The host injury involved in multiorgan system failure during severe inflammation is mediated, in part, by massive infiltration and sequestration of hyperactive neutrophils in the visceral organ. A recombinant form of human activated protein C (rhAPC) has shown cytoprotective and anti-inflammatory functions in some clinical and animal studies, but the direct mechanism is not fully understood. Recently, we reported that, during endotoxemia and severe polymicrobial peritonitis, integrin VLA-3 (CD49c/CD29) is specifically upregulated on hyperinflammatory neutrophils and that targeting the VLA-3high neutrophil subpopulation improved survival in mice. In this article, we report that rhAPC binds to human neutrophils via integrin VLA-3 (CD49c/CD29) with a higher affinity compared with other Arg-Gly-Asp binding integrins. Similarly, there is preferential binding of activated protein C (PC) to Gr1highCD11bhighVLA-3high cells isolated from the bone marrow of septic mice. Furthermore, specific binding of rhAPC to human neutrophils via VLA-3 was inhibited by an antagonistic peptide (LXY2). In addition, genetically modified mutant activated PC, with a high affinity for VLA-3, shows significantly improved binding to neutrophils compared with wild-type activated PC and significantly reduced neutrophil infiltration into the lungs of septic mice. These data indicate that variants of activated PC have a stronger affinity for integrin VLA-3, which reveals novel therapeutic possibilities.

Neutrophils play a critical role in the host defense system by participating in the phagocytosis and killing of infectious organisms with powerful antimicrobial substances (1). During a severe inflammatory response caused by an infection, the activation of neutrophils with proinflammatory cytokines and microbial components induces reduced apoptosis, altered chemotaxis, and excessive infiltration into the visceral organs, leading to collateral tissue injury (1). Integrins are heterodimeric, transmembrane proteins that mediate adhesion and signaling between neutrophils and the extracellular matrix proteins, and facilitate their intravascular and interstitial migration. Recently, we showed that during endotoxemia and severe polymicrobial peritonitis, there is significant upregulation of integrin VLA-3 (CD49c/CD29) on the surface of hyperactive neutrophils both in mice and patients (2). Gr1highCD11bhighVLA-3high neutrophils, isolated from the bone marrow (BM) of septic mice, produce significantly higher amounts of proinflammatory cytokines and show elevated myeloperoxidase functions compared with those of the Gr1highCD11bhighVLA-3low subset. We further demonstrated that deletion of integrin VLA-3, specifically from the neutrophils, significantly improved the survival of septic animals.

Activated protein C (PC), a vitamin K–dependent serine protease with strong anticoagulant functions, is derived from the thrombin-mediated cleavage of circulating PC. In addition to its natural anticoagulant functions, activated PC also possesses cytoprotective and anti-inflammatory activities, which include protecting the endothelial barrier, inhibiting cell apoptosis, reducing secretion of proinflammatory mediators, and inhibiting leukocyte migration (3). Although the antiapoptotic and endothelial barrier functions of activated PC require the activation of the endothelial PC receptor (EPCR)–dependent protease-activated receptor-1 (PAR-1), its anti-inflammatory effects are mediated by both EPCR-PAR-1–dependent and –independent pathways and may involve cell adhesion receptors, such as integrins (3).

Data from Sturn et al. (4) and Nick et al. (5) showed that neutrophils express receptors for activated PC, and that neutrophil chemotaxis is inhibited by exposure to recombinant form of human activated PC (rhAPC). We also showed that rhAPC blocks integrins and inhibits neutrophil adhesion and migration on extracellular matrix proteins (6). In this study, we report that, among the leukocyte integrins, VLA-3 (α3β1; CD49c/CD29), which is a unique cell surface marker for the hyperinflammatory neutrophil subpopulation arising during severe systemic inflammation, is a novel high-affinity cellular receptor for rhAPC. Consistently, rhAPC preferentially binds to the VLA-3high, a proinflammatory neutrophil population isolated from inflamed mice. The binding of rhAPC to human neutrophils was significantly reduced by a VLA-3 antagonistic peptide. Finally, we describe a genetic modification approach using a yeast surface display system to develop recombinant activated PCs with high affinity for VLA-3 as potential therapeutic candidates to treat severe inflammation with a higher selectivity and an improved potency.

Endotoxemia and cecal ligation and puncture (CLP) were performed according to the Animal Resource Protocol approved by the Committee at University of Rochester. For the endotoxemia assay, 8- to 12-wk-old C57BL/6 (Harlan) male mice were weighed, and LPS (Escherichia coli O55:B; Sigma-Aldrich) was administered by an i.p. injection to achieve a LD90 mortality rate. The CLP survival surgery was performed under isoflurane inhalation anesthesia. The cecum was ligated and punctured through and through with a 21-gauge needle. All the mice were resuscitated with 1 ml of lactated Ringers injected s.c. Activated PC was administered via the tail vein (10 μg) at 1 h and via retro orbital venous plexus at 4, 24, and 72 h (5 μg) after the CLP surgery. Survival was monitored for 120 h. For the analysis of neutrophil migration into the inflamed lungs at 12 h after endotoxemia, wild-type (WT) activated PC and R177G–activated PC were administered at 1 (via tail vein) and 4 h (via retro orbital venous plexus) after the LPS injection.

Blood was collected from healthy volunteers via antecubital vein puncture in heparin-containing vacutainers. The granulocytes and erythrocytes were separated from the whole blood by centrifugation through a one-step polymorphs (Fresenius Kabi Norge AS) density gradient. The remaining erythrocytes were removed by hypotonic lysis, yielding a neutrophil purity of >98%. The Human Research Studies Review Board of the University of Rochester approved this study, and informed consent was obtained in accordance with the Declaration of Helsinki. The neutrophils were stimulated with PMA (20 ng/ml), TNF-α (20 ng/ml), LPS (100 μg/ml), or fMLF (1 μM) for 1 or 3 h in L15 (Leibovitz) medium with glucose at 37°C.

The soluble integrin binding assay was performed using purified soluble human α3β1, α5β1, and αVβ3 (United States Biological, Swampscott, MA); 1 μg/ml of the soluble integrins was mixed with an mAb against the β1 or β3 subunit (mAb TS2/6 for β1 and mAb D3 for β3 integrins; kind gift from L. Jennings, University of Tennessee) plus rhAPC in the presence of 1 mM Mn2+ in L15 medium for 1 h at room temperature. The protein complexes were precipitated with protein A/G and were then subjected to SDS-PAGE and a Western blot with a rat anti-human activated PC Ab (Cell Science, Norwood, MA) or silver staining.

For the activated PC binding inhibition assay, the neutrophils were isolated from healthy donors as described earlier and were incubated with 0, 0.1, 1, and 10 μg activated PC along with blocking Abs for integrin α3, α5, αv, or αM (Millipore, Billerica, MA) in 1 ml of L15 medium for 30 min at 4°C. After washing, the cells were fixed with 3.7% formaldehyde for 10 min at room temperature. After fixing, the cells were stained with rat anti–activated PC primary Ab and a PE-labeled goat anti-rat (BioLegend, San Diego, CA) secondary Ab for 30 min at 4°C in the dark and were then washed and resuspended in PBS for flow cytometry analysis. All of the samples were collected on a FACSCalibur flow cytometer (BD Biosciences, San Diego, CA), and the data were analyzed using FlowJo software.

For the flow cytometry measurement of the expression integrins on the neutrophils, the BM, peritoneal lavage (PL), peripheral blood, and lungs were isolated from naive and septic mice at the indicated time points; subsequently, single-cell suspensions were prepared. An RBC lysis was performed using ACK lysing buffer (Invitrogen, San Diego, CA). The Fc receptors were blocked with unconjugated anti-CD16/32 (eBioscience, San Diego, CA) for 30 min. Samples were stained with Alexa Fluor 488–labeled anti-Gr1 (Life Technologies, Camarillo, CA). Allophycocyanin-labeled Ly6G (BD Biosciences), PerCp-Cy5.5–labeled anti-CD11b (eBioscience), FITC anti-F4/80 (eBioscience), purified goat anti-mouse integrin α3/CD49c (R&D Systems), and PE-conjugated donkey anti-goat IgG (Santa Cruz) Abs were used. For the EPCR surface expression studies, the samples were blocked with anti-CD16/32 and stained with FITC–anti-7/4 (AbD Serotec), PerCP–Cy5.5–anti-Ly6G (BD Biosciences), goat anti-mouse integrin α3/CD49c (R&D Systems), PE-conjugated donkey anti-goat IgG (Santa Cruz), and activated PC–EPCR (eBioscience). For comparing the WT activated PC R177G–activated PC, the lung cells were stained with propidium iodide, Brilliant violet anti-Ly6G (BioLegend), PerCp/Cy5.5 anti-CD11b (BioLegend), and anti–VLA-3 Ab (R&D Systems) as described earlier. For the ex vivo activated PC binding experiments, anti-human allophycocyanin mAb (Hycult) was repurified to remove BSA using the AbSelect purification kit (Innova Biosciences) and was directly conjugated to allophycocyanin using the Lightning-Link labeling kit (Innova Biosciences). The BM cells were blocked with anti-CD16/32 and were then incubated with 100 μg/ml human allophycocyanin (Xigris; Eli Lilly) for 1 h on ice in staining buffer (HBBS supplemented with 1.5 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, and 3% FBS) with 1 mM MnCl2 to achieve integrin activation and were stained with FITC–anti-7/4 (AbD Serotec), PerCP-Cy5.5–anti-Ly6G (BD Biosciences), goat anti-mouse integrin α3/CD49c (R&D Systems), PE-conjugated donkey anti-goat IgG (Santa Cruz), and allophycocyanin–anti-human allophycocyanin conjugated as described earlier. All of the samples were fixed with 3.7% formaldehyde and were collected on a FACSCalibur flow cytometer (BD Biosciences). The data were analyzed using FlowJo software.

Human PC (coding for Gla-domain, L chain, and H chain) was used as a template for the following PCR. To randomize three residues, Lys146, Arg147, and Arg177, we applied a two-step overlap PCR. The outer-forward primer (5′-GGT GGT GGT GGT TCT GGT GGT GGT GGT TCT GGT GGT GGT GGT TCT GCT AGC-3′) and the focus-reverse primer (5′-ATC TAC TTG GTC TTC TTG GTC TTC TGT GTC TCG TTT CAG GTG ACT GCG CTT CTT CTC CAT SNN SNN CCA GGG CCT CCC ACA AGG GAA-3′) were used to randomize Lys147 and Arg148. The focus-forward primer (5′-AAA CGA GAC ACA GAA GAC CAA GAA GAC CAA GTA GAT CCG CGG CTC ATT GAT GGG AAG ATG ACC NNS CGG GGA GAC AGC CCC TGG CAG-3′) and the outer-reverse primer (5′-GCC GCC GAG CTA TTA CAA GTC TTC TTC AGA AAT AAG CTT TTG TTC GGA TCC-3′) were used to randomize Arg177, where N represents 25% of A, G, C, or T, and S represents 50% of G or C. The two amplified DNA fragments were used as templates for the overlap PCR. The overlap PCR was performed with the outer-forward and outer-reverse primers. The final PCR product was transformed into the yeast strain EBY100 with NheI/BamHI-restricted pNL6. The transformed yeasts were selected by culturing in selective media at 30°C and induced to express the library proteins by culturing them in induction media at 25°C for 20 h (7). To activate PC on the yeast surface, we washed 2.5 × 107 inducing media-cultured yeast cells with PBS and incubated them with 10 μM bovine thrombin in 250 μl of 5 mM EDTA TBS (pH 7.4) at 37°C for 3 h.

HEK293 cells were grown to confluence on six-well plates. Before panning, the HEK293 cells and yeast cells were washed twice with HEPES buffer, containing 1 mM Ca2+/Mg2+/Mn2+, 5 μg/ml TS2/16 Ab, and 0.1% BSA. The mutant activated PC yeast cells were resuspended in the same buffer and were pipetted onto the HEK293 cells. The plates containing the yeasts and HEK293 cells were incubated at 25°C for 3 h. After the incubation, each well was washed twice with pH 7.5 HEPES buffer, containing 1 mM Ca2+/Mg2+/Mn2+, by gently swirling the plate 10 times in each direction. Then the yeast-bound HEK293 cells were scraped off, resuspended in selective medium, and cultured at 30°C overnight to expand the recovered yeast clones. In parallel, a small aliquot of the mixture was plated on selective medium to quantify the number of recovered yeast cells. The yeast mixtures were then induced again for the next round of panning. This process was repeated until the number of binder yeast cells reached a plateau (8).

Purified WT PC or R177G PC variant were incubated with human thrombin (10:1 mol/mol) in 20 mM Tris-HCl, 100 mM NaCl (pH 7.4) at 37°C for 2 h in the presence of 5 mM EDTA, followed by the addition of hirudin (Sigma) to inactivate the thrombin. Q Sepharose fast flow chromatography was used to remove thrombin. Activated PC concentrations were estimated by measurement of absorbance at 280 nm. Amidolytic activities were determined using chromogenic substrate S-2366 (Chromogenix) in a microplate reader and expressed in absorbance change at 405 nm. S-2366 (ranged between 33 and 667 μM) was added to activated PCs (67 nM) in 50 mM Tris-HCl, 130 mM NaCl, 10 mM CaCl2 (pH 8) at room temperature. Michaelis constant (Km) and maximal velocity (Vmax) values were obtained from Lineweaver–Burk plots. The experiments were performed in triplicate wells for each condition.

The adhesion assay was carried out essentially as previously described (6). Coverslips were coated with LXY2 (10 μg/ml). The residual binding sites were blocked by incubating the wells with 0.1% (w/v) polyvinylpyrrolidone in PBS for 30 min at room temperature; 2.5 × 105/250 μl neutrophils was suspended in L15 medium plus 2 mg/ml glucose and was pretreated for 15 min at 37°C with rhAPC or R177G–activated PC. The coverslips were aspirated and washed with L15 medium; 250 μl of L15/2 mg/ml of the glucose medium containing rhAPC or R177G–activated PC, with or without 20 nM fMLF, was placed on each coverslip and prewarmed for 15 min at 37°C. The cells (250 μl) were then immediately added and were further incubated at 37°C for 15 min. The unbound cells were then washed with warm L15 medium. The bound cells were then fixed with formaldehyde. For each experimental condition from three independent donors, five random phase-contrast images were obtained, and the number of cells in each well was scored from the printed micrographs.

All of the values are expressed as the mean ± SEM. All of the statistics were performed using the GraphPad Prism 4.0 software. A p value <0.05 was considered significant.

Emerging evidence suggests that activated PC inhibits neutrophil migration both in vitro and in vivo, at least in part, by directly interacting with cell surface integrins through an Arg-Gly-Asp (RGD) motif (46). Human neutrophils express several RGD-binding integrins, including VLA-3 (α3β1), VLA-5 (α5β1), and αVβ3 (9, 10). To determine the specific neutrophil integrin that interacts with activated PC with a high affinity, we performed a soluble integrin-binding assay. To this end, increasing concentrations of activated PC were incubated with equal amounts of activated, soluble VLA-3, VLA-5, or αVβ3 integrins. An analysis of the integrin immunoprecipitates revealed that VLA-3 manifested a significantly higher affinity for activated PC compared with the other two integrins (Fig. 1A). To further validate the strong and selective interaction between activated PC and VLA-3 on human neutrophils, we performed flow cytometry–based activated PC-binding assays. The amount of surface-bound activated PC was determined by measuring the mean florescence intensity (MFI) of a labeled Ab. The assay buffer, containing 1 mM Ca2+ and 1 mM Mg2+, was supplemented with 1 mM Mn2+ to activate all of the cell-surface integrins, and this condition significantly increased the binding of activated PC to the neutrophil surface (Fig. 1B). The addition of blocking mAbs against VLA-5, αVβ3, or Mac-1 (CD11b/CD18; αMβ2) did not influence the binding of rhAPC to the neutrophil surface. Consistent with the soluble integrin-binding assay, the presence of a blocking mAb against VLA-3 significantly displaced activated PC from the neutrophil surface (Fig. 1B). Collectively, these results suggest that the major mechanism controlling the binding of activated PC to human neutrophils is through a direct interaction between activated PC and VLA-3.

FIGURE 1.

Integrin VLA-3 is a receptor for activated PC. (A) Binding assay of soluble VLA-3, VLA-5, and αVβ3 to activated PC. Soluble integrins were incubated with the indicated amount of rhAPC in the presence of 1 mM MnCl2 + integrin-activating Abs. The protein complexes were precipitated and subjected to SDS-PAGE and Western blotting with an activated PC Ab (upper). The total amounts of soluble integrins precipitated were shown by silver staining (lower). The black lines indicate where parts of the image are joined. (B) rhAPC was incubated with human neutrophils in the presence of 1 mM MnCl2. The cells were pretreated with control IgG (Con IgG) or blocking Abs against integrins VLA-3 (α3), VLA-5 (α5), αVβ33), and Mac-1 (αM). Bound activated PC was detected by flow cytometry. Activated PC binding in the presence of integrin blocking Abs is expressed as the percentage increase in MFI, compared with the cells not treated with activated PC (control) in the presence of the same blocking Ab. Data show the mean ± SEM of three donors. Asterisk indicates significance of Con IgG versus anti–VLA-3 (α3): *p < 0.05 (Student t test).

FIGURE 1.

Integrin VLA-3 is a receptor for activated PC. (A) Binding assay of soluble VLA-3, VLA-5, and αVβ3 to activated PC. Soluble integrins were incubated with the indicated amount of rhAPC in the presence of 1 mM MnCl2 + integrin-activating Abs. The protein complexes were precipitated and subjected to SDS-PAGE and Western blotting with an activated PC Ab (upper). The total amounts of soluble integrins precipitated were shown by silver staining (lower). The black lines indicate where parts of the image are joined. (B) rhAPC was incubated with human neutrophils in the presence of 1 mM MnCl2. The cells were pretreated with control IgG (Con IgG) or blocking Abs against integrins VLA-3 (α3), VLA-5 (α5), αVβ33), and Mac-1 (αM). Bound activated PC was detected by flow cytometry. Activated PC binding in the presence of integrin blocking Abs is expressed as the percentage increase in MFI, compared with the cells not treated with activated PC (control) in the presence of the same blocking Ab. Data show the mean ± SEM of three donors. Asterisk indicates significance of Con IgG versus anti–VLA-3 (α3): *p < 0.05 (Student t test).

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We recently showed that, in septic mice, there is a significant upregulation of integrin VLA-3 on CD11bhighGr1high neutrophils (2). Consistently, both mouse activated PC (mAPC) and LXY2, a specific blocking peptide for VLA-3, reduced the levels of the IL-6 cytokine, a measure of sepsis severity, in the serum from septic mice (Fig. 2A, 2B). The fact that activated PC binds to VLA-3 with a high affinity and that the expression of VLA-3 dramatically increases in the hyperinflammatory neutrophil subtype suggests that activated PC may selectively target Gr1highCD11bhighVLA-3high proinflammatory neutrophils during sepsis. To assess the relative binding of activated PC to Gr1highCD11bhighVLA-3high versus Gr1highCD11bhighVLA-3low neutrophils, we performed ex vivo binding assays using neutrophils isolated from septic mice. As shown in Fig. 2C, the cell-surface binding of activated PC to Gr1highCD11bhighVLA-3high cells was significantly enhanced, suggesting the preferential binding of activated PC to the proinflammatory neutrophil subtype. We previously showed that activated PC binds to EPCR and integrins simultaneously at the neutrophil surface, where EPCR provides support for integrin binding (6). The cell-surface expression of EPCR was measured by flow cytometry in the neutrophils from septic mice, thereby revealing that the Gr1highCD11bhighVLA-3high neutrophils expressed significantly higher levels of surface EPCR compared with the Gr1highCD11bhighVLA-3low cells (Fig. 2D). Thus, these data suggest that the enhanced expression of both VLA-3 and EPCR at the neutrophil surface allows activated PC to selectively target the Gr1highCD11bhighVLA-3high proinflammatory neutrophil subpopulation during sepsis.

FIGURE 2.

Preferential binding of activated PC to Gr1highCD11bhighVLA-3high neutrophils. (A and B) Serum levels of IL-6 were measured by a sandwich ELISA. The graph shows the OD values at 450 nm. Asterisk indicates significance versus Con (PBS): *p < 0.05 (Mann–Whitney U test). (C) BM cells from LPS-induced septic mice were isolated at 12 h and incubated with rhAPC in the presence of 1 mM MnCl2. Surface-bound activated PC on the Ly6GhighVLA-3high and Ly6GhighVLA-3low populations among the mature and stimulated neutrophils was detected by flow cytometry. Activated PC binding is expressed as the percentage increase in MFI compared with a no activated PC control. Data are the mean ± SEM of three mice. Asterisk indicates significance of VLA-3high versus VLA-3low: *p < 0.05 (Student t test). (D) BM and peripheral blood cells from LPS-induced septic mice were isolated at 12 h, and the surface expression of EPCR was measured in the Ly6GhighVLA-3high and Ly6GhighVLA-3low populations by flow cytometry. Data are the mean ± SEM of six mice. Asterisk indicates significance of VLA-3high versus VLA-3low: *p < 0.01 (Mann–Whitney U test). (E and F) The mice were injected with PBS or rhAPC (10 μg, i.v.). The lungs and PL were harvested after 8 h and were digested with collagenase to prepare a single-cell suspension. The cells were stained with CD11b, Ly6G, and VLA-3. The bar graphs show the frequencies in the total granulocyte population (gated on Gr1highCD11bhigh) and the total number of VLA-3high and VLA-3low cells in the PL (E) and lungs (F). n = 3 per group. Data are the mean ± SEM of six mice. Asterisk indicates significance of PBS versus activated PC: *p < 0.05 (Mann–Whitney U test).

FIGURE 2.

Preferential binding of activated PC to Gr1highCD11bhighVLA-3high neutrophils. (A and B) Serum levels of IL-6 were measured by a sandwich ELISA. The graph shows the OD values at 450 nm. Asterisk indicates significance versus Con (PBS): *p < 0.05 (Mann–Whitney U test). (C) BM cells from LPS-induced septic mice were isolated at 12 h and incubated with rhAPC in the presence of 1 mM MnCl2. Surface-bound activated PC on the Ly6GhighVLA-3high and Ly6GhighVLA-3low populations among the mature and stimulated neutrophils was detected by flow cytometry. Activated PC binding is expressed as the percentage increase in MFI compared with a no activated PC control. Data are the mean ± SEM of three mice. Asterisk indicates significance of VLA-3high versus VLA-3low: *p < 0.05 (Student t test). (D) BM and peripheral blood cells from LPS-induced septic mice were isolated at 12 h, and the surface expression of EPCR was measured in the Ly6GhighVLA-3high and Ly6GhighVLA-3low populations by flow cytometry. Data are the mean ± SEM of six mice. Asterisk indicates significance of VLA-3high versus VLA-3low: *p < 0.01 (Mann–Whitney U test). (E and F) The mice were injected with PBS or rhAPC (10 μg, i.v.). The lungs and PL were harvested after 8 h and were digested with collagenase to prepare a single-cell suspension. The cells were stained with CD11b, Ly6G, and VLA-3. The bar graphs show the frequencies in the total granulocyte population (gated on Gr1highCD11bhigh) and the total number of VLA-3high and VLA-3low cells in the PL (E) and lungs (F). n = 3 per group. Data are the mean ± SEM of six mice. Asterisk indicates significance of PBS versus activated PC: *p < 0.05 (Mann–Whitney U test).

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To investigate whether the preferential binding of activated PC to the Gr1highCD11bhighVLA-3high cells results in delayed tissue infiltration by these cells during sepsis, we measured the percentage and number of cells in the peritoneum and lungs of septic animals after activated PC injection. The administration of activated PC significantly inhibited the infiltration of Gr1highCD11bhighVLA-3high cells into the peritoneal tissues, whereas the migration of Gr1highCD11bhighVLA-3low cells was not altered in the presence of activated PC (Fig. 2E). In the lungs, a reduction in the number of both infiltrating Gr1highCD11bhighVLA-3high and Gr1highCD11bhighVLA-3low cells in the presence of activated PC was observed (Fig. 2F). The activated PC–induced decrease in the number of Gr1highCD11bhighVLA-3high cells in the lung was more pronounced compared with the Gr1highCD11bhighVLA-3low cell population (Fig. 2F). These data indicate that the specific binding of activated PC to VLA-3high granulocytes is sufficient to cause a delay in tissue infiltration by the proinflammatory Gr1highCD11bhighVLA-3high neutrophil population during sepsis.

In the crystal structure of the integrin αVβ3 bound to the RGD peptide, the calculated buried surface area of the RGD motif is 373 Å (11). The solvent accessible surface area of the rhAPC RGD motif, which is comparable with the buried area of the complex, is 140 Å. This small-buried area is mainly attributed to the fact that the L chain covers a portion of the RGD motif in the catalytic domain (Fig. 3A). This property suggests that the accessibility of the RGD motif in WT rhAPC is not optimal for integrin binding. We also compared the structures of the rhAPC RGD motif and the RGD peptide in complex with αVβ3. We found that the rhAPC RGD motif has different amino acid side-chain orientations than the integrin-bound RGD motif, suggesting the possibility of a steric clash when the WT rhAPC RGD motif binds to VLA-3.

FIGURE 3.

Development of a mutant activated PC variant with a higher affinity for integrin VLA-3. (A) The crystal structure of activated PC without the Gla domain (1AUT.pbd) is depicted in a ribbon diagram showing the EGF-like domain 1 (EGF1) in red, EGF2 in blue, and the H chain in green (left). The region encircled by dots from the bottom view is enlarged (right), representing the RGD sequence (residues 178–180) in the sphere and the mutational target residues (Arg177 and Lys146) on the mesh surface. (B) Schematic depiction of the yeast display. Aga2, hemagglutinin (HA), PC, and cMyc are expressed in order from the pNL6 vector. (C) Flow cytometry analysis of the expression of HA, c-Myc, and PC. The samples were compared before and after activation with bovine thrombin. Note that the binding of anti-PC Ab decreased after the surface PC was activated by thrombin. (D) Microscopic analysis of each round of biopanning on a confluent HEK293 cell monolayer during the enrichment of yeasts expressing the high-affinity mutant activated PC. The yeast cells were labeled with a fluorescent conjugated anti-HA Ab (left), and the fluorescence signal (red) was overlapped with the bright-field image of HEK293 cell monolayer. (E) The recovered yeast cell numbers from each round of biopanning. (F) Flow cytometry analysis of WT and mutant activated PCs in the yeast cell surface with an anti–activated PC Ab. (G) The recovered yeast cell numbers from the binding assay (similar to biopanning) with HEK293 cells using WT activated PC– and mutant activated PC–expressing yeast cells. The control condition was HEPES buffer without Mn2+/TS2-16. The binding was measured in three independent experiments and is presented as the mean ± SEM. Asterisk indicates significance of control versus Mn2+/TS2-16: *p < 0.05 (Student t test).

FIGURE 3.

Development of a mutant activated PC variant with a higher affinity for integrin VLA-3. (A) The crystal structure of activated PC without the Gla domain (1AUT.pbd) is depicted in a ribbon diagram showing the EGF-like domain 1 (EGF1) in red, EGF2 in blue, and the H chain in green (left). The region encircled by dots from the bottom view is enlarged (right), representing the RGD sequence (residues 178–180) in the sphere and the mutational target residues (Arg177 and Lys146) on the mesh surface. (B) Schematic depiction of the yeast display. Aga2, hemagglutinin (HA), PC, and cMyc are expressed in order from the pNL6 vector. (C) Flow cytometry analysis of the expression of HA, c-Myc, and PC. The samples were compared before and after activation with bovine thrombin. Note that the binding of anti-PC Ab decreased after the surface PC was activated by thrombin. (D) Microscopic analysis of each round of biopanning on a confluent HEK293 cell monolayer during the enrichment of yeasts expressing the high-affinity mutant activated PC. The yeast cells were labeled with a fluorescent conjugated anti-HA Ab (left), and the fluorescence signal (red) was overlapped with the bright-field image of HEK293 cell monolayer. (E) The recovered yeast cell numbers from each round of biopanning. (F) Flow cytometry analysis of WT and mutant activated PCs in the yeast cell surface with an anti–activated PC Ab. (G) The recovered yeast cell numbers from the binding assay (similar to biopanning) with HEK293 cells using WT activated PC– and mutant activated PC–expressing yeast cells. The control condition was HEPES buffer without Mn2+/TS2-16. The binding was measured in three independent experiments and is presented as the mean ± SEM. Asterisk indicates significance of control versus Mn2+/TS2-16: *p < 0.05 (Student t test).

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To increase the affinity of activated PC for VLA-3 by altering the protein structure surrounding the RGD motif, we used a yeast surface display system that was based on random mutagenesis and coupled it with a functional affinity screen (12). First, mutagenesis of the three amino acids adjacent to the RGD motif (Lys146, Arg147, and Arg177) of WT human PC was performed using site-specific randomized primers and two-step overlap PCR (Fig. 3A). Second, the mutant PC variants were expressed and activated PC on the yeast surface (Fig. 3B, 3C). Third, the progressive enrichment of the cells encoding high-affinity activated PC was achieved by biopanning (8) (Fig. 3D, 3E) using HEK293T cells that do not express EPCR but express high levels of VLA-3 (data not shown). Fourth, the genes isolated from the remaining yeast colonies were sequenced. A total of 68 yeast colonies, selected by panning, was sequenced. Based on the amino acid substitution frequency at residue 177 and in an effort to minimize a possible steric hindrance by bulky positively charged side chains at residues 146 and 147 (Table I), we designed three mutant activated PC variants (R177G, K146G/R147G/R177G, and K146G/R177G) for further analysis. The yeast surface expression levels of these three mutant activated PCs were identical (Fig. 3F). However, only the R177G–activated PC–expressing yeast showed a significantly increased binding to HEK293 cells in the presence of Mn2+ and mAb TS2/16 (β1 integrin–activating Ab) (Fig. 3G).

Table I.
Frequency of mutant amino acid substitutions in the target residue
Lys146
Arg147
Arg177
MutationCountsMutationCountsMutationCounts
11 Ra 14 16 
L, Ra 
A, S 
A, G H, M 
L, T T, V 
I, N, P, Q, W 
C, W, Y F, Q, W E, Y 
D, I, K,aG, H, V   
F, H, N, Q C, E, I, K   
Lys146
Arg147
Arg177
MutationCountsMutationCountsMutationCounts
11 Ra 14 16 
L, Ra 
A, S 
A, G H, M 
L, T T, V 
I, N, P, Q, W 
C, W, Y F, Q, W E, Y 
D, I, K,aG, H, V   
F, H, N, Q C, E, I, K   

Sixty-eight clones were sequenced.

a

WT amino acid.

R177G–activated PC was purified from a mammalian cell culture system (HEK293), and enzyme assay using the chromogenic substrate, S-2366, indicated that the R177G mutation did not perturb amidolytic functions of activated PC, although it showed a slight decreased enzymatic activity compared with that of WT activated PC (Supplemental Fig. 1). It is important to note, however, that Kerschen et al. (13) showed that activated PC variants with reduced anticoagulant activities retain efficacy while reducing the risk for bleeding in severe sepsis. To determine whether R177G–activated PC binds to VLA-3 with a high affinity, we performed a soluble integrin-binding assay. An analysis of the immunoprecipitates by silver staining revealed that R177G–activated PC had a significantly higher affinity for VLA-3 compared with WT activated PC (Fig. 4A). To further demonstrate the strong and specific interaction between R177G–activated PC and VLA-3 on human neutrophils, we assessed cell adhesion to a cover glass coated with the VLA-3–specific peptide LXY2. Human neutrophils were allowed to adhere to immobilized LXY2 in the presence of fMLF. Compared with WT activated PC, the addition of R177G–activated PC significantly enhanced the inhibition of fMLF-induced adhesion (Fig. 4B). Furthermore, in the LPS-induced endotoxemia model, the systemic administration of R177G–activated PC significantly reduced the infiltration of CD11bhiLy6Ghi neutrophils into the inflamed lungs compared with WT activated PC (Fig. 4C). These data further support a specific interaction between activated PC and VLA-3, and suggest that our approach using a yeast surface display system can serve as a blueprint for engineering better targeted and safer activated PC variants to treat septic patients.

FIGURE 4.

R177G–activated PC inhibits VLA-3–mediated neutrophil adhesion. (A) Binding assay of soluble VLA-3 to WT activated PC and R177G–activated PC. Soluble integrins were incubated with the indicated amounts of WT activated PC or R177G–activated PC in the presence of 1 mM MnCl2 and the mAb TS2/16 (β1 integrin–activating Ab). The protein complexes were precipitated and subjected to SDS-PAGE and silver staining. The total amounts of activated PC (upper) and soluble VLA-3 (β1 subunit) (lower) precipitated are shown. (B) The binding of 10 nM fMLF-treated neutrophils to immobilized LXY2 in the presence of various concentrations of WT rhAPC or R177G–activated PC. For each condition, the binding was measured in triplicate and is presented as the mean ± SEM. *p < 0.05 versus fMLF-treated cells in the presence of WT activated PC (Student t test). Data are representative of at least three independent experiments. (C) The mice were injected with PBS, WT activated PC (5 or 10 μg, i.v.) or R177G–activated PC (5 or 10 μg, i.v.) 1 and 4 h after the LPS injection. The lungs were harvested after 12 h, and a single-cell suspension was prepared after digestion with collagenase-1. The cells were stained with propidium iodide and CD11b, Ly6G, and VLA-3 Abs. The line diagram shows the total neutrophils (gated on Gr1highCD11bhigh granulocytes) in the lungs (n = 3–4 mice per group; *p < 0.05, two-way ANOVA with a Bonferroni posttest).

FIGURE 4.

R177G–activated PC inhibits VLA-3–mediated neutrophil adhesion. (A) Binding assay of soluble VLA-3 to WT activated PC and R177G–activated PC. Soluble integrins were incubated with the indicated amounts of WT activated PC or R177G–activated PC in the presence of 1 mM MnCl2 and the mAb TS2/16 (β1 integrin–activating Ab). The protein complexes were precipitated and subjected to SDS-PAGE and silver staining. The total amounts of activated PC (upper) and soluble VLA-3 (β1 subunit) (lower) precipitated are shown. (B) The binding of 10 nM fMLF-treated neutrophils to immobilized LXY2 in the presence of various concentrations of WT rhAPC or R177G–activated PC. For each condition, the binding was measured in triplicate and is presented as the mean ± SEM. *p < 0.05 versus fMLF-treated cells in the presence of WT activated PC (Student t test). Data are representative of at least three independent experiments. (C) The mice were injected with PBS, WT activated PC (5 or 10 μg, i.v.) or R177G–activated PC (5 or 10 μg, i.v.) 1 and 4 h after the LPS injection. The lungs were harvested after 12 h, and a single-cell suspension was prepared after digestion with collagenase-1. The cells were stained with propidium iodide and CD11b, Ly6G, and VLA-3 Abs. The line diagram shows the total neutrophils (gated on Gr1highCD11bhigh granulocytes) in the lungs (n = 3–4 mice per group; *p < 0.05, two-way ANOVA with a Bonferroni posttest).

Close modal

The mechanism involved in the beneficial effects of rhAPC in severe inflammation is associated primarily with its cytoprotective attributes via EPCR and PAR-1 receptors (14). Previously, we showed that hyperinflammatory subsets of neutrophils, with the potential to cause tissue injury during sepsis, express high levels of VLA-3 on their surface (Gr1highCD11bhighVLA-3high) (2). In this study, we showed that the enhanced expression of both VLA-3 and EPCR at the neutrophil surface allows activated PC to selectively target the Gr1highCD11bhighVLA-3high proinflammatory neutrophil subpopulation during sepsis (Fig. 2C, 2D). Therefore, we hypothesized that activated PC might bind more strongly to the Gr1highCD11bhighVLA-3high proinflammatory neutrophil subset in vivo, and thus show a superior suppression of migration in this population than in Gr1highCD11bhighVLA-3low neutrophils. As expected, administration of activated PC significantly inhibited the infiltration of Gr1highCD11bhighVLA-3high cells into the peritoneum and lung (Fig. 2E, 2F). Thus, our data suggest that rhAPC selectively targets VLA-3–expressing hyperactive neutrophils and improves survival in sepsis possibly by limiting their ability to infiltrate into the tissues of visceral organs.

Loss of endothelial barrier function is a prominent feature in the pathogenesis of severe inflammatory diseases. An increase in vascular permeability results in the massive infiltration of inflammatory cells into adjacent tissues, ultimately leading to organ failure during sepsis (15). EPCR was the first activated PC receptor identified on the endothelium (16). The antiapoptotic and vascular-protective effects of activated PC are mediated by EPCR engagement and the secondary activation of G protein–coupled PAR-1 by the activated PC–EPCR complex (1719). Importantly, many cell types that express EPCR, including endothelial cells, also express VLA-3. We showed that activated PC binds to EPCR and integrins simultaneously on the cell surface because the integrin binding site on activated PC is distinct from the EPCR binding site (6). Thus, activated PC may bind to EPCR and VLA-3 simultaneously at the endothelial cell surface, and this interaction could synergistically impact activated PC–mediated EPCR–PAR-1 signaling. It is also important to note that neutrophils express EPCR at their surface (Fig. 2D) (6). These observations also suggest that activated PC may not only prevent the massive infiltration of hyperinflammatory neutrophils, but may also improve EPCR-mediated vascular barrier protection through a simultaneous interaction with VLA-3.

Consistent with our findings in human neutrophils, a study in mice using mAPC demonstrated that activated PC binds to integrins on macrophages (20). In addition, the interaction between activated PC and integrin suppresses proinflammatory cytokine production. The authors proposed that Mac-1 is a novel activated PC receptor on macrophages that mediates the anti-inflammatory functions. In stark contrast, our study revealed that blocking Mac-1 in human neutrophils had no effect on activated PC binding (Fig. 1B). This discrepancy may be because of the different binding affinity of mouse Mac-1 to activated PC or because of the use of a different activated PC (human versus mouse) in the experiments. In the aforementioned study, cell adhesion assays were performed mainly using surfaces coated with mAPC, in which nonspecific binding was blocked by pretreating the plates with BSA, which is a strong Mac-1 ligand (21).

Our mutational studies demonstrated that R177G–activated PC showed a higher binding affinity to VLA-3 and inhibited the specific interaction between VLA-3 and its peptide ligand, LXY2, more effectively than WT activated PC did. According to the crystal structure of activated PC lacking the Gla-domain, there are hydrogen bonds between Arg177 and Asp180 (22). The bulky and positively charged side chains of the two adjacent arginines (residue Arg177 and Arg178) are positioned apart from each other, possibly because of the repulsive electrical force. Therefore, it is likely that the substitution of Arg177 with Gly may release the electrical tension between the two arginines and provide enough flexibility to cause a conformational change in the RGD motif that is more favorable for a high-affinity binding to VLA-3.

The combination of the anticoagulant and cytoprotective properties of activated PC has made it an important clinical adjuvant for the treatment of severe sepsis. However, the anticoagulant properties have also led to increased bleeding events and resulted in a very narrow therapeutic window for this drug, ultimately leading to a voluntary withdrawal of the drug by the manufacturer. In this study, we demonstrated that VLA-3, which is specifically upregulated in a hyperinflammatory neutrophil subpopulation in both human and murine sepsis, is a novel cellular receptor for activated PC. Moreover, the specific interaction of activated PC with VLA-3 selectively inhibits the migration of the proinflammatory neutrophil subpopulation during sepsis. In support, R177G–activated PC was more efficient at reducing the infiltration of neutrophils (CD11bhiLy6Ghi) into the lungs of LPS-treated endotoxemic mice. Pursuing this alternative mechanism and optimizing activated PC binding to neutrophils will enable the development of better targeted, more effective, and safer sepsis therapies.

We thank Jennifer Wong for technical assistance.

This work was supported by National Institutes of Health Grants HL101917 (to A.R.R.), HL125265 (to M.K.), and T32 DA007232 (to Y.V.L.) and Indian Institute of Technology Roorkee IITR/PDA/100643 (to P.P.S.).

The online version of this article contains supplemental material.

Abbreviations used in this article:

     
  • BM

    bone marrow

  •  
  • CLP

    cecal ligation and puncture

  •  
  • EPCR

    endothelial PC receptor

  •  
  • mAPC

    mouse activated PC

  •  
  • MFI

    mean florescence intensity

  •  
  • PAR-1

    protease-activated receptor-1

  •  
  • PC

    protein C

  •  
  • PL

    peritoneal lavage

  •  
  • RGD

    Arg-Gly-Asp

  •  
  • rhAPC

    recombinant form of human activated protein C

  •  
  • WT

    wild-type.

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The authors have no financial conflicts of interest.

Supplementary data