Identification of the α Chain Lysine Donor Sites Involved in Factor XIIIa Fibrin Cross-linking

Abstract

Biochemical studies of fibrin cross-linking were conducted to identify the specific Aα chain lysine residues that potentially serve as Factor XIIIa amine donor substrates during α polymer formation. A previously characterized Factor XIIIa fibrin lysine labeling system was employed to localize sites of donor activity based on their covalent incorporation of a synthetic peptide acceptor substrate analog modelled after the NH2-terminal cross-linking domain of α2 antiplasmin. Peptide-decorated fibrin was prepared using purified fibrinogen as the starting material. Cyanogen bromide digestion, immunoaffinity chromatography, high pressure liquid chromatography (HPLC), and enzyme-linked immunosorbent assay (anti-peptide) methodologies were employed to isolate purified CNBr fibrin fragments whose structures included the acceptor probe in cross-linked form and, therefore, represented regions of (amine) donor activity. Five α chain CNBr fragments (within Aα 208-610) and one γ chain CNBr fragment (γ 385-411) were the only portions of fibrin found associated with the acceptor peptide, based on collective sequencing, mass, and compositional data. Trypsin digestion, HPLC, and enzyme-linked immunosorbent assay (anti-peptide) methodologies were used to isolate smaller derivatives whose structures included an α chain tryptic cleavage product (the donor arm) cross-linked to the trypsin-resistant synthetic peptide (the acceptor arm). Biochemical characterization and quantitative peptide recovery data revealed that 12 of the 23 potential lysine donor residues within α 208-610 had incorporated the peptide probe, whereas γ chain donor activity was due solely to peptide cross-linking at (γ) Lys406; the α chain lysines, Lys556 and Lys580, accounted for 50% of the total α chain donor cross-linking activity observed, with Lys539, Lys508, Lys418, and Lys448 contributing an additional 28% and Lys601, Lys606, Lys427, Lys429, Lys208, Lys224, and/or Lys219 responsible for the remaining proportion (2-5%, each). The collective findings extend current models proposed for the mechanism of α polymer formation, raise questions concerning the physiological role of multiple α chain donor sites, and, most importantly, provide specific information that should facilitate future efforts to identify the respective lysine and glutamine partners involved in native fibrin α chain cross-linking. Biochemical studies of fibrin cross-linking were conducted to identify the specific Aα chain lysine residues that potentially serve as Factor XIIIa amine donor substrates during α polymer formation. A previously characterized Factor XIIIa fibrin lysine labeling system was employed to localize sites of donor activity based on their covalent incorporation of a synthetic peptide acceptor substrate analog modelled after the NH2-terminal cross-linking domain of α2 antiplasmin. Peptide-decorated fibrin was prepared using purified fibrinogen as the starting material. Cyanogen bromide digestion, immunoaffinity chromatography, high pressure liquid chromatography (HPLC), and enzyme-linked immunosorbent assay (anti-peptide) methodologies were employed to isolate purified CNBr fibrin fragments whose structures included the acceptor probe in cross-linked form and, therefore, represented regions of (amine) donor activity. Five α chain CNBr fragments (within Aα 208-610) and one γ chain CNBr fragment (γ 385-411) were the only portions of fibrin found associated with the acceptor peptide, based on collective sequencing, mass, and compositional data. Trypsin digestion, HPLC, and enzyme-linked immunosorbent assay (anti-peptide) methodologies were used to isolate smaller derivatives whose structures included an α chain tryptic cleavage product (the donor arm) cross-linked to the trypsin-resistant synthetic peptide (the acceptor arm). Biochemical characterization and quantitative peptide recovery data revealed that 12 of the 23 potential lysine donor residues within α 208-610 had incorporated the peptide probe, whereas γ chain donor activity was due solely to peptide cross-linking at (γ) Lys406; the α chain lysines, Lys556 and Lys580, accounted for 50% of the total α chain donor cross-linking activity observed, with Lys539, Lys508, Lys418, and Lys448 contributing an additional 28% and Lys601, Lys606, Lys427, Lys429, Lys208, Lys224, and/or Lys219 responsible for the remaining proportion (2-5%, each). The collective findings extend current models proposed for the mechanism of α polymer formation, raise questions concerning the physiological role of multiple α chain donor sites, and, most importantly, provide specific information that should facilitate future efforts to identify the respective lysine and glutamine partners involved in native fibrin α chain cross-linking.