Alterations of phospholipid asymmetry in the membrane of spontaneously aggregated platelets in diabetes

Abstract The changes of asymmetric distribution of anionic phospholipids of human platelets in diabetic patients were studied by fluorescent and freeze fracture cytochemistry, using merocyanine 540 (MC 540) and polymyxin B (P×B) as specific markers. The membrane anionic phospholipids were detected with P×B, a membrane nonpermeant probe, used either in native form for freeze fracture electron microscopy or as dansylated or iodinated derivative for fluorescence microscopy or gamma counting, respectively. MC 540 is a naturally fluorescent probe which reportedly inserts into less packed hilayer domains. Both in platelet rich plasma and in washed platelets obtained from diabetic patients, some small platelet aggregates were observed, their number being generally dependent on the level of hyperglycemia. In contrast with single platelets, the aggregated ones bind P×B as revealed by all assay methods. The fluorescence microscopic studies with dansyl P×B and MC 540 displayed a strong binding of the fluorescent markers to aggregated platelets. The electron microscopic examination of freeze fracture replicas showed the appearance of characteristic PxB-induced deformations in the plasmalemma of aggregated platelets. The gamma counting of 125 I-P×B incubated samples indicates significant differences on the platelets of diabetic patients as compared to those obtained from healthy subjects. Our data provide evidence that in diabetic patients, the spontaneous aggregated platelets are a result of the appearance of the anionic phospholipids in the outer half of plasmalemma. These changes may enhance the procoagulant activity and should represent a determinant of activated platelet recognition and their removal from circulation by splenic macrophages.