Figure 1. FIXa interacts with PS in vitro and ex vivo.

(A) Flow cytometry was used to quantify the binding of fluorescently labeled FIXa and PS to resting platelets (black bars) and convulxin/PAR-4 peptide-activated platelets (blue bars). Minimal interactions with Gla-containing proteins are seen on the resting platelets. In contrast, binding of both FIXa and PS is observed upon platelet activation, as reflected by increased mean fluorescent intensity (MFI). (B) A forward scatter of resting and activated platelets exposed to fluorescently tagged FIXa (top) and PS (bottom) demonstrated that platelet size decreases upon activation. These smaller, activated, “coated,” platelets showed an increase in fluorescence when binding to FIXa or PS. (C) Flow cytometry studies of resting and activated platelets were performed with fluorescently tagged FIXa (1 nmole/liter) and PS (140 nmole/liter) (labeled above each plot). A fluorescence shift into Q1 indicates FIXa binding to platelets, whereas a shift into Q4 suggests PS binding. Movement into Q2 indicates co-localization of the two proteins on the same platelet (see plot of ‘PS + FIXa’). All panels in (C) were derived from experiments that utilized activated platelets, except for the upper left panel, which describes resting platelets exposed to both FIXa and PS. (D) The flow cytometry studies in (C) were repeated on activated platelets with des-Gla FIXa in place of wild type FIXa. A shift to Q2 is observed in the Des-Gla FIXa + PS experiment, despite the fact that des-Gla FIXa alone is unable to bind platelets; this result demonstrated that PS was required to recruit des-Gla FIXa to the platelet surface. (E) Co-immunoprecipitation of FIXa with PS, and vice versa, revealed that FIXa interacts with PS in plasma. FIXa (1 nmole/liter) was added (lanes with +) or was not added (lanes with -) to FIX-deficient plasma. This plasma was immunoprecipitated with an IgG control antibody (both blots IgG lane) or with an anti-FIXa or anti-PS antibody (top blot and bottom blot, respectively), and immunoblotted for PS or FIXa (top blot and bottom blot, respectively). Initial plasma samples which were supplemented with FIXa are shown on both blots in the ‘Input’ lane. Molecular weights of bands (kDa) are shown to the right of the blots. The values in Figure 1A are expressed as mean ± STDEV (n=3 replicates). *, p<0.05 vs resting platelet (by t-test).