Abstract
Human plasminogen, a glycoprotein with NH2-terminal Glu, is rapidly converted by traces of plasmin to proteolytic derivatives with NH2-terminal Met 68, Lys 77, or Val 78 ("Lys-plasminogen"), which are much more readily activated to plasmin than is Glu-plasminogen. It has, therefore, been proposed that physiological activation of Glu-plasminogen occurs mainly via Lys-plasminogen intermediates (Wiman, B., and Wallén, P. (1973) Eur. J. Biochem. 36, 25-31). In the present study we have characterized a murine monoclonal antibody (LPm1) directed against an epitope exposed in Lys-plasminogen but not in Glu-plasminogen. The antibody was secreted by a hybridoma obtained by fusion of mouse myeloma cells (P3X63-Ag8-6.5.3) with spleen cells of a mouse immunized with purified Lys-plasmin-alpha 2-antiplasmin complex. Coupling of the alpha-amino groups of Lys-plasminogen with phenylisothiocyanate resulted in complete loss of immunoreactivity for LPm1, which was, however, fully restored by cleavage of the derivatized NH2-terminal amino acid. After a second cycle, immunoreactivity was not restored, indicating that the LPm1 antibody-binding site depends on the presence of Lys 77 and/or Val 78 as NH2-terminal amino acids. The immunoreactivity of Lys-plasminogen with LPm1 is abolished by reduction of the protein, suggesting that conversion of Glu-plasminogen to Lys-plasminogen is associated with a conformational alteration exposing the epitope for the LPm1 monoclonal antibody. In order to investigate the pathways of plasminogen activation in vivo, total plasmin-alpha 2-antiplasmin and Lys-plasmin-alpha 2-antiplasmin complexes were measured with sandwich-type micro enzyme-linked immunosorbent assays. Therefore, microtiter plates were coated with monoclonal antibodies against alpha 2-antiplasmin, and bound antigen was quantitated with horseradish peroxidase-conjugated LPm1 or a monoclonal antibody reacting equally well with Glu-plasmin as with Lys-plasmin. In 25 healthy subjects the plasmin-alpha 2-antiplasmin levels in plasma were undetectable (less than 0.1 nM). Infusion of tissue-type plasminogen activator in patients with thromboembolic disease resulted in generation of high concentrations of Glu-plasmin-alpha 2-antiplasmin complex (620 +/- 150 nM, n = 7) whereas neither Lys-plasmin-alpha 2-antiplasmin complex nor Lys-plasminogen were consistently detected. It is, therefore, concluded that activation of the fibrinolytic system in vivo occurs by direct cleavage of the Arg 560-Val 561 bond in Glu-plasminogen and not via formation of the Lys-plasminogen intermediates.
Highlights
Human plasminogen, a glycoprotein with NHz-ter- in Glu-plasminogen and not via formation of the Lysminal Glu,is rapidly converted by traces of plasmin to plasminogen intermediates
Microtiter plates were coated with monoclonal antibodies against az-antiplasmin, and bound antigen was quantitated with horseradish peroxidase-conjugatedLPml oar monoclonal antibody reacting well with Glu-plasmin as with Lysminogen, proteolytically modified forms of plasminogen with NH2terminal lysine, valine, or methionine (M, = 85,000), obtained by hydrolysis of the Arg 68-Met 69, Lys 76-Lys 77, or Lys 77-Val 78 peptide bonds, LBS I, a fragment of plasminogen isolated after digestion with elastase, consisting of triple-loop structures 1-3 ofthe plasmin A-chain ( M,= 35,000),previously shown to consist plasmin
Gluplasminogen, Lys-plasminogen, and Lys-plasmin were digested with elastase coupled to Sepharose, and the fractions containing low". plasminogen, LBS I, and LBS I1 were purified by gel filtration on Sephadex G-75 and affinity chromatography on lysine-Sepharose [14]
Summary
From the Center for Thrombosisand Vascular Research, University of Leuven, Leuven, Belgium. Microtiter plates were coated with monoclonal antibodies against az-antiplasmin, and bound antigen was quantitated with horseradish peroxidase-conjugatedLPml oar monoclonal antibody reacting well with Glu-plasmin as with Lysminogen, proteolytically modified forms of plasminogen with NH2terminal lysine, valine, or methionine (M, = 85,000), obtained by hydrolysis of the Arg 68-Met 69, Lys 76-Lys 77, or Lys 77-Val 78 peptide bonds, LBS I, a fragment of plasminogen isolated after digestion with elastase, consisting of triple-loop structures (kringles) 1-3 ofthe plasmin A-chain ( M ,= 35,000),previously shown to consist plasmin. Min-az-antiplasmin complex, but not with Glu-plasminogen or Glu-plasmin-az-antiplasmicnomplex With theuse of this antibody it was shown that activation of plasminogen in vivo yields Glu-plasmin-a2-antiplasmincomplex, indicating that physiological plasminogen activation does not occur via the Lys-plasminogen intermediate
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