Background: The serine protease activated protein C (aPC) plays a role in innate immunity through both its anticoagulant and signaling properties via protease activated receptors (PARs). We recently established a function of aPC and the signaling incompetent receptor (PAR3) in adaptive immunity (GvHD). aPC has been shown to interact with RGD-binding-integrins as co-receptors. However, whether aPC and PAR3 can modulate T-cell integrin signaling, a key mechanism regulating T-cell activation, adhesion and migration, is unknown.

Aims: We aim to investigate whether aPC regulates T-cell activation, adhesion and migration via integrins on T-cells.

Methods: GvHD was induced in APC^high, APC^high/RGE, and C57BL/6 (WT) mice. Disease severity was assessed by studying survival, clinical scores and cell death in gastrointestinal tract. In-vivo migration of aPC pre-incubated T-cells was studied in a murine DTH model by two-photon-microscopy. T-cell activation and adhesion were studied in-vitro. The effect of aPC on integrin signaling was studied using immunoblotting and FACS. A T-cell(Jurkat)-B-cell(Raji) conjugation assay was performed using dual color flow cytometry to study the effect of aPC on immunological synapse.

Results: Mice with endogenous high levels of aPC (APC^high), but not mice expressing a mutant aPC lacking the integrin binding site (APC^high/RGE), were protected from GvHD in comparison to WT mice. Surprisingly, we observed in-vitro that aPC additionally regulates the activation of non-RGD integrin. aPC modulated global T-cell phosphorylation. aPC reduced T-cell activation, and adhesion to ICAM1 and fibronectin while increased migration (in-vivo) and phosphorylation of p-PLCγ-1 along with a decline of active-Rap1. A reduction in T-cell B-cell conjugation was observed upon aPC incubation of T-cells.

Conclusions: aPC reduces global phosphorylation and thus modulates inside-out signaling of integrins. In addition, aPC binds via its RGD domain to RGD-binding integrins, affecting T-cell function via outside-in signaling. In future studies we will dissect these independent molecular pathways and evaluate the pathophysiological relevance of these findings.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/cross-talk-of-activated-protein-capc-with-integrin-and-its-effect-on-t-cell-signaling/