PROCEDURE FOR DETERMINING PLASMIN ACTIVITY

Abstract: A procedure for quantitative plasmin activity determination from a body fluid, the plasmin sample being reacted with an amino acid sequence to which a coumarin derivative has been coupled, so that through hydrolysis a reaction product is obtained, which is fluorometrically assayed.

Description: PROCEDURE FOR DETERMINING PLASMIN ACTIVITY The present invention concerns a procedure for quantitative determination of plasmin in a body fluid, as defined in the claims. In addition, the invention concerns a diagnos¬ tic means for quantitative determination of plasmin activity from body fluids, in particular from tear fluid. Normal tear fluid contains plasminogen activi¬ ty activator (Thδrig et al., 1983, Hayashi and Suie- ishi, 1988), but this plasmin activity is very low or totally absent (Salonen et al. 1987, Tervo et al., 1989, van Setten et al., 1990). Degradation of fibrin and fibronectin by effect of plasmin seems to contribu¬ te to formation of corneal ulcers or to their abnormal slow healing (Berman et al., 1983). On experimental lesion of the corneal epithelium and stroma (keratec- tomy) ensue short-term elevation of plasmin in the tear fluid and, simultaneously, decrease of plasminogen activator activity. The plasmin activity of the tear fluid goes down rapidly, and the epithelium heals in 1- 2 days (van Setten et al., 1990). On the other hand, patients with poorly healing corneal ulcers present elevated tear fluid plasmin activity during up to ten weeks (Salonen et al. , 1987). Topical administration of a plasmin inhibitor, aprotinin (Trasylol R , Bayer) is followed by normalization of tear fluid plasmin activi¬ ty as well as healing of the ulcer (Salonen et al., 1987). A successful study on corneal epithelial ulcer therapy has recently been reported in which plasmin inhibition therapy was applied (Tervo and van Setten, 1989) (US 4,849,406, July 18, 1989). Initiation of aprotinin treatment should be based on demonstration of plasmin activity in the tear fluid (Salonen et al. 1987). However, all problems re¬ lating to healing of the corneal surface are not neces- sarily associated with elevated plasmin activity in the tear fluid (Tervo et al., 1988, Tδzser et al., 1989). Salonen et al. (1987) originally used Saksela's (1981) modification of radial caseinolysis assay (Remmert and Cohen, 1949) to measure plasmin activity. This method is however not applicable in clinical use because many proteolytic enzymes display caseinolytic activity (cf. Cawston and Murphy, 1981) . A specificity check can be made, using caseinolysis inhibition with antiplasmin antibodies or aprotinin, or employing sodium dodecyl- sulphate polyacrylamide gel electrophoresis. The last- mentioned procedure (Salonen et al., 1987) is expensive and time-consuming, and it is usually hard to imple¬ ment, owing to very small quantities of tear fluid. The development of lytic zones on casein agar takes 24 to 48 hrs, and the reaction is not linear in relation to incubation time. Therefore, the caseinolytic method is not usable in outpatient praxis, where most of the oph- thalmologic patients are seen. Moreover, the method is semi-quantitative only. Wear of contact lenses, especially soft con¬ tact lenses, also leads to elevated tear fluid plasmin activity in part of the patients. Normalization of val¬ ues occurs after discontinuation of contact lens use (Tervo et al., 1989). Monitoring the tear fluid plasmin activity might also be a means to assess contact lens fit, pathological changes of the normal eye surface, or therapy applied in treatment of corneal ulcer (Tervo and van Setten, 1989). High values suggest corneal damage, allergy, poor contact lens fit or inflammation from microbes. Derangements of proteolytic activity analogous to those just described in association with corneal damage (Berman et al., 1980, Salonen et al., 1S87, Tervo et al., 1989, Tδzser et al., 1989) are also seen in muscle and mucous membrane lesions caused by acci¬ dents, inflammations or other pathologic states (Danδ et al., 1985, Reich et al. , 1975, Barret et al. , 1980, Vaheri and Salonen, 1988). Coronary artery thromboses are nowadays treat¬ ed with intravenously administered streptokinase (ISIS- 2, 1988, Chesebro et al., 1987), tissue plasminogen activator (Fox et al., 1987, Magnani, 1989, Wilcox et al., 1988) and urokinase (Chesebro et al., 1987, PRIMI Trial Study Group, 1989). Thrombolytic therapy of coro¬ nary artery occlusion is usually implemented without monitoring the serum fibrinolytic system and, particu¬ larly, the serum plasma activity. This would however be important since lysis of the thrombus is achieved by activating the plasminogen present therein, to become plasmin, which disperses the matrix in the thrombus, made up of fibrin and fibronectin (Bergman et al., 1983, Fox et al., 1984). Human serum contains a plural¬ ity of plasmin inhibitors (serpins) (Linjen and Collen, 1986), which form complexes with plasmin, and inacti¬ vate it. Collen and Verstraete (1979) showed that dur- ing thrombolytic therapy with streptokinase systemic fibrinogenolysis only occurs when the circulating alpha-2-antiplasmin concentration decreases. Complica¬ tions ensuing in connection with thrombolytic therapy (e.g. cerebral haemorrhages) are rare but dangerous. It might be possible to optimize the treatment if the com¬ ponents of the fibrinolytic system could be monitored during its course, in particular plasmin and alpha-2- antiplasmin, or the components of these (Linjen and Collen, 1986). Patients with bronchial asthma have ulcers in the bronchial mucosa (Laitinen et al., 1985). Their morphology and fibronectin localization are greatly similar to those of corneal ulcers (Laitinen et al., in preparation) . Sputum samples of such patients ϋrequent- ly contain proteolytic activity measurable by the case- inolytic method. Since many bacteriogenic proteases can inactivate serpins (Catanase and Kress, 1986), septic inflammations may have a relationship to high plasmin levels in the serum or in certain organs, such as the brain. An appropriate monitoring system enables this imbalance to be detected and treatment instituted. Activation of fibrinolysis is reported to take place in certain renal and hepatic diseases, in arthritis and in Bechet's disease (Linjen and Collen, 1988). Application of the principle of local fibrino¬ lysis, e.g. utilization of a tissue plasminogen activa- tor containing sodium hyaluronate, has been suggested to prevent postoperative scar and adhesion formation, and to protect soft tissue implants. Therapeutic met¬ hods of this type have been studied, and used, not only in intraocular surgery but also in articular disorders, abdominal surgery and surgical trauma. A fast diagnostic procedure for monitoring the components of the fibrinolytic system could be helpful in assessment of such therapies; for instance, elevated plasmin activity in the anterior chamber fluid of the eye could dissolve postsurgical fibrin clots, while it could, at the same time, be deleterious to poorly re¬ generating corneal endothelial cells, causing their separation from the substrate. Whenever high proteolytic activity due to plasmin can be established, specific therapy with a plasmin inhibitor or with a combination of such inhibi¬ tor and e.g. hyaluronic acid and/or certain growth fac¬ tors can be initiated. The caseinolytic procedure (Saksela, 1981, also described in USP 4,849,406) has been employed to¬ wards measurement of plasmin activity in tear fluid. The drawbacks of this technique have been discussed in the foregoing. Chromogenic substrates can be used in measurement both of plasminogen activator and of plas- minogen-independent amidolytic activity (Karlan et al., 1987, Toezer et al., 1989). Plasmin-alpha-2-antiplasmin complex can be demonstrated by an enzyme-linked immuno- sorbent assay (Holvoet et al., 1986) and by radio- immunochemical analysis (Plow et al., 1980), and by Latex agglutination test (Collen et al., 1977). Esters and amides of basic amino acids may be used for synthetic substrates for plasmin (e.g. Bell et al., 1974). The derivatives most often used have been 4-methoxy-2-naphthylamine (Clavin et al., 1977) and p-nitroanilide (Soris et al., 1978). Selection of a suitable amino acid sequence has enabled highly speci- fie substrates to be found for certain proteinases. The most specific substrate for plasmin has been the amino acid triplet D-Val-L-Leu-L-Lys derivative (Soria et al., 1978). The hydrolyzed reaction product can be di¬ rectly measured photometrically (Soria et al., 1978), or indirectly by coupling it with certain dyes, or fluorometrically, based on the product's native fluo¬ rescence (Clavin et al., 1977). Certain coumarins dis¬ play even stronger fluorescence than 4-methoxy-2-naph- thylamine. Coupled to an appropriate amino acid trip- let, they can serve as highly sensitive and specific plasmin substrates (Smith et al., 1980). It is also an object of the invention, to dis¬ close a novel quantitative procedure for determination of plasmin activity in body fluids. It is also an object of the invention, to dis¬ close a diagnostic means with the aid of which said determination can be carried out. The invention discloses a fast and high-speci¬ ficity, clinically usable procedure for quantitative determination of the components of the fibrinolytic system, in particular of plasmin, from body fluids. The procedure is potentially important in preventing plasmin-induced tissue destruction in many pathologic states, as has been described in the foregoing. Such states include delayed healing of corneal epithelium, or other cutaneous or mucosal ulcers or erosions. Thanks to the invention, e.g. monitoring of the fibrin- olytic system becomes feasible when thrombolytic ther¬ apy is applied in coronary or cerebral or peripheral venous occlusion or after intraocular surgery. The pro¬ cedure is also applicable for ascertaining start-up of systemic fibrinolysis. This may be due not only to the therapy just mentioned but also to septic conditions, severe shock, trauma or inflammation. In the present invention a study has been made of the characteristics of a certain 7-amino-4-methyl- coumarin (AMC) peptide as a suitable substrate for plasmin activity assay from body fluids, e.g. from tear fluid, in various physiologic and pathophysiologic states. The peptide is an amino acid triplet. The procedure constituting the object of the invention is based on reaction of plasmin with a 7- amino-4-methylcoumarin (AMC) peptide, resulting in a hydrolyzed reaction product which is determined by fluorometry. A suitable peptide coupled to AMC is the amino acid triplet D-Val-L-Leu-L-Lys. Plasmin activity may be determined from dif¬ ferent body fluids, among others from tear fluid with diagnostic intent or in monitoring ulcer therapy, from anterior chamber fluid in connection with intraocular surgery and inflammations, from the vitreous body in connection with operations and inflammations, from syn- ovial fluid during inflammations, from sputum samples in inflammations and in bronchial asthma, in cutaneous lesions in connection with inflammations and burns, from serum in monitoring fibrinolytic treatment, in sepsis and various injuries, and from spinal fluid in severe inflammations or injuries and in monitoring thrombolytic treatment. The invention is described in the following in detail, with embodiment examples, referring to the tables hereto attached. Example 1, Determination of plasmin activity from tear fluid. 50 μl reagent solution containing 250 mmol/1 Tris-HCl buffer, pH 8.0, and 1.5 mmol/1 D-Val-L-Leu-L- Lys-AMC are freeze-dried into small cuvettes. The cu¬ vettes are then closed and stored, protected from light (keeps for prolonged periods at room temperature, for instance) . For plasmin activity determination, 10 μl tear fluid are pipetted into a cuvette, together with 500 μl Millipore-filtered water. 10 μl plasmin standard (10 IU/1 plasmin, Human plasmin, National Institute for Biological Standards and Controls, London, England) are added into a calibration cuvette, together with 500 μl water. An initial reading is then taken, and after 10 min. incubation at room temperature a second reading is taken, and the plasmin activity is calculated by com¬ paring the difference of the second and first readings with the respective reading of the plasmin standard. Spontaneous hydrolysis of the substrate is minimal dur¬ ing 10 min. incubation, to the degree that its subtrac¬ tion is not indicated. If the plasmin activity in the sample exceeds 125 IU/1, the sample is diluted 1:10 with 50 mmol/1 Tris-HCl buffer, pH 8.0, and the activi- ty is determined once more. The plasmin activity de¬ termination is a fluorometric assay and was done with a Transcon 102 FN analyzer. The results are shown in Tables 1-5. Table 1 presents the characteristics of the D-Val-L-Leu-L-Lys-AMC substrate in plasmin activity determination. Table 2 presents the plasmin activity in tear fluid of healthy persons. Table 3 presents the plasmin activity during recovery in corneal ulcer pa¬ tients. Table 4 presents the plasmin activity in the rabbit eye anterior chamber fluid during mechanical irritation of the iris, and Table 5 presents the plas¬ min activity in the rabbit's anterior chamber fluid in neurogenic inflammatory reaction induced by a capsaicin injection given into the anterior chamber (axon ref¬ lex) . Table 1. The characteristics of the D-Val-L-Leu-L-Lys derivative of 7-amino-4-methylcoumarin (AMC) in deter¬ mination of plasmin activity at 25°C, fluorometer: Transcon 102 FN. Excitation wavelength, max. 377 nm Emission wavelength, max. 440 nm Selected excitation wavelength 380 nm (Corning filter 7-54, cut-off 370 nm) Selected emission wavelength 480 nm (490 nm, cut¬ off 420 nm) Optimum pH 7.85 Km 0-25 mmol/1 Detection limit 0.3 IU/1 Linear range 1 to 125 IU/1 Table 2. Plasmin activity (IU/1) in tear fluid of healthy persons. Substrate used: D-Val-L-Leu-L-Lys-AMC, 25°C, and fluorometer: Transcon 102 FN. Detection limit = 0.3 IU/1 F = Female M = Male Table 3♦ Plasmin activity (IU/1) during recovery in corneal ulcer patients after topical aprotinin treat¬ ment. Activity measured on D-Val-L-Leu-L-Lys-AMC sub¬ strate by fluorometric method, fluorometer: Transcon 102 FN, 25°C. Pat. Sex Age Diagnosis Microbe Treatment No. ND = No determination = Symptoms still present FA = Fluorometric assay CA = Caseinolytic assay Table 4. Plasmin activity (IU/1) in the rabbit's anteri chamber fluid after mechanical iris irritation. Rabbit Prior to 20 min. after 60 min. after No. irritation irritation irritation CA = Caseinolytic assay FA = Fluorometric assay (D-Val-L-Leu-L-Lys-AMC, 25°C) N.D. = Not determined Caseinolytic and fluorometric assay were carried o immediately after sampling. Table 5. Plasmin activity (IU/1) in the rabbit's chamb fluid after neurogenic iris irritation induced by intr cameral capsaicin injection. CA = Caseinolytic assay FA = Fluorometric assay (D-Val-L-Leu-L-Lys-AMC, 25°C) References Bell PH, Dziobkowski, CT ja Englert, ME: A sensitive fluorometric assay for plasminogen, plasm and streptokinase Anal Biochem. 61:200-208, 1974 Bermann SR, Fox KA, Ter-Pogossian MM, Sobel BE, Collen Clot-selective coronary thrombolysis with tissue-t plasminogen activator Science 220:1181-1183, 1983 Berman MB: Collagenase and corneal ulceration Collagenase in normal and pathological connective tissu John Wiley & Sons Ltd New York, NY, 1980 Berman M, Manseau E, Law M & Aiken D: Ulceration is correlated with degradation of fibrin fibronectin at the corneal surface Invest Ophthamol Vis Sci 24:1358-1366, 1983 Catanese J & Kress LJ: Enzymatic inactivation of human plasma Cl-inhibitor and antichymotrypsin by Pseudomonas aeruginosa proteinase a elastase Biochem Biophys Acta 789:37-43, 1984 Caustoa TE & Murphy G: Mam alion collagenases Methods Enzymol 80, pt C:711-722, 1981 Chesebro JH, Knatterud G, Roberts R et. al: Thrombolysis in myocardiai infarction (TIMI) trial, pha 1: a comparison between intravenous tissue plasminog activator and intravenous streptokinase Circulation 76:142-154, 1987 Clavin SA, Bobbit JL, Schuman RT and Smithwick EL Use of peptidyl-4-methoxy-2-nepthylamides to assay plasmi Anal. Biochem 80:355-365, 1977 Collen D & Verstraete M: α 2 -antiplasmin consumption and fibrinogen breakdown duri thrombolytic therapy Thromb. Res. 14:631-639, 1979 Dano/ K, Andreasen PA, Gro/ndahl-Hansen J, Kristensen Nielsen LS, Skriver L: Plasminogen activators and cancer Adv. Cancer Res. 44:139-266, 1985 Fox KA, Bergmann SR, Sobel BE: Coronary thrombolysis: pharmacological considerations? wi emphasis on tissue-type plasminogen activator (t-PA) Biochem Pharmacol 33 (12) :1831-1838, 1984 Hayashi, K & Sueishi K: Fibrinolytic activity and species of plasminogen activat in human tears Exp. Eye Res. 46:131-137, 1988 Holvoet P, de Boer A, Verstreken M & Collen D: An enzyme-linked i munosorbent assay (ELISA) for the meas rement of plasmin-α 2 -antiplasmin complex in human plasm application to the detection of in vivo activation of t fibrinolytic system. Thrombosis and Haemostasis, 56 (2):124-127, 1986 ISIS-2 (Second International Study of Infarct Survival) collaborative group; Randomised trial of intravenous stre tokinase, oral aspirin, both, or neither among 17187 cas of suspected acute myocardiai infarction?: ISIS-2 Lancet 2:349-360, 1988 Joutsimo L, van Setten G-B, Renkonen OV, Tarkkanen Paivarinta H, Tervo T: On the proteolytic activity of contact lenses and bacte Acta Ophthalmol (Kbh), (in press) Karlamo BY, Clark AS, Littlefield BA: A highly sensitive chromogenic microtiter plate assay plasminogen activators which quantitatively discrimina between the urokinase and tissue-type activators Biochem. Biophys. Res. Commun. 142:147-154, 1987 Kress LJ: Inactivation of human plasma serine proteinase inhibito (serpins) by limited proteolysis of the reactive site lo with snake venom and bacterial metalloproteinases J Cell Biochem 32:51-58, 1986 Laitinen LA, Heino M, Kava T & Haahtela T: Damage of the airway epithelium and bronchial reactivity patients with asthma Am. Rev. Resp. Dis. 131:599-606, 1985 Lijnen HR & Collen D: Alpha-2-antiplasmin J Med. 16:225-84, 1985 Magnani, B: Plasminogen Activator Italian Multicenter Study (PAIMS) : Comparison of intravenous recombinant single-chain hum tissue-type plasminogen activator (rt-PA) with intraveno streptokinase in acute myocardiai infarction J Am. Coll. Cardiol 43:19-26, 1989 Plow EF, Wiman B & Collen D: Changes in antigenic structure and conformation of α -ant plasmin induced by interaction with plasmin. J. Biol. Chem. 255:2902-2906, 1980 Reich E, Rifkin DB, Shaw E (eds.): Proteases and biological control p. 1021 Cold Spring Harbor Laboratory, 1975 Remmert 0. , LeMar F & Chen PP: Partial purifiction and properties of a proteolytic enz of human serum J biol Chem. 181:432-448, 1949 Saksela 0: Radial caseinolysis in agarose: a simple method for det tion of plasminogen activator in the presence of inhibit substance and serum. Ann. Biochem. 111:276-282, 1981 Salonen EM, Tervo T, Tδrma E, Tarkkanen A & Vaheri A: Plasmin in tear fluid of patients with corneal ulce basis for new therapy Acta Ophthalmol. 65:3-12, 1987 Sherry S, Alkjaersig N & Fletcher AP: Comparative activity of thrombin on substituted argin and lysine eslers Am. J Physiol 209:577-583, 1965 Smith RE, Bissell ER, Mitchell AB and Pearson KW: Direct photometric or fluorometric assay of proteina using substrates containing 7-amino-4-trifluromethylcou rin Trombos. Res. 17:393-402, 1980 Soria J, Soria C and Sama a C: A Plasminogen assay using chromogenic synthetic substra Results from clinical work and from studies of thromboly in progress in Chemical Fibrinolysis and Thrombolysis Vo ed. Davidson JF, Rowan RM, Samama MM ja Desnoyers PC Raven Press, New York 1978, 337-346. Tervo T, Salonen EM, Vaheri A, Immonen I, van Setten Himberg JJ, Tarkkanen A: Elevation of tear fluid plasmin in corneal disease Acta Ophthalmol (Copenh.) 66:393-399, 1988 Tervo T, van Setten GB: Aprotinin for inhibition of plasmin on the ocular surfac principles and clinical observations In: Healing of the processes of the cornea Beuermann RW, Crosson CE & Kaufman HE (eds.), Portfolio Publishing Company of Texas, Inc., The Woodland Texas, ss. 151-163, 1989 Tervo T, van Setten G-B, Andersson R, Salonen E-M, Vahe A, Immonen I, Tarkkanen A: Contact lens wear is associated with the appearance plasmin in the tear fluid-preliminary results Graefe's Arch Clin Exp Ophthalmol 227:42-44, 1989 Thδrig L, Wijngaards G, van Haeringen NJ: Immunological characterization and possible origin plasminogen activator in human tear fluid Ophthalmol Res. 15:268-276, 1983 Tδzser J, Berta A & Punyiczki M: Plasminogen activator activity and plasminogen independe amidolytic activity in tear fluid from healthy persons a patients with anterior segment inflammation Clinica Chi ica Acta 183:323-332, 1989 Vaheri A & Salonen E-M: Fibronectin and regulation of proteolysis in cancer an tissue destruction Proc. 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Claims: CLAIMS 1. A procedure for quantitative plasmin activity determination from a body fluid, c h a r - a c t e r i z e d in that the plasmin sample is reacted with a peptide of 7-amino-4-methylcoumarin (AMC), to form through hydrolysis a reaction product, which is fluorometrically assayed. 2. Procedure according to claim 1, c h a r- a c t e r i z e d in that the peptide is the amino acid triplet D-Val-L-Leu-L-Lys. 3. Procedure according to claim 1 or 2, c h a r a c t e r i z e d in that the reagent is buffe¬ red with a buffer appropriate for plasmin activity mea- surement, particularly with Tris-HCl buffer. 4. Procedure according to any one of claims 1- 3, c h a r a c t e r i z e d in that the reagent is freeze-dried and admixed to the diluted body fluid. 5. Diagnostic means for quantitative plasmin activity determination from a body fluid, c h a r a c ¬ t e r i z e d in that the means comprises a buffer and a reagent. 6. Means according to claim 5, c h a r a c¬ t e r i z e d in that the reagent is a peptide to which has been coupled a compound, suitably a coumarin deriva¬ tive. 7. Means according to claim 5 or 6, c h a r¬ a c t e r i z e d in that the peptide is the amino acid triplet D-Val-L-Leu-L-Lys. 8. Means according to any one of claims 5-8, c h a r a c t e r i z e d in that the compound coupled to the amino acid is 7-amino-4-methylcoumarin (AMC) . 9. Means according to any one of claims 5-8, c h a r a c t e r i z e d in that the buffer is Tris- HC1 buffer.

Inventors: HAERKOENEN MATTI, SUOVANIEMI OSMO, TERVO TIMO, HAERKOENEN, MATTI, SUOVANIEMI, OSMO, TERVO, TIMO
Applicants: ELOMIT OY
Application year: 1991
Main classification: C07K5
IPC codes: C07K5, C12Q1