Atrial natriuretic factor receptors on human platelets.
Specific binding sites for ANF have been identified on human platelets. To determine maximal binding (Bmax) and dissociation constant (Kd), we adapted the only original method by developing a specific sequence of platelet preparation. From venous blood collected on citrated anticoagulant, platelets were prepared by successive centrifugations at 20 degrees C (blood centrifugated at 1500 rpm for 10 min., supernatant centrifugated 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 microliters at 5.10(5) platelets/microliters). Competition experiments [incubation of platelets with fixed concentration (20-25 pM) of labeledhuman ANF (125Ih ANF) and increasing concentrations (10(-12) to 10(-6) pM) of unlabeled hANF] led to the drawing of a mean displacement curve (n = 8), usable as reference, and to verification of the specificity of binding assay (cross-reactivity with ratANF, no cross-reactivity with arginine-vasopressine). From saturation experiments [incubation of platelets with increasing concentrations (3.5 to 63.7 pM) of labeled hANF and with (10(-8) M) or without unlabeled hANF], we determined (n = 11): Bmax (m +/- SEM) = 4.5 +/- 0.7 pM or 5.4 +/- 0.8 sites per cell and Kd (m +/- SEM) = 10.84 +/- 1.70 pM.
Blaise
V
V
Faculté de Médecine, Laboratoire de Physiologie, Besançon, France.
Wolf
J P
JP
Regnard
J
J
Berthelay
S
S
D009320:Atrial Natriuretic Factor / Q000378:metabolism; D001667:Binding, Competitive; D001792:Blood Platelets / Q000737:chemistry*; D005260:Female; D006801:Humans; D007700:Kinetics; D008297:Male; D011485:Protein Binding; D017461:Receptors, Atrial Natriuretic Factor / Q000032:analysis*; D015203:Reproducibility of Results
C472955:NPPA protein, human; D009320:Atrial Natriuretic Factor; D017461:Receptors, Atrial Natriuretic Factor