Abstract
Biochemical studies of fibrin cross-linking were conducted to identify the specific Aalpha chain lysine residues that potentially serve as Factor XIIIa amine donor substrates during alpha 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 alpha2 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 alpha chain CNBr fragments (within Aalpha 208-610) and one gamma chain CNBr fragment (gamma 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 alpha 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 alpha 208-610 had incorporated the peptide probe, whereas gamma chain donor activity was due solely to peptide cross-linking at (gamma) Lys406; the alpha chain lysines, Lys556 and Lys580, accounted for 50% of the total alpha 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 alpha polymer formation, raise questions concerning the physiological role of multiple alpha 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 alpha chain cross-linking.
Highlights
Methodologies were used to isolate smaller derivatives whose structures included an ␣ chain tryptic cleavage product cross-linked to the trypsin-resistant synthetic peptide
The collective findings extend current models proposed for the mechanism of ␣ polymer formation, raise questions concerning the physiological role of multiple ␣ 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 ␣ chain cross-linking
We describe a series of biochemical studies to identify the potential lysine donor sites involved in native ␣ chain cross-linking, taking advantage of the fact that in the lysine labeling methodology susceptible lysine residues are all cross-linked to the same acceptor peptide partner
Summary
Methodologies were used to isolate smaller derivatives whose structures included an ␣ chain tryptic cleavage product (the donor arm) cross-linked to the trypsin-resistant synthetic peptide (the acceptor arm). The structure-function relationships involved in fibrinogen’s transition to the cross-linked fibrin gel that forms the hemostatically active portion of a thrombus has been the subject of intense investigation for more than two decades During this time, the complete primary structure of this large molecule has been elucidated [1,2,3,4,5,6], its domainal architecture has been characterized [7,8,9], and the mechanisms involved in the initial events of the transition, i.e. thrombin cleavage and fibrin polymerization, have been defined (as reviewed in Ref. 10). We describe a series of biochemical studies to identify the potential lysine donor sites involved in native ␣ chain cross-linking, taking advantage of the fact that in the lysine labeling methodology susceptible lysine residues are all cross-linked to the same acceptor peptide partner. The identification of the specific lysines involved in peptide-␣ chain cross-linking here should facilitate future efforts to characterize the respective glutamine and lysine partner residues involved in native fibrin cross-linking
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