Fluorescence polarization assay of plasmin, plasminogen, and plasminogen activator

A chromatographic method involving medium-pressure liquid chromatography on alumina impregnated with silver nitrate is described for the separation of a series of closely related C₂₇ sterol precursors of cholesterol differing only in the number and location of olefinic double bonds. The features of the described system are compared with those of previously described thin-layer, gas-liquid, gravity column, and high-pressure liquid chromatographic methods.     

A covalent molecular weight approximately 92,000 hybrid plasminogen activator derived from human plasmin fibrin-binding and tissue plasminogen activator catalytic domains

A covalent hybrid plasminogen activator was prepared from the sulfhydryl forms of the NH2-terminal heavy (A) chain of human plasmin (PlnA) containing the fibrin-binding domain and the COOH-terminal B chain of tissue plasminogen activator (t-PAB) containing the catalytic domain. The sulfhydryl form of PlnA [PlnA(SH)2] was isolated from reduced Lys-2-plasmin on an L-lysine-substituted Sepharose column, and the sulfhydryl form of t-PAB [t-PAB(SH)] was prepared from reduced two-chain tissue plasminogen activator (t-PA) by removing the tissue plasminogen activator NH2-terminal A chain (t-PAA) on an L-lysine-substituted Sepharose column from the chain mixture. The specific plasminogen activator activity, with soluble fibrin, of the isolated t-PAB(SH) chain was determined to be 62,700 international units (IU)/mg of protein, about 13% of the specific plasminogen activator activity of the parent t-PA. The PlnA(SH)2 and the t-PAB(SH) chains were mixed in a 1:1 molar ratio, and hybridization (reoxidation) was allowed to proceed by first dialyzing out the reducing agent at 4 degrees C and then concentrating the mixture. The time for maximum hybridization, or formation of the covalent hybrid activator, was 6 days, as determined by both specific plasminogen activator activity, with soluble fibrin, and specific amidolytic activity; sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the continual formation of an Mr approximately 92,000 hybrid. The covalent PlnA-t-PAB hybrid activator was isolated from the 6-day hybridization mixture by a two-step affinity chromatography method.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tissue plasminogen activator catalyzed Lys-plasminogen activation on heparin-inserted phospholipid liposomes

We prepared heparin-inserted phospholipid liposomes as a functional model of heparan sulfate present on the vascular surface and examined tissue plasminogen activator (t-PA) catalyzed plasminogen activation on the liposome surface. Kinetic analyses showed a marked increase in the affinity of t-PA for Lys-plasminogen in the presence of heparin-inserted phosphatidylcholine (PC) liposomes. The catalytic efficiency (kcat/Km) of t-PA for the plasminogen activation on the surface of heparin-inserted PC liposomes was 5.4 times that on the surface of heparin-free PC liposomes. This stimulatory action of immobilized heparin was apparently affected by changing the phospholipid component of liposomes. Phosphatidylethanolamine or stearylamine, having a positively charged group, reduced the catalytic efficiency of t-PA by raising its Km value (10-fold), whereas negatively charged phospholipids, phosphatidylserine and phosphatidylinositol, did not affect the efficiency. t-PA and generated plasmin bound to the liposome surface heparin were protected from inhibition by plasminogen activator inhibitor type 1 and alpha 2-plasmin inhibitor, respectively. t-PA-induced clot lysis of euglobulin or whole plasma, which contained native (Glu-) plasminogen and the above inhibitors, was also accelerated by addition of heparin-inserted PC liposomes. These results suggest that the vascular surface heparin-like molecules may play an important role in modulating fibrinolytic events. The principles of conjugation of t-PA with a biologically active liposome will be applied to the construction of better thrombolytic agents.     

Synthesis of recombinant human single-chain urokinase-type plasminogen activator variants resistant to plasmin and thrombin

Single-chain urokinase-type plasminogen activator (scu-PA), a potential therapeutic reagent for thrombosis, is activated in plasma by plasmin. The activated enzyme is further digested by plasmin to generate low-molecular-weight urokinase (LMW-UK), which has no affinity for fibrin. To circumvent this dual effect of plasmin, we synthesized in Escherichia coli a variant of scu-PA, which is not converted to LMW-UK on treatment with plasmin. In another variant, the activation cleavage site was modified such that activation by plasmin was slowed down and that inactivation by thrombin was greatly diminished. The combination of these variants may be applicable as an effective thrombolytic reagent for clinical use.     

Actin accelerates plasmin generation by tissue plasminogen activator.

Actin has been found to bind to plasmin's kringle regions, thereby inhibiting its enzymatic activity in a noncompetitive manner. We, therefore, examined its effect upon the conversion of plasminogen to plasmin by tissue plasminogen activator. Actin stimulated plasmin generation from both Glu- and Lys-plasminogen, lowering the Km for activation of Glu-plasminogen into the low micromolar range. Accelerated plasmin generation did not occur in the presence of epsilon-amino caproic acid or if actin was exposed to acetic anhydride, an agent known to acetylate lysine residues. Actin binds to tissue plasminogen activator (t-Pa) (Kd = 0.55 microM), at least partially via lysine-binding sites. Actin's stimulation of plasmin generation from Glu-plasminogen was inhibited by the addition of aprotinin and was restored by the substitution of plasmin-treated actin, indicating the operation of a plasmin-dependent positive feedback mechanism. Native actin binds to Lys-plasminogen, and promotes its conversion to plasmin even in the presence of aprotinin, indicating that plasmin's cleavage of either actin or plasminogen leads to further plasmin generation. Plasmin-treated actin binds Glu-plasminogen and t-PA simultaneously, thereby raising the local concentration of t-PA and plasminogen. Together, but not separately, actin and t-PA prolong the thrombin time of plasma through the generation of plasmin and fibrinogen degradation products. Actin-stimulated plasmin generation may be responsible for some of the changes found in peripheral blood following tissue injury and sepsis.     

Tissue plasminogen activator mediates amyloid-induced neurotoxicity via Erk1/2 activation

Tissue plasminogen activator (tPA) is the main activator of plasminogen into plasmin in the brain where it may have beneficial roles but also neurotoxic effects that could be plasmin dependent or not. Little is known about the substrates and pathways that mediate plasmin-independent tPA neurotoxicity. Here we show in primary hippocampal neurons that tPA promotes a catalytic-independent activation of the extracellular regulated kinase (Erk)1/2 signal transduction pathway through the N-methyl-D-aspartate receptor, G-proteins and protein kinase C. This results in GSK3 activation in a process that requires de novo synthesis of proteins, and leads to tau aberrant phosphorylation, microtubule destabilization and apoptosis. Similar effects are produced by amyloid aggregates in a tPA-dependent manner, as demonstrated by pharmacological treatments and in wt and tPA-/- mice neurons. Consistently, in Alzheimer's disease (AD) patients' brains, high levels of tPA colocalize with amyloid-rich areas, activated Erk1/2 and phosphorylated tau. This is the first demonstration of an intracellular pathway by which tPA triggers kinase activation, tau phosphorylation and neurotoxicity, suggesting a key role for this molecule in AD pathology.     

Fibrin and plasminogen structures essential to stimulation of plasmin formation by tissue-type plasminogen activator

Plasminogen activation catalysed by tissue-type plasminogen activator (t-PA) has been examined in the course of concomitant fibrin formation and degradation. Plasmin generation has been measured by the spectrophotometric method of Petersen et al. (Biochem. J. 225 (1985) 149-158), modified so as to allow for light scattering caused by polymerized fibrin. Glu1-, Lys77- and Val442-plasminogen are activated in the presence of fibrinogen, des A- and des AB-fibrin and the rate of plasmin formation is found to be greatly enhanced by both des A- and des AB-fibrin polymer. Plasmin formation from Glu1- and Lys77-plasminogen yields a sigmoidal curve, whereas a linear increase is obtained with Val442-plasminogen. The rate of plasmin formation from Glu1- and Lys77-plasminogen declines in parallel with decreasing turbidity of the fibrin polymer effector. In order to study the effect of polymerization, this has been inhibited by the synthetic polymerization site analogue Gly-Pro-Arg-Pro, by fibrinogen fragment D1 or by prior methylene blue-dependent photooxidation of the fibrinogen used. Inhibition of polymerization by Gly-Pro-Arg-Pro reduces plasmin generation to the low rate observed in the presence of fibrinogen. Antipolymerization with fragment D1 or photooxidation has the same effect on Glu1-plasminogen activation, but only partially reduces and delays the stimulatory effect on Lys77- and Val442-plasminogen activation. The results suggest that protofibril formation (and probably also gelation) of fibrin following fibrinopeptide release is essential to its stimulatory effect. The gradual increase and subsequent decline in the rate of plasmin formation from Glu1- or Lys77-plasminogen during fibrinolysis may be explained by sequential exposure, modification and destruction of different t-PA and plasminogen binding sites in fibrin polymer.     

Tissue plasminogen activator and acute pulmonary embolism

We assessed the efficacy and safety of peripheral intravenous recombinant human tissue-type plasminogen activator (rt-PA) in 47 patients with angiographically documented pulmonary embolism (PE). We administered 50 mg/2 h and, if necessary, an additional 40 mg/4 h. By 6 hours, 94% of the patients had angiographic evidence of clot lysis that was slight in 5, moderate in 12, and marked in 27 patients. Among the 34 patients with pulmonary hypertension prior to treatment, average pulmonary artery pressure decreased from 43/17 (27) to 31/13 (19) mm Hg (P less than 0.0001). The average lung scan perfusion defect decreased from 37% before therapy to 16% (P less than 0.01) after therapy among the 19 patients who had pre- and post-treatment lung scans. Of 7 patients with pre- and post-treatment imaging and Doppler echocardiograms, hypokinetic right ventricular wall movement (mild in 1, moderate in 2, and severe in 4) normalized in 5 and improved to mild hypokinesis in 2. Right ventricular diameter decreased from 3.9 +/- 1.0 to 2.0 +/- 0.5 cm (P less than 0.005). Fibrinogen decreased 33% from baseline at 2 h and 42% from baseline at 6 h. However, patients with the greatest degree of angiographic clot lysis at 2 h had a preponderance of fibrinogenolysis over fibrinolysis, demonstrated by a lower ratio of cross-linked fibrin degradation products to fibrin(ogen) degradation products (0.14 +/- 0.09 vs. 0.54 +/- 0.82) (P less than 0.04). Among selected patients, peripheral intravenous rt-PA is associated with rapid lysis of PE, improved pulmonary perfusion, and improved right ventricular function.     

Follicular plasminogen and plasminogen activator and the effect of plasmin on ovarian follicle wall.

Plasminogen, plasminogen activator, protease inhibitors, and a proteolytic activity are shown to be present in bovine follicular fluid. Much of the proteolytic activity appears to be due to plasmin. In addition, plasminogen activator activity can be demonstrated in follicle wall homogenates. Evidence that plasmin decreases the tensile strength of follicle wall preparations is also reported. The potential for the involvement of these substances in ovulation is discussed.     

Tissue plasminogen activator is a potent activator of PDGF-CC

Tissue plasminogen activator (tPA) is a serine protease involved in the degradation of blood clots through the activation of plasminogen to plasmin. Here we report on the identification of tPA as a specific protease able to activate platelet-derived growth factor C (PDGF-C). The newly identified PDGF-C is secreted as a latent dimeric factor (PDGF-CC) that upon proteolytic removal of the N-terminal CUB domains becomes a PDGF receptor alpha agonist. The CUB domains in PDGF-CC directly interact with tPA, and fibroblasts from tPA-deficient mice fail to activate latent PDGF-CC. We further demonstrate that growth of primary fibroblasts in culture is dependent on a tPA-mediated cleavage of latent PDGF-CC, generating a growth stimulatory loop. Immunohistochemical analysis showed similar expression patterns of PDGF-C and tPA in developing mouse embryos and in tumors, indicating both autocrine and paracrine modes of activation of PDGF receptor-mediated signaling pathways. The identification of tPA as an activator of PDGF signaling establishes a novel role for the protease in normal and pathological tissue growth and maintenance, distinct from its well-known role in plasminogen activation and fibrinolysis.     

Activation with plasmin of tow-chain urokinase-type plasminogen activator derived from single-chain urokinase-type plasminogen activator by treatment with thrombin

Thrombin converts single-chain urokinase-type plasminogen activator (scu-PA) to an inactive two-chain derivative (thrombin-derived tcu-PA) by hydrolysis of the Arg-156--Phe-157 peptide bond. In the present study, we show that inactive thrombin-derived tcu-PA (specific activity 1000 IU/mg) can be converted with plasmin to active two-chain urokinase-type plasminogen activator (specific activity 43,000 IU/mg) by hydrolysis of the Lys-158--Ile-159 peptide bond. This conversion follows Michaelis-Menten kinetics with a Michaelis constant Km of 37 microM and a catalytic rate constant k2 of 0.013 s-1. The catalytic efficiency (k2/Km) for the activation of thrombin-derived tcu-PA by plasmin is about 500-fold lower than that for the conversion of intact scu-PA to tcu-PA. tcu-PA, generated by plasmin treatment of thrombin-derived tcu-PA, has similar properties to tcu-PA obtained by digestion of intact scu-PA with plasmin (plasmin-derived tcu-PA); its plasminogen activating potential and fibrinolytic activity in an in vitro plasma clot lysis system appear to be unaltered. These observations confirm that the structure of the NH2-terminal region of the B chain of u-PA is an important determinant for its enzymatic activity, whereas that of the COOH-terminal region of the A chain is not.