The RGD sequence of rhAPC is critical for inhibition of neutrophil migration in vivo. (A) Neutrophil counts in BALF from LPS-treated mice. Mice were given rhAPC or RGE-APC (10 μg/mouse) or PBS 2 hours after LPS treatment. Results are expressed as mean (± SEM); n = 5 per group. (B) Survival curves for mice (C57BL/6, male, 6-8 weeks) challenged with an LD₉₀ of LPS; 200 μg of the RGD peptide or the control (RAD) peptide was given via intravenous injection before receiving 37 to 40 mg/kg LPS intraperitoneally (n = 15-25 per group). The statistical significance of mortality was determined by the Kaplan-Meier log-rank test.

Direct binding of rhAPC to neutrophil integrins. (A) Binding of rhAPC to human neutrophils was assayed using chromogenic substrate S-2366 in the presence of 1 mM MnCl₂ ± cyclic RGD peptide (20 μg/mL), β₁ blocking Ab, β₃ blocking Ab, or β₂ blocking Ab (10 μg/mL each). *P < .05 versus MnCl₂-treated cells. (B) Binding of control or 10 nM fMLP-treated neutrophils to immobilized FN in the presence of rhAPC (10 μg/mL) or Gla-less APC (10 μg/mL), or an equivalent amount of PBS. (C) Binding of control or 10 nM fMLP-treated neutrophils to immobilized FN in the presence of rhPC (10 μg/mL) or an equivalent amount of PBS. (D) Induction of LIBS epitopes by rhAPC. Control (PBS) or rhAPC-treated neutrophils were incubated with the indicated concentrations of MgCl₂ and CaCl₂. The LIBS of β₁ and β₃ integrins were detected by B44 and D3 mAb, respectively. *P < .05 versus PBS-treated cells. (E) Solid-phase binding of immobilized α₃β₁, α₅β₁, and α_Vβ₃ to FN or rhAPC. (F) Solid-phase binding of immobilized α₃β₁, α₅β₁, and α_Vβ₃ to wild-type or mutant rhAPC (RGE-APC). (G) Migration of human neutrophils on FN-coated cover glass in the presence of fMLP ± RGE-APC, rhPC, Gla-less APC, or S360A-APC. Experiments were repeated on neutrophil preparations from 3 independent donors. Results in panels A and C through F are expressed as mean (± SEM) of 3 independent experiments.

rhAPC inhibits neutrophil adhesion and migration. (A) Binding of 10 nM fMLP-treated neutrophils to immobilized FN in the presence of various concentrations of rhAPC. For each condition, binding was measured in triplicate and stated as mean (± SEM). *P < .05 versus fMLP-treated cells in the absence of rhAPC. (B) Migration of human neutrophils on FN-coated cover glasses in the presence of fMLP ± rhAPC. Cells were tracked over a 30-minute period, and each line represents one cell. Experiments were repeated on neutrophil preparations from 3 independent donors. (C) Directional migration of neutrophils, as measured by the underagarose migration assay. The number of control or rhAPC-treated neutrophils migrating to fMLP (1 and 2) or to PBS (3) was counted. Results are expressed as mean ± SEM of 3 experiments from 2 independent donors. (D) Center-zeroed tracks of control or rhAPC-treated neutrophils migrating toward microtips containing fMLP. The scale of each graph is in microns. The speed (S, μm s⁻¹), migratory index (MI), and X^D (mean direction in which the population is moving, in degrees) are shown (mean ± SEM). (E) Migration of human neutrophils on FN-coated cover glasses in the presence of IL-8 ± rhAPC. (F) Cytosolic Ca²⁺ levels in stirred Fluo-4–labeled neutrophils were continuously measured in a fluorometer. Control (PBS) or rhAPC-treated neutrophils in Ca²⁺-containing buffer were sequentially stimulated with 10 nM fMLP and 40 μM digitonin. The data are representative of at least 3 independent experiments.

Simultaneous binding of rhAPC to neutrophil integrins and EPCR. (A) EPCR RT-PCR on human neutrophils. Reverse-transcribed cDNAs from human heart and lung served as positive controls (top). FACS analysis of cell-surface EPCR using mAb RCR252 (bottom). IgG control and fMLP-treated cells were exposed to fMLP for 30 minutes before staining with Rat IgG₁ isotype and RCR252, respectively. As a negative control (Untreated), cells were incubated without fMLP and labeled with RCR252. (B) Binding of 10 nM fMLP-treated neutrophils to immobilized FN in the presence of rhAPC (10 μg/mL) ± EPCR mAb (50 or 100 μg/mL). (C) A hypothetical model for rhAPC binding. Cells expressing hEPCR-mCFP and β₁-mYFP will exhibit FRET only when these 2 molecules are brought into close proximity (100 Å) after rhAPC binding. (D) Whole-cell lysates of HEK293 cells transiently transfected with hEPCR-mCFP and β₁-mYFP K562 were subjected to SDS-PAGE and Western blotting with the indicated antibodies. (E) Fluorescence images of transiently transfected HEK293 cells with hEPCR-mCFP and β₁-mYFP demonstrate membrane localization of CFP and YFP signals. (F) HEK293 cells were transfected with hEPCR-mCFP and β₁-mYFP in a delta T dish. FRET images under TIRF microscopy were extracted from videos (time above images, Videos S3–S5). FRET was measured by sensitized emission method and analyzed by AutoQuant software. FRET signals are shown as rainbow colors (red indicates highest and blue, lowest).