Substitution of the gamma-chain Asn308 disturbs the D:D interface affecting fibrin polymerization, fibrinopeptide B release, and FXIIIa-catalyzed cross-linking.
Crystallographic structures indicate that gamma-chain residue Asn308 participates in D:D interactions and indeed substitutions of gammaAsn308 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: gammaAsn308 changed to lysine (gammaN308K), isoleucine (gammaN308I), and alanine (gammaN308A). 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 gammaN308K 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 gammaN308K. Factor XIIIa-catalyzed gamma-gamma dimer formation was delayed with fibrinogen (in absence of thrombin), whereas with fibrin (in presence of thrombin) gamma-gamma dimer formation of only gammaN308K 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.
Okumura
Nobuo
N
Laboratory of Clinical Chemistry, Department of Biomedical Laboratory Sciences, School of Health Sciences, Shinshu University, 3-1-1 Asahi, Matsumoto 390-8621, Japan. nobuoku@gipac.shinshu-u.ac.jp
Gorkun
Oleg V
OV
Terasawa
Fumiko
F
Lord
Susan T
ST
D019943:Amino Acid Substitution; D002118:Calcium / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000378:metabolism; D026122:Factor XIIIa / Q000378:metabolism*; D005337:Fibrin / Q000378:metabolism*; D005340:Fibrinogen / Q000235:genetics* / Q000378:metabolism*; D005345:Fibrinopeptide B / Q000378:metabolism*; D006490:Hemostatics / Q000494:pharmacology; D006801:Humans; D011108:Polymers / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology
D003432:Cross-Linking Reagents; D006490:Hemostatics; D011108:Polymers; D005345:Fibrinopeptide B; D005337:Fibrin; D005340:Fibrinogen; D026122:Factor XIIIa; D013917:Thrombin; D002118:Calcium