Proteolytic processing of human coagulation factor IX by plasmin.
Previous studies have shown that thrombin generation in vivo caused a 92% decrease in factor IX (F.IX) activity and the appearance of a cleavage product after immunoblotting that comigrated with activated F.IX (F.IXa). Under these conditions, the fibrinolytic system was clearly activated, suggesting plasmin may have altered F.IX. Thus, the effect(s) of plasmin on human F.IX was determined in vitro. Plasmin (50 nM) decreased the 1-stage clotting activity of F.IX (4 microM) by 80% and the activity of F.IXa (4 microM) by 50% after 30 minutes at 37 degrees C. Plasmin hydrolysis of F.IX yields products of 45, 30, 20, and 14 kd on reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 2 products of 52 and 14 kd under nonreducing conditions. Plasmin-treated F.IX did not bind the active site probe, p-aminobenzamidine, or form an SDS-stable complex with antithrombin. It only marginally activated human factor X in the presence of phospholipid and activated factor VIII. Although dansyl-Glu-Gly-Arg-chloromethyl ketone inactivated-F. IXa inhibited the clotting activity of F.IXa, plasmin-treated F.IX did not. Plasmin cleaves F.IX after Lys43, Arg145, Arg180, Lys316, and Arg318, but F.IXa is not appreciably generated despite cleavage at the 2 normal activation sites (Arg145 and Arg180). Tissue plasminogen activator-catalyzed lysis of fibrin formed in human plasma results in generation of the 45- and 30-kd fragments of F.IX and decreased F.IX clotting activity. Collectively, the results suggest that plasmin is able to down-regulate coagulation by inactivating F.IX.
Samis
J A
JA
Departments of Pathology, Biochemistry, and Medicine; Queen's University, Kingston, Ontario, Canada. samisj@post.queensu.ca
Ramsey
G D
GD
Walker
J B
JB
Nesheim
M E
ME
Giles
A R
AR
D019943:Amino Acid Substitution; D000990:Antithrombin III / Q000378:metabolism; D001777:Blood Coagulation / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005164:Factor IX / Q000378:metabolism*; D015949:Factor IXa / Q000378:metabolism; D005337:Fibrin / Q000378:metabolism; D005341:Fibrinolysin / Q000378:metabolism*; D005342:Fibrinolysis; D006493:Heparin / Q000378:metabolism; D006801:Humans; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis / Q000302:isolation & purification; D020539:Sequence Analysis, Protein; D010959:Tissue Plasminogen Activator / Q000378:metabolism
D010446:Peptide Fragments; D000990:Antithrombin III; D005164:Factor IX; D005337:Fibrin; D006493:Heparin; D015949:Factor IXa; D010959:Tissue Plasminogen Activator; D005341:Fibrinolysin