Cross-linking of PECAM-1 inhibits collagen-stimulated platelet aggregation.
(A) PECAM-1 cross-linking on platelet surfaces results in its tyrosine phosphorylation and does not stimulate platelet aggregation. (i) PECAM-1 was immunoprecipitated from washed human platelets under resting conditions or after PECAM-1 cross-linking. Proteins were separated by SDS-PAGE and immunoblotted to detect phosphotyrosine residues (upper panel). Immunoprecipitation was verified by reprobing for PECAM-1 (lower panel). (ii) PECAM-1 was cross-linked on washed platelets (as described in “Materials and methods”), and aggregation was monitored using optical aggregometry. (B) (i) Platelets were incubated with isotype-matched control IgG for 5 minutes before the addition of F(ab′)2 cross-linker for 90 seconds and then were stimulated with collagen (100 μg/mL). Aggregation was monitored using optical aggregometry. (ii) Cross-linking of PECAM-1 inhibits collagen-stimulated platelet aggregation. Platelets were stimulated with collagen (100 μg/mL) for 90 seconds with and without first cross-linking PECAM-1 and platelet aggregation monitored by optical aggregometry. Data are representative of 3 separate experiments. Tyr(P), tyrosine phosphorylation; PECAM-1 XL, PECAM-1 cross-linking.
