Role of ascorbic acid in the modulation of inhibition of platelet aggregation by polymorphonuclear leukocytes


      Objectives: We investigated the modulatory effect of ascorbate on the inhibition of platelet aggregation response by polymorphonuclear leukocytes (PMNs) and characterized the mechanism of the inhibitory response. Background: PMNs have been reported to play a significant role in vascular homeostasis by releasing various factors including short-lived reactive oxygen species (ROS) and nitric oxide (NO). NO prevents the activation of circulating platelets and plays a significant role in hemostasis. In addition, PMNs also have the capacity to store very high concentrations of ascorbate. The physiological implications of storing such high concentrations of an antioxidant by a cell-releasing free radicals is unknown, viz. a viz. hemostatic regulation. Methods: ADP-induced aggregation in human, monkey and rat platelet-rich plasma (PRP) was monitored in the presence of PMNs treated with varying concentrations of ascorbate/dehydroascorbate. NO generation from rat and human PMNs treated with ascorbate was monitored on a FACS Calibur flow cytometer and intraplatelet cyclic guanosine 3′,5′-monophosphate (cGMP) levels was also measured. Results: PMNs induced a cell number and time-dependent inhibition of ADP-induced aggregation. The PMNs dependent inhibition was enhanced significantly at 30 min by ascorbate (300 μM). Ascorbate seemed to exert its effects through its oxidized product, dehydroascorbate, as the effects was prevented in the presence of d-glucose (10 mM). Dehydroascorbate elicited significant potentiation of the PMNs induced inhibitory responses and these effects were mediated by the release of NO and subsequent activation of platelet guanylyl cyclase. Flow cytometry experiments with human and rat PMNs confirmed the release of NO and the elevated platelet cGMP levels confirmed NO-mediated activation of guanylyl cyclase. Conclusions: Ascorbate in circulation seems to prevent the activation of platelets by enhancing the release of antiaggregatory NO, from neighbouring or cohabitant PMNs. The ascorbate effect is mediated through its conversion to dehydroascorbate, subsequently, gets taken up by the cell and converted back to ascorbate. Intracellular ascorbate potentiates the release of NO from the PMNs and subsequently activates guanylyl cyclase in the platelets.
      Condensed Abstract
      The thrombotic process involves platelets and polymorphonuclear leukocytes (PMNs). PMNs release both reactive oxygen species (ROS) and nitric oxide (NO). The present study was conducted to investigate the physiological significance of the ascorbate in circulation, by using rat, human and monkey platelets and PMNs. Ascorbate significantly potentiated the PMNs-mediated inhibition of aggregation. Ascorbate-mediated effects were mediated by dehydroascorbate and subsequently releases NO. NO, then activates the guanylyl cyclase in the platelets and elevates the platelet cGMP levels. It seems likely that ascorbic acid in PMNs play a significant role in vascular homeostasis by elevating levels of NO and subsequently maintaining the platelet in an inactivated state, preventing the initiation of thrombotic process.



      PMNs (polymorphonuclear leukocytes), ROS (reactive oxygen species), NO (nitric oxide), cGMP (cyclic guanosine 5′-monophosphate), PRP (platelet-rich plasma), DAF (diaminofluorescein diacetate), DHA (dehydroascorbate), ADP (adenosine-5′-diphosphate), PTIO (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide), ODQ (1H-[1,2,4]oxadiazolo[4,3]quinoxalin-1-one), cAMP (cyclic adenosine 5′-monophosphate), HBSS (Hanks balanced salt solution)
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