Cellular Expression of Tissue Factor, CD40 Ligand and P-Selectin in Normal Healthy Subjects during Hyperglycemia and Hyperinsulinemia.

Type 2 diabetes mellitus (DM) patients have increased atherosclerotic and acute vascular complications. Both, high glucose, high insulin levels are independently associated risk factors for increased mortality. We have previously reported elevated circulating tissue factor procoagulant activity (TF-PCA) during hyperglycemia and hyperinsulinemia in normal subjects. To evaluate the effects of hyperglycemia and hyperinsulinemia on blood cells activation we measured monocyte tissue factor (TF) expression, monocyte-platelet aggregates, neutrophil-platelet aggregates, platelet P- selectin and CD40 ligand (CD40L) expression using flow cytometry. We studied 4 groups of healthy individuals at baseline and 24 hr after being subjected to following clamps. Hyperglycemia and Hyperinsulinemia Group, HG+HI: 10 subjects were infused with glucose to maintain levels at 200 mg/dl. Euglycemia and Hyperinsulinemia Group, EG+HI: 7 subjects were infused with insulin to maintain serum levels at 1000 pmol/l. Selective Hyperglycemia and Euinsulinemia Group, HG+EI: 6 subjects were infused with glucose (~200 mg/dl) and somatostatin to block endogenous insulin secretion. Placebo Group: 5 subjects received saline. TF expressing monocytes were increased from 20 to 32% (p < 0.002) and from 13 to 18% (p < 0.02) with HG+HI and EG+HI, respectively. Percent positive cells for monocyte-platelet aggregates increased in the HG+HI group, from 72 to 85% (p < 0.05). Platelet CD40L expression increased with HG+HI (33 to 46%, p < 0.001), EG+HI (31 to 38%, p = 0.01) and HG+EI (33 to 37 %, p = 0.05), whereas P-selectin and neutrophil-platelet aggregates did not increase in any group. There was no change in mean fluorescence intensity (MFI) for any of the measurements. No changes were noted in placebo group at 24 hrs.

In addition, we studied the effect of activation of whole blood with ADP (1, 5 and 25μM) and thrombin peptide SFLLRN (25 μM). Upon stimulation with ADP and SFLLRN the percent positive cells and MFI for monocyte-platelet and neutrophil-platelet aggregates, and platelet P-selectin expression were elevated compared to untreated sample in all groups, both at baseline and 24 hr. Percent positive TF expressing monocytes and CD40L expressing platelets were also increased with stimulation.

Conclusions: Combination of hyperglycemia with hyperinsulinemia increases circulating TF expression, monocyte-platelet aggregates, and platelet CD40L expression but not neutrophil-platelet aggregates or platelet P- selectin. Hyperinsulinemia and hyperglycemia alone also increased monocyte TF expression and platelets CD40L expression. Stimulation with ADP and SFLLRN increased TF, CD40L, P-selectin and monocyte-platelet aggregates compared to untreated cells. These studies provide evidence for platelet activation and enhanced monocyte TF expression by hyperglycemia- hyperinsulinemia. CD40L and P-selectin are recognized to increase monocyte TF production. The increase in platelet CD40L expression without increase in P-selectin suggests a role of CD40L in the enhanced TF expression. These studies provide evidence for a procoagulant and proinflammatory state that contributes to both the acute vascular events and atherogenesis in DM.