title: Two-wavelength near-infrared fluorescence for the quantitation of drug antiplatelet effects in large animal model systems.
abstract: OBJECTIVE
  Intraoperative imaging of intravascular thrombi is limited by the inability of visible light to penetrate thick-walled vessels. Near-infrared (NIR) light has relatively high tissue penetration and low autofluorescence and scatter, offering significant advantages. We hypothesized that the development of 700-nm NIR fluorophores for platelet labeling, in conjunction with existing 800-nm NIR fluorophores, would permit simultaneous and separable quantitation of intravascular thrombi and measurement of the antiplatelet effect of drugs.
  
  
  METHODS
  We synthesized a series of lipophilic, cationic, polymethine indocyanine dyes (MHI-86, 94, 106, and 114) that emit at approximately 700 nm. Platelet uptake was optimized in vitro and the bioactivity and blood half-life of labeled platelets was characterized in vitro and in vivo. FeCl(3)-induced injury of the femoral arteries and intravascular thrombus formation was performed in 35-kg Yorkshire pigs. A combination of 700-nm and 800-nm NIR fluorophore-labeled platelets was used in conjunction with the fluorescence-assisted resection and exploration imaging system to image and quantify the antiplatelet effect of cilostazol and acetylsalicylic acid.
  
  
  RESULTS
  MHI-114 was incorporated at nearly 4.1 × 10(6) molecules per platelet without affecting platelet function. When infused into pigs, the signal-to-background ratio of MHI-114-labeled platelets exhibited a blood half-life of 16.4 ± 2.2 (mean ± SEM; n = 3) minute and generated a signal-to-background ratio of 2.5 ± 0.5 (mean ± SEM; n = 3) at the site of thrombi. Using dual-NIR-labeled platelet populations, cilostazol and acetylsalicylic acid were found to cause a reduction in platelet incorporation into thrombi of 51 ± 2% and 10 ± 1% (mean ± SEM; n = 3), respectively, relative to vehicle-only treated control thrombi.
  
  
  CONCLUSIONS
  New platelet-avid 700-nm NIR fluorophores permit simultaneous two-wavelength NIR fluorescence imaging and quantitation of intravascular thrombi in intact vessels approaching the size of humans and can be used to study the antiplatelet effect of drugs.
authors.affiliation: Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, Boston, MA 02215, USA.
authors.forename: Yoshitomo
authors.initials: Y
authors.lastname: Ashitate
authors.forename: Soon Hee
authors.initials: SH
authors.lastname: Kim
authors.forename: Eiichi
authors.initials: E
authors.lastname: Tanaka
authors.forename: Maged
authors.initials: M
authors.lastname: Henary
authors.forename: Hak Soo
authors.initials: HS
authors.lastname: Choi
authors.forename: John V
authors.initials: JV
authors.lastname: Frangioni
authors.forename: Robert
authors.initials: R
authors.lastname: Flaumenhaft
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D001241:Aspirin / Q000494:pharmacology*; D001792:Blood Platelets / Q000378:metabolism*; D000077407:Cilostazol; D004195:Disease Models, Animal; D005260:Female; D005263:Femoral Artery; D005456:Fluorescent Dyes / Q000138:chemical synthesis*; D006207:Half-Life; D006801:Humans; D015394:Molecular Structure; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology*; D019265:Spectroscopy, Near-Infrared / Q000379:methods*; D013552:Swine; D013777:Tetrazoles / Q000494:pharmacology*; D013927:Thrombosis / Q000175:diagnosis* / Q000601:surgery
chemical_list: D005456:Fluorescent Dyes; D010975:Platelet Aggregation Inhibitors; D013777:Tetrazoles; D000077407:Cilostazol; D001241:Aspirin