A branched fluorescent peptide probe for imaging of activated platelets

Novel fluorescent probes for thrombi and activated-platelet detection were developed that were based on the glycoprotein IIb/IIIa (GP-IIb/IIIa) binding sequence, Pro-Ser-Pro-Gly-Asp-Trp. Linear, Pro-Ser-Pro-Gly-Asp-Trp-Aha-Gly-Cys(Cy5.5)-NH(2) (1PF), and branched, (Pro-Ser-Pro-Gly-Asp-Trp-Aha)(2)-Lys-Gly-Cys(Cy5.5)-NH(2) (2PF), fluorescent-labeled peptide probes were synthesized. A third probe, also branched, (Pro-Ser-Pro-Gly-Glu-Trp-Aha)(2)-Lys-Gly-Cys(Cy5.5)-NH(2) (2CF), was synthesized as control. The platelet-binding activity of the probes was tested in clots generated from human platelet-rich plasma. Fluorescence reflectance imaging results showed that 2PF has a 16-fold increase in fluorescence intensity compared to the autofluorescence of clots. The linear conjugate, 1PF, and free dye did not show appreciable fluorescence enhancement. 2PF fluorescence was also found 5.5-fold higher than that of the control probe, 2CF. Overall, our results suggest that 2PF binds tightly to GP-IIb/IIIa and potentially can be used for in vivo imaging of thrombosis.