Substitution of the gamma-chain Asn308 disturbs the D:D interface affecting fibrin polymerization, fibrinopeptide B release, and FXIIIa-catalyzed cross-linking.

Abstract:

: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.

journal_name

Blood

journal_title

Blood

authors

Okumura N,Gorkun OV,Terasawa F,Lord ST

doi

10.1182/blood-2003-12-4296

subject

Has Abstract

pub_date

2004-06-01 00:00:00

pages

4157-63

issue

11

eissn

0006-4971

issn

1528-0020

pii

2003-12-4296

journal_volume

103

pub_type

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