Recombinant human thrombospondin 1

Leuko-depleted RBC concentrates were provided by Canadian Blood Services (CBS) Network Centre for Applied Development (netCAD, Vancouver, BC, Canada) after prior approval from the CBS Research Ethics Board (#2015.022). For some experiments, these concentrates were provided by the blood bank of the University of Tübingen (#184/2003 V), Germany, or by the blood bank of Norrlands University Hospital, Umeå, Sweden. Donor RBCs, drawn from refrigerated blood bags containing SAG-M additive solution, were washed twice in PBS (1000×g for 10 min) and subsequently incubated in vitro (1% hematocrit unless indicated otherwise) at 37 °C in Ringer’s solution (pH 7.4) containing 125 mM NaCl, 5 mM KCl, 1 mM MgSO₄, 32 mM HEPES, 5 mM glucose, and 1 mM CaCl₂. Sample sizes (number of RBC units; n) for control and treatment groups used in individual experiments are indicated in the figure legends. Where indicated, RBCs were incubated with recombinant human thrombospondin-1 (1–50 μg/mL; R&D Systems, Minneapolis, MN, USA) or with anti-human CD47 mAb 1F7 (mouse IgG1), which was purified from hybridoma supernatants [29 (link)–31 (link)]. In some experiments, RBCs were treated with sodium nitroprusside (Sigma Aldrich, Taufkirchen, Germany) or amiloride (Sigma Aldrich), as described in the figure legends.

The drugs used were obtained from the following sources: L-phenylephrine hydrochloride, acetylcholine chloride (Sigma-Aldrich, St. Louis, MO, USA), native human thrombospondin 1 (Novus Biologicals, Littleton, CO, USA; Athens Research & Technology, Athens, GA, USA; Thermo Fisher Scientific, Anthem, AZ, USA), recombinant human thrombospondin 1 (R&D Systems, Minneapolis, MN, USA; BioVision, Milpitas, CA, USA), recombinant human CD47 peptide (Bon Opus, previously NovoProtein, Millburn, NJ; Abcam, Cambridge, MA, USA). All drugs were dissolved and diluted in physiological salt solution. All animal studies were performed under a protocol approved (16 August 2018) by the Midwestern University Institutional Animal Care and Use Committee (IACUC) in accordance with NIH guidelines. Male C57BL/6 mice were obtained from the Jackson Laboratory (Bar Harbor, ME, USA).

Recombinant human thrombospondin-1 was purchased from R&D. Recombinant human VEGF₁₆₅ was obtained from Peprotech. Fibronectin(FN) was obtained from BD Bioscience.