Platelet dysfunction in diabetes mellitus : impact of hyperglycemia and glycoprotein GPIIb/IIIa inhibition

Abstract

Diabetes mellitus (DM) is associated with increased cardiovascular morbidity and mortality, due to diabetic angiopathy. Hyperglycemia is one of the factors that may cause platelet dysfunction in diabetic patients. This work investigated mechanisms underlying hyperglycemia-induced platelet dysfunction, and its impact in DM patients. Antiplatelet treatment by glycoprotein (GP) IIb/IIIa blockade provides more efficient thrombotic protection in diabetic than in non-diabetic subjects. We therefore also examined the effect(s) of GPIIb/IIIa blockade on platelet procoagulant activity in vitro, and their possible impact in type 2 DM (T2DM) patients.

High glucose dose-dependently enhanced agonist-induced platelet activation in vitro through increased osmolality, as shown by experiments with different sugars. High glucose enhanced platelet P-selectin expression but not fibrinogen binding in ADP-stimulated samples, and enhanced both platelet P-selectin expression and fibrinogen binding in thrombin receptor activating peptide (TRAP)-stimulated whole blood. Protein kinase C (PKC) blockade did not counteract the enhancement of platelet P-selectin expression by high glucose, but abolished the enhancement of TRAP-induced platelet fibrinogen binding. Superoxide anion scavenging by superoxide dismutase attenuated the enhancement by high glucose of ADP- and TRAPinduced platelet P-selectin expression, but did not influence platelet fibrinogen binding.

The influence of a standardized carbohydrate-rich meal on platelet reactivity was assessed in T2DM patients and matched healthy controls; the patients also received oral antidiabetic treatment with repaglinide and glibenclamide in a cross-over study. In T2DM patients food intake caused postprandial hyperglycemia and markedly augmented platelet P-selectin expression and platelet-leukocyte aggregation induced by the thromboxane A2 (TxA2) analogue U46619, and slightly enhanced ADP-induced platelet P-selectin expression. The meal caused no postprandial hyperglycemia or enhancement of platelet reactivity in the healthy controls. Both repaglinide and glibenclamide treatment only mildly reduced the postprandial hyperglycemia, and did not attenuate the meal-induced enhancement of platelet reactivity in the T2DM patients.

Platelet procoagulant activity was assessed by annexin V binding, reflecting phosphatidylserine appearance on the platelet surface membrane (PS exposure), and effects of GPIIb/IIIa blockade were examined in washed platelets from healthy volunteers. Thrombin-induced PS exposure was cell-cell contact dependent. GPIIb/IIIa blockade inhibited this by enhancing translocase activity and inhibiting scramblase activity. Thrombin-induced platelet derived microparticle (PDMP) formation was not influenced by GPIIb/IIIa blockade.

Platelet procoagulant activity, i.e, PS and factor Va (FVa) expression, and PDMP generation, was compared in T2DM patients and healthy subjects. The unstimulated platelet procoagulant activity was only slightly higher in patients compared to controls. TRAP enhanced the PS exposure and FVa expression of platelets, and PDMP generation more markedly among T2DM patients. These hyperprocoagulant alterations in T2DM patients were associated with elevated thrombin generation and a shortened plasma clotting time. GPIIb/IIIa blockade with c7E3 or SR121566 reduced the platelet PS exposure and FVa expression, and also reduced the procoagulant activity seen in T2DM patients.

In conclusion, high glucose levels enhance the platelet reactivity to agonist stimulation through elevated osmolality. This occurs via superoxide anion production that enhances platelet P-selectin expression (secretion), and PKC signalling that enhances TRAP-induced fibrinogen binding (aggregablity). Food intake markedly enhances TxA2-induced platelet activation in type 2 diabetic patients but not in healthy subjects, presumably due to postprandial hyperglycemia. The procoagulant PS exposure is cell-cell contact dependent and it is inhibited by GPIIb/IIIa blockade. The inhibition of PS exposure by GPIIb/IIIa blockade occurs via increased translocase and reduced scramblase activity. Moreover, GPIIb/IIIa blockade attenuates the platelet hyperprocoagulant activity which is seen in T2DM patients. This thesis provides additional information on mechanisms that probably contribute to the clinical benefits of glycemic control, and of GPIIb/IIIa blocker treatment, which might be useful in shaping more efficient antithrombotic therapy for T2DM patients.