Novel Injury Site Targeted Fusion Protein Comprising Annexin V and Kunitz Inhibitor Domains Ameliorates Ischemia-Reperfusion Injury and Promotes Survival of Ischemic Rat Abdominal Skin Flaps.
Appropriate antithrombotic therapy is critical for successful outcomes in reconstructive microsurgical procedures involving free tissue transfer. The annexin V-6L15 (ANV-6L15) fusion protein was developed as a targeted antithrombotic reagent. Annexin V specifically binds to exposed phosphatidylserine on apoptotic or injured cells, and prevents coagulation and cell adhesion, whereas 6L15 inhibits tissue factor-VIIa pathway within the coagulation cascade. The treatment efficacy of ANV-6L15 on rat island muscle and pedicled abdominal fasciocutaneous flaps following ischemic injury and ischemia-reperfusion injury (IRI) was evaluated.


METHODS
The effects of ANV-6L15 on survival of rat abdominal fasciocutaneous flaps subjected to 10 hours of critical ischemia were assessed on day 5. Near-IR imaging was applied to evaluate the distribution of ANV-6L15 and flap perfusion. The rat cremaster muscle island flap was used to evaluate the effect of ANV-6L15 on IRI-induced leukocyte-endothelial interactions via intravital microscopy. 2,3,5 triphenyl-tetrazolium chloride assay was used to determine the ratio between live-versus-dead tissue.


RESULTS
ANV-6L15 significantly increased the ratio of viable tissue (68.5 ± 9.79% vs 84.8 ± 5.14%, P < 0.05), and promoted survival of rat pedicled abdominal flaps (59.3 ± 6.86 vs. 47.0 ± 8.67, P < 0.05). Intravital microscopy demonstrated a significant decrease in the number of adhesive leukocytes (1.8 ± 1.64 vs. 10.0 ± 6.32, P < 0.05), and the percentage change of functional capillaries (16.4 ± 15.1 vs. 47.3 ± 18.3, P < 0.05) in ANV-6L15-treatment group.


CONCLUSIONS
ANV-6L15 promoted survival of ischemic rat cremaster muscle and abdominal fasciocutaneous flaps and ameliorated leukocyte-related IRI. Future evaluation of potential clinical application of ANV-6L15 is warranted as a flap treatment adjunct.
From the *Center for Vascularized Composite Allotransplantation, †Department of Plastic and Reconstructive Surgery, Chang Gung Memorial Hospital; ‡School of Medicine, Chang Gung University; Taoyuan, Gueishan, Taiwan; §EVAS Therapeutics, LLC, Ballwin; and ∥Department of Radiology, Washington University School of Medicine, St Louis, MO.
Victor Bong-Hang
VB
Shyu
Chung En
CE
Hsu
Chih-Jen
CJ
Wen
Tze-Chein
TC
Wun
Rui
R
Tang
Samuel
S
Achilefu
Fu-Chan
FC
Wei
Hui-Yun
HY
Cheng
D000005:Abdomen; D000818:Animals; D017304:Annexin A5 / Q000494:pharmacology*; D007611:Aprotinin / Q000494:pharmacology*; D004195:Disease Models, Animal; D006083:Graft Occlusion, Vascular / Q000517:prevention & control; D006439:Hemodynamics; D008297:Male; D051381:Rats; D011917:Rats, Inbred Lew; D011993:Recombinant Fusion Proteins / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000494:pharmacology*; D015427:Reperfusion Injury / Q000517:prevention & control*; D013524:Surgical Flaps / Q000098:blood supply*
D017304:Annexin A5; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C098973:recombinant Kunitz protease inhibitor; D007611:Aprotinin