Thrombin-induced GPIb signaling requires cooperation of PARs. (A-D) Calcium mobilization in control CHO cells, 1b9, and Δ605 CHO cells pretreated with DMSO control or PAR1 inhibitor SCH79797 (20 µM). Cells were stimulated with (S) wild-type (WT) thrombin or (B) low or (C) high concentrations of S195A mutant thrombin and then recorded for calcium mobilization as described in the Methods. (D) Aggregation of human platelets stimulated with increasing doses of S195A mutant thrombin, PPACK-treated S195A thrombin, and 1000 nM PPACK-treated WT thrombin. (E) In vitro enzymatic activity of WT, S195A mutant thrombin, PPACK-treated S195A mutant thrombin, and PPACK-treated WT thrombin. The y-axis is shown as a log scale of relative fluorescence units (RFUs). (F) Western blot analysis of LIMK1 phosphorylation in human platelets stimulated with increasing doses of PPACK-treated S195A mutant thrombin or wild-type thrombin (Thr). (G) LIMK1 phosphorylation in control CHO, 1b9, and Δ605 cells preincubated with vehicle control (0.1% DMSO) or 20 μM PAR1 antagonist SCH 79797 and stimulated with WT thrombin. (H) LIMK1 phosphorylation in mouse platelets preincubated with 10 μM MCsC (control peptide), 10 µM MPαC, and/or 2 mM PAR4 antagonist tcY-NH₂ and then stimulated with 3 nM WT thrombin. LIMK1 phosphorylation was detected using an anti-LIMK1 phospho-Thr^505/508 antibody. Loading was determined using a phosphorylation-independent anti-LIMK1 antibody. (I) A schematic of signaling-mediated cooperativity between GPIb-IX and PARs. This cooperativity requires a unique GPIb-IX–dependent signaling pathway involving 14-3-3. Rac1 and LIMK1 and activation of this pathway requires thrombin binding to GPIb-IX and stimulation of PARs.