Nonclottable fibrin obtained from partially reduced fibrinogen: characterization and tissue plasminogen activator stimulation.
Out of 29 disulfide bonds in human fibrinogen, 7 were cleaved during limited reduction under nondenaturing conditions in calcium-free buffer: 2 A alpha 442Cys-A alpha 472Cys and 2 gamma 326Cys-gamma 339Cys intrachain disulfide bonds in the carboxy-terminal ends of the A alpha- and gamma-chains and the symmetrical disulfide bonds at gamma 8Cys, gamma 9Cys, and A alpha 28Cys. We studied the loss of thrombin clottability that followed limited reduction and the increase in the susceptibility of the fibrinogen A alpha 19-A alpha 20 bond to hydrolysis by thrombin. Using differential scanning calorimetry, we show that the extent of unfolding and denaturation of specific domains following limited reduction is small. Heat absorption peaks corresponding to the melting of the major regions of compact structure give high calorimetric enthalpies, as in untreated nonreduced fibrinogen, indicating that substantial regions of native structure are still present in partially reduced fibrinogen. Thrombin releases fibrinopeptide A at an identical rate as in nonreduced fibrinogen while fibrinopeptide B release is slower. Sedimentation velocity studies show that thrombin treatment leads to complex formation; however, gelation does not occur. Amino-terminal analysis indicates that the second thrombin cleavage in the A alpha-chain at A alpha 19-A alpha 20 takes place only after fibrinopeptide A release. Thus, the loss of clottability appears to result from perturbation of carboxy-terminal polymerization sites, probably a consequence of gamma 326Cys-gamma 339Cys intrachain disulfide bond cleavage. The thrombin-treated partially reduced fibrinogen remains soluble in buffered saline and fully expresses at least one epitope, B beta 15-21, unique to fibrin. Furthermore, this nonclottable form accelerates the tissue plasminogen activator dependent conversion of plasminogen to plasmin.
Procyk
R
R
Blood Coagulation Biochemistry, New York Blood Center 10021.
Medved
L
L
Engelke
K J
KJ
Kudryk
B
B
Blombäck
B
B
D000478:Alkylation; D000595:Amino Acid Sequence; D001777:Blood Coagulation / Q000187:drug effects*; D004220:Disulfides / Q000737:chemistry; D005337:Fibrin / Q000737:chemistry / Q000187:drug effects / Q000302:isolation & purification*; D005340:Fibrinogen / Q000737:chemistry* / Q000187:drug effects; D006801:Humans; D008969:Molecular Sequence Data; D011108:Polymers; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000494:pharmacology; D010959:Tissue Plasminogen Activator / Q000494:pharmacology*
D004220:Disulfides; D011108:Polymers; D005337:Fibrin; D005340:Fibrinogen; D013917:Thrombin; D010959:Tissue Plasminogen Activator