Protein C Inhibitor (PCI) Binds to Phosphatidylserine Exposing Cells with Implications in the Phagocytosis of Apoptotic Cells and Activated Platelets

Abstract

Protein C Inhibitor (PCI) is a secreted serine protease inhibitor, belonging to the family of serpins. In addition to activated protein C PCI inactivates several other proteases of the coagulation and fibrinolytic systems, suggesting a regulatory role in hemostasis. Glycosaminoglycans and certain negatively charged phospholipids, like phosphatidylserine, bind to PCI and modulate its activity. Phosphatidylerine (PS) is exposed on the surface of apoptotic cells and known as a phagocytosis marker. We hypothesized that PCI might bind to PS exposed on apoptotic cells and thereby influence their removal by phagocytosis. Using Jurkat T-lymphocytes and U937 myeloid cells, we show here that PCI binds to apoptotic cells to a similar extent at the same sites as Annexin V, but in a different manner as compared to live cells (defined spots on ∼10–30% of cells). PCI dose dependently decreased phagocytosis of apoptotic Jurkat cells by U937 macrophages. Moreover, the phagocytosis of PS exposing, activated platelets by human blood derived monocytes declined in the presence of PCI. In U937 cells the expression of PCI as well as the surface binding of PCI increased with time of phorbol ester treatment/macrophage differentiation. The results of this study suggest a role of PCI not only for the function and/or maturation of macrophages, but also as a negative regulator of apoptotic cell and activated platelets removal.

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

“…Plasma-derived MPs of healthy individuals did not bind any exogenously added pPCI-Cy3 as determined by flow cytometry. As positive control we studied the binding of pPCI-Cy3 to apoptotic Jurkat cells known to bind pPCI-Cy3 [ 45 ]. In contrast to MPs from healthy individuals, about 20% of apoptotic Jurkat cells bound pPCI-Cy3 ( Fig 2D ).…”

“…In previous studies we have shown that annexin V and PCI were co-localized in atherosclerotic plaques [ 41 ]. Furthermore, binding of PCI and annexin V occurred at the same sites on apoptotic Jurkat cells and U937 cells [ 45 ]. The presence of PCI on annexin V binding MPs is therefore an additional indication that PCI and PS interact in vivo .…”

“…Human Jurkat T-lymphocytes and U937 myeloid cells (ATCC, American Type Culture Collection, Manassas, USA) were cultured in RPMI-1640 medium (Sigma-Aldrich, Austria) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich, Austria), 1% penicillin/streptomycin/fungizone (PSF, Lonza, Austria) and 1% L-glutamine (Lonza, Austria). Cells were maintained in a humidified incubator with 5% CO 2 at 37°C as published earlier [ 45 ]. Cells were harvested by centrifugation (500xg, 5 min, 20°C).…”

“…MPs were washed with PBS, stained with annexin V-EGFP as indicated earlier and subjected to flow cytometry. Apoptotic Jurkat cells [ 45 ] were incubated with 100 nM pPCI-Cy3 in parallel to MPs. In vitro generated MPs derived from Jurkat cells upon serum starvation or STS treatment (1 μM, 18 hours, 37°C) were incubated with pPCI-Cy3 (80 nM) in the absence or presence of heparin (10 U/ml) in PBS for 45 min at RT.…”

“…Immunohistochemical staining of atherosclerotic plaques suggested that co-localization of PCI with PS on apoptotic cells also occurs in an in vivo environment [ 41 ]. A recent study published by our group has shown that PCI inhibited phagocytosis of activated, PS-exposing platelets by human blood-derived monocytes [ 45 ].…”