Platelet and leukocyte activation, and platelet-leukocyte cross-talk : Mechanistic aspects with special reference to diabetes mellitus

Abstract: Platelet-leukocyte cross-talk modulates platelet and leukocyte function, and appears to be important in thrombosis and haemostasis. Type 1 diabetes mellitus (DM) is associated with platelet and leukocyte dysfunction and an increased risk of cardiovascular complications. The present work investigated mechanisms of platelet-leukocyte cross-talk, and the possible role of platelet-leukocyte cross-talk in the pathophysiology of type 1 DM. Activation of leukocytes with fMLP evoked platelet activation in whole blood. The effect did not depend on platelet-leukocyte aggregate (PLA) formation, but was markedly inhibited by platelet activating factor (PAF), 5lipoxygenase, or glycoprotein (GP) IIb/IIIa antagonism. Collagen-activated platelets evoked leukocyte activation. The effect was inhibited by blocking PLA formation. Thus, leukocyte initiated crosstalk appears to rely on soluble mediators, while platelet initiated cross-talk largely depends on direct cellcell contact. A follow-up study was carried out to elucidate why GPIIb/IIIa blockade attenuates leukocyteplatelet cross-talk. GPlIb/Illa inhibitors were found to attenuate PAF-induced platelet P-selectin expression in whole blood, via attenuation of PAFinduced protein kinase C (PKC) activation. GPIIb/Illa blockade also enhanced thrombin-induced MEK 1/2 and ERK 1/2 phosphorylation. Effects of shear stress on PLA formation were also evaluated. Shear stress per se increased PLA formation. ADPstimulated PLA formation was reduced, whilst fMLP-induced PLA formation was enhanced by shear stress. Pselectin blockade abolished, whilst GPIIb/IIIa blockade enhanced shear- and agonist-induced PLA formation. Simultaneous blockade of GPIIb/IIIa, CD11b and CD18 increased PLA formation at low shear rate, but this increase was reduced under high shear stress. Thus, P-selectin is essential for the initiation of PLA formation, while integrins contribute importantly to the stability of PLAs under high shear. Type 1 DM was found to be associated with platelet and leukocyte hyperreactivity to in vitro stimuli, especially in patients with microangiopathy. fMLP induced more pronounced leukocyte-platelet cross-talk in DM patients (both with and without microangiopathy) than in healthy subjects. Correlation analysis revealed that enhanced leukocyte-platelet cross-talk was associated with diabetic platelet hyperreactivity in type 1 DM patients with microangiopathy. A stress model of strenuous exercise was also employed to study whether type I DM is associated with enhanced prothrombotic responses to stress. Stress induced platelet and leukocyte activation, and increased PLA formation in vivo, and enhanced platelet and leukocyte sensitivity to agonist-stimulation in vitro. However, responses to stress were similar in metabolically well controlled DM patients and healthy subjects. Thus, despite hyperactive platelets/leukocytes and enhanced leukocyte-platelet cross-talk in type 1 DM, platelet and leukocyte hyperactivity was not augmented by stress in metabolically well controlled DM patients. Effects of insulin and proinsulin C-peptide on platelet activation were evaluated in type 1 DM patients and healthy subjects. Insulin enhanced ADP-induced platelet fibrinogen binding (reflecting platelet aggregability) similarly in both patients and healthy subjects, but did not affect P-selectin expression. Proinsulin C-peptide did not influence platelet fibrinogen binding or P-selectin expression in either type 1 DM patients (who lack Cpeptide) or healthy subjects. Taken together, platelet-leukocyte cross-talk involves soluble mediators and cell adhesion molecules. Plateletleukocyte cross-talk occurs mainly via heterotypic conjugation, which is initiated by P-selectin bridging and stabilized by integrins. Leukocyte-platelet cross-talk occurs mainly via soluble mediators, such as PAF and leukotrienes, while a blockade of platelet GPIIb/IIIa inhibited this cross-talk by reducing PAFinduced PKC activity in platelets. Type 1 DM is associated with enhanced leukocyte-platelet cross-talk, which may contribute to diabetic platelet hyperactivity and diabetic microangiopathy. However, despite platelet/leukocyte hyperactivity at rest, responses of platelets and leukocytes to stress were not augmented in metabolically well-controlled type 1 DM patients. Insulin treatment may attenuate platelet activation via indirect mechanisms in type 1 DM patients. Therapies aimed at reducing platelet activation and plateletleukocyte cross-talk should be considered, in addition to good metabolic control, and further investigated in type 1 DM.