Fig. 6.

aPC inhibits allogenic T-cell activation via PAR3. a Expression of PARs and EPCR on human and mouse pan T-cells (T) and regulatory T-cells (T_reg) was determined by immunoblotting. GAPDH was used as loading control. Exemplary immunoblots of three independent repeat experiments. b Expression of PAR3 in human pan T-cells, CD8⁺ T-cells, CD4⁺ T-cells, and T_regs (CD4⁺CD25⁺). Exemplary immunoblots of three independent repeat experiments. c Preincubation of human peripheral blood T-cells (T) with N-terminal binding antibodies against PAR3 (αPAR3), but not against PAR1 (αPAR1), PAR2 (αPAR2), or PAR4 (αPAR4), or with an EPCR blocking antibody (αEPCR) prior to aPC-preincubation and MLR abrogates aPC’s inhibitory effect in regard to T-cell reactivity. d Preincubation of T-cells (T) from C57BL/6 PAR3-deficient (PAR3^−/−) mice with aPC and subsequent allogenic stimulation (T-PAR3^−/−(aPC) + AgPC) abrogates aPC’s inhibitory effect on T-cell reactivity. e Proteolytic fragments (indicated by red lines) observed following incubation of the N-terminal end of mouse PAR3 (sequence shown at the top) with human thrombin (10 nM) or human aPC (500 nM). Proteolysis was determined at indicated time-points (left, t(h)) and estimated cleavage efficiency is shown in percentage (right). Mean value ± SEM (c, d), results of at least three repeat experiments each with three biological disjunct donors. **P < 0.01 (c, d: ANOVA)