Prabhakar Deonikar

Modulation of ADP-Induced Platelet Activation by Aspirin and Pravastatin: Role of Lectin-Like Oxidized Low-Density Lipoprotein Receptor-1, Nitric Oxide, Oxidative Stress, and Inside-Out Integrin Signaling

Lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1), a receptor for oxidized-LDL, is up-regulated in activated endothelial cells, and it plays a role in atherothrombosis. However, its role in platelet aggregation is unclear. Both aspirin and HMG CoA reductase inhibitors (statins) reduce LOX-1 expression in endothelial cells. In this study, we investigated the effect of aspirin and pravastatin on LOX-1 expression on plate-lets. After ADP stimulation, mean fluorescence intensity of LOX-1 expression on platelets increased 1.5- to 2.0-fold. Blocking LOX-1 inhibited ADP-induced platelet aggregation in a concentration- and time-dependent manner. We also established that LOX-1 is important for ADP-stimulated inside-out activation of platelet αIIbβ3 and α2β1 integrins (fibrinogen receptors). The specificity of this interaction was determined by arginine-glycine-aspartate-peptide inhibition. Furthermore, we found that LOX-1 inhibition of integrin activation is mediated by inhibition of protein kinase C activity. In other experiments, treatment with aspirin (1–10 mM) and pravastatin (1–5 μM) reduced platelet LOX-1 expression, with a synergistic effect of the combination of aspirin and pravastatin. Aspirin and pravastatin both reduced reactive oxygen species (ROS) released by activated platelets measured as malonyldialdehyde (MDA) release and nitrate/nitrite ratio. Aspirin and pravastatin also enhanced nitric oxide (NO) release measured as nitrite/nitrite + nitrate (NOx) ratio in platelet supernates. Small concentrations of aspirin and pravastatin had a synergistic effect on the inhibition of MDA release and enhancement of nitrite/NOx. Thus, LOX-1 is important for ADP-mediated platelet integrin activation, possibly through protein kinase C activation. Furthermore, aspirin and pravastatin inhibit LOX-1 expression on platelets in part by favorably affecting ROS and NO release from activated platelets.

Extracellular diffusion and permeability effects on NO–RBCs interactions using an experimental and theoretical model

Nitric oxide (NO) is a potent vasodilator and its homeostasis depends on interaction with RBCs. A key factor in understanding NO-RBC interactions in vascular lumen is a comprehensive analysis of product identification and quantification. In this context, administration of NO during in vitro NO-RBC interactions becomes a crucial variable. In this study, we designed a bioreactor that maintains a precise NO concentration in the headspace that diffuses to RBCs suspension to study the quantitative effect of NO concentration and hematocrit (Hct) on NO-RBC interactions. The products of NO-RBC reaction (nitrite and total nitrogen species (total NOx)) were measured by chemiluminescence assay. A mathematical model simulating NO biotransport to a single RBC was developed to (1) estimate NO-RBC reaction rate constant; (2) predict the NO concentrations in the bulk RBC suspension and at the RBC membrane for RBC membrane NO permeability (P(m)) values of 0.0415-40 cm/s. Measured nitrite and total NOx concentrations increased with increase in headspace NO concentration whereas nitrite concentrations decreased with hematocrit and total NOx concentrations increased with hematocrit. This indicates that the extracellular resistance is a controlling factor for RBC uptake of NO. Modeling results showed that the effective reaction rate constant (k(eff)) for NO-RBC interactions was 2.32 x 10(4)-1.08 x 10(6) M(-1) s(-1). Results also predict that the membrane permeability in the range of 0.0415-0.4 cm/s is required to maintain physiologically relevant levels of NO at the smooth muscle cell layer. The effective reaction rate constant increased with increase in P(m) and magnitude of increase was higher at 45% Hct. For all P(m) values, the k(hb)/k(eff) ratios were lower for 45% Hct as compared to 5% Hct indicating extracellular resistance is important for RBC NO uptake. Our experimental and mathematical analyses of NO-RBC interactions indicate that both unstirred layer and RBC membrane have a significant effect on NO transport to RBCs. In addition, the membrane permeability in the range of 0.0415-0.4 cm/s is required to maintain sufficient NO concentrations at the smooth muscle cell layer.