Key Points• The K d for the association of FXIII subunits is in the range of 10 210 M, and in plasma approximately 1% of FXIII-A 2 exists in free form.• The binding site for FXIII-A is located within the 2 N-terminal sushi domains of FXIII-B.Coagulation factor XIII (FXIII) is a heterotetramer consisting of 2 catalytic A subunits (FXIII-A 2 ) and 2 protective/inhibitory B subunits (FXIII-B 2 ). FXIII-B, a mosaic protein consisting of 10 sushi domains, significantly prolongs the lifespan of catalytic subunits in the circulation and prevents their slow progressive activation in plasmatic conditions. In this study, the biochemistry of the interaction between the 2 FXIII subunits was investigated. , it can cross-link fibrin. In cerebrospinal fluid and tears with much lower FXIII subunit concentrations, >80% of FXIII-A 2 existed in free form. A monoclonal anti-FXIII-B antibody that prevented the interaction between the 2 subunits reacted with the recombinant combined first and second sushi domains of FXIII-B, and its epitope was localized to the peptide spanning positions 96 to 103 in the second sushi domain. (Blood.

To cite this article: Pé nzes K, Kö vé r KE, Fazakas F, Haramura G, Muszbek L. Molecular mechanism of the interaction between activated factor XIII and its glutamine donor peptide substrate. J Thromb Haemost 2009; 7: 627-33.Summary. Background: Activated factor XIII (FXIII), a dimer of truncated A-subunits (FXIII-A 2 *), is a transglutaminase that crosslinks primary amines to peptide-bound glutamine residues. Because in the few natural substrates of FXIII-A 2 * no consensus sequence could be identified around the reactive glutamine, studying the interaction between individual substrates and FXIII-A 2 * is of primary importance. Most of the a 2 -plasmin inhibitor (a 2 PI) molecules become truncated by a plasma protease, and the truncated isoform (N1-a 2 PI) is an important substrate of FXIII-A 2 *. The crosslinking of N1-a 2 PI to fibrin plays a major role in protecting fibrin from fibrinolysis. Methods: We studied the interaction of FXIII-A 2 * with its dodecapeptide glutamine donor substrate, N1-a 2 PI(1-12), the sequence of which corresponds to the N-terminal sequence of N1-a 2 PI. Kinetic parameters for N1-a 2 PI(1-12) and for its truncated or synthetic mutants were determined by a spectrophotometric assay. The interaction of N1-a 2 PI(1-12) with FXIII-A 2 * was investigated by proton nuclear magnetic resonance (NMR) and saturating transfer difference (STD) NMR. Results and Conclusions: Kinetic experiments with peptides in which the Asn1 residue was either truncated or replaced by alanine and proton NMR analysis of the FXIII-A 2 *-N1-a 2 PI(1-12) complex demonstrated that Asn1 is essential for effective enzyme-substrate interaction. Experiments with C-terminally truncated peptides proved that amino acids 7-12 are essential for the interaction of N1-a 2 PI(1-12) with the enzyme, and suggested the existence of a secondary binding site on FXIII-A 2 *. Hydrophobic residues, particularly Leu10 and the C-terminal Lys12, seemed to be especially important in this respect, and direct interaction between hydrophobic C-terminal residues and FXIII-A 2 * was demonstrated by STD NMR.

Summary. Background: The consensus thrombin aptamer C15-mer is a single-stranded DNA of 15 nucleotides [d(GGTTGGTGTGGTTGG)] that was identified by the selection of thrombin-binding molecules from a large combinatorial library of oligonucleotides. It is capable of inhibiting thrombin at nanomolar concentrations through binding to a specific region within thrombin exosite 1. As has been shown in our earlier studies, the 4-thio-deoxyuridylate (s4dU)-containing oligonucleotides have high affinity for a number of proteins, due to the reduced hydrophilic character of the modified oligonucleotide. Methods: Three different analogs of the original thrombin-inhibiting sequence, in which some of the thymidylate residues were replaced by 4-thio-deoxyuridylates, were synthesized. The inhibitory effect of modified aptamers was tested on thrombin-catalyzed fibrin clot formation and fibrinopeptide A release from fibrinogen, thrombin-induced platelet aggregation/ secretion, and the formation of thrombus on coverslips coated with human collagen type III, thrombin-treated fibrinogen or subendothelial matrix of human microvascular endothelial cells. Results: As compared with the C15-mer, the analog with the sequence GG(s4dU)TGG(s4dU)G(s4dU)GGT(s4dU)GG (UC15-mer) showed a 2-fold increased inhibition of thrombincatalyzed fibrin clot formation, fibrinopeptide A release, platelet aggregation and secretion in human plasma and thrombus formation on thrombin-treated fibrinogen surfaces under flow conditions. Concerning the inhibition of thrombininduced fibrin formation from purified fibrinogen and activation of washed platelets, UC15-mer was 3-fold and twelve-fold more effective than C15-mer, respectively. Conclusion: The replacement of four thymidylate residues in C15-mer by 4-thio-deoxyuridylate resulted in a new thrombin aptamer with increased anticoagulant and antithrombotic properties.