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Activated Protein C inhibits platelet activation during xenogeneic GalTKO.hCD39 pig lung perfusion

Elana Rybak^1,4, Lars Burdorf^1,4, Tianshu Zhang^1,4, Xiangfei Cheng^1,4, Amal Laaris^1,4, Emily Welty^1,4, Christopher Avon^1,4, David Ayares², Simon Robson³, Agnes M. Azimzadeh^1,4, Richard N. Pierson III^1,4

¹Department of Surgery, University of Maryland, Baltimore, MD; ²Revivicor Inc., Blacksburg, VA; ³BI/Deaconess Hospital, Harvard University, Boston, MA; ⁴Department of Surgery, VA Maryland Health Care System, Baltimore, MD; United States

Purpose: Expression of human CD39 by GalTKO pig lungs is a genetic modification designed to inhibit platelet activation and vasoconstriction by accelerating adenosine diphosphate metabolism. Activated Protein-C (Xigris®; APC) inhibits Factors Va and VIIIa, inhibiting thrombin-driven amplification of the coagulation cascade and is profibrinolytic. Prolific coagulation cascade activation is typical of GalTKO pig lung perfusion with xenogeneic human blood, and xeno lung injury is closely associated with rapid platelet sequestration and activation. Here we evaluated effects of APC on platelet activation during perfusion of GalTKO pig lungs expressing variable amounts of hCD39.

Methods: GalTKO.hCD39 (n=6) transgeneic pig lungs were perfused with heparinized fresh human blood for 4h, or until graft failure as determined by pre-defined criteria. In 6 paired experiments using hCD39-expressing GalTKO pigs, one lung in each pair was treated with APC added to the perfusate (0.7mg bolus followed by 0.45mg/h/L of blood continuous infusion, total 2.5mg APC), while the other was left untreated. Beta thromboglobulin (BTG) was measured as an index of platelet activation.

Results: Median survival time (MST) in both treated and untreated GalTKO.hCD39 lungs (225’) was longer than MST in prior work with GalTKO lungs (120’; p=0.19). hCD39 expression levels were heterogeneous in the GalTKO.hCD39 lungs studied. Platelet sequestration was significantly delayed in one of two lungs that exhibited high levels of hCD39 expression but not in lungs expressing lower levels of hCD39, and was not significantly affected by APC treatment. However, BTG release was significantly lower in the APC-treated group at multiple time points compared to the GalTKO.hCD39 paired lungs (e.g., BTG at 30’: 433±88 vs. 821±251, p=0.004).

Conclusion: APC significantly decreases platelet activation during the initial phase of GalTKO pig lung perfusion with human blood. Although expression of hCD39 on a GalTKO background appears to prolong survival time of xenogeneically-perfused pig lungs, APC treatment is not associated with additional survival prolongation, and neither approach (alone or together) consistently prevents platelet sequestration. Methods to inhibit complement activation as well as other previously identified non-physiologic cross-species platelet and coagulation pathways will likely be required.