Fibrin biomaterial is widely used in the clinic as a tissue sealant and in pre-clinical research as a carrier material for growth factor delivery. In these applications, premature fibrin degradation leads to suboptimal tissue adhesion, recurrent bleeding and limited regenerative efficacy. Therefore, fibrinolytic inhibitors are commonly added into fibrin formulations, such as the potent inhibitor aprotinin. Nevertheless, the use of aprotinin in the clinic has been associated to some important side-effects, including some immunogenic reactions related to the bovine origin of the drug. In this project, we characterized the use of an endogenous human fibrinolytic inhibitor, α2-antiplasmin (α2PI), as a potential substitute for aprotinin. We first tested α2PI as an antifibrinolytic agent for applications in tissue sealants and for growth factor delivery in diabetic wound healing. Then, we assessed α2PI as a stand-alone hemostatic agent to reduce blood loss during surgery.
We produced α2PI as a recombinant his-tagged protein in Human Embryonic Kidney (HEK) 293 mammalian cells and purified it by affinity-based chromatography. The longevity of α2PI-supplemented fibrin hydrogels was assessed in vitro in presence of plasmin, and in vivo upon subcutaneous implantation in mice using an in vivo imaging system (IVIS). Next, the delivery of angiogenic growth factors via α2PI-supplemented fibrin hydrogels was tested in a wound healing model in the db/db diabetic mouse and was quantified by histomorphometric analyses. More specifically, wound regeneration was assessed in terms of wound re-epithelialization, granulation tissue formation and wound angiogenesis. Finally, we used a tail vein bleeding model in mice to evaluate α2PI hemostatic properties upon intravenous injection.
Incorporation of recombinant human α2PI into fibrin biomaterials significantly prolonged their duration in vitro and in vivo. Upon subcutaneous implantation, α2PI-supplemented fibrin implants remained present for over 30 days, thus vastly outperforming the implants supplemented by aprotinin. In the diabetic wound healing model, the delivery of angiogenic growth factors by α2PI-supplemented fibrin significantly enhanced granulation tissue formation and wound angiogenesis as compared to the delivery of growth factors in absence of α2PI. In addition, we observed positive trend toward enhanced wound re-epithelialization (p-value = 0.09). Finally, we demonstrated that α2PI had similar hemostatic properties than aprotinin in the mouse tail vein bleeding model in vivo, significantly reducing blood coagulation time and blood loss as compared to non-treated animals.
In conclusion, α2PI showed strong efficacy in vivo, both as an anti-fibrinolytic and as a hemostatic agent, therefore appearing as a highly competitive human-derived substitute to the bovine aprotinin. Indeed, α2PI successfully increased the longevity of fibrin implants, enhanced growth factor delivery in diabetic wound healing and reduced bleeding time and loss upon intravenous delivery, respectively mimicking the 3 main applications of aprotinin in fibrin sealants, fibrin-mediated drug delivery and in surgery."