The Link Between Zinc, Platelets And The Control Of Haemostasis

Introduction: Thrombosis linked to cardiovascular diseases is one of the leading causes of mortality worldwide despite the development of effective pharmacological interventions. Platelets are essential in thrombus formation and as such are a therapeutic target for numerous anti-thrombotic medications. Several studies have indicated that zinc deficiency in men and rodents is linked to increased bleeding times, impaired platelet aggregation and dysfunctional coagulation (1, 2). However, the role of zinc in platelet reactivity remains poorly defined. We therefore aimed to characterise and define the roles of zinc in the regulation of platelet reactivity.

Methods: Blood was obtained by venepuncture from healthy volunteers (NHS St. Thomas Hospital Research Ethics Committee; Ref 07/Q0702/24) into 3.2% sodium citrate. Citrated whole blood was centrifuged for 15 minutes (175 x g) to obtain platelet rich plasma (PRP). To prepare washed platelets, PRP was centrifuged (1000 x g, 10minutes) with prostacyclin (2µg/ml) and apyrase (0.02U/ml) and resuspended in modified Tyrode’s HEPES buffer. Light transmission aggregometry was used to determine the effect of zinc chloride (0.1µM-300µM) on washed platelet responses alone as well in conjunction with arachidonic acid (0.03mM-0.1mM), ADP (0.1µM-30µM), collagen (0.1µg/ml-30µg/ml), epinephrine (0.001µM-100μM), ristocetin (0.2µg/ml-3µg/ml), TRAP-6 (0.1µM-30µM), U46619 (0.1µM-30µM) and thrombin (0.01mM-1mM). Platelet adhesion to 96-well plates under static conditions was determined by measurement of intracellular cellular acid phosphatases as the conversion of p-nitro phenyl phosphate to p-nitro phenol following platelet lysis. Platelet activation in response to zinc chloride was measured by P-selectin (anti-CD62P) expression by flow cytometry using anti-CD61 APC to identify platelets. Data is expressed at the mean ±SEM and a significant difference was established at p<0.05.

Results:Aggregation in washed platelets increased in a concentration-dependent manner in response to zinc chloride, beingsignificant at concentrations of >30µM (p<0.001). At a sub-maximal concentration of 60µM zinc chloride augmented platelet aggregation, particularly that induced by low concentrations of platelet agonists. Platelet adhesion in the presence of zinc chloride increased in a concentration-dependent manner and 100µM zinc chlorideenhanced the adhesion of platelets to both collagen (24±2% to 32±3%) and fibrinogen (23±2% to 41±5%) coated plates (p<0.01). Flow cytometry analysis indicated that zinc chloride at 60µM, 100µM and 300µM induced the formation of platelet micro aggregates with an elevated expression of P-selectin (37±5, 45±5 and 60±7 geometric mean fluorescence respectively).

Conclusions: Zinc chloride induces platelet aggregation, adhesion and activationin vitro, both as a direct stimulator and as an augmenter of responses to other platelet agonists. The optimal concentration of zinc chloride to produce these effects isbetween 60µM and100µM.. This data may be relevant both to the effects of circulating zinc and of zinc released in higher concentrations from the granules of activated platelets. Understanding these interactions could provide opportunities for novel therapeutic interventions to decrease thrombotic risk.

References:

(1) Gordon PR and O’Dell BL (1980) Rat platelet aggregation impaired by short-term zinc deficiency. J Nutr 110: 2125-9.

(2) Gordon PR et al. 1982) Effect of acute zinc deprivation on plasma zinc and platelet aggregation in adult males. Am J Clin Nutr 35: 113-119.