The protein C pathway is a primary regulator of blood coagulation and a critical component of the host response to inflammatory stimuli. The most recent member of this pathway is the endothelial protein C receptor (EPCR), a type I transmembrane protein with homology to CD1d/MHC class I proteins. EPCR accelerates formation of activated protein C, a potent anticoagulant and antiinflammatory agent. The current study demonstrates that soluble EPCR binds to PMA-activated neutrophils. Using affinity chromatography, binding studies with purified components, and/or blockade with specific Abs, it was found that soluble EPCR binds to proteinase-3 (PR3), a neutrophil granule proteinase. Furthermore, soluble EPCR binding to neutrophils was partially dependent on Mac-1 (CD11b/CD18), a β2 integrin involved in neutrophil signaling, and cell-cell adhesion events. PR3 is involved in multiple diverse processes, including hemopoietic proliferation, antibacterial activity, and autoimmune-mediated vasculitis. The observation that soluble EPCR binds to activated neutrophils via PR3 and a β2 integrin suggests that there may be a link between the protein C anticoagulant pathway and neutrophil functions.

Proteinase-3 (PR3)4 is a serine proteinase stored in neutrophil granules and released to the cell surface upon activation (1, 2, 3). PR3 is generally thought of as a soluble enzyme, in the sense of being released into the local inflammatory milieu upon neutrophil activation and disgorgement of granular contents. However, several recent studies have observed that most of the neutrophil PR3 remains associated with the cell membrane upon activation, with very little released into the medium (4). PR3 is possibly best known as the primary target Ag of the PR3 anti-neutrophil cytoplasmic Abs (PR3-ANCA) in Wegener’s granulomatosis, a debilitating autoimmune disease characterized by necrotizing vasculitis (5, 6). PR3-ANCA are also found in patients with microscopic polyangiitis, a systemic vasculitic disease (7). PR3 has activities that include degradation of extracellular matrix proteins (8), regulation of myeloid differentiation (9, 10), potentiation of platelet activation (11), and antibacterial action that is independent of its enzymatic activity (12). Structurally, PR3 is very similar to neutrophil elastase (13), but it does have unique substrates, including the membrane-bound precursors of the proinflammatory TNF-α and IL-1β cytokines (14, 15). Thus, PR3 expression near a vascular surface would very likely contribute to local tissue destruction and inflammation.

In ongoing studies with the primate model of Escherichia coli-mediated septic shock, we found that blocking the endothelial protein C receptor (EPCR) in vivo with specific mAbs transformed a transient response to sublethal E. coli into a lethal response (16). EPCR is a type 1 transmembrane protein expressed on endothelium that binds protein C zymogen and facilitates formation of activated protein C, a potent anticoagulant (17) and antiinflammatory protein (18, 19). A soluble form of EPCR, which exists in plasma, retains full ligand-binding capability (20), and levels increase in patients with sepsis or systemic lupus erythematosus (21) either from vascular injury or through a regulated proteolytic release of soluble receptor (22). Of particular interest in the primate study was the observation of a massive polymorphonuclear cell influx into specific areas of the adrenal, liver, and kidney, suggesting that EPCR may be involved in neutrophil interactions and leukocyte trafficking.

In the present study, we examined whether EPCR could interact directly with neutrophils. It was found that recombinant soluble EPCR (sEPCR) binds to activated neutrophils via PR3 and supported, in part, by CD11b/CD18 (Mac-1), a β2 integrin involved in cell-cell adhesion and neutrophil-signaling events. The results are discussed with respect to their potential importance in modulating vascular damage due to inflammatory stimuli.

sEPCR (3) was a recombinant soluble human EPCR and was prepared as described previously (23). The construct codes for the extracellular domain of EPCR truncated immediately above the transmembrane domain at residue 210, with a 12-residue HPC4 epitope tag at the carboxyl terminus for calcium-dependent HPC4-immunoaffinity purification (24). sEPCR was labeled with Oregon Green 488 carboxylic acid, succinimidyl ester 5-isomer (Molecular Probes, Eugene, OR), or Cy3 (Pharmacia-Amersham, Uppsala, Sweden) using standard methods. Human neutrophil PR3 was obtained from Athens Research and Technology (Athens, GA) and biotinylated with sulfo-NHS-LC-biotin (sulfosuccinimidyl 6-(biotinamido)hexanoate; Pierce, Rockford, IL).

The preparation of and screening methods for mAbs against human EPCR have been described elsewhere (20). The PL1 mAb against neutrophil P-selectin glycoprotein ligand-1 was a kind gift from Kevin Moore (Cardiovascular Biology Research, Oklahoma Medical Research Foundation, Oklahoma City, OK). The IB4 (IgG2a) hybridoma cell line was obtained from American Type Culture Collection (Manassas, VA), and the TS1 (IgG1) hybridoma cell line was obtained from the Developmental Studies Hybridoma Bank, Department of Biological Sciences, University of Iowa (Iowa City, IA). Abs were purified from hybridoma supernatants by protein G affinity chromatography using standard methods. mAbs against CD11a (clone HI111; IgG1) and CD11b [clone ICRF44 (44); IgG1] were purchased from PharMingen (San Diego, CA). Hybridoma supernatant containing Ab against CD11c (IgG1) was obtained from Serotec (Oxford, U.K.). The Ab preparations against CD11b and CD11c α-chains were reported by their respective manufacturers to inhibit β2 integrin-mediated adhesion functions. V189 (IgG2a), an anti-human factor V mAb, served as an isotype control for IB4.

Blood was collected by venipuncture into vacutainer tubes (Becton Dickinson, San Jose, CA) containing heparin (100 U.S.P. units), buffered sodium citrate (3.8%), or EDTA (7.5% K3EDTA). For anticoagulation with hirudin (Sigma, St. Louis, MO), blood was collected into a sterile tube containing 50 U/ml hirudin and mixed.

For experiments using purified neutrophils, heparinized blood was mixed with an equal volume of 6% Dextran 70 in 0.9% NaCl (McGaw, Irvine, CA). RBC were allowed to sediment for 45–60 min at room temperature. The supernatant was removed and centrifuged at 400 × g (10 min, 4°C). The pellet was resuspended in 25 ml of ice-cold 0.2% NaCl with mixing for 25 s, followed by 25 ml of ice-cold 1.6% NaCl. The cells were centrifuged and the pellet gently resuspended in 5 ml of HHB buffer (HBSS, 10 mM HEPES, pH 7.5, 1% BSA). This was transferred to a 15-ml conical centrifuge tube and underlayered with 5 ml of Lymphocyte Separation Media (density 1.077 ± 0.001 g/ml at 22°C; Cellgro, Herndon, VA). The sample was centrifuged at 400 × g for 30 min at 4°C. The pellet containing purified neutrophils was resuspended in 5–10 ml HHB buffer. A typical yield was ∼10 × 106 neutrophils per preparation.

Leukocytes were purified from 50 ml of citrated blood by sedimentation through dextrose and hypotonic lysis, as described above. The cell pellet was washed and resuspended in HBSS, 10 mM HEPES (pH 7.5), 3 mM CaCl2, and 0.6 mM MgCl2 and surface biotinylated with 0.5 mg/ml sulfo-NHS-LC-biotin for 30 min at room temperature. The cells were washed, then lysed with 1% Nonidet P-40 in 10 mM HEPES (pH 7.5) and 0.1 mM EDTA, and centrifuged. The supernatant was mixed with 100 μl of sEPCR-AffiGel 10 (Bio-Rad; ∼1 mg sEPCR/ml resin) or Tris-inactivated AffiGel 10 (control) in HHB/CaMg buffer (HHB with 3 mM CaCl2, 0.6 mM MgCl2; total volume 600 μl). The samples were mixed overnight at 4°C, washed five times with buffer (without albumin), and eluted with HBSS, 50 mM HEPES (pH 7.5) and 5 mM EDTA. The samples were centrifuged and the supernatant was analyzed by SDS-PAGE on a 4–20% gradient gel. The samples were transferred to a polyvinylidene difluoride membrane, and the membrane was blocked with a 10% (w/v) nonfat dry milk solution, washed, and incubated with streptavidin-alkaline phosphatase conjugate. Bound conjugate was detected with ECF substrate (Amersham-Pharmacia), and image analysis was performed using a Storm 860 imager (Molecular Dynamics, Sunnyvale, CA).

The procedure was scaled up so that purified neutrophils from 400 ml citrated blood were washed and lysed with 1% Nonidet P-40, 10 mM HEPES (pH 7.5), and 0.1 mM EDTA. The lysate was centrifuged, and the supernatant was diluted 5-fold and adjusted to 3 mM CaCl2, 0.6 mM MgCl2. This was applied to an sEPCR-Affi-Gel 10 affinity column (25 ml of resin coupled at ∼1 mg sEPCR/ml resin) equilibrated in 20 mM Tris-HCl, 0.1 M NaCl, 0.2% Nonidet P-40, 3 mM CaCl2, and 0.6 mM MgCl2 (pH 7.5), with a 10-ml precolumn of Tris-inactivated Affi-Gel 10 to reduce nonspecific interactions. The cell pellet was extracted twice more with lysing buffer, and the supernatants were applied. The column was washed to baseline OD280 and eluted with buffer containing 50 mM HEPES (pH 7.5) and 5 mM EDTA. The eluate fractions were pooled, concentrated, and analyzed by SDS-PAGE on a 10% resolving gel. The band corresponding to the 33-kDa band observed previously was cut out and sent to the Harvard Microchemistry Facility (Cambridge, MA) for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis, trypsin digestion, and amino-terminal sequencing.

Data acquisition by flow cytometry and subsequent analysis was done with a FACScaliber flow cytometer using CellQuest software (Becton Dickinson, San Jose, CA). Neutrophils were gated according to their relative size (forward scatter) and relative granularity (side scatter) properties.

For experiments using blood, 50-μl aliquots were incubated at 37°C for 10 min with 500 nM Oregon Green-sEPCR and 0.1 μg/ml PMA (Sigma). Samples were placed on ice, and RBC were lysed with 1 ml FACS Lysing Solution (Becton Dickinson). After centrifugation, pellets were resuspended in 1 ml of ice-cold HHB/CaMg buffer and centrifuged. The washed pellet was resuspended in ice-cold HHB/CaMg buffer and analyzed by flow cytometry. Purified neutrophils were evaluated for sEPCR binding in a similar fashion, with the exception of the lysing solution.

Heparinized blood (50 μl) was preincubated at room temperature for 15 min with purified Ab (IB4, TS1, PL1, V189, anti-CD11a, anti-CD11b, anti-CD11c), 10 μl of anti-CD11c hybridoma culture supernatant, or 10 μl of nonconditioned media. The final concentration of the purified Abs was 40 μg/ml. Oregon Green-sEPCR (500 nM) was added in the absence or presence of PMA (100 nM), and the samples were incubated at 37°C for 15 min. RBC were removed by hypotonic lysis, the cells were washed, and cell-associated fluorescence was determined by flow cytometry, as described above.

The PR3-ANCA, perinuclear ANCA (P-ANCA), and sEPCR interaction with ethanol-fixed neutrophils was evaluated with a Leica TCS NT confocal system equipped with four-laser excitation, four fluorescent detectors, and a transmitted detector (Heidelberg, Germany). These studies were performed at the Flow and Image Cytometry Laboratory, University of Oklahoma Health Sciences Center, William K. Warren Medical Research Institute (Oklahoma City, OK). Slides with ethanol-fixed neutrophils, P-ANCA, and FITC anti-human IgG were from The Binding Site (Birmingham, U.K.). PR3-ANCA was from the Varelisa Autoimmune Analysis kit (Pharmacia & UpJohn, Kalamazoo, MI). The cells were incubated with the autoantibodies (30 min) and washed with HHB buffer. Bound Ab was detected with FITC anti-human IgG reagent according to the manufacturer’s directions. The slides were washed once in HHB and twice in HHB/CaMg, and then incubated with Cy3-sEPCR (1 μM) in HHB/CaMg buffer (30 min). The slides were washed with HHB/CaMg and mounted with a coverslip and Slow Fade reagent (Molecular Probes). The slides were then visualized for FITC and Cy3 fluorescence.

Microtiter plate wells were coated overnight at 4°C with 4 μg/ml of mAbs against sEPCR (1494, 1500, or HPC4 at 50 μl/well). All subsequent steps were done at room temperature. The wells were washed with wash buffer (20 mM Tris-HCl, pH 7.5, 0.1 M NaCl, 0.05% Tween 20, 3 mM CaCl2, 0.6 mM MgCl2), blocked, washed again, and then incubated with 100 nM sEPCR (50 μl/well) for 30 min. This approach was used to create a binding surface because EPCR tends to be conformationally sensitive and, at least for screening mAbs, immobilization of EPCR directly to plastic surfaces destroys a majority of the available epitopes. After washing, increasing concentrations of biotin-PR3 were added (30 min). The plates were washed, and bound biotin-PR3 was detected with streptavidin-alkaline phosphatase (1 μg/ml, 30 min) and Blue Phos substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD). Color development was stopped with 2.5% EDTA, and the absorbance at 650 nm was read in a Vmax microplate reader (Molecular Devices, Sunnyvale, CA). The background absorbance observed in the absence of coating Ab was subtracted from all samples.

Microtiter plates precoated with human PR3 (The Binding Site) were used to assess the ability of excess unlabeled sEPCR to inhibit binding of biotin-sEPCR to purified PR3. sEPCR (0–5 μM) was added to the PR3-coated wells (30 min, room temperature), followed by 500 nM biotin-sEPCR (30 min). The wells were washed (HHB/CaMg), and bound biotin-sEPCR was detected with streptavidin-alkaline phosphatase and Blue Phos substrate, as described above.

Sera from five patients were kindly provided for this study by Judith James-Wood (Arthritis & Immunology Department, Oklahoma Medical Research Foundation). Four of the patients (P1, P2, P3, and P5) have biopsy-proven Wegener’s granulomatosis, as well as anti-PR3 autoantibodies by commercial ELISA (PR3-ANCA), and were not on aggressive immunosuppressive therapy at the time of collection. The fifth patient (P4) has anti-PR3 autoantibodies, but only eye involvement and not systemic disease, and does not fulfill the criteria for Wegener’s. These samples were used to evaluate the influence of serum containing anti-PR3 autoantibodies on the binding of sEPCR and PR3 in vitro. Normal sera were obtained from three apparently healthy adult volunteers (N1, N2, and N3) using standard venipuncture techniques. IgG was purified for some experiments on protein G columns using standard methods.

Serum samples were diluted 1/200 and 1/1000 and preincubated for 30 min at room temperature on plates precoated with PR3 (The Binding Site). Biotinylated-sEPCR was added (80 nm final), and incubation continued for an additional 30 min. The plates were washed and bound biotin signal detected with streptavidin-HRP and tetramethylbenzidine substrate. The absorbance at 450 nm was read on a Vmax microplate reader. The maximal binding (100%) was the level of bound sEPCR after preincubation of the PR3 wells with buffer (10 mM HEPES, pH 7.4, 0.1 M NaCl, 0.05% Tween 20) instead of serum.

As an initial approach to evaluate the possibility of direct EPCR-neutrophil interactions, sEPCR was labeled with fluorescent Oregon Green and mixed with purified neutrophils, and cell-bound fluorescence was monitored by flow cytometry. As shown in Fig. 1, the fluorescently labeled sEPCR bound to PMA-activated neutrophils. Without activation (no PMA), the cell-associated fluorescence was only slightly higher than the background cell autofluorescence (neutrophils alone). In contrast, the fluorescence associated with the PMA-activated neutrophils (solid black) was at least an order of magnitude higher than the controls.

FIGURE 1.

sEPCR binds to activated neutrophils. Purified neutrophils were incubated with Oregon Green-sEPCR (500 nM) at 37°C for 10 min in the absence (no PMA) or presence (solid black) of 0.1 μg/ml PMA. The autofluorescence of neutrophils alone is indicated.

FIGURE 1.

sEPCR binds to activated neutrophils. Purified neutrophils were incubated with Oregon Green-sEPCR (500 nM) at 37°C for 10 min in the absence (no PMA) or presence (solid black) of 0.1 μg/ml PMA. The autofluorescence of neutrophils alone is indicated.

Close modal

To test whether the above phenomenon could be observed in the presence of all the components of blood, fluorescently labeled sEPCR was added to blood samples anticoagulated with citrate, EDTA, heparin, or hirudin, and binding was evaluated by flow cytometry after lysis of RBC (Fig. 2). Essentially the same results were obtained, and activation of the neutrophils again greatly enhanced sEPCR binding. An EDTA buffer system supported about half the sEPCR binding relative to the others, the difference with citrate probably attributable to the stronger chelating properties of EDTA. These experiments were repeated multiple times with blood from three different donors with essentially identical results.

FIGURE 2.

sEPCR binds to neutrophils activated in blood. Anticoagulated blood (as indicated) was incubated with Oregon Green-sEPCR (500 nM) at 37°C for 10 min in the absence (gray) or presence (black) of 0.1 μg/ml PMA. RBC were lysed, and the remaining washed cells were analyzed for cell-bound fluorescence (MCF) by flow cytometry. Neutrophils were gated according to their forward and side scatter properties.

FIGURE 2.

sEPCR binds to neutrophils activated in blood. Anticoagulated blood (as indicated) was incubated with Oregon Green-sEPCR (500 nM) at 37°C for 10 min in the absence (gray) or presence (black) of 0.1 μg/ml PMA. RBC were lysed, and the remaining washed cells were analyzed for cell-bound fluorescence (MCF) by flow cytometry. Neutrophils were gated according to their forward and side scatter properties.

Close modal

To search for the neutrophil-binding protein(s) on an analytical scale, surface-biotinylated leukocytes were lysed and incubated with sEPCR immobilized on a resin. As a control, an equal volume of lysate was incubated with Tris-inactivated resin (no sEPCR) and treated identically to the affinity resin sample. After extensive washing, the resins were eluted with buffer containing EDTA. The eluates were electrophoresed on 4–20% gradient gels, transferred to a membrane, blocked, and then processed for biotin signal. A single major band was observed at about 33–35 kDa on the gel from the sample incubated with the sEPCR affinity resin (Fig. 3, lane 2). More bands were observed with longer exposure times, but correlated with those in the control sample (lane 1).

FIGURE 3.

sEPCR affinity chromatography. The lysate of surface-biotinylated leukocytes was incubated with sEPCR-affinity resin (lane 2) or Tris-inactivated control resin (lane 1). EDTA eluates were processed for biotin signal by SDS-PAGE, transfer to polyvinylidene difluoride membranes, and incubation with streptavidin-alkaline phosphatase and substrate. A single major band of ∼33 kDa is observed in the eluate from the sEPCR affinity resin.

FIGURE 3.

sEPCR affinity chromatography. The lysate of surface-biotinylated leukocytes was incubated with sEPCR-affinity resin (lane 2) or Tris-inactivated control resin (lane 1). EDTA eluates were processed for biotin signal by SDS-PAGE, transfer to polyvinylidene difluoride membranes, and incubation with streptavidin-alkaline phosphatase and substrate. A single major band of ∼33 kDa is observed in the eluate from the sEPCR affinity resin.

Close modal

The procedure was scaled up, and the corresponding band from the EDTA eluate of an sEPCR-affinity column was cut from the gel and sent to the Harvard Microchemistry Facility for MALDI-MS analysis, trypsin digestion, and microsequencing. From one peptide of the digest, the sequence identified was LVNVVLGAHNVR, which was used in a search of the Swiss-Protein database. The sequence was a unique match to residues of Leu70-Arg81 of PR3 (molecular mass ∼30 kDa), a serine proteinase stored in the secretory vesicles and primary and azurophilic granules of neutrophils. This sequence of PR3 is distinct from the homologous region of neutrophil elastase, azurocidin, or cathepsin G, which are closely related structurally and also stored in azurophilic granules (25).

To test directly whether sEPCR was binding to PR3, in vitro studies were done using purified components. To create a binding surface, microtiter plate wells were coated with mAbs to EPCR, and the Ab surface was then saturated with sEPCR. This approach was used to create a binding surface because direct immobilization of EPCR on plastic surfaces was found to destroy the conformation of EPCR. Purified biotin-PR3 was then added and, in each case, the Ab-sEPCR surface supported biotin-PR3 binding in a saturable manner (Fig. 4,A). These are nonoverlapping Abs that either block protein C/activated protein C binding to EPCR (1494) (17, 26), do not block ligand binding (1500), or bind to the carboxyl-terminal tag (HPC4). None of the anti-sEPCR mAbs screened to date appear to inhibit the sEPCR-PR3 interaction. In addition, increasing concentrations of unlabeled sEPCR decreased biotin-sEPCR binding to PR3-coated wells (Fig. 4 B). Thus, the sEPCR-PR3-binding interaction was saturable and dissociable with excess, unlabeled ligand using purified components.

FIGURE 4.

sEPCR binds to purified PR3. A, Wells coated with anti-sEPCR mAb 1494 (•), 1500 (□), or HPC4 (▵) were blocked and then saturated with sEPCR. Biotin-PR3 was added as indicated, and bound probe was detected with streptavidin-alkaline phosphatase and Blue Phos substrate (absorbance at 650 nm). B, PR3-coated wells were preincubated with increasing concentrations of unlabeled sEPCR. Biotin-sEPCR (500 nM) was added, and bound biotin was detected with streptavidin-alkaline phosphatase and Blue Phos substrate.

FIGURE 4.

sEPCR binds to purified PR3. A, Wells coated with anti-sEPCR mAb 1494 (•), 1500 (□), or HPC4 (▵) were blocked and then saturated with sEPCR. Biotin-PR3 was added as indicated, and bound probe was detected with streptavidin-alkaline phosphatase and Blue Phos substrate (absorbance at 650 nm). B, PR3-coated wells were preincubated with increasing concentrations of unlabeled sEPCR. Biotin-sEPCR (500 nM) was added, and bound biotin was detected with streptavidin-alkaline phosphatase and Blue Phos substrate.

Close modal

ANCA directed against PR3 (PR3-ANCA) are found in patients with some autoimmune vasculitides, including Wegener’s granulomatosis and microscopic polyangiitis. ANCA react with neutrophil granular proteins, and two common ANCA types are distinguished by indirect immunofluorescence on ethanol-fixed neutrophils. One type, P-ANCA, produces an artifactual perinuclear staining and results from Ab binding primarily to myeloperoxidase, and occasionally to other cationic granule proteins, which diffuses during fixation to the negatively charged nucleus. The other type, PR3-ANCA, produces a diffuse, cytoplasmic staining directed toward PR3. The PR3-ANCA pattern is highly specific for Wegener’s granulomatosis, and typically >80% of Wegener’s patients test positive for PR3-ANCA by both immunofluorescence and ELISA against purified PR3 (5, 27, 28).

This technique was used in two-color confocal microscopy colocalization studies with ethanol-fixed neutrophils (Fig. 5). sEPCR was labeled with Cy3, a fluorescent probe that emits a red color. The PR3-ANCA (or P-ANCA) was detected with FITC anti-human IgG that emits a green color. When the two signals overlap, a yellow color is observed. Cy3-sEPCR staining was diffuse and distributed throughout the cytoplasm (Fig. 5, A and E), and the PR3-ANCA produced the typical green cytoplasmic staining (Fig. 5,B). When the images were overlaid, extensive areas of diffuse, yellow color were observed in the cell cytoplasms (Fig. 5,C). In contrast, similar experiments with P-ANCA (Fig. 5,D) and Cy3-sEPCR (Fig. 5,E) did not show colocalization (Fig. 5 F). Thus, the sEPCR localized to neutrophil cytoplasmic sites in parallel with the PR3-ANCA, whose target Ag is PR3.

FIGURE 5.

Coincubation of sEPCR and PR3-ANCA on ethanol-fixed neutrophils. Neutrophils were incubated with PR3-ANCA- or P-ANCA-positive serum, and bound autoantibody was detected with FITC anti-human IgG. The diffuse granular cytoplasmic staining (A) usually corresponds to Ab against PR3. The perinuclear pattern (D) results from Ab against primarily myeloperoxidase. Cy3-sEPCR produced a diffuse cytoplasmic staining (B and E). Incubation with both PR3-ANCA and sEPCR produced extensive areas of yellow in the cytoplasm, indicating colocalization of the fluorescent signals (C). Little overlap of signals was observed when P-ANCA and sEPCR were incubated with the cells (F).

FIGURE 5.

Coincubation of sEPCR and PR3-ANCA on ethanol-fixed neutrophils. Neutrophils were incubated with PR3-ANCA- or P-ANCA-positive serum, and bound autoantibody was detected with FITC anti-human IgG. The diffuse granular cytoplasmic staining (A) usually corresponds to Ab against PR3. The perinuclear pattern (D) results from Ab against primarily myeloperoxidase. Cy3-sEPCR produced a diffuse cytoplasmic staining (B and E). Incubation with both PR3-ANCA and sEPCR produced extensive areas of yellow in the cytoplasm, indicating colocalization of the fluorescent signals (C). Little overlap of signals was observed when P-ANCA and sEPCR were incubated with the cells (F).

Close modal

Since the PR3-ANCA and sEPCR colocalized on the cells, the effect of patient sera containing PR3-ANCA on sEPCR binding to purified PR3 was tested (Fig. 6). The PR3 was immobilized on the surface of microtiter plate wells and preincubated with buffer (100%), normal serum samples (N1, N2, and N3), or patient sera containing PR3-ANCA (P1-P5). Although the normal serum samples did not affect sEPCR binding to PR3, preincubation with three of the patient sera diluted 1/200 resulted in reduced sEPCR binding (P2, P3, and P4). Further dilution of these sera to 1/1000 resulted in sEPCR binding similar to the normal samples. Incubation with the P1 and P5 sera did not appreciably alter sEPCR binding to the PR3-coated wells. The relative ability to reduce sEPCR binding did not correlate with the PR3-ANCA titer of the patient samples (data not shown) and is more likely related to the PR3 epitope specificity of the autoantibodies. A similar result was observed using confocal microscopy in which pretreatment of the ethanol-fixed neutrophils with PR3-ANCA, but not P-ANCA (positive and negative control sera with the commercial PR3 ELISA), reduced the signal observed from Oregon Green-labeled sEPCR binding by ∼50%, relative to untreated cells (data not shown).

FIGURE 6.

PR3-ANCA inhibits the sEPCR-PR3 interaction. PR3-coated wells were pretreated for 30 min with normal human serum (N1, N2, and N3) or PR3-ANCA-positive serum from patients (P1-P5) at dilutions of 1/200 (black) and 1/1000 (white). Biotin-sEPCR (80 nM) was added, and bound biotin signal was detected after 30 min with streptavidin-HRP and substrate. The OD at 450 nm was read. Maximal binding (100%) refers to bound biotin-sEPCR in buffer-treated wells.

FIGURE 6.

PR3-ANCA inhibits the sEPCR-PR3 interaction. PR3-coated wells were pretreated for 30 min with normal human serum (N1, N2, and N3) or PR3-ANCA-positive serum from patients (P1-P5) at dilutions of 1/200 (black) and 1/1000 (white). Biotin-sEPCR (80 nM) was added, and bound biotin signal was detected after 30 min with streptavidin-HRP and substrate. The OD at 450 nm was read. Maximal binding (100%) refers to bound biotin-sEPCR in buffer-treated wells.

Close modal

Other potential modulators of this system were tested as well, including the known EPCR ligands, protein C, and activated protein C. Our preliminary data indicate that neither protein C, activated protein C, nor α1-antitrypsin (a natural inhibitor of PR3 protease activity) significantly alters the sEPCR-PR3 interaction (data not shown).

The ability of serum containing PR3-ANCA to reduce sEPCR binding to PR3 raised the possibility that sEPCR may modulate the protein-protein interactions in this system. Although it is generally accepted that PR3-ANCA binds to neutrophil PR3, it is less widely appreciated that accessory molecules may be involved as well. A previous study indicated that elastase and azurocidin, and possibly PR3, are ligands for the β2 integrin, Mac-1 (CD11a/CD18), on the neutrophil surface (29). Mac-1 is a member of the β2 integrin family, consisting of LFA-1 (CD11a/CD18), Mac-1 (CD11b/CD18), p150,95 (CD11c/CD18), and a newly described fourth member (αd/CD18) (30). These are heterodimeric complexes with a common β-chain (CD18) and unique α-chains (CD11a, b, c, and αd), and are involved in neutrophil adhesion and signaling (30, 31, 32).

These observations raised the possibility that sEPCR may be binding to a preassembled PR3-β2 integrin complex on the cell surfaces. To address this, sEPCR binding to neutrophils activated in whole blood was evaluated in the presence of buffer or mAbs to the β2 integrins (Fig. 7). IB4 Ab (IgG2a) binds to the common β-chain of the β2 integrin family (CD18) and blocks β2 integrin-mediated adhesion (33). This Ab reduced sEPCR binding to PMA-activated neutrophils by 81% (p < 0.0001). Interestingly, TS-1 Ab (IgG1) had either no effect, or slightly increased, sEPCR binding to the cells. TS-1 also binds to CD18, but to a different epitope, and does not inhibit neutrophil adhesion to matrix proteins. As controls, PL-1 (IgG1), which binds to neutrophil P-selectin glycoprotein ligand-1 (34), had no effect on sEPCR binding, nor did an irrelevant isotype-matched Ab (V189; IgG2a). Essentially identical results were observed when IB4 was added to purified neutrophils and sEPCR binding to PMA-activated neutrophils was determined (data not shown).

FIGURE 7.

Mac-1 (CD11b/CD18) supports sEPCR binding to neutrophils. Heparinized blood (50 μl) was incubated with buffer (no mAb), 40 μg/ml Abs (IB4, TS1, PL1, anti-CD11a, anti-CD11b, isotype controls), or 10 μl anti-CD11c hybridoma supernatant. Oregon Green-sEPCR was added (500 nM) in the absence or presence of activation with PMA at 37°C for 15 min. RBC were removed by hypotonic lysis, and binding of fluorescent sEPCR to the washed cells was analyzed by flow cytometry. The IB4 Ab against CD18 and the anti-CD11b Ab significantly inhibited sEPCR binding to activated neutrophils. ∗, p < 0.0001 compared with no mAb or nonconditioned media controls (control for anti-CD11c).

FIGURE 7.

Mac-1 (CD11b/CD18) supports sEPCR binding to neutrophils. Heparinized blood (50 μl) was incubated with buffer (no mAb), 40 μg/ml Abs (IB4, TS1, PL1, anti-CD11a, anti-CD11b, isotype controls), or 10 μl anti-CD11c hybridoma supernatant. Oregon Green-sEPCR was added (500 nM) in the absence or presence of activation with PMA at 37°C for 15 min. RBC were removed by hypotonic lysis, and binding of fluorescent sEPCR to the washed cells was analyzed by flow cytometry. The IB4 Ab against CD18 and the anti-CD11b Ab significantly inhibited sEPCR binding to activated neutrophils. ∗, p < 0.0001 compared with no mAb or nonconditioned media controls (control for anti-CD11c).

Close modal

To address which β2 integrin supports sEPCR binding, the neutrophils were preincubated with mAbs against CD11a, CD11b, or hybridoma culture supernatant containing Ab against CD11c. Only the Ab against CD11b inhibited sEPCR binding to the activated neutrophils (p < 0.0001) (Fig. 7). This indicates that at least Mac-1 is involved in the neutrophil receptor complex that supports sEPCR binding, probably as a PR3/Mac-1 heterocomplex.

The question then arose as to whether IB4, the anti-CD18-blocking Ab, could alter PR3-ANCA IgG binding to activated neutrophils. Neutrophils were preincubated with IB4 (10 μg/ml) in the absence or presence of each patient PR3-ANCA IgG (100 μg/ml) and activated with cytochalasin B/fMLP, and bound Ab was detected with fluorescein-anti-human IgG by flow cytometry. There was no difference in the amount of patient IgG bound in the presence of IB4 Ab (data not shown). The simplest interpretation is that the ability of IB4 to block sEPCR binding to the cells (and presumably to PR3) is a steric effect, rather than a direct inhibition of a ligand interaction. Interestingly, IgG from P1 and P5, which did not reduce sEPCR binding to activated neutrophils, also bound very little to the activated neutrophils relative to the other three autoantibodies.

The current data demonstrate that sEPCR binds to activated neutrophils, and PR3, the Wegener’s autoantigen, was identified as a component of the binding site based on sEPCR affinity chromatography results and in vitro binding studies using the purified enzyme. Furthermore, the β2 integrin Mac-1 was identified as contributing to sEPCR binding to the activated neutrophils. These observations, in combination with an earlier study indicating that PR3 forms heterocomplexes with Mac-1 on neutrophils (29), led us to our working model in which sEPCR binds to PR3 and PR3/Mac-1 complexes on activated neutrophils.

Although a causative role for the PR3-ANCA autoantibodies in the etiology of autoimmune vasculitis remains controversial, many in vitro studies have demonstrated that they are capable of propagating an inflammatory response by cross-linking Fcγ receptors as well as by PR3 binding to F(ab′)2 regions (35, 36, 37, 38, 39, 40). In Wegener’s patients, PR3 is expressed on the surface of circulating neutrophils (41, 42), and PR3-ANCA binding to PR3 on the surface of TNF-α-primed neutrophils sets off a full-blown activation response, including degranulation and production of oxygen radicals (35). However, mechanisms that may modulate this process of vasculitis are poorly understood. The current study suggests that sEPCR may modulate the ability of PR3-ANCA to bind to PR3 on neutrophils, potentially regulating the inflammatory response and vasculitic injury. However, this potential regulatory role is speculative and it must be recognized that there is no in vivo data defining a pathogenic role for ANCA, and the possibility remains that the presence of ANCA in the vasculitis patients is an epiphenomenona.

One prerequisite for the current model of vascular injury is local accumulation of primed neutrophils that will eventually adhere to the endothelium to provide a local source of inflammatory mediators. The β2 integrins, which include Mac-1 (CD11b/CD18), are expressed on granulocytes and monocytes and are involved in cell-cell adhesion and signaling events. Integrin expression is inducible, and circulating leukocytes from patients with active Wegener’s granulomatosis show significantly increased surface expression of Mac-1 that declines to normal levels upon treatment (43). Expression of two other β2 integrins (LFA-1 and p150,95) was normal in the Wegener’s patients, as was Mac-1 expression on cells from patients with other systemic diseases (systemic lupus erythematosus, myeloperoxidase-positive systemic vasculitis, sepsis). An additional study found that CD18 on TNF-α-primed neutrophils was required for the respiratory burst activation induced by an anti-PR3 mAb (40). These observations are consistent with the current data demonstrating Mac-1 participation in the sEPCR binding site on neutrophils, possibly as a PR3/Mac-1 heterocomplex (29). A recent study further demonstrated that PR3-ANCA was capable of a transient activation of rolling neutrophils under flow conditions with resultant firm adhesion to a platelet selectin surface (44). This activation and adhesion was Mac-1 mediated and dependent on interaction of the ANCA with Fcγ receptors. One prediction from our working model is that sEPCR may modulate Mac-1 integrin-mediated events, such as outside-in signaling or adhesion, and these studies are in progress. Based on current models, neutrophils play a role in the early stages of vasculitic injury, and the later fulminant lesions typically consist of lymphocytes and macrophages.

The current studies demonstrating Mac-1 participation in the binding complex for sEPCR are uniquely limited by the epitope specificity of the Abs used. Mac-1 has a diverse ligand repertoire (ICAM-1, fibrinogen, complement iC3b, coagulation factor X, neutrophil elastase, azurocidin, PR3), and there is no particular reason to believe that PR3 has a singular preference for binding to only one β2 integrin. It is quite possible that the epitopes of the Abs against CD11a and CD11c may not have overlapped the ligand binding site. Additional studies with other Abs are ongoing to further evaluate LFA-1 or p150,95 as potential partners in the sEPCR-binding complex. Participation of Mac-1 in the sEPCR-binding complex does provide insight into the partial metal dependence observed for sEPCR binding to the activated neutrophils since β2 integrin function is metal ion dependent (45).

The current studies were done with sEPCR, raising the question of whether EPCR anchored in the endothelial membrane shares this ability to bind PR3. In this regard, EPCR mRNA levels increase rapidly in response to endotoxin challenge in a rat model of septic shock (22). Although the tissue EPCR Ag levels do not rise appreciably, there is a substantial increase in sEPCR levels in the plasma, suggesting a regulated pathway for EPCR synthesis and sEPCR release, possibly through thrombin stimulation of the endothelium and subsequent metalloproteinase activity (46). This concept is supported by observations that significantly elevated levels of circulating soluble EPCR are observed in patients with sepsis or systemic lupus erythematosus (21). Regulated release of soluble EPCR may play a role in Wegener’s patients in whom selective microvascular beds in the nose, sinuses, lungs, and kidneys are the initial targets of the vasculitis (7), sites in which membrane-bound EPCR expression is relatively poor (47). However, unlike its membrane-bound parent, soluble EPCR is not restricted to a particular vascular bed and could be recruited for binding to neutrophils.

These observations represent novel additions toward understanding the fundamental mechanisms of inflammation and the molecular basis for the vasculitis in Wegener’s granulomatosis and related vasculitides. Additional studies are required to further characterize the effect of PR3-ANCA and other modulators on the pair-wise interactions between PR3, soluble EPCR, and CD11b/CD18 on activated neutrophils.

We thank Dr. Rodger McEver (University of Oklahoma), for support and helpful discussions. We gratefully acknowledge the assistance of Jim Henthorn with the confocal microscopy and image analysis and Noriko Hidari and Kandice Swindle for technical assistance.

1

This research was supported by a scientist development grant from the American Heart Association (to S.K.), an Atorvastation Research Award (to S.K.), and grants from the National Heart, Lung, and Blood Institute of the National Institutes of Health (Grant R01 HL64787-01 to S.K.; Grants PO1 HL54804 and R37 HL30340 to C.T.E.). C.T.E. is an Investigator of the Howard Hughes Medical Institute.

4

Abbreviations used in this paper: PR3, proteinase-3; ANCA, anti-neutrophil cytoplasmic Ab; EPCR, endothelial protein C receptor; P-ANCA, perinuclear ANCA; sEPCR, recombinant soluble EPCR.

1
Jenne, D. E., J. Tschopp, J. Ludemann, B. Utecht, W. L. Gross.
1990
. Wegener’s autoantigen decoded.
Nature
346
:
520
2
Labbaye, C., P. Musette, Y. E. Cayre.
1991
. Wegener autoantigen and myeloblastin are encoded by a single mRNA.
Proc. Natl. Acad. Sci. USA
88
:
9253
3
Csernok, E., M. Ernst, W. Schmitt, D. F. Bainton, W. L. Gross.
1994
. Activated neutrophils express proteinase 3 on their plasma membrane in vitro and in vivo.
Clin. Exp. Immunol.
95
:
244
4
Witko-Sarsat, V., E. M. Cramer, C. Hieblot, J. Guichard, P. Nusbaum, S. Lopez, P. Lesavre, L. Halbwachs-Mecarelli.
1999
. Presence of proteinase 3 in secretory vesicles: evidence of a novel, highly mobilizable intracellular pool distinct from azurophil granules.
Blood
94
:
2487
5
Bajema, I. M., E. C. Hagen, F. J. van der Woude, J. A. Bruijn.
1997
. Wegener’s granulomatosis: a meta-analysis of 349 literary case reports.
J. Lab. Clin. Med.
129
:
17
6
Hoffman, G. S. 1998. Wegener’s granulomatosis. In Rheumatology. J. Klippel and P. Dieppe, eds. Mosby International, London, pp. 22.1–22.10.
7
Jennette, J. C..
1998
. Renal involvement in systemic vasculitis. J. C. Jennette, and J. L. Olson, and M. M. Schwartz, and F. G. Silva, eds.
Heptinstall’s Pathology of the Kidney
1059
-1095. Lippincott-Raven Publishers, Philadelphia.
8
Rao, N. V., N. G. Wehner, B. C. Marshall, W. R. Gray, B. H. Gray, J. R. Hoidal.
1991
. Characterization of proteinase-3 (PR-3), a neutrophil serine proteinase: structural and functional properties.
J. Biol. Chem.
266
:
9540
9
Bories, D., M. C. Raynal, D. H. Solomon, Z. Darzynkiewicz, Y. E. Cayre.
1989
. Down-regulation of a serine protease, myeloblastin, causes growth arrest and differentiation of promyelocytic leukemia cells.
Cell
59
:
959
10
Skold, S., B. Rosberg, U. Gullberg, T. Olofsson.
1999
. A secreted proform of neutrophil proteinase 3 regulates the proliferation of granulopoietic progenitor cells.
Blood
93
:
849
11
Renesto, P., L. Halbwachs-Mecarelli, P. Nusbaum, P. Lesavre, M. Chignard.
1994
. Proteinase 3: a neutrophil proteinase with activity on platelets.
J. Immunol.
152
:
4612
12
Campanelli, D., P. A. Detmers, C. F. Nathan, J. E. Gabay.
1998
. Azurocidin and a homologous serine protease from neutrophils: differential antimicrobial and proteolytic properties.
J. Clin. Invest.
85
:
904
13
Fujinaga, M., M. M. Chernaia, R. Halenbeck, K. Koths, M. N. James.
1996
. The crystal structure of PR3, a neutrophil serine proteinase antigen of Wegener’s granulomatosis antibodies.
J. Mol. Biol.
261
:
267
14
Coeshott, C., C. Ohnemus, A. Pilyavskaya, S. Ross, M. Wieczorek, H. Kroona, A. H. Leimer, J. Cheronis.
1999
. Converting enzyme-independent release of tumor necrosis factor α and IL-1β from a stimulated human monocytic cell line in the presence of activated neutrophils or purified proteinase 3.
Proc. Natl. Acad. Sci. USA
96
:
6261
15
Robache-Gallea, S., V. Morand, J. M. Bruneau, B. Schoot, E. Tagat, E. Realo, S. Chouaib, S. Roman-Roman.
1995
. In vitro processing of human tumor necrosis factor-α.
J. Biol. Chem.
270
:
23688
16
Taylor, F. B., Jr, D. J. Stearns-Kurosawa, S. Kurosawa, G. Ferrell, A. Chang, Z. Laszik, G. Peer, C. T. Esmon.
2000
. The endothelial protein C receptor aids in host defense against E. coli sepsis.
Blood
95
:
1680
17
Stearns-Kurosawa, D. J., S. Kurosawa, J. S. Mollica, G. L. Ferrell, C. T. Esmon.
1996
. The endothelial cell protein C receptor augments protein C activation by the thrombin-thrombomodulin complex.
Proc. Natl. Acad. Sci. USA
93
:
10212
18
Fijnvandraat, K., B. Derkx, M. Peters, R. Bijlmer, A. Sturk, M. H. Prins, S. J. H. van Deventer, J. Wouter ten Cate.
1995
. Coagulation activation and tissue necrosis in meningococcal septic shock: severely reduced protein C levels predict a high mortality.
Thromb. Haemostasis
73
:
15
19
Smith, O. P., B. White.
1999
. Infectious pupura fulminans: diagnosis and treatment.
Br. J. Haematol.
104
:
202
20
Kurosawa, S., D. J. Stearns-Kurosawa, N. Hidari, C. T. Esmon.
1997
. Identification of functional endothelial protein C receptor in human plasma.
J. Clin. Invest.
100
:
411
21
Kurosawa, S., D. J. Stearns-Kurosawa, C. W. Carson, A. D’Angelo, P. Della Valle, C. T. Esmon.
1998
. Plasma levels of endothelial cell protein C receptor are elevated in patients with sepsis and systemic lupus erythematosus: lack of correlation with thrombomodulin suggests involvement of different pathological processes.
Blood
91
:
725
22
Gu, J.-M., Y. Katsuura, G. L. Ferrell, P. Grammas, C. T. Esmon.
1999
. Endotoxin and thrombin elevate rodent endothelial cell protein C receptor mRNA levels and increase receptor shedding in vivo.
Blood
95
:
1687
23
Fukudome, K., S. Kurosawa, D. J. Stearns-Kurosawa, X. He, A. R. Rezaie, C. T. Esmon.
1996
. The endothelial cell protein C receptor: cell surface expression and direct ligand binding by the soluble receptor.
J. Biol. Chem.
271
:
17491
24
Rezaie, A. R., M. M. Fiore, P. F. Neuenschwander, C. T. Esmon, J. H. Morrissey.
1992
. Expression and purification of a soluble tissue factor fusion protein with an epitope for an unusual calcium-dependent antibody.
Protein Expression Purif.
3
:
453
25
Jenne, D. E..
1994
. Structure of the azurocidin, proteinase 3, and neutrophil elastase genes: implications for inflammation and vasculitis.
Am. J. Respir. Crit. Care Med.
150
:
S147
26
Fukudome, K., C. T. Esmon.
1994
. Identification, cloning and regulation of a novel endothelial cell protein C/activated protein C receptor.
J. Biol. Chem.
269
:
26486
27
Schultz, D. R., E. C. Tozman.
1995
. Antineutrophil cytoplasmic antibodies: major autoantigens, pathophysiology, and disease associations.
Semin. Arthritis Rheum.
25
:
143
28
Wong, R. C., R. A. Silvestrini, J. A. Savige, D. A. Fulcher, E. M. Benson.
1999
. Diagnostic value of classical and atypical antineutrophil cytoplasmic antibody (ANCA) immunofluorescence patterns.
J. Clin. Pathol.
52
:
124
29
Cai, T. Q., S. D. Wright.
1996
. Human leukocyte elastase is an endogenous ligand for the integrin CR3 (CD11b/CD18, Mac-1, αM β2) and modulates polymorphonuclear leukocyte adhesion.
J. Exp. Med.
184
:
1213
30
Carlos, T. M., J. M. Harlan.
1994
. Leukocyte-endothelial adhesion molecules.
Blood
84
:
2068
31
Clark, E. A., J. S. Brugge.
1995
. Integrins and signal transduction pathways: the road taken.
Science
268
:
233
32
Van der Vieren, M., H. Le Trong, C. L. Wood, P. F. Moore, T. St. John, and D. E. G. W. M. Staunton. 1995. A novel leukointegrin, α-d β-2, binds preferentially to ICAM-3. Immunity 683.
33
Wright, S. D., P. E. Rao, W. C. Van Voorhis, L. S. Craigmyle, K. Iida, M. A. Talle, E. F. Westberg, G. Goldstein, S. C. Silverstein.
1983
. Identification of the C3bi receptor of human monocytes and macrophages by using monoclonal antibodies.
Proc. Natl. Acad. Sci. USA
80
:
5699
34
Moore, K. L., K. D. Patel, R. E. Bruehl, F. Li, D. A. Johnson, H. S. Lichenstein, R. D. Cummings, D. F. Bainton, R. P. McEver.
1995
. P-selectin glycoprotein ligand-1 mediates rolling of human neutrophils on P-selectin.
J. Cell Biol.
128
:
661
35
Falk, R. J., R. S. Terrell, L. A. Charles, J. C. Jennette.
1990
. Anti-neutrophil cytoplasmic autoantibodies induce neutrophils to degranulate and produce oxygen radicals in vitro.
Proc. Natl. Acad. Sci. USA
87
:
4115
36
Kettritz, R., J. C. Jennette, R. J. Falk.
1997
. Crosslinking of ANCA-antigens stimulates superoxide release by human neutrophils.
J. Am. Soc. Nephrol.
8
:
386
37
Ralston, D. R., C. B. Marsh, M. P. Lowe, M. D. Wewers.
1997
. Antineutrophil cytoplasmic antibodies induce monocyte IL-8 release: role of surface proteinase-3, α1-antitrypsin, and Fcγ receptors.
J. Clin. Invest.
100
:
1416
38
Tomer, Y., O. Lider, B. Gilburd, R. Hershkoviz, P. L. Meroni, A. Wiik, Y. Shoenfeld.
1997
. Anti-neutrophil cytoplasmic antibody-enriched IgG induces adhesion of human T lymphocytes to extracellular matrix proteins.
Clin. Immunol. Immunopathol.
83
:
245
39
Mayet, W. J., Z. B. K. Meyer.
1993
. Antibodies to proteinase 3 increase adhesion of neutrophils to human endothelial cells.
Clin. Exp. Immunol.
94
:
440
40
Reumaux, D., P. J. Vossebeld, D. Roos, A. J. Verhoeven.
1995
. Effect of tumor necrosis factor-induced integrin activation on Fcγ receptor II-mediated signal transduction: relevance for activation of neutrophils by anti-proteinase 3 or anti-myeloperoxidase antibodies.
Blood
86
:
3189
41
Csernok, E., W. H. Schmitt, M. Ernst, D. F. Bainton, W. L. Gross.
1993
. Membrane surface proteinase 3 expression and intracytoplasmic immunoglobulin on neutrophils from patients with ANCA-associated vasculitides.
Adv. Exp. Med. Biol.
336
:
45
42
Muller Kobold, A. C., C. G. Kallenberg, J. W. Tervaert.
1998
. Leukocyte membrane expression of proteinase 3 correlates with disease activity in patients with Wegener’s granulomatosis.
Br. J. Rheumatol.
37
:
901
43
Haller, H., J. Eichhorn, K. Pieper, U. Gobel, F. C. Luft.
1996
. Circulating leukocyte integrin expression in Wegener’s granulomatosis.
J. Am. Soc. Nephrol.
7
:
40
44
Radford, D. J., C. O. Savage, G. B. Nash.
2000
. Treatment of rolling neutrophils with antineutrophil cytoplasmic antibodies causes conversion to firm integrin-mediated adhesion.
Arthritis Rheum.
43
:
1337
45
Bohnsack, J. F., X. N. Zhou.
1992
. Divalent cation substitution reveals CD18- and very late antigen-dependent pathways that mediate human neutrophil adherence to fibronectin.
J. Immunol.
149
:
1340
46
Xu, J., D. Qu, N. L. Esmon, C. T. Esmon.
2000
. Metalloproteolytic release of endothelial protein C receptor.
J. Biol. Chem.
275
:
6038
47
Laszik, Z., A. Mitro, F. B. Taylor, Jr, G. Ferrell, C. T. Esmon.
1997
. Human protein C receptor is present primarily on endothelium of large blood vessels: implications for the control of the protein C pathway.
Circulation
96
:
3633