Western blot identification of platelet proteins that bind normal serum immunoglobulins. Characteristics of a 95-Kd reactive protein

Abstract

We have found that Western blots (WBs) of whole platelets exposed to normal autologous or homologous sera commonly have bands at 90 to 95 (95) Kd, and less often at 100 to 110, 80 to 85, 60 to 75, and 50 to 60 Kd when developed with antiglobulins. The percentages of normal sera producing a 95-Kd band with anti-immunoglobulin G (IgG), -IgA, and -IgM are 85, 50, and 30, respectively. Antiglobulin reagents alone also produce background bands on WBs that we have shown correspond to levels of platelet-associated Igs (PAIgs) or their derivatives. Titers of 95 Kd-reactive IgG in normal sera range from 10 to 1,280 (85% less than or equal to 50), and the reaction appears to be partially F(ab')2- mediated. The 95-Kd protein is internal and differs in many respects from surface glycoproteins IIIa, IV, and V of similar apparent molecular weight. In thrombocytopenic patients there was no correlation between severity of thrombocytopenia or PAIgG of platelet eluates and corresponding serum titers of 95 Kd-reactive IgG. Some WB reactions previously reported as evidence of autoimmunity may represent normal variations in reactions of Igs with internal platelet proteins. These reactions may be immunospecific or analogous to nonspecific, partially F(ab')2-dependent binding of Igs by certain bacterial proteins.

Detection and Effects on Platelet Function of Anti-Platelet Antibody in Mule Foals with Experimentally Induced Neonatal Alloimmune Thrombocytopenia

“…Additional work is warranted to further characterize these protein bands. The protein bands may have resulted from a reaction between the antiglobulin reagent and IgG from donkey platelet alpha-granules 14,34 and not from a reaction between the antiglobulin reagent and antibodies bound to the surface of platelets (platelet-bound antibodies).…”

“…[3][4][5][6][7][8][9] Definitive diagnosis of IMTP requires demonstration of anti-platelet antibodies, either in the serum or bound to platelets or bone marrow megakaryocytes. 10 Laboratory techniques used to detect anti-platelet antibody include platelet factor-3 test, 1,10,11 enzyme-linked immunosorbent assays (ELISAs), 12,13 western immunoblotting, [14][15][16] indirect immunofluorescence of megakarocytes, 17 and flow cyto-metric assays. 18,19 Reliable tests for detection of anti-platelet antibodies in the horse are not readily available.…”