies done using an aggregometer, thromboxane-A2 in the platelets determined by radioimmunoassay, (125)I-insulin radioligand binding studies done using normal subject platelets. The purified nitric oxide synthase inhibitor protein was ~66 kDa, concentration in AMI subjects' plasma varied from 114 to 9,090 μM and was undetected in normal subjects' plasma. The inhibitor protein competes with insulin for insulin receptor binding sites. The Incubation of the normal subject PRP with 5.0 μM inhibitor for 30 min followed by 0.4 μM ADP addition caused platelet aggregation in vitro, 130 μM aspirin or 400 μU insulin/ml addition was able to abrogate 0.4 μM ADP induced platelet aggregation even in the presence of 5.0 μM inhibitor. A potent inhibitory protein against insulin activated nitric oxide synthase in platelets appears in circulation of AMI subjects impairing nitric oxide production, potentiating ADP induced platelet aggregation and increasing the thromboxane-A2 level in platelets.