Crossed immunoelectrophoresis of human platelet membranes.

Human platelet membranes were prepared by the glycerol-lysis technique of Barber and Jamieson (Barber, A. J., and Jamieson, G. A. (1970) J. Biol. Chem 245, 6357-6365), solubilized in 1% Triton X-100 and subjected to crossed immunoelectrophoresis, in 1% agarose, employing a rabbit anti-human platelet membrane antibody in the second dimension. Approximately 20 immunoprecipitates (numbered 1 to 20) could be detected in normal subjects. At least 10 different antigens were observed fairly consistently with different membrane preparations. One could be identified as albumin (12A), the other as fibrinogen (1F). Four membrane antigens were also present in the cell sap, two of which were 12A and 1F. Absorption of the antibody with increasing concentrations of washed platelets revealed the disappearance of at least six different antigens (lF, 7-8, 10, 13, 14, and 18) at a constant rate, suggesting an external surface location. Four of these surface antigens (lF, 10, 13, and 18) reacted with concanavalin A when this was employed as an intermediate spacer gel, indicating that they are glycoproteins. At least six antigens did not react with concanavalin A. A major surface antigen, 10, was present on all preparations and had lines of identity with two other antigens, 13 and 18, which moved more cathodally. Platelets from subjects with full 10 antigen peaks had absent or diminished 13 and 18 antigen peaks, whereas platelets from subjects with absent to incomplete cathodal arms had increased 13 and 18 antigen peaks. Furthermore, digestion of intact washed platelets with achymotrypsin resulted in a decrease in the 10 antigen peak and an increase in the 13 and 18 antigen peaks, suggesting a structural or organizational relationship among these three antigens. This is supported by studies with neuraminidase digestion of intact washed platelets, which resulted in a cathodal shift of the major antigen and the disappearance of antigens 13 and 18. Platelet membranes from three patients with Glanzmann’s thrombasthenia (a disorder in which platelets do not aggregate with aggregating agents) revealed the absence or marked reduction of the major antigen 10, as well as of 13,18, and lF, and the exposure of a nonglycoprotein antigen, normally hidden “behind” 10 and designated loa. Platelet membranes from two patients with Bernard-Soulier syndrome, a disorder in which platelets do not adhere to injured subendothelial surfaces, the receptor for von Willebrand factor is absent,