Isolation of Platelet Specific Glycoprotein Antigen

Abstract

Hanna and Nelken in 1971 observed two specific antigens on human platelets. One is a glycoprotein, m.w. 110,000, with α-electrophoretic mobility and the other is protein, m.w. 30,000. We have isolated a platelet specific glycoprotein antigen m.w. 70,000, with β-globulin mobility. Differentially centrifuged and Sepharose 2B gel filtered platelets were suspended in deionized water, frozen, thawed and sonicated. One volume of 1.2 M perchloric acid (PCA) was added and mixture centrifuged and the supernatant dialysed against water and lyophilized. Antiserum was obtained by s.c. injection of this extract to a rabbit. After absorbtion with 0.6M PCA extract of human plasma and erythrocytes, this antiserum showed one precipitin line with platelet PCA extract, but no line with PCA extract of plasma, erythrocyte or lymphocyte on double diffusion. Furthermore, it formed no precipitin line with pus, liver, colon, stomach or kidney, but a weak line with lung and a strong line with spleen which fused with the platelet line. By gel filtration of crude platelet PCA extract on Sephadex G-200, antigenic activity was observed in the third fraction. On 7.5% Polyacrylamide disc electrophoresis of this fraction, three bands stained with amidoblack and PAS. Main band was the middle one of m.w. 70,000 with β-globulin mobility. Positive immunofluorescent staining was found on platelets of human buffy coat smear, but not on erythrocytes or lymphocytes. The absorbed antiserum showed precipitin line with serum from whole blood and recalcified platelet rich plasma, but not with serum from recalcified platelet poor plasma. β-thromboglobulin is released from platelets during coagulation. We are investigating the immunological identity of β-thromboglobulin with our antigen. The result will be discussed.