Recombinant BbetaArg14His fibrinogen implies participation of N-terminus of Bbeta chain in desA fibrin polymerization.
We synthesized BbetaArg14His fibrinogen with histidine substituted for arginine at the Bbeta thrombin-cleavage site. This substitution led to a 300-fold decrease in the rate of thrombin-catalyzed fibrinopeptide B (FpB, Bbeta 1-14) release, whereas the rate of FpA release was normal with either thrombin or the FpA-specific enzyme, batroxobin. Both thrombin- and batroxobincatalyzed polymerization of BbetaArg14His fibrinogen were significantly impaired, with a longer lag time, slower rate of lateral aggregation, and decreased final turbidity. Moreover, desA monomer polymerization was similarly impaired, demonstrating that the histidine substitution itself, and not the lack of FpB cleavage, caused the abnormal polymerization of BbetaArg14His fibrin. Scanning electron microscopy showed BbetaArg14His fibrin fibers were thinner than normal (BbetaArg14His, approximately 70 nm; normal, approximately 100 nm; P <.0001), as expected from the decreased final turbidity. We conclude that the N-terminus of the Bbeta chain is involved in the lateral aggregation of normal desAprotofibrils and that the Arg-->His substitution disrupts these interactions in BbetaArg14His fibrinogen.
Moen
Jennifer L
JL
Department of Chemistry, University of North Carolina at Chapel Hill, USA.
Gorkun
Oleg V
OV
Weisel
John W
JW
Lord
Susan T
ST
D019943:Amino Acid Substitution / Q000502:physiology; D000818:Animals; D012103:Batroxobin / Q000378:metabolism / Q000494:pharmacology; D001777:Blood Coagulation / Q000187:drug effects / Q000502:physiology; D006224:Cricetinae; D005337:Fibrin / Q000378:metabolism* / Q000648:ultrastructure; D005340:Fibrinogen / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006490:Hemostatics / Q000494:pharmacology; D006801:Humans; D008855:Microscopy, Electron, Scanning; D016296:Mutagenesis / Q000502:physiology; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D011108:Polymers / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology
D006490:Hemostatics; D010446:Peptide Fragments; D011108:Polymers; D011994:Recombinant Proteins; C415348:desA-profibrin; D005337:Fibrin; D005340:Fibrinogen; D012103:Batroxobin; D013917:Thrombin