High Affinity For Fibrin Research Articles

High-performance chromatographic method for the purification of tissue-type plasminogen activator

High-performance affinity chromatography was performed on five ligand-bound columns in an attempt to purify tissue-type plasminogen activator (t-PA), which is a glycoprotein with a high affinity for fibrin and also has two Kringle structures and finger-domain in its molecule. The five columns were concanavalin A-5PW, p-aminobenzamidine-5PW, imidinodiacetic acid-5PW, boric acid-5PW and lysine-5PW. All five were able to rapidly separate t-PA from contaminating proteins, with high resolution and recovery.

Proteolytic cleavage of single-chain pro-urokinase induces conformational change which follows activation of the zymogen and reduction of its high affinity for fibrin.

A plasminogen activator secreted from human kidney cells was highly purified by affinity chromatography on an anti-urokinase IgG-Sepharose column. The purified plasminogen activator was inactive and had a single-chain structure and a Mr of 50,000. It not only did not incorporate diisopropyl fluorophosphate, which reacts with active site serine residue in urokinase, but also did not bind to p-aminobenzamidine-immobilized CH-Sepharose, to which urokinase bind via its side-chain binding pocket present in active center. The plasminogen activator was converted to the active two-chain form with the same Mr by catalytic amounts of plasmin. Its potential enzymatic activity was quenched completely by anti-urokinase IgG, but not by anti-tissue plasminogen activator Ig. These results indicate that the plasminogen activator is an inactive proenzyme form of human urokinase. Therefore, the plasminogen activator was termed single-chain pro-urokinase. The cleavage of single-chain pro-urokinase by plasmin induced conformational change which followed the generation of reactive serine residue at active site, the increase enzyme activity and the reduction of its high affinity for fibrin. These findings suggest that conformational change occurs in both regions responsible for enzyme activity and affinity for fibrin upon activation of single-chain pro-urokinase.

Primary structure of single-chain pro-urokinase.

Single-chain pro-urokinase is an inactive proenzyme form of human urokinase with a single-chain structure and a Mr of 50,000 and converted to the active two-chain form by catalytic amounts of plasmin. It was isolated from culture fluid of human kidney cells and subjected to chemical (CNBr) and proteolytic (lysyl endopeptidase) degradation. The resulting peptides were separated by reverse-phase high performance liquid chromatography and subjected to automated sequence analysis. Amino acid sequence of 85% of the 411 residues recovered in 17 peptides were found to be consistent with those of the A chain (157 amino acids) and B chain (253 amino acids) of human urokinase reported by Günzler and co-workers (Günzler, W. A., Steffens, G.J., Otting, F., Kim, S.-M., A., Frankus, E., and Flohé, L. (1982) Hoppe-Seyler's Z. Physiol. Chem. 363, 133-141; 1155-1165; Steffens, G.J., Günzler, W.A., Otting, F., Frankus, E., and Flohé, L. (1982) Hoppe-Seyler's Z. Physiol. Chem. 363, 1043-1058). It revealed the presence of Lys at position 158 in single-chain pro-urokinase through which the two polypeptide chains of human urokinase are unified into one molecule. In addition, firm evidence was found that upon activation by plasmin single-chain pro-urokinase is cleaved at the Lys-Ile bond between residues 158 and 159, resulting in the formation of a two-chain urokinase molecule held together by one disulfide linkage. These results indicate that the cleavage at the Lys-Ile bond between residues 158 and 159 is responsible for conformational change, appearance of enzyme activity and reduction of its high affinity for fibrin.

Production of monoclonal antibodies to the high fibrin-affinity, tissue- type plasminogen activator of human plasma. Demonstration of its endothelial origin by immunolocalization

Monoclonal antibodies (MAbs) to vascular plasminogen activator (vPA), the tissue-type plasminogen activator (tPA) in human plasma, were produced to be used as probes for immunochemical analysis. Human tissue sections and one of these MAbs were used to demonstrate the endothelial origin of plasma-tPA by immunohistochemistry. To produce MAbs, mice were immunized with semipurified vPA isolated from postocclusion human venous blood. Primed spleen cells were fused with the mouse myeloma cell line NS-1. Screening for MAb-producing hybridomas was performed with postocclusion euglobulins as a source of antigen by means of a solid-phase fibrin-vPA immunoassay. The selective and high-affinity binding of vPA for fibrin ensures the specificity and sensitivity of this test. Thus, eight hybridomas secreting MAbs to vPA were selected, cloned, and established as permanent hybridoma cell lines. Immunohistochemical analysis of cryostat sections of human tissues was performed with EA-delta 12D, a MAb having no inhibitory effect against vPA activity but binding to vPA with a high affinity. Thus, the only structures immunostained were endothelial cells of venules, capillaries, and arterioles. The EA-delta 12D monoclonal localization of plasma vPA in the endothelial lining of blood vessels provides evidence that tPA in plasma originates from the vascular wall and validates its designation as vascular plasminogen activator, ie, vPA. Also, our results are consistent with the fact that vPA in blood and tPA in tissues are immunologically identical and have a common endothelial origin.

Production of Monoclonal Antibodies to the High Fibrin-Affinity, Tissue-Type Plasminogen Activator of Human Plasma. Demonstration of Its Endothelial Origin by Immunolocalization

Monoclonal antibodies (MAbs) to vascular plasminogen activator (vPA), the tissue-type plasminogen activator (tPA) in human plasma, were produced to be used as probes for immunochemical analysis. Human tissue sections and one of these MAbs were used to demonstrate the endothelial origin of plasma-tPA by immunohistochemistry. To produce MAbs, mice were immunized with semipurified vPA isolated from postocclusion human venous blood. Primed spleen cells were fused with the mouse myeloma cell line NS-1. Screening for MAb-producing hybridomas was performed with postocclusion euglobulins as a source of antigen by means of a solid-phase fibrin-vPA immunoassay. The selective and high-affinity binding of vPA for fibrin ensures the specificity and sensitivity of this test. Thus, eight hybridomas secreting MAbs to vPA were selected, cloned, and established as permanent hybridoma cell lines. Immunohistochemical analysis of cryostat sections of human tissues was performed with EA-delta 12D, a MAb having no inhibitory effect against vPA activity but binding to vPA with a high affinity. Thus, the only structures immunostained were endothelial cells of venules, capillaries, and arterioles. The EA-delta 12D monoclonal localization of plasma vPA in the endothelial lining of blood vessels provides evidence that tPA in plasma originates from the vascular wall and validates its designation as vascular plasminogen activator, ie, vPA. Also, our results are consistent with the fact that vPA in blood and tPA in tissues are immunologically identical and have a common endothelial origin.

Expression in E. coli of finger-domain lacking tissue-type plasminogen activator with high fibrin affinity

Tissue-type plasminogen activator (t-PA) has a high affinity for fibrin and induces lysis of fibrin (fibrinolysis) on the surface of fibrin without degrading circulating fibrinogen. cDNA for t-PA which lacks the ‘fingerdomain’ (the site for fibrin affinity) was isolated from Detroit 562 cells. Analysis of the nucleotide sequence revealed a lack of the sequences which code for the finger-domain. A plasmid (pDPAT 1) containing the Escherichia coli tac promoter/operator and the cDNA sequence coding for ‘finger-domain lacking t-PA’ was constructed for expression in E. Coli. The polypeptide so produced was a new type of t-PA lacking finger-domain, but revealed plasminogen activator activity with the function of fibrin affinity. Tissue-type plasminogen activator Finger-domain Fibrin affinity Plasmid E. coli tac promoter/operator Detroit 562 cell

Thrombolytic properties of an inactive proenzyme form of human urokinase secreted from human kidney cells.

The relative fibrin-binding, fibrinolytic and fibrinogenolytic properties of single-chain pro-urokinase, an inactive proenzyme form of human urokinase purified from cultured human kidney cells, and urokinase were compared. The affinity of single-chain pro-urokinase for fibrin was much higher than that of urokinase. In Vitro thrombolytic studies showed that single-chain pro-urokinase is approximately three times more potent in fibrinolysis than urokinase and that it does not degrade fibrinogen in the plasma at a concentration, at which complete plasma clot lysis takes place; whereas, urokinase extensively degrades the fibrinogen in the plasma. These specific, potent thrombolytic properties of single-chain pro-urokinase seem to be due to its high affinity for fibrin and to its conversion from the inactive single-chain form to the active two-chain form on the thrombus by the catalytic amount of plasmin generated during coagulation. This single-chain pro-urokinase obtained from human kidney cells by tissue culture should prove advantageous than urokinase in thrombolytic therapy.

The fibrin-binding site of human plasminogen. Arginines 32 and 34 are essential for fibrin affinity of the kringle 1 domain.

Kringle 1 (Tyr 79/Leu 80-His 167 and Tyr 79/Leu 80-Tyr 173), a chymotryptic fragment of human plasminogen that has high affinity for fibrin and omega-aminocarboxylic acids, has been subjected to modification with 1,2-cyclohexanedione to identify arginine residues essential for ligand binding. Reaction of 1,2-cyclohexanedione with kringle 1 was found to rapidly abolish the fibrin-Sepharose affinity of the fragment, whereas the affinity for lysine-Sepharose was lost at a significantly slower rate. Successive affinity chromatography of modified kringle 1 on fibrin- and lysine-Sepharose was used to separate kringle 1 that lost affinity for fibrin-, but retained affinity for lysine-Sepharose from kringle 1 that lost affinity for both affinants. The modified proteins were subjected to structural studies in order to locate the labeled arginine residues in kringle 1. These studies have revealed that modification of Arg 34 leads to the loss of both the fibrin- and lysine-Sepharose affinities of kringle 1, whereas reaction of Arg 32 abolishes fibrin affinity but leaves lysine-Sepharose affinity unaltered. The results suggest that Arg 32 and Arg 34 are both involved in fibrin binding and that Arg 34 is also involved in binding omega-aminocarboxylic acids. Previous NMR studies on kringles have indeed shown that the segment containing residue 34 is in the proximity of and interacts with the omega-aminocarboxylic acid-binding site. This interaction may explain the influence of omega-aminocarboxylic acids on fibrin binding by kringle 1.

Sequence of formation of molecular forms of plasminogen and plasmin-inhibitor complexes in plasma activated by urokinase or tissue-type plasminogen activator.

The pathway of plasminogen transformation was studied in plasma, particularly in relation to fibrin formation and the subsequent stimulation of plasminogen activation. Plasminogen was activated by urokinase (low fibrin-affinity) or tissue-type plasminogen activator (high fibrin-affinity). Formation of 125I-labelled free and inhibitor-bound plasminogen derivatives was quantified after their separation by acetic acid/urea/polyacrylamide-gel electrophoresis. In plasma activator converted Glu-plasminogen (residues 1-790) into Glu-plasmin, which was complexed to alpha 2-plasmin inhibitor. When this inhibitor was saturated, Glu-plasmin was autocatalytically converted into Lys-plasmin (residues 77-790). No plasmin-catalysed Lys-plasminogen formation was observed. Upon fibrin formation, activation initially followed the same Glu-plasminogen-into-Glu-plasmin conversion pathway, and stimulation of plasminogen activation was only observed with tissue-type plasminogen activator. In agreement with the emergence of novel effector function, on early plasmin cleavage of fibrin [Suenson, Lützen & Thorsen (1984) Eur. J. Biochem. 140, 513-522] the fibrin-binding of Glu-plasminogen increased when solid-phase fibrin showed evident signs of degradation. This was associated with the formation of considerable amounts of the more easily activatable Lys-plasminogen, most of which was fibrin-bound. At the same time the rate of plasmin formation with urokinase increased over that in unclotted plasma and the rate of plasmin formation with tissue-type plasminogen activator accelerated. Altogether these processes favoured enhanced fibrin degradation. The rates of Lys-plasminogen and plasmin formation abruptly decreased after lysis of fibrin, probably owing to a compromised effector function on further fibrin degradation.

Purification and partial characterization of a single-chain high-molecular-weight form of urokinase from human urine

Affinity chromatography on fibrin/celite, a previously described technique for isolating plasminogen activators with a high fibrin affinity ( S. Husain, B. Lipinski, and V. Gurewich, 1981, Proc Nat. Acad. Sci. USA 78, 4265–4269), was performed on freshly voided urine. About 10–25% of the plasminogen activator activity present was adsorbed whereas less than 1% was adsorbed when urine was stored at room temperature for 12–24 h. The adsorbed activator was isolated by elution with arginine (0.1 m) and further purified by gel filtration. This plasminogen activator was similar in molecular weight (56,000) to the known high-molecular-weight form of urokinase but differed from it by its binding affinity for fibrin/celite, single-chain subunit structure, lower specific activity (approximately 20,000 IU/mg), and slower reaction with diisopropylfluorophosphate. The enzymatic activity of the purified activator was quenched by anti-urokinase antibody and therefore established it to be a new form of urokinase. The relationship of this high affinity, single-chain urokinase to the previously described forms of urokinase is unclear.

Studies on the mechanism of the antifibrinolytic action of tranexamic acid

The effect of the antifibrinolytic agent tranexamic acid on the solubilization of 125 I-labeled fibrin by plasmin or by mixtures of plasminogen and plasminogen activator (tissue activator or urokinase) was studied. The time required to solubilize half of the radioactivity ( S 50 ) decreased curvilinearly with the logarithm of the concentration of plasmin or plasminogen. Tranexamic acid caused a concentration-dependent retardation of fibrinolysis. When corresponding S 50 values were converted to apparent concentrations of plasmin or plasminogen in the absence of tranexamic acid, sigmoidal relationships were obtained between the apparent plasmin(ogen) concentration and the logarithm of the concentration of tranexamic acid. When tissue activator (with a high affinity for fibrin) was used, the shape of these curves was compatible with single association reactions between plasminogen and tranexamic acid. A 50% decrease of the apparent plasminogen concentration was obtained at 1.2 μM tranexamic acid for native plasminogen (Glu-plasminogen) and 2.3 μM for proteolytically degraded plasminogen (Lys-plasminogen). The dissociation constants of the interaction between tranexamic acid and the high affinity lysine-binding site of Glu-plasminogen or Lys-plasminogen have previously been estimated at 1.1 and 2.2 μM, respectively. Direct measurements of the binding of 125 I-labeled plasminogen to fibrin clots revealed that tranexamic acid displaced plasminogen from the fibrin surface; a 50% displacement was obtained at 1.3 μM tranexamic acid for Glu-plasminogen and 5.0 μM for Lys-plasminogen. These observations are compatible with the interpretation that saturation of the high affinity lysine-binding site of plasminogen with tranexamic acid results in its displacement from the fibrin surface and abolishes its activation by fibrin-bound plasminogen activator. When native or degraded plasminogen was activated with urokinase (with a low affinity for fibrin), tranexamic acid also caused a concentration-dependent retardation of fibrinolysis but of a much more complex nature. Saturation of the high affinity lysine-binding site in plasminogen with tranexamic acid had no significant influence on the solubilization rate of fibrin, indicating that binding of plasminogen to fibrin is of little importance for its activation by urokinase. At higher tranexamic acid concentrations an enhancement of Glu-plasminogen activation was observed, followed by interference of tranexamic acid with fibrinolysis by plasmin. These effects have already been described previously. Tranexamic acid caused a concentration-dependent retardation of fibrinolysis by plasmin; a 50% reduction of the apparent plasmin concentration was obtained at 45 μM tranexamic acid.

Studies on the mechanism of the antifibrinolytic action of tranexamic acid

The effect of the antifibrinolytic agent tranexamic acid on the solubilization of 125I-labeled fibrin by plasmin or by mixtures of plasminogen and plasminogen activator (tissue activator or urokinase) was studied. The time required to solubilize half of the radioactivity ( S 50) decreased curvilinearly with the logarithm of the concentration of plasmin or plasminogen. Tranexamic acid caused a concentration-dependent retardation of fibrinolysis. When corresponding S 50 values were converted to apparent concentrations of plasmin or plasminogen in the absence of tranexamic acid, sigmoidal relationships were obtained between the apparent plasmin(ogen) concentration and the logarithm of the concentration of tranexamic acid. When tissue activator (with a high affinity for fibrin) was used, the shape of these curves was compatible with single association reactions between plasminogen and tranexamic acid. A 50% decrease of the apparent plasminogen concentration was obtained at 1.2 μM tranexamic acid for native plasminogen (Glu-plasminogen) and 2.3 μM for proteolytically degraded plasminogen (Lys-plasminogen). The dissociation constants of the interaction between tranexamic acid and the high affinity lysine-binding site of Glu-plasminogen or Lys-plasminogen have previously been estimated at 1.1 and 2.2 μM, respectively. Direct measurements of the binding of 125I-labeled plasminogen to fibrin clots revealed that tranexamic acid displaced plasminogen from the fibrin surface; a 50% displacement was obtained at 1.3 μM tranexamic acid for Glu-plasminogen and 5.0 μM for Lys-plasminogen. These observations are compatible with the interpretation that saturation of the high affinity lysine-binding site of plasminogen with tranexamic acid results in its displacement from the fibrin surface and abolishes its activation by fibrin-bound plasminogen activator. When native or degraded plasminogen was activated with urokinase (with a low affinity for fibrin), tranexamic acid also caused a concentration-dependent retardation of fibrinolysis but of a much more complex nature. Saturation of the high affinity lysine-binding site in plasminogen with tranexamic acid had no significant influence on the solubilization rate of fibrin, indicating that binding of plasminogen to fibrin is of little importance for its activation by urokinase. At higher tranexamic acid concentrations an enhancement of Glu-plasminogen activation was observed, followed by interference of tranexamic acid with fibrinolysis by plasmin. These effects have already been described previously. Tranexamic acid caused a concentration-dependent retardation of fibrinolysis by plasmin; a 50% reduction of the apparent plasmin concentration was obtained at 45 μM tranexamic acid.

Effect of Local Urokinase on Arterial Occlusion of Lower Limbs

Sixty nine patients were treated with local intra-arterial urokinase (37,500 U/CTA. hr-1) for recent severe ischemia of lower limbs: 27 (40%) ultimately required amputation. The difference of amputation rate between the groups with and without thrombolysis was not significant (33% v. 42%). A biological study in 6 patients showed that local arterial plasminemia occurred in only 1 patient. Local urokinase does not strongly stimulate "endogenous" thrombolysis and enhances "exogenous" thrombolysis only very inconstantly. A better adaptation of urokinase dosage or the use of an agent with higher affinity for fibrin might improve the efficiency of local thrombolytic therapy.

Differences in thrombolytic effects in accordance with dosing- resimens of tissue- type plasminogen activator in experimental pulmonary embolism.

Background: Tissue-type plasminogen activator is a physiologic activator, which has high affinity for fibrin and is activated by fibrin. Because of these properties, t-PA has the potential to induce effective thrombolysis without producing a systemic lytic state. In practice, however, therapeutically efficacious doses of t-PA has been associated with the development of a systemic lytic state. As experience with t-PA has accumulated, it has suggested that the fibrin selectivity is influenced by the dose and duration of t-PA infusion, and many studies have performed in an attempt to optimize the duration of t-PA regimen. Methods: This study was designed to assess the thrombolytic efficacy of t-PA and the differences of two dosing regimens of t-PA (infusion of 1 mg/kg t-PA over 15 or 180 minutes) in a canine model of pulmonary embolism, induced by injection of radioactive autologous blood clots. By continuously counting over both lung fields with a external gamma counter, we correlated rate and extent of pulmonary thrombolysis with corresponding pulmonary hemodynamics in addition to the gas analyses of arterial and mixed venous blood. Results: 1) While total clot lysis was similar ( and respectively, p>0.05) when t-PA was infused over 15 or 180 minutes, the rate of lysis during infusion was markedly increased with the shorter infusion ( vs , p minutes although t-PA was administered over 15 minutes, and it was only minutes in case of the infusion over 180 minutes (p