Abstract
This experiment was designed and carried out to isolate and identify the membrane receptor proteins and antigens in association with platelet functions.
The platelet specific membrane proteins against rabbit anti-human platelet antibody, rabbit anti-β2 microglobulin, platelet antibodies 4 SLE and ITP patients were examind. The O type healthy human ACD blood (200-600ml) was collected and the platelet suspension was prepared. The washed platelets were labelled with 125I-Na by lactoperoxydase method, and then washed 3 times with the same buffer. The 125I-labelled platelets were sonicated or disrupted by glycerol loading method, and this lysed platelet solution was layered on the top of a solution of 27% sucrose and centrifuged at 4°C at 23, 000rpm for 4 hours. The membrane fraction remained as a narrow band above the sucrose interface, and this band was isolated, suspended with phosphate buffer for washing, and centrifuged at 4°C at 23, 000rpm for 2 hours. These isolated 125I-labelled membrane was solubilized with 0.1% Emalgen 911 (HLB 13.7) for 12-16 hours at 4°C, and centrifuged at 78, 000G for 2 hours. The supernatant was used for double antibody immunoprecipitation method. This method was performed as follows: solubilized, iodinated platelet membrane solution was incubated with IgG fraction of antiserum (rabbit anti-human platelet serum, SLE patient's serum, ITP patient's serum, normal rabbit and human serum or rabbit anti-β2 microglobulin) for 1 hour at 37°C and 18 hours at 4°C. The IgG fraction of rabbit anti-human IgG or goat anti-rabbit IgG was added and the mixtures were again incubated in the same way. The immunoprecipitation was prepared by centrifugation, washed 3 times with PBS, then solubilized in 2% SDS solution and analyzed by 5% SDS polyacrylamide gel electrophoresis. The gel was cut into 1mm slices and their radioactivities were counted.
The 125I-labelled solubilized platelet membrane was precipitated as five radioactive peaks by rabbit anti-human platelet antibody and their molecular weights (M. W.) were 18×104, 16.5-15×104, 10.5-9×104, 6.8-6.0×104 and 5.2-5.0×104, respectively. Two of five peaks corresponded to glycoprotein I and III. When SLE serum was used, the radioactivities of glycoprotein III and M. W. 6×104 membrane protein were greater than those obtained by using normal human serum, but ITP serum did not show remarkable changes compared to normal. The anti-β2 microglobulin bound to the glycoprotein ha remarkably.
These results may suggest that the platelet has various specific antigen determinant sites against anti-human platelet antibody, platelet antibody-positive SLE serum and anti-β2 microglobulin.
But to decide if these membrane proteins bind to each serum specifically or not, F (ab)2 fragment of IgG from each serum must be used instead of IgG.
