Factor VII and single-chain plasminogen activator-activating protease: activation and autoactivation of the proenzyme.
Structural and biological characteristics of a recently described plasma serine protease, which displayed factor VII as well as pro-urokinase-activating properties in vitro, indicated a dual role for this factor VII-activating protease (FSAP) in hemostasis. Only the active protease (two-chain FSAP) has been isolated from plasma and from a prothrombin complex concentrate, whereas activators of the proenzyme have not been identified so far. After purification of the FSAP proenzyme from cryo-poor plasma by adsorption to an immobilized mAb and subsequent ion-exchange chromatography, activation to generate two-chain FSAP was followed by a direct chromogenic assay as well as by the ability of two-chain FSAP to activate pro-urokinase. Purified single-chain FSAP underwent autoactivation leading to the typical protease two-chain pattern and subsequent degradation products, as demonstrated by Western-blotting analysis using a site-specific mAb. This autoactivation was significantly enhanced in the presence of heparin, whereas Ca2+ ions stabilized single-chain FSAP (the proenzyme) resulting in slower autoactivation kinetics. Correspondingly, the heparin-augmented reaction, which was associated with autodegradation particularly of the protease domain, was slowed down by co-incubation with Ca2+. Of the other proteases and cofactors tested, only urokinase (uPA) was able to generate the typical two-chain FSAP pattern. Studies with different forms of uPA suggest that the catalytic activity of pro-urokinase/uPA is needed to activate single-chain FSAP, indicating that it is the only hemostatic protease that can act as a physiological activator of FSAP.
Kannemeier
C
C
Aventis Behring GmbH, Research, Marburg, Germany.
Feussner
A
A
Stöhr
H A
HA
Weisse
J
J
Preissner
K T
KT
Römisch
J
J
D001777:Blood Coagulation; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005167:Factor VII / Q000378:metabolism*; D005342:Fibrinolysis; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D009005:Monosaccharides / Q000032:analysis; D011499:Protein Processing, Post-Translational; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D004792:Enzyme Precursors; D009005:Monosaccharides; D005167:Factor VII; C433550:HABP2 protein, human; D012697:Serine Endopeptidases