Interaction of Protein C Inhibitor with the Type II Transmembrane Serine Protease Enteropeptidase

The serine protease inhibitor protein C inhibitor (PCI) is expressed in many human tissues and exhibits broad protease reactivity. PCI binds glycosaminoglycans and certain phospholipids, which modulate its inhibitory activity. Enteropeptidase (EP) is a type II transmembrane serine protease mainly found on the brush border membrane of epithelial cells in the duodenum, where it activates trypsinogen to initiate the digestion of food proteins. Some active EP is also present in duodenal fluid and has been made responsible for causing pancreatitis in case of duodeno-pancreatic reflux. Together with its substrate trypsinogen, EP is furthermore present in the epidermis and in some cancer cells. In this report, we show that PCI inhibited EP with an apparent 2nd order rate constant of 4.48×104 M−1 s−1. Low molecular weight (LMWH) and unfractionated heparin (UFH) slightly reduced the inhibitory effect of PCI. The SI (stoichiometry of inhibition) value for the inhibition of EP by PCI was 10.8 in the absence and 17.9 in the presence of UFH (10 U/ml). By inhibiting trypsin, chymotrypsin, and additionally EP, PCI might play a role in the protection of the pancreas from autodigestion. Furthermore the interaction of PCI with EP may influence the regulation of epithelial differentiation

Phospholipid Binding Protein C Inhibitor (PCI) Is Present on Microparticles Generated In Vitro and In Vivo.

Protein C inhibitor is a secreted, non-specific serine protease inhibitor with broad protease reactivity. It binds glycosaminoglycans and anionic phospholipids, which can modulate its activity. Anionic phospholipids, such as phosphatidylserine are normally localized to the inner leaflet of the plasma membrane, but are exposed on activated and apoptotic cells and on plasma membrane-derived microparticles. In this report we show by flow cytometry that microparticles derived from cultured cells and activated platelets incorporated protein C inhibitor during membrane blebbing. Moreover, protein C inhibitor is present in/on microparticles circulating in normal human plasma as judged from Western blots, ELISAs, flow cytometry, and mass spectrometry. These plasma microparticles are mainly derived from megakaryocytes. They seem to be saturated with protein C inhibitor, since they do not bind added fluorescence-labeled protein C inhibitor. Heparin partially removed microparticle-bound protein C inhibitor, supporting our assumption that protein C inhibitor is bound via phospholipids. To assess the biological role of microparticle-bound protein C inhibitor we performed protease inhibition assays and co-precipitated putative binding partners on microparticles with anti-protein C inhibitor IgG. As judged from amidolytic assays microparticle-bound protein C inhibitor did not inhibit activated protein C or thrombin, nor did microparticles modulate the activity of exogenous protein C inhibitor. Among the proteins co-precipitating with protein C inhibitor, complement factors, especially complement factor 3, were most striking. Taken together, our data do not support a major role of microparticle-associated protein C inhibitor in coagulation, but rather suggest an interaction with proteins of the complement system present on these phospholipid vesicles

Stoichiometry of inhibition of EP by PCI.

<p>EP (0.3 nM) was incubated overnight with varying concentrations of recombinant human PCI in the absence and presence of 10 U/ml UFH. Remaining EP activity was determined and plotted as relative activity against [PCI]₀/[EP]₀. The x-intercept of each linear regression line represents the SI value. The SI values were 10.8±0.7 without and 17.9±0.6 (mean ± S.D., n = 3) with 10 U/ml UFH, respectively. The results of one experiment are shown in the figure. Each single experiment was done in duplicates.</p

Inhibition of EP by antithrombin.

<p>EP (0.3 nM) was incubated for 60 min at 37°C with varying concentrations of AT in the absence and presence of UFH or LMWH (as indicated in the figure). For comparison, inhibition by PCI (incubation time 45 min) is also included. Remaining EP activity was quantified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039262#s4" target="_blank">Materials and Methods</a>. The activity of EP in the absence of serpins was set as 1. n = 3; error bars show standard deviation. Each single experiment was done in duplicates.</p

Bovine EP cleaves human PCI and complex formation is observed.

<p>PCI (0.05 g/l) and bovine EP (0.04 g/l) were incubated at 37°C in a volume of 10 µl TCNB buffer for the time periods indicated. Thereafter, an equal amount of Laemmli buffer with (<b>A</b>) or without (<b>B</b>) 10% 2-mercaptoethanol was added. For time point zero, the reagents were applied directly into Laemmli buffer. SDS-PAGE and Western blotting using mouse anti-PCI IgG (<b>left panel in A and B</b>) or SDS-PAGE followed by Coomassie staining (<b>right panel in A and B</b>) using PageBlue solution (Fermentas, St. Leon-Rot, Germany) were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039262#s4" target="_blank">Materials and Methods</a>. The position of the molecular mass standard (kDa) is indicated on the left and also visible as first lane on the right panel in A and B.</p

PCI forms complexes with EP and is cleaved by EP.

<p>Recombinant human PCI (440 nM) and EP (60 nM) were incubated at 37°C in a volume of 10 µl TCNB buffer for the time periods indicated. Thereafter, an equal amount of Laemmli buffer with (<b>A, B</b>) or without (<b>C</b>) 10% 2-mercaptoethanol was added. For time point zero, the reagents were applied directly into Laemmli buffer. SDS-PAGE and Western blotting were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039262#s4" target="_blank">Materials and Methods</a> using either monoclonal mouse anti-PCI IgG (<b>A</b>), rabbit anti-EP (light chain) IgG (<b>B</b>) or mouse anti-EP (heavy chain) IgG (<b>C</b>), respectively. The position of the molecular mass markers (kDa) is indicated on the left. For control purposes, the left 2 lanes contained PCI alone or EP alone, respectively.</p

Heparin removes PCI from MPs.

<p>Plasma-derived MPs from healthy donors were incubated without or with different heparin concentrations (0.05, 0.5, 5, 50 U/ml) for 30 min (panels A and B) at RT. Surface-bound PCI was analyzed by flow cytometry after staining with anti-PCI-IgG-AF633 and annexin V-EGFP. (A) % of PCI positive MPs and (B) MFI of PCI positive MPs. Data represent means + SEM of four independent experiments performed with MPs isolated from pooled plasma (equal aliquots from 4 healthy individuals), each performed in duplicates. (C) Western blot probed with rabbit anti-PCI-IgG showing supernatants of MPs after incubation with heparin (50 U/ml) for the time periods indicated. PCI in supernatants was quantified and normalised to supernatants with heparin at time point 0. Data represent means and range of two independent experiments performed with MPs isolated from pooled plasma (equal aliquots from 4 healthy individuals).</p