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Blood, 1 June 2004, Vol. 103, No. 11, pp. 4157-4163.
Prepublished online as a Blood First Edition Paper on February 5, 2004; DOI 10.1182/blood-2003-12-4296.
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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Substitution of the  -chain Asn308 disturbs the D:D interface affecting fibrin polymerization, fibrinopeptide B release, and FXIIIa-catalyzed cross-linking
Nobuo Okumura,
Oleg V. Gorkun,
Fumiko Terasawa, and
Susan T. Lord
From the Laboratory of Clinical Chemistry, Department of Biomedical Laboratory Sciences, School of Health Sciences, Shinshu University, Matsumoto, Japan; and the Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill.
Crystallographic structures indicate that  -chain residue Asn308 participates in D:D interactions and indeed substitutions of Asn308 with lysine or isoleucine have been identified in dysfibrinogens with impaired polymerization. To probe the role of Asn308 in polymerization, we synthesized 3 variant fibrinogens: Asn308 changed to lysine (N308K), isoleucine (N308I), and alanine (N308A). We measured thrombin-catalyzed polymerization by turbidity, fibrinopeptide release by high-performance liquid chromatography, and factor XIIIa–catalyzed cross-linking by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. In the absence of added calcium, polymerization was clearly impaired with all 3 variants. In contrast, at 0.1 mM calcium, only polymerization of N308K remained markedly abnormal. The release of thrombin-catalyzed fibrinopeptide B (FpB) was delayed in the absence of calcium, whereas at 1 mM calcium FpB release was delayed only with N308K. Factor XIIIa–catalyzed - dimer formation was delayed with fibrinogen (in absence of thrombin), whereas with fibrin (in presence of thrombin) - dimer formation of only N308K was delayed. These data corroborate the recognized link between FpB release and polymerization. They show fibrin cross-link formation likely depends on the structure of protofibrils. Together, our results show substitution of Asn308 with a hydrophobic residue altered neither polymer formation nor polymer structure at physiologic calcium concentrations, whereas substitution with lysine altered both.
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