This suggests an interaction of PCI (46 kDa) with the light chain of EP (~50 kDa), on which the serine protease domain is localized. To confirm this finding, we used an antibody to the light chain of EP. We could observe complex formation comparable to the results when anti-PCI IgG was used (Figure 1B). We performed the same experiment definitely under non-reducing conditions, using an antibody to the heavy chain of EP. Here, it was possible to see complex formation at about 215 kDa (Figure 1C). Figure 1 PCI forms complexes with EP and is cleaved by EP. Effect of Cofactors on the Inhibition of EP by PCI In the EP activity assay, PCI strongly inhibited the amidolytic activity of EP (Figure 2). UFH (Figure 2A) and LMWH (Figure 2B) slightly reduced the inhibitory effect of PCI.

Inhibition of EP with purified plasma PCI showed similar results as with recombinant PCI (data not shown). The phospholipid PAPS (1-palmitoyl-2-arachidonoyl-sn-glycero-3-phospho-L-serine) had no effect on the inhibition of EP by PCI. Consistent with previous results [43], PAPS strongly increased the inhibition of activated protein C in a parallel experiment. Recombinant mouse PCI showed a similar inhibitory effect on human EP as human PCI. With mouse PCI, the inhibitory effect was also slightly reduced in the presence of LMWH (data not shown). Figure 2 Inhibition of EP by PCI in the absence or presence of heparin. The apparent 2nd order rate constants (kapp) for the inhibition of EP by human recombinant PCI were calculated under pseudo 1st order conditions as outlined in Materials and Methods (Figure 3).

PCI inhibited EP with an apparent 2nd order rate constant of 4.48��0.32��104 M?1 s?1 (mean �� S.D., n=3). In the presence of 10 U/ml UFH, the constant was reduced to 8.83��0.75��103 M?1 s?1 (mean �� S.D., n=3). The SI value for the inhibition of EP by PCI was 10.8��0.7 (mean �� S.D., n=3). In the presence of 10 U/ml UFH, it increased to 17.9��0.6 (mean �� S.D., n=3) (Figure 4). The kapp values were corrected for SIs by multiplication (kapp �� SI), as this provides a measure for the overall rate of association [13], [44]. Corrected rate constants (kapp �� SI) were 4.84��105 M?1 s?1 in the absence and 1.58��105 M?1 s?1 in the presence of 10 U/ml UFH, respectively. Figure 3 Determination of the apparent 2nd order rate constant for the inhibition of EP by PCI. Figure 4 Stoichiometry of inhibition of EP by PCI.

Human PCI Forms Complexes with EP from Natural Sources In addition to recombinant human EP, the interaction of PCI with two additional EPs from natural sources has been studied. EP purified from porcine intestine (data not shown) as well as bovine EP (bEP) purified from calf intestine formed complexes with PCI, as demonstrated by Anacetrapib SDS-PAGE analysis followed by Western blotting (Figure 5). Functional assays showed strong inhibition of bEP by PCI.