ATRIAL-NATRIURETIC-FACTOR RECEPTORS ON HUMAN PLATELETS

Specific binding sites for ANF have been identified on human platelets . To determine maximal binding (B-max) and dissociation constant (Kd), we adapted the only original method by developing a specific sequence of platelet preparation. From venous blood collected on citrated anti coagulant, platelets were prepared by successive centrifugations at 20 degrees C (blood centrifugated at 1500 rpm for 10 min., supernatant c entrifugated at 3000 rpm for 1 min., supernatant centrifugated at 2800 rpm for 10 min, the inner platelet-rich layer resuspended in citrated solution) and aliquoted (200 mu l at 5.10(5) platelets/mu l). Competi tion experiments [incubation of platelets with fixed concentration (20 -25 pM) of labeled (human)ANF ((125)I(h)ANF) and increasing concentrat ions (10(-12) to 10(-6) pM) of unlabeled (h)ANF] led to the drawing of a mean displacement curve (n = 8), usable as reference, and to verifi cation of the specificity of binding assay (crossreactivity with (rat) ANF, no cross-reactivity with arginine-vasopressine). From saturation experiments [incubation of platelets with increasing concentrations (3 .5 to 63.7 pM) of labeled (h)ANF and with (10(-8) M) or without unlabe led (h)ANF], we determined (n = 11): B-max (m +/- SEM) = 4.5 +/-: 0.7 pM or 5.4 +/- 0.8 sites per cell and Kd (m +/- SEM) = 10.84 +/- 1.70 p M.