Identification of the a Chain Lysine Donor Sites Involved in Factor

Biochemical studies of fibrin cross-linking were conducted to identify the specific Aa chain lysine residues that potentially serve as Factor XIIIa amine donor substrates during a 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 a2 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 a chain CNBr fragments (within Aa 208‐610) and one g chain CNBr fragment (g 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 a 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 a 208‐610 had incorporated the peptide probe, whereas g chain donor activity was due solely to peptide cross-linking at (g) Lys 406 ; the a chain lysines, Lys 556 and Lys 580 , accounted for 50% of the total a chain donor cross-linking activity observed, with Lys 539 , Lys 508 , Lys 418 , and Lys 448 contributing an additional 28% and Lys 601 , Lys 606 , Lys 427 , Lys 429 , Lys 208 , Lys 224 , and/or Lys 219 responsible for the remaining proportion (2‐5%, each). The collective findings extend current models proposed for the mechanism of a polymer formation, raise questions concerning the physiological role of multiple a 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 a chain cross-linking.