Radioimmunoassay of human platelet thrombospondin: different patterns of thrombospondin and beta-thromboglobulin antigen secretion and clearance from the circulation.
A method for radioimmunoassay of human thrombospondin was developed. Monospecific precipitating anti-human thrombospondin antibody was raised in rabbits after injection of thrombospondin purified by fibrinogen-agarose chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The linear portion of the thrombospondin radioimmunoassay standard curve was 0.5 to 20 ng/ml. Normal platelets and platelet-poor plasma contained 28,900 +/- 14,500 ng thrombospondin per 10(9) platelets and 60.6 +/- 10.7 ng/ml (mean +/- SD), respectively. Using radioimmunoassays for beta-thromboglobulin and thrombospondin antigens, we compared platelet location and secretion of these proteins. Both antigens shared similar distributions in platelet subcellular fractions with the largest amount localized to platelet alpha-granules. With thrombin (0.25 U/ml) as a platelet agonist, 62.4% and 19.5% of total beta-thromboglobulin and thrombospondin, respectively, were secreted from suspensions of washed human platelets. Because only 20% of the total platelet thrombospondin was secreted, further studies were initiated to determine whether the remaining thrombospondin became localized on the activated platelets membrane. 125I-Fab antithrombospondin specifically bound to activated platelets but not to unstimulated platelets. In contrast, 125I-Fab anti-beta-thromboglobulin did not bind to activated platelets. Plasma clearance of human beta-thromboglobulin (half-life fast 7.6 minutes, slow 56.6 minutes) and of human thrombospondin (half-life fast 29.9 minutes, slow 190 minutes) followed a biphasic exponential curve. In conclusion, both beta-thromboglobulin and thrombospondin are located in platelet alpha-granules, but they show a different pattern of secretion and expression on the platelet membrane and plasma clearance.
H I
HI
Switalska
S
S
Niewiarowski
G P
GP
Tuszynski
B
B
Rucinski
A H
AH
Schmaier
T A
TA
Morinelli
C S
CS
Cierniewski
D000818:Animals; D001609:Beta-Globulins / Q000032:analysis*; D001792:Blood Platelets / Q000032:analysis* / Q000187:drug effects; D002458:Cell Fractionation; D006023:Glycoproteins / Q000097:blood* / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007106:Immune Sera / Q000302:isolation & purification; D010949:Plasma / Q000032:analysis; D011817:Rabbits; D011863:Radioimmunoassay / Q000379:methods*; D013917:Thrombin / Q000494:pharmacology; D019699:Thrombospondins; D001620:beta-Thromboglobulin / Q000032:analysis* / Q000097:blood / Q000276:immunology / Q000378:metabolism
D001609:Beta-Globulins; D006023:Glycoproteins; D007106:Immune Sera; D019699:Thrombospondins; D001620:beta-Thromboglobulin; D013917:Thrombin