The interaction of soluble Glu-Pg with α_Mβ₂-expressing cells and the recombinant α_MI-domain. (A) The α_Mβ₂-expressing HEK 293 cells (10⁶) were incubated with mAb 44a (40 μg/mL), mAb IB4 (30 μg/mL), or tranexamic acid (5 mM) in HBSS containing 0.1% BSA and 2 mM MnCl₂ for 20 minutes at 22° C. Pg (10 μg/mL, 5 × 10⁵ cpm/mL) was added, and the incubation was continued for another 20 minutes. Cell suspensions (50 μL) were layered over 300 μL of 20% sucrose in HBSS-BSA followed by centrifugation at 12 400g for 3 minutes. Radioactivity was measured as described in “Materials and methods.” (B) Binding of the radiolabeled Glu-Pg to immobilized α_MI-domain.¹²⁵I-Glu-Pg (20 μg/mL) was pretreated with P2 (100 μM), ϵ-ACA (50 mM), or tranexamic acid (5 mM) for 20 minutes at 22° C before being added to microtiter wells coated with α_MI-domain (5 μg/mL) in the GST-free form and postcoated with 0.4% PVA. Binding of¹²⁵I-Glu-Pg to the α_MI-domain was quantitated. (C) Equilibrium analysis of the interaction of the α_MI-domain with Pg. (i) Representative profiles of the SPR responses (RU) for Glu-Pg concentrations ranging from 0.1 μMto11 μM binding to the α_MI-domain coupled to the chip surface. Pg was inactivated by incubating with diisopropylfluorophosphate. RU indicates response units. (ii) Saturable binding curve and (inset) Scatchard plot. Req indicates the response at equilibrium; C, the Glu-Pg concentration.