A platelet membrane protein expressed during platelet activation and secretion. Studies using a monoclonal antibody specific for thrombin-activated platelets.
To identify structures on the platelet surface which become expressed after platelet activation, we have prepared murine monoclonal antibodies specific for thrombin-activated platelets. Hybridomas were screened for clones producing antibodies which bound to thrombin-activated platelets but not to resting platelets. Clone KC4 was identified. The binding of purified I-labeled KC4 antibody, an IgG1k, to thrombin-activated platelets was saturable. Minimal binding was observed to resting platelets. The interaction of antibody with thrombin-activated platelets was characterized by a binding constant, KD, of 7.2 +/- 0.4 nM and revealed 13,400 +/- 3,000 binding sites per platelet. The presence of Ca2+ or EDTA, a pH ranging from 4 to 10, or high ionic strength had no influence on antigen-antibody interaction. The KC4 antigen was expressed on the platelet surface after activation with ADP, collagen, epinephrine, or thrombin. The extent of [14C] serotonin release during activation was directly proportional to the availability of antigen on the platelet surface regardless of agonist or platelet aggregation. The antibody is directed against a single protein which migrated between GPIIb and GPIIa after sodium dodecyl sulfate gel electrophoresis. This protein was purified from platelet membranes by immunoaffinity chromatography using KC4 antibody-agarose and demonstrated an apparent molecular weight of 140,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under both nonreducing and reducing conditions. Of the cells examined, only platelets contained this protein. These results indicate that platelet secretion is associated with the expression of an Mr = 140,000 integral membrane protein composed of a single polypeptide chain. This protein may be component of the internal granule membrane which is fused with the plasma membrane during activation.
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D001792:Blood Platelets / Q000502:physiology*; D002462:Cell Membrane / Q000502:physiology; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000096:biosynthesis / Q000097:blood* / Q000302:isolation & purification; D008970:Molecular Weight; D010974:Platelet Aggregation; D011863:Radioimmunoassay; D013917:Thrombin / Q000502:physiology*
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D008565:Membrane Proteins; D013917:Thrombin