Purification and characterization of a plasminogen activator inhibitor 1 binding protein from human plasma. Identification as a multimeric form of S protein (vitronectin)

A binding protein for plasminogen activator inhibitor 1 (PAI-1-BP) was isolated from human plasma by a four-step procedure. 1) The 7 S globulin fraction of plasma was isolated by gel filtration on Sephacryl S-300. 2) Human endothelial cell-type plasminogen activator inhibitor (PAI-1), pretreated with 12 M urea, was added to this fraction (22 micrograms of PAI-1/ml of plasma), and a PAI-1 antigen peak with apparent mass 450 kDa (representing 65% of PAI-1 antigen and 85% of PAI activity) was isolated by gel filtration of this mixture. 3) The PAI-1.PAI-1-BP complex was further purified by immunoadsorption on an immobilized murine monoclonal antibody directed against PAI-1 (MA-7D4) and by elution with 4 M KSCN. 4) The complex was then dissociated by addition of excess human tissue-type plasminogen activator (t-PA), and t-PA and PAI-1 antigen (t-PA.PAI-1 complexes and free t-PA and PAI-1) were removed by immunoadsorption on monoclonal antibodies directed against t-PA (MA-62E8) and against PAI-1 (MA-7D4 and MA-12A4). Sodium dodecyl sulfate-gel electrophoresis of the purified material under nonreducing conditions revealed two bands with apparent mass approximately equal to 150 kDa and two bands with mass 74 and 68 kDa. Reduced sodium dodecyl sulfate-gel electrophoresis displayed two main bands with apparent masses of 73 and 64 kDa. The PAI-1-BP reacts with urea-treated, but not with inactive PAI-1. t-PA dissociates the complex between PAI-1 and PAI-1-BP. PAI-1 in complex with PAI-1-BP is 2-3-fold more stable at 37 degrees C than purified PAI-1, suggesting that PAI-1-BP may stabilize PAI-1 in blood. The concentration of PAI-1-BP in plasma determined by titration with PAI-1 is approximately 130 mg/liter. The isolated PAI-1-BP was shown to be identical to S protein (vitronectin) both by cross-reactivity with monospecific rabbit antisera and by NH2-terminal amino acid sequence analysis. The gel filtration behavior, mobility on sodium dodecyl sulfate-gel electrophoresis, and concentration in plasma suggest that PAI-1-BP is a multimer (presumably a dimer) of S protein accounting for approximately 35% of the S protein in plasma.     

Identification of the human plasma protein which inhibits fibrinolysis associated with malignant cells

Mixed cultures of mouse fibroblasts and mouse fibroblasts transformed with Kirsten murine sarcoma virus were grown in petri dishes and overlayed with casein. The appearance of focal lysis zones required the presence of transformed cells in the culture and plasminogen in the overlay, indicating that caseinolysis was due to plasminogen activator released by the malignant cells. Caseinolysis was inhibited by addition of human plasma or bovine pancreatic trypsin inhibitor to the overlay, 1 ml of plasma being equivalent to 67 ± 18 (mean ± S.E.) kallikrein inhibitor (KI) units of trypsin inhibitor.The culture fluid of a human melanoma line induced lysis of a fibrin clot, 1 ml of culture fluid being equivalent to 250 CTA units of urokinase (EC 3.4.99.26). Fibrinolysis was inhibited by addition of human plasma or trypsin inhibitor, 1 ml of plasma being equivalent to 94 ± 34 KI units of trypsin inhibitor.Specific removal of antiplasmin, the fast-reacting plasmin inhibitor (Collen, D. (1976) Eur. J. Biochem. 69, 209), from plasma by immunoabsorption completely abolished its inhibitory activity, both in the caseinolytic and fibrinolytic assays. It is therefore concluded that antiplasmin is the only protein in human plasma capable of inhibiting the fibrinolytic activity associated with oncogenic transformation or neoplasia. Whether this effect is exclusively due to inhibition of formed plasmin or also to interference with plasminogen activvtion remains unsettled.     

Critical role of glutamic acid 202 in the enzymatic activity of stromelysin-1 (MMP-3).

To test the hypothesis that Glu202, adjacent to the His201 residue that participates in the coordination of Zn(2+) in matrix metalloproteinase-3 (MMP-3 or stromelysin-1), plays a role in its enzymatic activity it was substituted with Ala, Lys or Asp by site-specific mutagenesis. Wild-type proMMP-3, proMMP-3(E202A), proMMP-3(E202K) and proMMP-3(E202D) were expressed in Escherichia coli and purified to apparent homogeneity. Whereas 33-kDa wild-type proMMP-3 (consisting of the propeptide and catalytic domains) was quantitatively converted to 24-kDa active MMP-3 by treatment with p-aminophenyl-mercuric acetate (APMA), proMMP-3(E202A) and proMMP-3 (E202K) were fully resistant to APMA and proMMP-3 (E202D) was quantitatively converted into a 14-kDa species. In contrast, treatment with plasmin quantitatively converted the wild-type and the three mutant proMMP-3 moieties into the corresponding 24-kDa MMP-3 moieties. Biospecific interaction analysis revealed comparable affinity for binding to plasminogen of wild-type and mutant proMMP-3 (K(a) of 2.6-6.3 x 10(6) M(-1)) or MMP-3 (K(a) of 33-58 x 10(6) M(-1)) moieties. The affinity for binding to single-chain urokinase-type plasminogen activator (scu-PA) was also similar for wild-type and mutant proMMP-3 (K(a) of 5.0-6.9 x 10(6) M(-1)) or MMP-3 (K(a) of 37-72 x 10(6) M(-1)) moieties. However, MMP-3(E202A) and MMP-3(E202K) did not hydrolyze plasminogen whereas MMP-3(E202D) showed an activity of 20--30% of wild-type MMP-3. All three mutants were inactive towards scu-PA under conditions where this was quantitatively cleaved by wild-type MMP-3. Furthermore, MMP-3(E202A) and MMP-3(E202K) were inactive toward a fluorogenic substrate and MMP-3 (E202D) displayed about 15% of the activity of wild-type MMP-3. Taken together, these data suggest that Glu202 plays a crucial role in the enzymatic activity of MMP-3.     

Abrupt rate accelerations or premature beats cause life-threatening arrhythmias in mice with long-QT3 syndrome

Deletion of amino-acid residues 1505-1507 (KPQ) in the cardiac SCN5A Na(+) channel causes autosomal dominant prolongation of the electrocardiographic QT interval (long-QT syndrome type 3 or LQT3). Excessive prolongation of the action potential at low heart rates predisposes individuals with LQT3 to fatal arrhythmias, typically at rest or during sleep. Here we report that mice heterozygous for a knock-in KPQ-deletion (SCN5A(Delta/+)) show the essential LQT3 features and spontaneously develop life-threatening polymorphous ventricular arrhythmias. Unexpectedly, sudden accelerations in heart rate or premature beats caused lengthening of the action potential with early afterdepolarization and triggered arrhythmias in Scn5a(Delta/+) mice. Adrenergic agonists normalized the response to rate acceleration in vitro and suppressed arrhythmias upon premature stimulation in vivo. These results show the possible risk of sudden heart-rate accelerations. The Scn5a(Delta/+) mouse with its predisposition for pacing-induced arrhythmia might be useful for the development of new treatments for the LQT3 syndrome.     

Inhibition of plasminogen activators or matrix metalloproteinases prevents cardiac rupture but impairs therapeutic angiogenesis and causes cardiac failure.

Cardiac rupture is a fatal complication of acute myocardial infarction lacking treatment. Here, acute myocardial infarction resulted in rupture in wild-type mice and in mice lacking tissue-type plasminogen activator, urokinase receptor, matrix metalloproteinase stromelysin-1 or metalloelastase. Instead, deficiency of urokinase-type plasminogen activator (u-PA-/-) completely protected against rupture, whereas lack of gelatinase-B partially protected against rupture. However, u-PA-/- mice showed impaired scar formation and infarct revascularization, even after treatment with vascular endothelial growth factor, and died of cardiac failure due to depressed contractility, arrhythmias and ischemia. Temporary administration of PA inhibitor-1 or the matrix metalloproteinase-inhibitor TIMP-1 completely protected wild-type mice against rupture but did not abort infarct healing, thus constituting a new approach to prevent cardiac rupture after acute myocardial infarction.     

Kinetics of the reactions between streptokinase, plasmin and alpha 2-antiplasmin.

Streptokinase reacts very rapidly with human plasmin (rate constant 5.4 S 10(7) M-1 s-1) forming a 1:1 stoichiometric complex which has a dissociation constant of 5 X 10(-11) M. This plasmin-streptokinase complex is 10(5) times less reactive towards alpha 2-antiplasmin than plasmin, the inhibition rate constant being 1.4 X 10(2) M-1 s-1. The loss of reactivity of the streptokinase-plasmin complex towards alpha 2-antiplasmin is independent of the lysine binding sites in plasmin since low-Mr plasmin, which lacks these sites, and plasmin in which the sites have been blocked by 6-aminohexanoic acid, are both equally unreactive towards alpha 2-antiplasmin on reaction with streptokinase. The plasmin-streptokinase complex binds to Sepharose-lysine and Sepharose-fibrin monomer in the same fashion as free plasmin, showing that the lysine binding sites are fully exposed in the complex. Bovine plasmin is rapidly inhibited by human alpha 2-antiplasmin (k1 = 1.6 X 10(6) M-1 s-1) and similarly loses reactivity towards the inhibitor on complex formation with streptokinase (50% binding at 0.4 microM streptokinase).     

A monoclonal antibody specific for Lys-plasminogen. Application to the study of the activation pathways of plasminogen in vivo

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.