CHOLESTEROL ENHANCES THE ADHESION OF HUMAN PLATELETS TO FIBRINOGEN - STUDIES USING A NOVEL FLUORESCENCE-BASED ASSAY

This communication reports investigations on the effect of platelet ch olesterol content on adhesion of platelets to a fibrinogen coated surf ace, The adhesion of platelets stimulated with thrombin or ADP was dra matically increased when the platelet cholesterol content was enriched by incubation with cholesterol containing phosphatidylcholine vesicle s, In contrast, ADP failed to promote the adhesion of platelets to fib rinogen after they had been depleted of cholesterol, either by incubat ion with phosphatidylcholine vesicles or by brief exposure to choleste rol oxidase, By comparison, the adhesion of resting platelets to fibri nogen coated surface was unaltered following either enrichment or depl etion of cholesterol, These data were obtained using a novel method of measuring the adhesion of platelets to a protein coated surface based upon the fluorescent detection of platelets containing the fluorescen t probe octadecyl rhodamine (R-18). R-18 was incorporated into platele t membranes using standard ethanol injection techniques at room temper ature for 30 min, The platelets were introduced into fibrinogen coated wells of a 96-well microtiter plate in the presence of various cation s and stimulatory or inhibitory ligands, The plate was then incubated at room temperature without agitation for various periods of time, Adh esion measured in this manner had characteristics similar to those rep orted using other methods, Thus the extent of adhesion ranged from 1-4 % under basal conditions, and was increased in a dose-dependent manner by Mg2+ and Ca2+, increased further by ADP, collagen or thrombin and not affected by prostacyclin.