FI91777C - Method for determining plasmin activity - Google Patents

Description

91777

METHOD FOR DETERMINING PLASMA VISIBILITY -FORFARANDE FOR BESTÅMNING AV PLASMINAKTIVITETEN

The invention relates to a method for the quantitative determination of plasmin activity in a body fluid as defined in the claims.

The invention further relates to a diagnostic device for the quantitative determination of plasmin activity in body fluids, in particular tear fluid.

10 Normal tear fluid contains plasminogen activator activity (Thorig et al., 1983 / Hayashi and Suieishi, 1988), but plasmin activity is very low or non-existent (Salonen et al., 1987; Tervo et al., 1989; van Setten et al. al., 1990).

Degradation of fibrin and fibronectin by plasmin appears to form myocardial corneal ulcers or their abnormally slow healing (Berman et al., 1983). An experimental wound in the corneal epithelium and superficial stroma (ke-20 ratectomy) is followed by a transient increase in plasmin in the tear fluid and a concomitant decrease in plasminogen activator activity. The plasmin activity of the tear fluid decreases rapidly and the epithelium improves in 1-2 days (van Setten et al., 1990). On the other hand, 25 patients with poorly healing corneal ulcers have increased tear fluid plasmin activity for up to 10 weeks (Salonen et al., 1987). Topical administration of the plasmin inhibitor, aprotinin (Trasylol®, Bayer), is followed by normalization of tear fluid plasmin activity and healing of the ulcer (Salonen et al., 1987). A successful study of the treatment of corneal epithelial ulcers using plasmin inhibitory therapy has recently been reported (Tervo and van Setten, 1989) (US 4,849,406, July 18, 35 1989).

Initiation of aprotinin therapy should be based on the demonstration of plasmin activity in tear fluid 777 2 (Salonen 1987). However, not all corneal surface healing problems are necessarily associated with an increase in plasmin activity in tear fluid (Tervo et al., 1988; Toezer et al., 1989). Salonen et al., (1987) 5 originally used a modification by Saksela (1981) from a radial-dysinolysis assay to measure plasmin activity (Renunert and Cohen 1949). However, this method is not suitable for clinical use because several proteolytic enzymes have caseinolytic activity (cf. Cawston and Murphy 1981). The latter method 15 (Salonen et al./877) is expensive, time consuming and usually difficult to implement due to very small tear fluid volumes. The formation of dissolved regions on casein agar takes 24 to 48 h, and the reaction is not linear with respect to the incubation time. Due to this, the caseinolytic method is not suitable for outpatient treatment / where ophthalmological patients are most often. In addition, the method is only semi-quantitative.

The use of contact lenses, especially soft glasses, also results in an increase in tear fluid plasmin activity in some 25 patients. Normalization of values occurs after discontinuation of contact lenses (Tervo et al., 1989). Monitoring the plasmin activity of tear fluid could also be a means of determining the suitability of contact lenses, pathological changes in the surface of the normal eye, or the treatment of corneal ulcers (Tervo and van Setten 1989). High values give reason to suspect corneal damage, allergies, poorly fitting contact lenses, or microbial inflammation.

Disorders of proteolytic activity 35 analogous to those described above in connection with corneal injury (Berman et al., 1980, Salonen et al. / 1987, Tervo et al., 1989, Toezer et 3 91 7 7 7 al., 1989 ) also occurs in skin and mucosal lesions caused by accidents, inflammation and other pathological conditions (Dano et al., 1985; Reich et al., 1975; Barret et al., 1980; Vaheri and Salonen 1988).

Coronary thrombosis is currently treated with intravenous streptokinase (ISIS-2 1988, Chesbro et al., 1987), tissue plasmonogen activator (Fox et al., 1987, Magnani 1989, Wilcox et al., 1988) and urokinase (Chesebro et al., 1988). ., 1987, 10 PRIMI Trial Study Group 1989). Thrombolytic treatment of coronary thrombosis is usually performed without a serum fibrinolytic system, and in particular serum plasmin activity is monitored. However, this would be important because the dissolution of the blockage is achieved by activating the plasminogen therein to plasmin, which degrades the matrix of fibrin and fibronectin in the blockage (Bergman et al., 1983; Fox et al., 1984). Human serum contains several plasmin inhibitors (serpins) (Linjen and 20 Collen 1986) that form complexes with plasmin, inactivating it. Collen and Verstraete (1979) showed that during thrombolytic therapy, systemic fibrinogenolysis with streptokinase occurs only with decreasing circulating alpha-2 antiplasmin levels.

Complications of thrombolytic therapy (eg cerebral haemorrhage) are rare but dangerous. Treatment could potentially be optimized if components of the fibrinolytic system, especially plasmin, and alpha-2 antiplasmin or their complexes could be monitored during treatment (Linjen. And Collen 1986).

Patients with asthma have ulcers on the bronchial mucosa (Laitinen et al., 1985). Their morphology and fibronectin localization are very similar to those of corneal ulcers (Laitinen et al., Pending). Sputum samples from female patients often contain proteolytic activity as measured by the caseinolytic method. Because many bacterial proteases can inactivate serpines (Catanase and Kress, 1984, Kress 1986), septic infections may be associated with high serum plasmin levels or in various organs, such as the brain. With a suitable monitoring system, this imbalance can be detected and treatment initiated. Activation of fibrinolysis has been reported to occur in some renal and hepatic diseases, arthritis, and 10 Bechet's disease (Linjen and Collen 1988).

The principle of local fibrinolysis, e.g., the utilization of a tissue plasminogen activator containing sodium hyaluronate, has been proposed as a kSytet to prevent postoperative scar and adhesion formation and to protect soft tissue transplantation. Such treatment methods have been studied and used, not only in intraocular surgery, but also in joint diseases, abdominal surgery, and injuries.

20 A rapid diagnostic method to monitor the components of the fibrinolytic system could help monitor such therapies; e.g., elevated plasmin activity in the anterior chamber fluid of the eye could dissolve fibrin clots after ocular surgery, but at the same time be detrimental to the poorly healing corneal endothelial cell, causing it to detach from its substrate.

When high proteolytic activity due to plasmin can be detected, specific treatment with a plasmin inhibitor or a combination thereof and e.g. hyaluronic acid and / or certain growth factors can be initiated.

The caseinolytic method (Saksela 1981, described in U.S. Pat. No. 4,849,406) has been used to measure the plasmin activity of tear fluid. The disadvantages of this technique have been described above. Chromogenic substrates can be used as well as plasminogen-activated! 91777 for measuring plasminogen-independent amidolytic activity (Karlan et al. / 1987; Toe2er et al., 1989). The plasmin-alpha-2-antiplasmin complex can be detected by enzyme-linked immunosorbent assay (Holvoet et al., 1986) and radioimmunochemical analysis (Plow et al., 1980) as well as the Latex agglomeration assay (Collen et al., 1977).

Esters and amides of basic amino acids can be used as synthetic substrates for plasmin 10 (e.g., Bell et al., 1974). The most frequently used derivatives have been 4-methoxy-2-naphthylamine (Clavin et al., 1977 and DK 155051 B) and p-nitroanilide (Soria et al., 1978). By selecting the appropriate amino acid sequence, highly specific substrates for certain proteinases have been found. The most specific substrate for plasmin has been the amino acid triplet D-Val-L-Leu-L-Lys derivative (Soria et al., 1978). The reaction product formed in the hydrolysis can be measured photometrically directly (Soria et al., 1978) or by cross-linking it with certain dyes or fluorometrically based on the product's own fluorescence (Clavin et al. 1977). Some coumarins have even stronger fluorescence than 4-methoxy-2-naphthylamine. When coupled to a suitable amino acid triplet, they can act as highly sensitive and specific plasmin substrates (Smith et al., 1980 and U.S. Patent No. 4,273,047 to Sakakibara).

It is also an object of the invention to describe a new quantitative method for the determination of plasmin activity in body fluids.

The object of the invention is also to describe a diagnostic device, a kit, by means of which this determination can be made.

For features of the invention, reference is made to the claims.

The invention describes a fast and highly specific method for the quantitative determination of components of a fibrinolytic system, in particular plasmin, in body fluids. The method is potentially important in preventing plasmin-induced tissue destruction in many pathological conditions, as described above. Such conditions include, for example, delayed healing of the corneal epithelium or other ulceration or heart failure of the skin or mucosa. Thanks to the invention, for example, the fibrinolytic system can be monitored in the use of thrombolytic therapy in coronary, cerebral, and femoral vein thrombosis or after intraocular surgery. The method can also be used to detect the initiation of systemic fibrinolysis. This may be due not only to the above treatment but also to septic conditions, severe shock, trauma 15 or inflammation.

The present invention has investigated the characteristics of a particular 7-amino-4-methylcoumarin (AMC) peptide derivative as a suitable substrate for determining plasmin activity from body fluids, e.g., tear-20 tea, in various physiological and pathophysiological conditions. The peptide is an amino acid triplet.

The process of the invention is based on the reaction of a plasmin sample with a peptide derivative of 7-amino-4-methylcoumarin (AMC), whereby hydrolysis yields a reaction product which is determined fluorometrically.

A suitable peptide coupled to AMC is the amino acid triplet D-Val-L-Leu-L-Lys.

Plasmin activity can be determined from a variety of body fluids, e.g. tear fluid for diagnostic purposes or for follow-up of ulcer treatment, anterior chamber fluid for intraocular surgery and inflammation, vitreous surgery and inflammation, synovial fluid for inflammation , sepsis and various injuries and spinal cord: 7 91777 fluid in severe inflammation or injury and in the follow-up of thrombolytic therapy.

The invention is described in detail below by way of exemplary embodiments with reference to the accompanying tables.

Example 1, Determination of plasmin activity in tear fluid.

50 μΐ of reagent solution with 250 mmol / l Tris-HCl buffer, pH 8.0, and 1.5 mmol / l D-Val-L-Leu-L-10 Lys-AMC are lyophilized in small cuvettes. Thereafter, the cuvettes are sealed and stored protected from light (e.g., stored at room temperature for long periods of time). To determine plasmin activity, 10 μΐ of tear fluid is pipetted together with 500 μΐ of Millipore filtered water into the cuvette. A 10 μΐ plasmin standard (10 IU / l plasmin, Human Plasmin, National Institute for Biological Standards and Controls, London, England) is added to the calibration cuvette together with 500 μΐ water. After this, an initial reading is taken, and after 10 min of in-20 incubation at room temperature, a second Luke-ma is taken, and plasmin activity is calculated by comparing the difference between the second and first readings to the corresponding reading of the plasmin standard. Spontaneous hydrolysis of the substrate during the 10 min incubation is so minimal that there is no reason to reduce it. If the plasmin activity in the sample is greater than 125 IU / l, the sample is diluted 1:10 with 50 mmol / l Tris-HCl buffer, pH 8.0 and the activity is re-measured. Plasmin activity was determined using a fluorometric mSari-30 test performed on a Transcon 102 FN analyzer. The results are shown in Tables 1 to 5. Table 1 shows the properties of the D-Val-L-Leu-L-Lys-AMC substrate in the plasmin activity assay. Table 2 address a plasmin activity in the tears of healthy person, 35 rolls, Table 3 address a plasmin activity during the pa-rantumisen sarveiskalvohaavaumapotilailla, Table 4 shows exemplary plasmin activity in the rabbit eye 91 777 8 humor during vårikalvon mechanical irritation, and Table 5 shows the plasmin on the rabbit aqueous humor into the anterior chamber Kápsas injection caused hermoperåisesså tulehdusreak -5 thion (axon reflex).

Table 1. Properties of 7-Amino-4-methylcoumarin (AMC) D-Val-L-Leu-L-Lys derivative for the determination of plasmin activity, 25 ° C, fluorometer

Transcon 102 FN

10

Wavelength of excitation light, max 377 nm

Wavelength of emission light, max 440 nm

Selected wake-up wavelength 380 nm (Cornig filter 7-54, 15 cut-off 370 nm)

Selected emission light wavelength 480 nm (490 nm, cut-off 420 nm) pH optimum 7.85

Km 0.25 mmol / l 20 Detection limit 0.3 IU / 1

Linear range 1-125 IU / 1

Table 2. Plasma activity of tear fluid (IU / 1) in healthy people. D-Val-L-Leu-L-25 Lys-AMC as substrate, 25 ° C and Transcon 102 FN as fluorometer

«

Subject Gender Age (v) Plasmin no activity 1 N 35 <0.3 30 2 N 28 <0.3 3 N 50 <0.3 4 N 59 <0.3 5 M 29 <0.3 35 Limit of detection = 0.3 IU / 1 N = female M = male i .

91777 9

Table 3. Plasmin activity (IU / 1) during healing in patients with corneal ulcers after topical aprotinin treatment. Activity was measured on D-Val-L-Leu-L-Lys-AMC substrate in a fluorometric 5 method, Transcon 102 FN fluorometer, 25 ° C.

Poti- Gen- Age Diagnosis Microbial Treatment Las No. 10 1 N 23 Cornea - Antibiotics erosion, diabetes and aprotinin 2 M 19 Corneal Staph.aureus Systemic ulcer, spring corticosteroid conjunctivitis treatment, antibiotic tivitis) locally 3 M 29 Chemical sar- - Antibiotics and bovine corpus aprotinin costly 20 4 M 25 Renewal cornea- Abrasion, antibiotic erosions and aprotinin topically

Plasmin activity (IU / 1)

Poti- Pv 1 Pv 2 Pv 3 Pv 4 Pv 22 Pv 26-28 25 las

No. FA CA FA CA FA CA FA CA FA CA FA CA

1 8.9 22.0 2.5 20.0 ND 0 2 2.7 6.2 1.8 2.0 0 0 3 15.5 ND 7.0 20.0 2.4 3.0 0 3, 8 "20.5 '30 4 8.9 56.0 8.7 ND 5.7 56.0 ND = not determined = symptoms still present FA = fluorometric method 35 CA = casein lytic method 91 777 10

Table 4. Plasmin activity (IU / 1) in rabbit anterior chamber fluid after mechanical iris irritation

Rabbit Before 20 min irritation 60 min irritation 5 No irritation after it

CA CA FA CA FA

1 <0.5 <0.5 <0.3 7 8 2 <0.5 <0.5 <0.5 30 10 10 3 <0.5 11 2 35 9 4 <0.5 18 N.D. 20 N.D.

5 <0.5 35 7.5 60 19 CA = caseinolytic method 15 FA = fluorometric method (D-Val-L-Leu-L-Lys-AMC, 25 ° C) N.D. = not determined

The casein lytic and fluorometric methods were performed immediately after sampling.

20

Table 5. Plasmin activity (IU / 1) in rabbit ventricular fluid after intraventricular capsaicin injection-induced neuritis 25 Rabbit Before injection 30 min after injection

No. CA CA FA

1 <0.5 16 7.1 2 <0.5 21 9.5 3 <0.5 25 4.5 30 4 <0.5 9 1.2 t CA = caseinolytic method FA = fluorometric method (D-Val -L-Leu-L-Lys-AMC, 25 ° C) to 35 91777 11

reference Publications

Bell PH, Dziobkowski, CT, and Englert, ME: A sensitive fluorometric assay for plasminogen, plas-5 min, and streptokinase

Anal Biochem. 61: 200-208, 1974

Bermann SR, Fox KA, Ter-Pogossian MM, Sobel BE, Collen D: 10 Clot-selective coronary thrombolysis with tissue-type plasminogen Activator Science 220: 1181-1183, 1983

Berman MB: 15 Collagenase and corneal ulceration

Collagenase in normal and pathological connective tissues

John Wiley & Sons Ltd New York, NY, 1980 20 Berman M, Manseau E, Law M & Aiken D:

Ulceration is correlated with the degradation of fibrin and fibronectin at the corneal surface

Invest Ophthamol Vis Sci 24: 1358-1366, 1983 25 Catanese J & Kress LJ:

Enzymatic inactivation of human plasma Cl-inhibitor and α-antichymotrypsin by Pseudomonas aeruginosa Proteinase and elastase

Biochem Biophys Acta 789: 37-43, 1984 30

Equipment TE & Murphy G:

Mammalion collagenases

Methods Enzymol 80, pt C: 711-722, 1981 35 Chesebro JH, Knatterud G, Roberts R et al:

Thrombolysis in myocardial infarction (TIMI) trial, phase 1: a comparison between intravenous tissue plas- 91777 12 minogen Activator and intravenous streptokinase Circulation 76: 142-154, 1987

Clavin SA, Bobbit JL, Schuman RT and Smithwick EL 5 Use of peptidyl-4-methoxy-2-nepthylamides to assay Plasmin.

Anal. Biochem 80: 355-365, 1977 Collen D & Verstraete M: 10 α2-antiplasmin consumption and fibrinogen breakdown during thrombolytic therapy Thromb. Res. 14: 631-639, 1979

Dano / K, Andreasen PA, Gro / ndahl-Hansen J, Kristensen 15 P, Nielsen LS, Skriver L:

Plasminogen activators and cancer Adv. Cancer Res. 44: 139-266, 1985

Fox KA, Bergmann SR, Sobel BE: 20 Coronary thrombolysis: Pharmacological considerations? with emphasis on tissue-type plasminogen Activator (t-PA)

Biochem Pharmacol 33 (12): 1831-1838, 1984 25 Hayashi, K & Sueishi K:

Fibrinolytic activity and species of plasminogen Activator in human Tears Exp. Eye Res. 46: 131-137, 1988 30 Holvoet P, de Boer A, Verstreken M & Collen D:

An enzyme-linked immunosorbent assay (ELISA) for the measurement of the Plasmin-α2-antiplasmin complex in human plasma-application to the detection of in vivo activation of the fibrinolytic system.

35 Thrombosis and Haemostasis, 56 (2): 124-127, 1986 ISIS-2 (Second International Study of Infarct Survival) i.

91777 13 Collaborative group: Randomized trial of intravenous streptokinase, oral aspirin, both, or neither among 17187 cases of suspected acute myocardial infarction ?: ISIS-2 5 Lancet 2: 349-360, 1988

Joutsimo L, van Setten G-B, Renkonen OV, Tarkkanen A, Påivarinta H, Tervo T:

On the proteolytic activity of contact lenses and bac-10 teria

Acta Ophthalmol (Kbh), (in press)

Karlamo BY, Clark AS, Littlefield BA: A highly sensitive chromogenic microtiter plate assay 15 for plasminogen activators which quantitatively discriminates between the urokinase and tissue-type activators

Biochem. Biophys. Res. Commun. 142: 147-154, 1987 20 Kress LJ:

Inactivation of human plasma Serine Proteinase inhibitors (serpins) by limited proteolysis of the reactive site loop with snake venom and bacterial metalloproteinases 25 J Cell Biochem 32: 51-58, 1986

Laitinen LA, Heino M, Kava T & Haahtela T:

Damage of the airway epithelium and bronchial reactivity in patients with asthma 30 Am. Rev. Resp. Dis. 131: 599-606, 1985

Lijnen HR & Collen D:

Alpha-2 antiplasmin J Med. 16: 225-84, 1985 35

Magnani, B:

Plasminogen Activator Italian Multicenter Study (PAIMS): 91777 14

Comparison of intravenous recombinant single-chain human tissue-type plasminogen Activator (rt-PA) with intravenous streptokinase in acute myocardial infarction 5 J Am. Coll. Cardiol 43: 19-26, 1989 Plow EF, Wiman B & Collen D:

Changes in antigenic structure and conformation of a2 ~ antiplasmin induced by interaction with Plasmin.

10 J. Biol. Chem. 255: 2902-2906, 1980

Reich E, Rifkin DB, Shaw E (eds.):

Proteases and Biological Control pp. 1021 Cold Spring Harbor Laboratory, 15, 1975

Remmert O., LeMar F & Chen PP:

Partial purification and properties of a proteolytic enzyme of human Serum J Biol Chem. 181: 432-448, 1949 20

Scissors 0:

Radial caseinolysis in Agarose: a simple method for the detection of plasminogen Activator in the presence of an inhibitory substance and Serum.

25 Ann. Biochem. 111: 276-282, 1981

Salonen EM, Tervo T, Torma E, Tarkkanen A & Vaheri A: Plasmin in tear fluid of patients with corneal ulcers: basis for new therapy 30 Acta Ophthalmol. 65: 3-12, 1987

Sherry S, Alkjaersig N & Fletcher AP:

Comparative activity of thrombin on substituted arginine and lysine unlers 35 Am. J Physiol 209: 577-583, 1965 I; 91777 15

Smith RE, Bisseil ER, Mitchell AB and Pearson KW:

Direct photometric or fluorometric assay of proteinases using substrates containing 7-amino-4-trifluoromethyl-coumarin 5 Thrombos. Res. 17: 393-402, 1980 Soria J, Soria C, and Samama C:

A Plasminogen assay using chromogenic synthetic substrate: Results from clinical work and from studies of 10 thrombolysis in progress in Chemical Fibrinolysis and Thrombolysis Vol 3 ed. Davidson JF, Rowan RM, Samama MM and Desnoyers PC

Raven Press, New York 1978, 337-346.

15 Tervo T, Salonen EM, Vaheri A, Immonen I, van Setten GB, Himberg JJ, Tarkkanen A:

Elevation of tear fluid Plasmin in corneal disease Acta Ophthalmol (Copenhagen) 66: 393-399, 1988 20 Tervo T, van Setten GB:

Aprotinin for inhibition of Plasmin on the ocular surface: principles and clinical observations In: Healing of the processes of the cornea Beuermann RW, Crosson CE & Kaufman HE (eds.), 25 Portfolio Publishing Company of Texas, Inc., The Woodlands, Texas , ss. 151-163, 1989

Tervo T, van Setten G-B, Andersson R, Salonen E-M, Vaheri A, Immonen I, Tarkkanen A: 30 Contact lens wear is associated with the appearance of. Plasmin in the tear fluid-preliminary results

Graefe’s Arch Clin Exp Ophthalmol 227: 42-44, 1989

Thorig L, Wijngaards G, van Haeringen NJ: 35 Immunological characterization and possible origin of plasminogen Activator in human tear fluid Ophthalmol Res. 15: 268-276, 1983 16 91 777

Tozser J, Berta A & Punyiczki M:

Plasminogen Activator activity and plasminogen indepen ^ -dent amidolytic activity in tear fluid from healthy persons and patients with anterior segment inflammation 5 Clinica Chimica Acta 183: 323-332, 1989

Vaheri A & Salonen E-M:

Fibronectin and regulation of proteolysis in cancer and tissue Destruction 10 Proc. Finn Dent. Soc. 84: 13-18, 1988 van Setten G-B, Salonen E-M, Vaheri A, Beuerman RW, Hietanen J, Tarkkanen A & Tervo T:

Plasmin and plasminogen Activator activities in tear 15 fluid during corneal wound Healing after anterior keratectomy

Curr. Eye Res. 8: 1293-1298, 1989 van Setten G-B, Tervo T, Andersson R, Perheentupa J, 20 Tarkkanen A: Effects of contact lens wear on the concentrations of Plasmin and EGF in human tear fluid Ophthalmic Res. (In press)

Wilcox RG, Olsson CG, Skene AM, von der Lippe G, Jensen 25 G, Hampton JR:

Trial of tissue plasminogen Activator for mortality reduction in acute myocardial infarction Anglo-Scandinavian Study of Early Thrombolysis (ASSET), Lancet, 11: 525-530, 1988 30 1,

Claims (5)

A method of quantitatively determining the plasma activity of body fluid, wherein a plasmin sample is reacted with a peptide derivative of 7-amino-4-methyl coumarin (AMC) and, via a hydrolysis, to form a reaction product which is determined fluorimetrically, , that the peptide is made up of the amino acid triplet D-Val-L-Leu-L-Lys. 2. A method according to claim 1, characterized in that peptide AMC reagent is used in the determination, which is buffered with a buffer suitable for plasma activity, specific Tris-HCl buffer. 3. A process according to claim 1 or 2, characterized in that the reagent in freeze-dried form is mixed with diluted body fluid. 4. Reagent for quantitative determination of the plasma activity of body fluid, which reagent is a peptide to which is joined 7-amino-4-methyl coumarin (AMC), characterized in that the peptide is constituted by the amino acid triplet D-Val. L-Leu-L-Lys. 5. Reagents according to claim 4, characterized in that the reagent is buffered with a buffer suitable for plasmin activity assays, especially Tris-HCl buffer.