Binding of platelet proteins to echicetin-Sepharose.

Platelet surface proteins were labeled with biotin, platelets were lysed by Triton X-100, and the lysate was added to echicetin-Sepharose. The proteins eluted from the echicetin-Sepharose were separated by SDS-PAGE, transferred to PVDF membrane, and detected with avidin-phosphatase conjugate (lanes 1-3) or with anti-GPIb monoclonal antibody (lanes 4-6). Lanes 1 and 4: platelet lysates. Lanes 2 and 5: eluate from Sepharose (negative control). Lanes 3 and 6: eluate from echicetin-Sepharose. Specific bands detected by anti-GPIb monoclonal antibody (Ib-4) are indicated by (nonreduced [NR]) GPIb and glycocalicin (GC; the extracellular proteolytic fragment of GPIbα) and (reduced [R]) GPIbα and macroglycopeptide (MG; the mucinlike proteolytic fragment of GPIbα). Under reducing conditions GC and GPIbα comigrated.