Abstract 168: Splice Variants of Tissue Factor Determine the Coagulant State of Endothelial Cells and Modulate Vascular Stabilization/Regression

Introduction: Recruitment of pericytes (PC), critical to microvascular maturation, requires deposition of basement membrane proteins such as von Willebrand factor (vWF). Loss of PCs ultimately leads to vessel regression and rarefaction. Hypothesis: Splice variants of tissue factor (TF) stabilize the vasculature by supporting EC-PC interactions. Results: We determined that alternatively spliced TF (asTF) resides in Weibel Palade bodies (WPB) of ECs. Overexpression of KLF-2 enhanced asTF protein levels in WPBs. In confluent EC monolayers, asTF was deposited into the extracellular matrix (ECM). In assays employing vascular plexus remodelling on 3D-basement membrane and purified recombinant asTF or full-length TF (flTF), only asTF improved EC-PC interactions. At sites of low KLF-2 expression (low or turbulent flow), total TF mRNA was diminished, leading to low asTF expression. However, upon EC activation by TNFα, total TF mRNA levels rapidly increased and TF protein expression shifted from the ECM-deposited asTF to luminally expressed flTF. Interestingly, flTF-driven EC conversion to a procoagulant state was associated with the release of vWF and asTF from WPBs, depleting the intracellular depots from these proteins. Immunohistochemical staining of kidney specimens of human living donors confirmed that asTF was present at high levels in WPBs of microvascular endothelium. Moreover, after 45 minutes of reperfusion of the transplanted kidney, asTF- and vWF-staining was markedly reduced, consistent with their release from WPB following an inflammatory insult. Conclusion: We show for the first time that quiescent, KLF-2 expressing ECs predominantly generate non-coagulant asTF, which is stored in WPBs and serves to stabilize the microvasculature by supporting EC-PC interactions. In contrast, in low-flow zones or sites with disturbed flow (e.g. at bifurcations), KLF-2 is downregulated, leading to decreased production of asTF and impaired stabilization of the vasculature. During inflammation, acute perturbation of ECs (e.g. exposure to TNFα) is associated with downregulation of KLF-2 and a post-transcriptionally regulated increase in the production of flTF, resulting in a pro-coagulant phenotype and loss of EC-PC interactions.