Fig. 1: Fibrinogen exists in multiple disulfide-bonded states in the circulation.

a Blood from healthy donors was drawn by venipuncture into citrate as anti-coagulant, plasma prepared by centrifugation and fibrinogen collected on antibody-coated magnetic beads. The unpaired cysteine thiols in bead-bound fibrinogen was alkylated with ¹²C-IPA, the protein resolved on SDS-PAGE and the disulfide-bonded cysteine thiols alkylated with ¹³C-IPA following reduction with DTT. The protein was digested with trypsin, 17 peptides (Table S1) encompassing cysteines representing 13 of the 17 fibrinogen disulfide bonds were analyzed by HPLC and mass spectrometry and the redox state of the disulfides quantified. b Example of ¹²C-IPA-labeled fibrinogen resolved on SDS-PAGE. Molecular mass standards are shown in the left-hand lane. Fibrinogen has a molecular mass of 340 kDa. c Ribbon structure of fibrinogen and the positions of the 13 disulfide bonds (red spheres for the D region disulfides and cyan spheres for the E region bonds) that were mapped¹³ (PDB identifier 3ghg). The α-chains are in wheat, β-chains in light blue and γ chains in light green. d Redox states of the five E region and eight D region disulfides in ten healthy human donors (six female—solid symbols; four male—open symbols). The bars and errors are mean ± SD. Source data are provided as a Source Data file.