Identification of Fc and F(ab')2 IgG receptors on human platelets.

The human platelet receptors for normal, nonimmune IgG and its F(ab')2 and Fc fragments were studied by the use of a cleavable, bifunctional, photoactivable, 125I-labeled cross-linking agent. Derivatization of the ligands with N-[4-(p-azido-m-[125I]iodophenylazo) benzoyl]-3-aminopropyl-N'-oxysuccinimide ester (Denny-Jaffe reagent) reduced their binding to platelets by greater than or equal to 20%. Cleavage of the azo linkage of the Denny-Jaffe reagent, which splits the molecule so that its 125I-labeled portion becomes associated with the receptor half of the cross-linked ligand-receptor complex, was utilized to directly identify receptors for the various immunoglobulin ligands. Specificity of the binding reaction could be demonstrated by suppressing the iodination of the receptors with excess nonderivatized ligand. Two principal IgG-related receptors could be identified by high-resolution NaDodSO4/PAGE and subsequent analysis of the electrophoretically transferred peptides to nitrocellulose filters for localization of radioactivity and immunological characterization. Intact monomeric IgG and F(ab')2 fragments derived from it appeared to have the glycoprotein IIIa as the major receptor, whereas Fc fragments bound predominantly to a peptide of Mr approximately 200,000 (Mr, approximately 50,000 under rigorous reducing conditions).

Asymmetrical loss of sialic acid from membrane glycoproteins during platelet aging.

Platelet cohorts prepared from less than or equal to 24 hour old rabbit platelets were infused into myelosuppressed recipient animals and allowed to age in them. Serial withdrawals of platelets at 24 hour intervals revealed a sharp drop in total platelet N-acetylneuraminic acid. Platelet membrane proteins were double labeled by reductive methylation of free amino groups in the presence of [14C]-formaldehyde and by metaperiodate oxidation of sialic acid residues followed by reduction with [3H]-NaBH4. Senescence of platelets in vivo is associated with a disproportionate loss of metaperiodate-oxidizable residues from a glycopeptide of M.W. 45,000.

A model for ultracentrifugal quantification of (35-S) bromosulphthalein-binding to plasma proteins in the presence of radioimpurities.

Sloutions of (35S)bromosulphthalein ((35S)BSP) in heparinized canine plasma, in the proportions established in vivo after injecting BSP intravenously to test liver function, were ultracentrifugated at 226,000 g for 24 hr at 5 degrees. Protein-free supernatant was replaced by Krebs-Ringer buffer (pH 7.40), the protein sediment resuspended, and the mixture recentrifugated. That process was repeated several times, and the radio-activity of each resulting supernatant was measured. Since (35-S)BSP could not be adequately purified, supernatant radioactivities reflected both (35-S)BSP and radioimpurity. Therfore, a model was derived that (i) interpreted the rapid decrease in supernatant radioactivities of initial centrifugations and the gradual fall therafter; and (ii) allowed us to determine picomoles of non-protein-bound (35-S)BSP. Results indicated that only 0.053% (SD .0013%) of BSP in our system was not protein-bound.