Rabbit anti human vwf

Group A (GalNAc α1 → 3 [Fuc α1 → 2] Galβ 1 → 4 GlcNAc β1 →) antigenic determinants on plasma were measured using a modified sandwich ELISA^{28 (link),30 (link)}. In brief, ELISA plates were coated with rabbit anti-human VWF (Dako), washed and blocked using TBS containing 1% BSA. After further washings, plasma samples were added and incubated for 2 h at room temperature. Each plasma was tested in duplicate at three dilutions. The plates were washed and then incubated with murine anti–A monoclonal antibody (Ortho Diagnostics) for 1 h. After a further three washes, the plates were incubated with goat anti-mouse IgM peroxidase conjugate (Sigma) for 1 h. After another TBS/Tween wash, peroxidase substrate solution was added. The reaction was stopped with 1 M H₂SO₄ and the optical density measured at wavelength 492 nm. Pooled group A plasma was assayed to produce a standard curve for each ELISA. Using the standard curve, a value for A antigen on VWF expression was determined for each plasma sample. Plasma VWF:Ag concentration strongly influenced the amount of A antigen detected in each ELISA well. To determine the amount of A antigen expressed per unit VWF, the amount of A antigen detected was divided by the amount of VWF:Ag present in the ELISA well^{28 (link)}. The pooled normal A plasma was assigned a value of 1U/ml for the amount of A antigen expressed per unit vWF.

Lipopolysaccharide derived from Escherichia coli O111:B4, TRAP-6, ADP and prostaglandin (PG) E1 were obtained from Sigma-Aldrich (St. Louis, MO, United States). Pam3CSK4 was purchased from InvivoGen (San Diego, CA, United States) and thrombin from Biopool (Umea, Sweden). Fluorescein isothiocyanate (FITC)-conjugated mouse anti-human P-selectin (CD62P), anti-CD40L (CD154), and CD63 and PE-conjugated mouse anti-human CD41, TLR2, and TLR4 were from BD Biosciences (San Jose, CA, United States). FITC-conjugated CD45 and Human CCL5 (RANTES) ELISA Max were purchased from BioLegend (San Diego, CA, United States) and Quantikine ELISA Human P-selectin/CD62P was from R&D Systems (Minneapolis, MN, United States). Rabbit anti-human VWF and HRP-conjugated anti-human VWF were from Dako (Glostrup, Denmark), mouse anti- p-ERK1/2 (Tyr 204) and rabbit anti ERK 1/2 were obtained from Santa Cruz Biotechnology (Dallas, TX, United States).

High molecular weight hyaluronic acid (HMW HA) was a kind gift from Professor Ivan Donati (Department of Life Sciences, University of Trieste, Italy). Fibronectin (FN) was purchased from Corning (Milan, Italy).
The following antibodies were used: mouse mAb anti-vimentin and mouse mAb anti-CD44, purchased from Sigma-Aldrich (St. Louis, MO, USA); rabbit anti-human vWF, rabbit anti-human CK8/18, rabbit anti-human FN and goat anti-mouse FITC-conjugated F(ab)’ from Dako (Milan, Italy); mouse mAb anti-human CD45-PE was bought from Immunotools (Friesoythe, Germany); rabbit anti-human MUC1 from Invitrogen (Monza, Italy); mouse anti-human CD44 was bought from Thermo Fisher Scientific (Milan, Italy); mouse anti-human β1-integrin from Merck Millipore (Darmstadt, Germania); mouse anti-human gC1qR was a kind gift from Professor Berhane Ghebrehiwet (Department of Medicine, State University of New York, Stony Brook, NY, USA); and FITC-conjugated goat anti-rabbit and Cy3-conjugated goat anti-rabbit were purchased from Jackson ImmunoResearch (Milan, Italy). All chemicals were purchased from Sigma-Aldrich.