Reactive oxygen species and human platelet GP IIb/IIIa receptor activation

Abstract

This study concerned reactive oxygen species for their potential to activate human platelet GP IIb/IIIa receptors. All cells produce reactive oxygen species - radicals that can abstract electrons and hydrogen atoms from biological molecules to alter cell function. In many cells, radicals contribute to cellular signaling. In platelets, the predominant oxidant effect is platelet activation. Less is known concerning oxidants and GP IIb/IIIa receptor activation. The first aim of the current study was to confirm that although both H(2)O(2) and tert butyl hydroperoxide both predispose platelets to aggregation; neither directly activates GP IIb/IIIa receptors. The second aim was to demonstrate that even in the presence of extracellular redox iron; H(2)O(2) does not activate GP IIb/IIIa receptors. The third aim was to determine if extracellular superoxide anions evoke GP IIb/IIIa activation. Finally, a role for intra-platelet iron in GP IIb/IIIa activation was examined. Intracellular superoxide anions are produced in excess during platelet activation and curiously, they are uniquely able to increase intracellular free iron. This iron can, in a redox manner, generate radicals and these iron dependent species modulate signaling systems, including systems associated with adhesion receptor activation. In the current studies, platelets in suspension were exposed to H(2)O(2) and to tert butyl hydroperoxide, to H(2)O(2) plus ferrous or ferric chloride (+/- ascorbate to enhance iron redox cycling) and to xanthine plus xanthine oxidase to generate extra-platelet superoxide anions. Intra-platelet iron was increased with iron ionophore 8-hydroxyquinoline. During flow cytometry, intra-platelet oxidant state was assessed with the redox sensitive fluorescent indicator H2DCF, while GP IIb/IIIa activation was assessed using fluorescent antibody PAC-1. Results showed that although all the oxidizing systems examined increased intra-platelet oxidant state, GP IIb/IIIa receptors were not activated by H(2)O(2), by tert butyl hydroperoxide, by H(2)O(2) plus iron (+/- ascorbate) or by xanthine plus xanthine oxidase. In contrast, iron plus ionophore 8-hydroxyquinoline evoked GP IIb/IIIa activation. Platelet positivity for PAC-1 increased from 2 +/- 0.2 to 28 +/- 7% (P < 0.005). However this response, although vigorous, was less than 56 +/- 8% (P < 0.001) evoked by thrombin 0.1 milliunit/ml. In conclusion, the results indicated that oxidant systems external to platelets did not activate GP IIb/IIIa receptors while increased intra-platelet iron was associated with appearance of cytosolic oxidizing species and with GP IIb/IIIa receptor activation.

An investigation of the antiplatelet effects of succinobucol (AGI-1067)

“…The concentrations of X/XO used in the current study are consistent with similar studies [24] and there are many reports in the literature which demonstrate that ROS derived either from platelets [25], from damaged red blood cells [26] or from addition of freeradical generating systems [27], can augment platelet aggregation in response to stimulating agents such as collagen and ADP. In this study, we found that whole blood aggregated more readily in response to collagen, suggesting that other cell types present in whole blood may contribute to platelet aggregation.…”

Clinically Relevant Concentrations of Dopamine Do Not Amplify Agonist-induced Human Platelet Ca2+ Mobilization or GP IIb IIIa Activation and Do Not Accelerate Acute Coronary Thrombosis in Dogs

“…Second, dogs were randomized to receive either intravenous dopamine 8 mgÁkg 21 Ámin 21 or epinephrine 0.2 mgÁkg 21 Ámin 21 (positive controls) and measurements repeated for 20 minutes. (Epinephrine 0.2 mgÁkg 21 Ámin 21 has been shown to increase spontaneous CFRs frequency and magnitude 14,26 ). Dopamine 8 mgÁkg 21 Ámin 21 dose was used because, when examined in a 4 dog's pilot study, this infusion rate approximately evoked the heart rate, arterial pressure, and cardiac index effects of epinephrine 0.2 mgÁkg 21 Ámin 21 .…”

“…Frequency and magnitude of these cycles are regarded as a semiquantitative measure of ongoing thrombosis. 14,25,26 CFR measurements were made during 4 periods in a crossover manner. First, spontaneous CFRs were assessed during 20-minute baseline control conditions.…”

“…Essential nonimmunostained negative controls, isotype controls, and PAC-1 controls in the presence of GP IIb IIIa blocker arginine-glycine-aspartic acid -serine were all done in a standard manner. 23,26 Data were collected as 5000-10,000 events per sample in dot plot format, each dot representing positivity above threshold for both PAC-1 and pan-platelet marker. Dopamine experiments were accompanied by positive controls with epinephrine 10 26 M. Positive controls were done to provide evidence that the experimental model was capable of capturing catecholamine-mediated platelet effects.…”

Bio-enriched Pleurotus mushrooms for deficiency control and improved antioxidative protection of human platelets?

“…Although the present study did not investigate the effect of extracts on platelet aggregation, it is known that ROS may play, along with nitric oxide, adenosine and prostacyclin, a profound role in platelet activation by scavenging nitric oxide (which has a preventive effect on platelet aggregation), triggering tyrosine phosphorylation of β3 or supporting the metabolism of collagen and arachidonic acid [23]. Considering that primary mechanism of tBHP action is generation of oxidative stress [45] and that tBHP has been previously demonstrated to support platelet activation cascade [46], it is plausible that the antioxidant properties of mushroom extracts may also prevent platelets from aggregation. Moreover, a recent study demonstrated that an extract of P. eous inhibited human platelet activation in vitro, the effect linked to the high mushroom content of phenolic compounds and flavonoids [47].…”