APC-β exhibits superior inhibition of cerebral injury in murine ischemic stroke. (A) 293T cells transfected with EPCR and PAR1-AP were incubated with mAPC or mAPC^N329Q (6.25-50 nM) for 3 hours. AP activity was assessed using QUANTI Blue AP substrate, as before. RAW264.7 macrophages were incubated with mAPC (black bars) or mAPC^N329Q (red bars) (12.5-50 nM) for 3 hours before stimulation with LPS (50 ng/mL) for 18 hours. Secretion of (B) TNFα and (C) IL-6 was measured by ELISA. (D) Schematic description of the ischemic stroke model and study protocol. (E) Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis under reducing conditions of purified recombinant mAPC^PS and mAPC^PS/N329Q. Proteins were stained with Coomassie Brilliant Blue. The heavy chain of mAPC^PS migrates as a diffuse band composed of the APC-α heavy chain and APC-β glycoform heavy chain fractions. The heavy chain of mAPC^PS/N329Q is composed solely of the APC-β fraction. (F) Brain infarct volumes post-stroke and t-PA administration were calculated in each mouse by staining 20-μm coronal brain sections with thionine, which is unable to stain lesion areas. For volume analysis, 1 section of every 10 covering the entire lesion was analyzed. Individual values and the median of each group are represented; n = 9-15; *P = .006, **P = .004. (G) Images corresponding to 2 thionine-stained sections of representative mice from each group are shown. Lesion areas are unstained, whereas remaining healthy tissue is stained purple.