Involvement of Nitric Oxide on Calcium Mobilization and Arachidonic Acid Pathway Activation during Platelet Aggregation with different aggregating agonists.

Platelet aggregation by different aggregating agonists is essential in the normal blood coagulation process, the excess of which caused acute coronary syndrome (ACS). In all cases, the activation of arachidonic acid by cycloxygenase was needed for the synthesis of thromboxane A2 (TXA2) but the mechanism of arachidonic acid release in platelets remains obscure. Studies were conducted to determine the role of nitric oxide (NO), if any, on the release of arachidonic acid in platelets. The cytosolic Ca(2+) was visualized and quantitated by fluorescent spectroscopy by using QUIN-2. NO was measured by methemoglobin method. Arachidonic acid was determined by HPLC. TXA2 was measured as ThromboxaneB2 (TXB2) by ELISA. Treatment of platelets in platelet-rich plasma (PRP) with different aggregating agents resulted in the inhibition of nitric oxide synthase (NOS) which inhibited the production of NO synthesis and increased TXA2 synthesis. Furthermore, the treatment of washed PRP with different platelet aggregating agents resulted in the increase of [Ca(2+)] in nM ranges. In contrast, the pre-treatment of washed PRP with aspirin increased platelet NO level and inhibited the Ca(2+) mobilization and TXA2 synthesis. These results indicated that the aggregation of platelets by different aggregating agonists was caused by the cytosolic Ca(2+) mobilization due to the inhibition of NOS.

The role of inhibition of nitric oxide synthesis in the aggregation of platelets due to the stimulated production of thromboxane A2.

The aggregation of platelets by ADP is reported to be mediated through prostaglandin synthesis. In contrast, nitric oxide is known to inhibit platelet aggregation through the synthesis of cyclic AMP and cyclic GMP. Studies were conducted to determine the role of ADP, if any, on the synthesis of nitric oxide in platelets. Both normal male and female volunteers between the ages of 30 and 45 years participated in the study. Thromboxane A2 (TXA2) was measured as thromboxane B2 by ELISA. Nitric oxide was measured by methhaemoglobin method. It was found that the treatment of platelet-rich plasma (PRP) with different concentrations of ADP (0-8.0 µmol/l) resulted in increased platelet aggregation, and at 8.0 µmol/l ADP, the basal nitric oxide level was found to be maximally decreased from 0.3 ± 0.10 nmol/10 platelets to 0 nmol/10 platelets in PRP (P < 0.0001; n = 10). Line-weaver-Burk plot of nitric oxide synthase (NOS) activity in the presence of 2.0 µmol/l ADP reduced the Vmax from 6.662 to 2.22 nmol nitric oxide/h per mg protein compared with control. Inhibition of nitric oxide synthesis by N-methyl-L-arginine acetate ester (L-NAME), an inhibitor of NOS, was found to aggregate platelets due to the reduction of platelet nitric oxide level (Pearson's coefficient of correlation, r =  -0.986, P < 0.001, n = 10). The treatment of PRP to L-NAME was found to increase TXA2 synthesis to 1.679 ± 0.05 from 0 pmol/10 platelets. These results suggested that inhibition of NOS in platelets resulted in platelet aggregation through TXA2 synthesis in PRP through a novel pathway.

Reduction of death rate due to acute myocardial infarction in subjects with cancers through systemic restoration of impaired nitric oxide.

INTRODUCTION: Excessive aggregation of platelets at the site of plaque rupture on the coronary artery led to the formation of thrombus which is reported to precipitate acute myocardial infarction (AMI). Nitric oxide (NO) has been reported to inhibit platelet aggregation and induce thrombolysis through the in situ formation of plasmin. As the plasma NO level in AMI patients from two different ethnic groups was reduced to 0 µM (median) compared to 4.0 µM (median) in normal controls, the effect of restoration of the NO level to normal ranges on the rate of death due to AMI was determined. METHODS AND RESULTS: The restoration of plasma NO level was achieved by a sticking small cotton pad (10×25 mm) containing 0.28 mmol sodium nitroprusside (SNP) in 0.9% NaCl to the abdominal skin of the participants using non-toxic adhesive tape which was reported to normalize the plasma NO level. The participants (8,283) were volunteers in an independent study who had different kinds of cancers and did not wish to use any conventional therapy for their condition but opted to receive SNP "pad" for their condition for 3 years. The use of SNP "pad" which normalized (≈4.0 µM) the plasma NO level that in consequence reduced the death rate due to AMI, among the participants, was found to be significantly reduced compared to the death due to AMI in normal population. CONCLUSION: Our data suggested that the use of SNP "pad" significantly reduced the death due to AMI. TRIAL REGISTRATION: www.ctri.nic.in CTRI/2013/12/004236.