Previously, we showed that labeled
bitistatin analogues possessed excellent characteristics for imaging both
deep-vein thrombosis and
pulmonary embolism. We hypothesized that the N-terminal amino acid sequence of
bitistatin, which is different from other
disintegrins, likely interacts with the binding site of platelets to confer desirable properties to
bitistatin for imaging. In this study, we present the design, synthesis, and initial
biological testing of a short-chain analogue of the native 83-amino-acid
bitistatin sequence. Our initial molecular modeling of the binding loop of
bitistatin showed that the minimal sequence that represented the binding region was a cyclic 10 amino acid sequence cyclo[Cys-Arg-Ile-Ala-
Arg-Gly-Asp-Trp-Asn-Cys(S)]. Systematic modeling of a truncated N-terminal sequence of
bitistatin fused with the optimized binding region having a
thioether sequence through a
Gaba spacer ultimately yielded the 24-amino
acid peptide, cyclo-[CH(2)CO-Arg-Ile-Ala-
Arg-Gly-Asp-Trp-Asn-Cys(S-)]-
Gaba-Gly-Asn-Glu-Ile-Leu-Glu-
Gln-Gly-
Glu-Asp-Ser-Asp-Ser-Lys-
OH, 1. The
peptide was then coupled to the hydrazino-
nicotinic acid bifunctional
chelating agent and the purified adduct labeled with (99m)Tc using
tricine as a coligand. Binding of the unlabeled and labeled
peptide to stimulated human platelets was assayed in vitro. The (99m)Tc labeling yield was > 90%. The in vitro binding assays showed that the IC(50) for inhibition of platelet aggregation was 3694 nM, while the Kd of the (99m)Tc labeled
peptide was 185 nM, indicating moderate affinity for the receptor. The (99m)Tc-labeled
peptide was able to identify sites of experimental thrombi and emboli in a canine model. The results suggest initial success in attempting to mimic the behavior of
bitistatin for imaging thrombi and emboli.