Purpose: Microvascular dysfunction post-reperfusion of the ischaemic endothelium manifests as cellular necrosis and enhanced endothelial permeability. Thrombin generation post-reperfusion of the ischaemic endothelium may be implicated in cellular activation and increased permeability due to cytoskeletal derangement. We examined the effect of endothelial pre-treatment with a novel, cytotopic direct thrombin inhibitor (Thrombalexin) on cellular injury.

Methods: Human microvascular endothelial cells were used in an in vitro model of ischaemia-reperfusion injury. Cell monolayers were exposed to hypoxic conditions at 4[deg]C, prior to reperfusion (normoxia, 37[deg]C). Apoptosis and necrosis were assessed using Annexin V/Propidium Iodide staining on flow cytometry. Endothelial permeability post-reperfusion was assessed by measuring Fluorescein isothiocyanate (FITC)-dextran passage through cells seeded on semi-permeable Transwell inserts.

Results: Pre-treatment with Thrombalexin prior to cold storage reduced the percentage of Annexin V/PI positive cells from 3.1% to 2.0% (p=0.0287). Annexin V/PI negative cell percentages increased in Thrombalexin treated cells also (0.47% to 14.4% respectively, p=0.0007). Treatment post cold storage, pre-reperfusion also resulted in a significant reduction in the percentage of Annexin V/PI positive cells from 4.4% to 1.7% (p=0.006). However, treatment at this interval had no significant effect on increasing viable cells (Annexin V/PI negative population, p=0.13) Exposure of the microvascular endothelium to IR resulted in a significant increase in endothelial permeability compared to cells maintained at resting conditions (119 vs. 36 Mean Relative Fluorescence Units respectively, p=<0.0001). Treatment of the endothelium prior to cold ischaemia with Thrombalexin abrogated this effect (IR untreated 119 vs. IR treated 38.5 RFU; p=<0.0001).

Discussion: Pre-treatment with cytotopic thromboregulation effectively protects the microvascular endothelium from deleterious reperfusion injury. The observed difference in treatment pre-reperfusion may be secondary to reduced adherence of the peptide to the injured ischaemic endothelium. This data also implicates thrombin generation post-reperfusion as a significant causative factor in the observed increased endothelial permeability, as direct thrombin inhibition restored permeability to near resting conditions.

CITATION INFORMATION: Sandhu B, Prendecki M, Crawley J, Galloway-Phillips N, Vallant N, Mason J, Dorling A, Smith R, Pusey C, Papalois V. Cytotopic Thrombin Inhibition Prior to Cold Ischaemia Attenuates Microvascular Endothelial Ischaemia-Reperfusion Injury. Am J Transplant. 2017;17 (suppl 3).

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