On the association of glycoprotein Ib and actin-binding protein in human platelets.
Glycoprotein (GP) Ib was purified from lysates of human platelets prepared in the presence or absence of inhibitors of the endogenous calcium-activated neutral protease (CANP) by immunoaffinity chromatography, employing the GPIb-specific murine monoclonal antibody, AP1, coupled to Sepharose CL4B. When derived from lysates prepared in the presence of EDTA or leupeptin, the eluate from the AP1-affinity column contained a 240,000-260,000-mol-wt protein in addition to GPIb. In SDS PAGE, this protein was stained by Coomassie Blue R, but not by the periodic acid-Schiff reagent, and it was not labeled with 125I in intact platelets by the lactoperoxidase-catalyzed method. When derived from lysates prepared in the absence of CANP inhibitors, the eluate contained only GPIb and its proteolytic derivative, glycocalicin. A change in the electrophoretic mobility of GPIb consistent with its association with the 240,000-260,000-mol-wt protein was confirmed by crossed immunoelectrophoresis. By an immunoblot technique involving transfer of proteins eluted from the AP1-affinity column and separated by SDS PAGE onto a nitrocellulose membrane, the 240,000-260,000-mol-wt protein bound polyclonal goat antibody raised against rabbit macrophage actin-binding protein (ABP). On the basis of these results, we conclude the GPIb is tightly associated with ABP under conditions in which the endogenous CANP is inhibited, and that this apparent transmembrane complex of GPIb-ABP can be isolated in lysates of nonactivated human platelets.
Okita
J R
JR
Pidard
D
D
Newman
P J
PJ
Montgomery
R R
RR
Kunicki
T J
TJ
D000199:Actins / Q000097:blood*; D001792:Blood Platelets / Q000378:metabolism; D002352:Carrier Proteins / Q000097:blood* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D018260:Gelsolin; D006023:Glycoproteins / Q000097:blood* / Q000302:isolation & purification; D006801:Humans; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000097:blood*; D008840:Microfilament Proteins; D008970:Molecular Weight; D010980:Platelet Membrane Glycoproteins; D011485:Protein Binding
D000199:Actins; D002352:Carrier Proteins; D018260:Gelsolin; D006023:Glycoproteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D008840:Microfilament Proteins; D010980:Platelet Membrane Glycoproteins; C016953:brevin