Figure 3.

Analyzing temporal crosstalk between GPIb and GPIIb-IIIa. (A,B) dBFP setup for switching between interactions of the target cell with two immobilized ligands. Two beads functionalized with distinct proteins were attached to the apexes of two RBCs respectively aspirated by LP1 and LP2 to form Probes 1 and 2. A platelet held by RP1 was aligned against one probe at a time to sequentially engage two ligands, designated as the pre- (A) and post- (B) switch positions, respectively. Scale bars are 5 μm. (C–E) Schematics of the switching assay for studying temporal crosstalk within a platelet dual-receptor system (GPIb to GPIIb-IIIa). In the pre-switch position, the platelet was driven to repeatedly engage the first receptor GPIb with its ligand VWF-A1 coated on Probe 1 and exert a controlled pulling force on the A1–GPIb bonds (C, top; D). Intraplatelet Ca²⁺ signals triggered by GPIb mechanoreception were observed for 200 s (C, color gradient transition, middle). The subsequent upregulation of GPIIb-IIIa was probed by its ligand fibronectin (FN) on Probe 2 (C, bottom; E). (F–H) Concurrent measurement of platelet GPIb (receptor 1) triggered Ca²⁺ flux and bond kinetics. Top: Representative epi-fluorescence pseudo-colored images of intraplatelet Ca²⁺ in a platelet touched by BSA (non-adhesive control) (F) and VWF-A1 under ramped (1000 pN/s) (G) and clamped (25 pN) (H) forces at indicated times. Bottom: Representative time courses of normalized Ca²⁺ intensity (color matched curves and legends). The concurrent measurements of bond lifetime events (symbol) and the cumulative lifetime (purple curve) for the platelet exhibiting Ca²⁺ fluxes are overlaid. I. Pre- and Post-stimulation adhesion frequencies of platelet GPIIb-IIIa (receptor 2) binding to beads functionalized with FN. n ≥ 5 platelets were tested for each condition. *denotes p < 0.01 by Student t-test.