Both PS and TFPI are expressed in murine synovium. (A) Immunostaining for PS and TFPI in the knee intra-articular space of injured knees from F8^−/−Pros1^+/+ mice previously treated with control siRNA or mPS siRNA. Arrowheads point to synovial tissue and arrows to vascular structures, all positive for both PS and TFPI. The boxed areas in the upper subpanels (scale bars: 200 µm) show the area enlarged in the subpanel below (scale bars: 50 µm). (B) Immunostaining for TFPI in the knee intra-articular space of uninjured knees from F8^−/−Pros1^+/+ and F8^−/−Pros1^−/− mice. Arrowheads point to synovial tissue and arrows to vascular structures, all positive for both PS and TFPI. The boxed areas in the upper subpanels (scale bars: 200 μm) show the area enlarged in the subpanel below (scale bars: 50 μm). (C-D) Western blot analysis of conditioned media from primary murine fibroblast-like synoviocyte (FLS) cultures using anti-PS (C) and anti-TFPI (D) antibodies. Platelet-free plasma, protein lysates from platelets, and murine PS were used as positive controls (C). TFPI isoform expression was determined by comparing the molecular weights of deglycosylated TFPI and of fully glycosylated TFPI. Murine placenta was used as a positive control for TFPIα. (E-F) Western blot analysis of total protein lysates isolated from FLS after 24 hours of culture in the presence of thrombin (+) or of a vehicle (−) using anti-TFPI (E) and anti-PS (F) antibodies. Human recombinant TFPI full length was used as a positive control for TFPIα. Blots are representative of 3 independent experiments. hrTFPI, human recombinant TFPI; PLT, protein lysates from platelets; Thr, thrombin.