The Role And Mechanism Of Vitamin D Deficiency In Platelet Activation And Thrombosis

Objective: High residual platelet reactivity(HRPR)plays a crucial role in influencing the clinical efficacy of dual antiplatelet therapy in patients with acute coronary syndrome(ACS)after percutaneous coronary intervention(PCI).Vitamin D deficiency has been associated with platelet hyperreactivity following dual antiplatelet therapy.However,the impact of vitamin D deficiency on platelet function and arterial thrombosis in vivo remains unclear.This study aimed to investigate the relationship between vitamin D deficiency and platelet hyperreactivity in ACS patients undergoing PCI.Additionally,we explored the role and specific mechanisms of vitamin D deficiency in platelet activation and thrombosis using platelets from vitamin D deficient animal models,Meg-01 cells with vitamin D receptor(VDR)knockdown,and platelets from clinical ACS patients.Methods:(1)Patients with ACS undergoing PCI and receiving regular dual antiplatelet therapy according to the guidelines were enrolled in the study.The plasma concentration of 25-hydroxyvitamin D3(25(OH)D3)was measured using HPLC-MS/MS,and the platelet response index(PRI)was assessed using VASP flow cytometry.(2)Thirty-six male SD rats weighing 140-160 g were randomly divided into three groups: control group(normal diet),vitamin D deficiency group(vitamin D deficiency diet),and vitamin D supplement group(1600 IU/day for one week continuously).The serum concentrations of 1,25-dihydroxyvitamin D3(1,25(OH)2D3),GPIIb/IIIa,and soluble Pselectin were measured using ELISA.Platelet aggregation rate was evaluated using a platelet function meter,platelet particle secretion was analyzed by transmission electron microscopy,and platelet membrane CD62 P expression was detected using flow cytometry.Blood vessel occlusion time was monitored using laser Doppler flow imager,and CD61 expression in thrombus was determined by immunohistochemistry.(3)Platelet proteins were extracted from animal models to detect the expressions of VDR,Akt,and p-Akt using western blotting.Meg-01 cells were transfected with VDR sh RNA,and the expression levels of P2Y12,Akt,and p-Akt were determined using western blotting.Platelet proteins from clinical samples were extracted for proteomic analysis.Results:(1)After adjusting for gender,age,genotype,and other risk factors,vitamin D deficiency was identified as an independent risk factor for HRPR in ACS patients after dual antiplatelet therapy(OR 4.21,P =0.001).Plasma concentration of 25(OH)D3 < 16.45 ng/m L was negatively correlated with PRI(R = 0.21,P = 0.031).Subgroup analysis revealed that low vitamin D levels mainly affected the incidence of HRPR in patients taking clopidogrel(0.49 vs 0.38,P < 0.05),whereas no significant effect was observed in patients taking ticagrelor(0.17 vs 0.14 vs 0.15 vs 0.16,P >0.05).Patients with vitamin D deficiency exhibited a significant increase in mean platelet volume(MPV)compared to the control group(10.35 vs10.38,P < 0.05).(2)Vitamin D deficiency enhanced ADP-induced platelet aggregation rate(30.60% vs 16.03,P < 0.05),increased platelet expansion on fibrinogen(P < 0.05),promoted the expression of GPIIb/IIIa and soluble P-selectin in plasma(P < 0.05),induced the release of α particles in platelets and the expression of CD62 P in plasma membrane(P < 0.05),and promoted platelet clot retraction(P < 0.05).Furthermore,the blood vessel occlusion time was significantly reduced in vitamin D deficiency group which accelerated the formation of arterial thrombosis in the body through establishing Fe Cl3-induced arterial thrombosis model(7.7 min vs 16.1 min,P < 0.05).The tail bleeding was also significantly shortened in vitamin D deficiency group(3.4 min vs 7.4 min,P < 0.05).(3)Compared to the control group,the expression of platelet surface VDR was significantly decreased(P < 0.05)in the vitamin D deficiency group,while p-Akt expression was significantly increased(P < 0.05).In vitro experiments confirmed that VDR sh RNA significantly increased P2Y12 expression in Meg-01 cells at the resting state or with ADP 100 n M stimulation compared to the control group(P < 0.05).Additionally,p-Akt expression was significantly increased(P < 0.05).Clinical proteomic analysis revealed a significant upregulation in the abundance of S100A8,S100A9,and IL-17 R proteins on platelets(P < 0.05),as well as increased expressions of plasma IL-17 and S100A12(P < 0.05)compared to the control group.Conclusion: Vitamin D deficiency is an independent risk factor for HRPR,potentially through decreased expression of platelet VDR receptors and subsequent activation of the Akt signaling pathway.Ultimately,this deficiency enhances platelet activity and exacerbates arterial thrombosis.The IL-17-S100A8/A9/A12 signaling pathway may be involved in regulating this process.