Plasma Fn inhibited Fg/VWF-independent platelet aggregation in vitro. (A top left) Platelet aggregation was enhanced in plasma Fn-depleted Cre⁺ TKO PRP compared with Cre⁻ control PRP when stimulated by 500 μM TRAP (P < .05). (Top right) Gel-filtered platelet aggregation, induced by 1 U/mL thrombin, was also enhanced in Cre⁺ TKO mice (P < .05). (Bottom) The Cre⁺ TKO platelets formed larger aggregates than did Cre⁻ control platelets when examined under a microscope. Pictures of aggregates were taken under a 10×/0.30 NA objective. (B) Platelet aggregation was induced in both Cre⁺ TKO PRP and gel-filtered platelets by collagen (5-10 μg/mL) but failed to be induced by ADP (20 μM) and U46619 (20 μM). No platelet aggregates were found in ADP- and U46619-treated Cre⁺ TKO samples when examined under a microscope (32×/0.40 NA objective). (C left) The enhancement of platelet aggregation in TKO conditions was diminished when Cre⁺ TKO platelets were aggregated in pFn-positive Cre⁻ control PPP in a parallel aggregation assay (top panel). (Right) The enhancement of platelet aggregation by pFn was partially restored in Cre⁻ control platelets when they were aggregated in pFn-depleted Cre⁺ TKO PPP. (D) Exogenous pFn significantly inhibited wild-type gel-filtered mouse platelet aggregation in a dose-dependent manner. (E top) Cre⁺ TKO platelet aggregation in PRP was inhibited by our newly generated mouse anti–mouse β₃ integrin monoclonal antibody M1 (left). Cre⁺ TKO platelet aggregation with gel-filtered platelets in PIPES buffer was also inhibited by polyclonal anti-β₃ integrin sera (right). (Bottom) Cre⁺ TKO platelet aggregation was not affected by a monoclonal antibody against GPIbα (left) and our mouse anti–mouse anti-GPIbα sera (right). All representative traces of platelet aggregation are shown from at least 3 independent experiments.