The present study shows that rats fed with high-fat diet (HFD) exhibit ex-vivo platelet hyperaggregability to ADP and thrombin, which is accompanied by higher intraplatelet ROS production. The platelet hyperaggregability was prevented by the antioxidant compounds PEG-catalase and NAC in HFD group indicating a critical role for intracellular ROS in this phenomenon. Moreover, the NO donors SNP and SNAP, as well as the soluble guanylyl cyclase stimulator BAY 41-2272 showed a lower efficacy in inhibiting the platelet aggregation in HFD rats, possibly as a consequence of lower platelet cGMP productions in this diet-induced obesity model.
Platelets, Hyperglycemia and Oxidative Stress
Evidences have shown that persistent hyperglycemia can activate alternative glucose metabolism pathways, which in turn result in the formation of deleterious products derived from protein or lipid structure alterations named advanced glycation end products (AGEs), which can deeply affect the function of the cardiovascular system . In vascular system, the interaction of AGES with their receptors (RAGE) can activate complex signaling pathways causing increased production of inflammatory mediators and massive ROS generation, resulting in reduced NO bioavailability and endothelium dysfunction , as well as alterations in coagulation system . In cardiac tissues, the hyperglycemia-induced ROS activate the MEK/ERK pathway to increase GATA-4 phosphorylation, which in turn generates cardiac hypertrophy . Hyperglycemia is also associated with dysregulation of sympathetic innervation to the myocardial tissues . Thus, the mechanistic event by which diet-induced obesity causes platelet dysfunction may therefore be associated with hyperglycemia, which is consistent with the abnormality of the OGTT and ITT in HFD group. Previous studies show that acute hyperglycaemia enhances collagen-induced platelet aggregation via increased mitochondrial O2- production . Acute hyperglycaemia following an oral glucose tolerance test or a carbohydrate-rich meal also promotes platelet activation in vivo [22, 23].
Radicals derived from oxygen represent the most important class of ROS generated in living systems. Superoxide anion (O2-) is considered the primary ROS, and it can further interact with other molecules, either directly or through enzyme- or metal-catalyzed processes, to generate other physiological relevant ROS such as hydrogen peroxyde (H2O2) and -OH, as well as peroxynitrite (ONOO-) . Adiposity in humans is reported to increase the risk of athero-thrombotic events due partly to increased oxidative stress, as evaluated by measurement of systemic biomarkers such as serum levels of lipid peroxidation, TNF-α, free fatty acids and oxidized LDL . Different sources, including platelets, may generate O2- including the NADPH-oxidase, xanthine oxidase and arachidonate-derived prostaglandin-like metabolites [25–29]. However, the contribution of intraplatelet ROS in modulating platelet reactivity in conditions of adiposity has not been explored. Therefore, this study was designed to explore the ex-vivo washed platelet aggregation in HFD rats, and the potential role of intraplatelet ROS production and NO bioavailability in modulating platelet reactivity. Our data showed that ADP- and thrombin-induced platelet aggregation were significantly higher in HFD group, which was accompanied by higher levels of ROS production, as assessed by fluorescence assays using DCFH . Moreover, prior incubation of platelets with the ROS scavengers PEG-catalase or NAC suppressed both the increased ROS production and the hyperaggregability in HFD rats. Altogether, our data indicate that ex vivo platelet hyperaggregability in HFD rats is closely linked with enhanced intraplatelet ROS production. A recent study showed that NAC, at concentrations attainable with oral dosing , significantly reduces ADP- and thrombin-induced platelet aggregation in whole blood of type 2 diabetic patients that is associated with an enhancement of its antioxidant activity .
Increased oxidative stress may also influence platelet function by decreasing NO bioavailability . Nitric oxide is a ROS involved in multiple biological functions essential for the cardiovascular system and platelet function. Accordingly, in our study the ADP-induced platelet aggregation was markedly reduced by the NO donors, SNP and SNAP, in SCD rats, that was accompanied by marked elevations in the cGMP levels, as expected. Interestingly, in HFD rats, platelets were resistant to the cGMP elevations in response to SNP and SNAP, as well as to the inhibitory actions of these agents on platelet aggregation. It is likely that excess of O2- production in platelets of HFD rats inactivates SNP- and SNAP-derived NO. This is consistent with studies performed in obese subjects and type 2 diabetic obese patients where platelets are resistant to glyceryl nitrate and SNP [33, 34].
Platelet Hyperaggregability and Role of the Cyclic Nucleotides
The soluble guanylyl cyclase (sGC) is a widely distributed signal transduction enzyme that, under activation by NO, converts GTP into the second messenger cGMP which in turn affects various downstream targets such as protein kinases, cyclic nucleotide-gated channels or phosphodiesterases . One of the crucial pre-requisites of the NO-mediated sGC activation is the presence of the reduced haem moiety where its oxidation or loss renders the enzyme insensitive to NO. Nitric oxide-independent sGC activators have emerged as valuable tools to elucidate the physiopathology of the NO-sGC-cGMP signaling pathway . The compound BAY 41-2272 was reported as a haem-dependent and potent NO-independent sGC stimulator . BAY 41-2272 directly stimulates sGC and increases the sensitivity of the enzyme to NO, generating significant amounts of cGMP by stimulating the sGC mostly via NO-independent mechanisms [38, 39]. Through this mechanism, BAY 41-2272 produces a variety of effects, including anti-aggregatory effects. In our study, BAY 41-2272 greatly elevated the cGMP levels and nearly abolished the platelet aggregation in SCD rats, as expected. However, the elevations of cGMP and inhibition of platelet aggregation by BAY 41-2272 in HFD rats were significantly lower compared with SCD group. This apparently indicates that sGC of platelets from HFD rats display a defect in producing appropriate amounts of intracellular cGMP. In rat platelets, under physiological conditions, inhibition of platelet aggregation by BAY 41-2272 requires the reduced form of sGC and the presence of NO . Furthermore, the free radical ONOO- is able to oxidize the prosthetic haem group of sGC to its NO-insensitive Fe3+ state [41–43]. If that takes place in platelets from HFD rats, then BAY 41-2272 would be indeed expected to be less effective in activating sGC. In this aspect, it would be worth trying haem-independent sGC activators such as HMR1766 and BAY 58-2667 because they prevent sGC from oxidation-induced degradation, as evidenced in chinese hamster ovary cell line and in primary porcine endothelial cells . Interestingly, the direct sGC activator HMR1766 has been shown to enhance the NO/cGMP-mediated signaling in platelets from streptozotocin-induced diabetic rats, reducing platelet-aggregates with other blood cells .
Besides the NO - cGMP - PDE5 pathway, the activation of platelets is inhibited by cAMP-elevating agents . Elevation of intracellular cAMP levels can be achieved through the activation of adenylate cyclase either directly or through appropriately coupled membrane receptors, as well as by preventing the hydrolysis of cAMP by the cyclic nucleotide phosphodiesterases. In our study, the cAMP-elevating agent iloprost (stable prostacyclin analogue) suppressed the ADP-induced platelet aggregation in both SCD and HFD groups, excluding that hyperaggregability in HFD rats reflect changes in the cAMP signaling pathway.