Release of calcium and P-selectin from intraplatelet granules is hampered by procaine

The inhibition of platelets by some local anaesthetics has been related to the modulation of platelet membrane lipid fluidity, and one of these compounds, procaine, has been proven to be particularly effective inhibitor. In the present study, we examined the effect of procaine on the mobilization of intracellular granule contents in isolated washed platelets. We revealed that the presence of 10 mg/ml procaine significantly hampered platelet release reaction, as demonstrated by the significant reduction in the expression of platelet P-selectin (CD62) on one hand, and significantly enhanced expression of GPIb alpha (CD42b) antigen on the other, following either 1 hour incubation of washed platelets at room temperature (%CD62: 37.1+/-6.8% of control incubated without procaine, p<0.0001; %CD42b: 116.2+/-6.3% of control, p<0.0001) or activation of whole blood platelets with ADP, TRAP, or thrombin. Procaine, which acted as a rigidizer, significantly decreased platelet membrane fluidity (ESR h(+1)/h0 ratio of 5-DOXYL-Ste reduced down to 93.1+/-3.7% of control, p<0.001). In washed Fura-2-loaded platelets procaine not only brought about the significantly reduced Ca2+ release from intraplatelet storage pools after platelet stimulation with 15 micromol/l ADP (25.3+/-12.5% of control, p<0.001), but also it significantly increased the reduction in Ca2+ concentration upon the addition of Ca2+ chelator, EDTAK2 (48.9+/-13.5% vs. 40.9+/-12.1% of initial Ca2+]i concentration, p(1,alpha)<0.025). Overall, procaine considerably reduced calcium mobilization from intraplatelet storage pools and Ca2+ efflux across platelet membrane. Based on these data, we suggest that the preventive effects of procaine on platelet release reaction and calcium mobilization might relate to the changes in the organization of membrane components embedded into a lipid bilayer, which are crucial in triggering of platelet release reaction. Procaine-mediated dislocations of some membrane components and/or distortion of lipid-protein interactions could generate a steric hindrance, which might interfere with platelet signal transduction, thus leading to impaired mobilization of Ca2+ and other components from intraplatelet storage pools.