Purification and characterization of a plasma factor which cleaves single-chain form of t-PA and u-PA.
Low molecular weight heparin (LMW-heparin) enhanced the amidolytic activity of plasma when the chromogenic substrate, H-D-Ile-Pro-Arg-pNA (S-2288), was used. The amidolytic activity increased in a time-dependent manner as the LMW-heparin concentration increased and reached its peak at around 15 mu/ml. Factor XII-deficient plasma increased the S-2288 amidolytic activity by LMW-heparin. In order to clarify the mechanism of the heparin-induced enhancement of the amidolytic activity, a plasma factor was purified. The plasma factor was obtained from human normal plasma by ammonium sulfate fractionation, followed by successive column chromatography with heparin-Sepharose, zinc chelate-Sepharose, aprotinin-Sepharose and protein A-Sepharose. The plasma factor so purified revealed a major band (88% of total protein) at 80 kD with several minor bands on analysis by SDS-PAGE. The plasma factor exhibited an intrinsic amidolytic activity, which was enhanced by heparin. The plasma factor further enhanced the amidolytic activity of sct-PA and scu-PA, the enhancement of which was of much greater degree than that for LMW-heparin. However, when the two-chain form of t-PA or u-PA was reacted with the plasma factor and LMW-heparin, no enhancement of the amidolytic activity of these enzymes was observed. The plasma factor cleaved a peptide bond of sct-PA and scu-PA and induced a structural change from a single-chain to a two-chain form. The amidolytic activity of the plasma factor was not inhibited by anti-t-PA IgG, anti-u-PA IgG, anti-plasminogen IgG, anti-factor XII IgG or anti-plasma prekallikrein IgG. These findings suggest an important role for the plasma factor in the activation of sct-PA and scu-PA in heparin-dependent fibrinolysis.
Okada
K
K
Department of Physiology, Kinki University School of Medicine, Osakasayama, Japan.
Fukao
H
H
Tanaka
H
H
Ueshima
S
S
Matsuo
O
O
D000577:Amides / Q000378:metabolism; D001685:Biological Factors / Q000037:antagonists & inhibitors / Q000097:blood / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D006493:Heparin / Q000494:pharmacology; D006495:Heparin, Low-Molecular-Weight / Q000494:pharmacology; D006801:Humans; D007120:Immunochemistry; D010958:Plasminogen / Q000302:isolation & purification; D010960:Plasminogen Activators / Q000302:isolation & purification / Q000378:metabolism*; D010959:Tissue Plasminogen Activator / Q000302:isolation & purification / Q000378:metabolism*; D014568:Urokinase-Type Plasminogen Activator / Q000302:isolation & purification / Q000378:metabolism*
D000577:Amides; D001685:Biological Factors; D006495:Heparin, Low-Molecular-Weight; D010958:Plasminogen; D006493:Heparin; D010960:Plasminogen Activators; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator