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 A␣ chain lysine residues that potentially serve as Factor XIII a amine donor substrates during ␣ polymer formation. A previously characterized Factor XIII a 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 NH 2 -terminal cross-linking domain of ␣ 2 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 ␣ chain CNBr fragments (within A␣ 208 -610) and one ␥ chain CNBr fragment (␥ 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 ␣ 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 ␣ 208 -610 had incorporated the peptide probe, whereas ␥ chain donor activity was due solely to peptide cross-linking at (␥) Lys , and/or Lys 219 responsible for the remaining proportion (2-5%, each). 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.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 (1-6), its domainal architecture has been characterized (7-9), 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). Although these aspects of fibrinogen biochemistry are well understood, less is known about the final stages of fibrin formation in which Factor XIII a cross-links are introduced between neighboring fibrin molecules to stabilize the alignments created during polymerization.Factor XIII a , or plasma transglutaminase, catalyzes the introduction of ⑀-(␥-glutamy...

The location of the carboxy-terminal region of γ chains in fibrinogen and fibrin D domains

“…Fraction I-9 fibrinogen was prepared as described (30). Fibrinogen molecules in this preparation lack Ϸ100 or more C-terminal A␣ chain residues (31), which contain almost all of the lysine amine donor sites (16,17), and therefore they do not participate in ␣ chain crosslinking of fibrin (32). ␥ chain dimerization is, however, slightly slower but otherwise normal (1).…”

“…␥ chain crosslinking occurs solely between these residues (2-4, 15, 16), whereas there are several amine donor lysine and amine acceptor glutamine sites in A␣ chains (16,17). Primary amine compounds like cadaverine are competitive amine donor substrates that become specifically incorporated in the presence of factor XIIIa at the single ␥ chain acceptor site or at one or more ␣ chain acceptor sites (2-4, 15, 18, 19).…”

Interaction of Recombinant Procollagen and Properdin Modules of Thrombospondin-1 with Heparin and Fibrinogen/Fibrin

“…The x-ray crystal structure of the D-fragment shows that the ␣-chain doubles back upon itself, apposing the A␣-chain residues of 113-126 of the TSP1 binding site to three potential cross-link sites, Lys 208 , Lys 219 , and/or Lys 224 (56). Lys 208 , Lys 219 , and Lys 224 all were found cross-linked to the ␣ 2 -antiplasmin peptide, although none were a preferred site (used only 2-5% of the time) (55). The results are consistent with P123 binding to region(s) within the D-domain as directed by P123 and cross-linking to Lys 208 , Lys 219 , and Lys 224 within the D-domain via the flexible amino-terminal tail derived from GE1.…”

Cross-linking of Plasminogen Activator Inhibitor 2 and α2-Antiplasmin to Fibrin(ogen)

“…A␣ chains then polymerize to form higher molecular mass polymers; this reaction occurs more slowly than ␥-␥ dimer formation (4). Many lysine residues have been implicated as substrates for crosslinking and subsequent generation of high molecular mass oligomers of A␣ chain (5,6). It has been found that multimerization of ␥-␥ dimers can occur over extended time periods (7).…”