Fitc conjugated rabbit anti human fibrinogen

Patients’ platelets were analysed for markers of activation using a whole blood flow cytometric method essentially as described previously^{10 (link),32 (link),33 (link)}. Briefly, 5 µl of citrated blood were added to 50 µl of HEPES-buffered saline (HBS; 10 mM HEPES; 150 mM NaCl; 5 mM LiCl; 1 mM MgSO₄; pH 6.0) containing either 5 µl of a FITC-conjugated rabbit anti-human fibrinogen (Dako) or a FITC-conjugated anti-human CD62P monoclonal antibody (R&D Systems) that detects surface expression of P-selectin; a marker of platelet degranulation. After 20 min incubation at 20–22 °C samples were diluted 100-fold with 0.2% (v/v) formyl saline and analysed within 2 h in a Gallios flow cytometer (Beckman Coulter, Milton Keynes, UK). Negative controls were set at 2% + ve (Supplementary Fig. 3). For fibrinogen binding the negative control comprised a parallel sample prepared in the presence of 2 mM EDTA to chelate calcium and thereby disrupt the binding of fibrinogen to the GPIIb-IIIa integrin. For P-selectin the negative control was an isotype control mouse antibody. Annexin V binding used a similar assay but with 2 mM Ca²⁺ and 0.5 mg ml⁻¹ GPRP peptide added to the HBS to allow, respectively, Annexin V binding to procoagulant phospholipids and to prevent clotting of the plasma. The negative control for these samples comprised a parallel sample that lacked calcium (Supplementary Fig. 3).

Oxylipin standards for LC–MS/MS were purchased from Cayman Chemical (Cayman Islands) or from Enzo Life Sciences (Exeter, UK). The thromboxane receptor antagonist (SQ29548) was from Alexis Biochemicals Corporation (San Diego, USA). FITC-conjugated rabbit anti-human fibrinogen was from Dako, (UK, Ltd), FITC-conjugated anti-CD62P monoclonal antibody and an isotype control were from R&D Systems (Abingdon, UK) as was Annexin V FITC. Unless otherwise stated, all other reagents were purchased from Sigma Aldrich (Dorset, UK).