A SOLUBILIZABLE, FIBRIN BASED COMPOSITION AND ITS USE, IN DETERMINATIONS OF FIBRINOLYSIS PARAMETERS

Abstract: A fully solubilizable fibrin based composition, which is characterized by the combination that the fibrin is desAA-fibrin or desAA-fibrin from which the C-terminal portions of the alpha -chains have been removed by enzymatic digestion, and that the solubilizing agent is a tetrapeptide containing the amino acid sequence -L-prolyl-L-arginyl-, preferably glycyl-L-prolyl-L-arginyl-L-prolin. The full solubility of the fibrin makes possible new uses within the area of determination of important fibrinolytical parameters, and the invention also relates to three such important alternative uses. A first use according to the invention is the use of the composition in connection with detection or quantification of the activity of the enzyme tissue plasminogen activator. A first alternative use means that the composition is used for the detection of soluble fibrin in biological fluids, and a second alternative use means that the composition is used for measuring or studying fibrinolytic activity in vitro or in vivo through the addition of the composition and registration of the degradation of the fibrin.

Description: TITLE OP INVENTION "A solubilizable, fibrinbased composition and its use, in determinations of fibrinolysis parameters". TECHNICAL HELP The preeent invention relateβ to a βolubiiizablβ, fibrin baβed compoβition which iβ based on a epecific combi¬ nation of a certain fibrin derivative and a low molecular peptide containing a certain amino acid sequence. The peptide referred to has, surprisingly, proved to give a complete βo- lubilization of said specific fibrin derivative, which opens, completely new possibilities of using such a compoβition when assaying important fibrinolytic parameters, or at eaβt dra¬ stically improves the opportunity to perform accurate and reproducible analyeee within thiβ field. Hence, the invention also relateβ to certain especially interesting applications or uses of said compoβition. BACKGROUND OF THE INVENTION Systematic studies in fibrinolysis (those own βyβtemβ of the body which are involved in the βolubilization of fib¬ rin, the protein from which blood clots are built up) has been hampered by difficultieβ in manipulating its target sub¬ stance, fibrin. For further information on the fibrinolytic system see Collen D, 1980, Thrombosis and heamoβtaβis 43: 77-86. The possibilities of replacing, for a model purpose, fibrin, which spontaneously forms a gel, with more easily handled substanceβ, such as caβein or albumin, are limited. Thiβ iβ due to the fact that fibrin, except from being the target substance, also has important regulatory functionβ in the fibrinolytic βyβtem. Theβe can not be replaced by model proteinβ. The demand for a soluble and hence easily handled fib¬ rin derivative iβ βubβtantial. Therefore, conβiderable re¬ search efforte have been made in order to βatiβfy thiβ de- mand. In 1955, Donβlly et al deβcribed in Arch. Biochem. Biophyβ. 56: 369-387, how fibrin can be βolubilizβd in 1 by Havβrkatβ and Timan in Progresβ in Chemical Fibrinolyβis and Thrombolysis 2: 67-71, that fibrin can be solubilized with 10 mole/1 of acetic acid, while in 1982 Ranby et al deβcribed in Thrombosis Research 27: 743-749, the eolubili- zation of desAA-fibrin in'3.5 mole/1 of urea. However, neither of theβe methodβ haβ been widely uβed which confirmβ that they are not fully βatiβfactory. Thuβ, the deβcribed methodβ cauβe denaturation of the fibrin, which ia expreββed aβ a tendency of the fibrin to form, after hav- ing been brought back to phyβiological conditions, a precipi¬ tate instead of a gel. Furthermore, the deβcribed compoβit- ionβ of soluble fibrin do not βtand being frozen/thawed or lyophilizβd/reconβtituted with preββrved propertieβ. As concerns prior art reference iβ alβo made to Chem. Abβtractβ 98 (1983), 69 554, which diβcloβeβ a retardation of the polymerization of deβAA-fibrin and deβAABB-fibrin in the presence of Gly-Pro-Arg. However, the present invention iβ based on the fact that it haβ very unexpectedly turned out that there iβ a special type of tetrapeptide which does not only retard but also completely and permanently prevents the polymerization of a certain type of fibrin, viz. deβAA-- fibrin. This prevention has made it possible to prepare a practical useful preparation of soluble fibrin and even to find completely new areaβ of applications. Furthermore, Chem. Abβtractβ 99 (1983), 118 035, dis¬ closes that "α-chain peptide" inhibits the polymerization of "thrombin-induced fibrin", i.e. dββAABB-fibriii. Thuβ, said article iβ not relevant in connection with the present inven¬ tion, according to which the above-mentioned tetrapeptide forms in combination with desAA-fibrin a stable, non-poly¬ merizing compoβition. Chem. Abβtractβ 101 <1984>, 106 025, merely discloβeβ that Gly-Pro-Arg-Pro retards the fibrin polymerization by being bound to fibrinopeptide A. Thuβ, thiβ article iβ not either relevant in connection with the present invention; see above with reference to C.A. 99 (1983), 118 035. SUMMARY OF THE INVENTION According to the present invention it haβ unexpectedly turned out that the addition of a low molecular weight pep¬ tide containing the βpβcific amino acid ββquβncβ -L-prolyl-L- -arginyl- to a certain fibrin derivative, namely dββAA- -fibrin, giveβ a fully βolubilizable compoβition, and ateuch a low concentration of the peptide that poββibilitieβ are opened for entirely new applicationβ in connection with the determination of fibrinolytic components or that the accuracy and reproducibility of applicationβ known per se are improved so that such applications can also come to a real practical Theβe reβultβ were not predictable for a perβon skilled in the art. It iβ true that in 1979 Laudano and Ooolittlβ diβcloβed in Procβedingβ of the National Academy of Science 75: 3085-3089, that certain peptideβ, especially glycyl-L- -prolyl-L-arginyl-L-prolin, have the ability to strongly re¬ duce the polymerization rate for fibrin, but based upon thiβ it was in no way possible to predict the extraordinary rβ- suits which the present invention haβ been shown to give. Thuβ, it haβ e.g. turned out that desAA-fibrin is fully solu- bilized in a compoβition according to the invention at pep¬ tide concentration levels where the same peptide does not show any detectable solubilizing effect at all on deaAABB- fibrin. In thiβ context, it could be mentioned that deβAA- fibrin differs from "normal" deβAABB-fibrin by having only one pair of the fibriopeptideβ (the A-peptideβ) split off. DeβAA-fibrin iβ commonly known as fibrin I, while desAABB- fibrin is named fibrin II, which denotations for simplicity will be used below. Another important difference that has been demonstrated between fibrin I and fibrin II is that in the preβencβ of the same peptide they differ essentially from each other as to the ability of stimulating the tisβue activator in activation of plaβ inogen. Thus, a compoβition according to the invent¬ ion containing fibrin I has been shown to be about 50% more effective than the corresponding compoβition containing fib¬ rin 11. Theβe and other propertiββ will be further illustrated below in connection with the definition of the invention and in connection with the working examples. More specifically the present invention relateβ to a βolubilizable, fibrin baβed compoβition, which iβ character¬ ized in that the fibrin ia deβAA-fibrin or dββAAfibrin from which the C-terminal parts of the -chainβ have been removed by enzymataic digeβtion, and that it compriβββ a βolubilizing amount of a tetrapeptide containing the amino acid ββquence -L-ρrolyl-L-arginyl-. The preferred tetrapeptide for uβe in the compoβition claimed ia glycyl-L-protyl-L-arginyl-L-prolin, but the in¬ vention iβ not limited thereto, aβ there should exist" other tetrapeptidββ containing the specified amino acid sequence which give eββentially the βame effect. The deβAA-fibrin used in the compositions according to the invention is preferably prepared by a, selective removal of merely fibrinopeptide A from fibrinogen'by enzymes, e.g. batroxobin extracted out of venom from snakes of the Bothropβ genus. The βolubilizing concentration of the tetrapeptide can of course be determined from case to case using routine tests. Generally, however, the lowest concentration for prac¬ tical uβe of the compoβition should be about 0.4 g per ml of a ready to uβe-compoβition or ready to use-preparation. The upper limit is more difficult to generalize and iβ influenced by different factors, such aβ economic considerations and intended u-.a of the compoβition. Thuβ, the economy may be decisive for. a suitable upper limit, aβ for financial reasons one will of course not use higher concentrations than are required for the intended purpose. However, in the use aβ a stimulator in determining the tiββue activator there iβ an upper limit, for the uβeful end concentration of the peptide. Thiβ iβ about 10 mg/ l and iβ Justified by the fact that the activation reaction iβ inhibited at peptide concen- trationβ exceeding about 0.03 mg/ml and by the fact that at least one threehundredthβ volume of fibrin preparation haβ to be added at the beginning of the reaction (0.033 x 300 = 10). Generally a suitable range iβ 0.4 - 10 mg/ml of ready to uβe- N V -preparation, an especially interesting range being 0.6 - 4 mg/ml. In many cases optimum results can be achieved at a concentration of about 2 mg/ml. The composition according to the invention comprising fibrin I βolubilized with the indicated peptide is preferably presented in a phyβiological buffer, which for inβtance meanβ that it can be administered without any inconvenience into the circulatory tract, e.g. injected intravβneouβly. The preparation of the compoβition according to the invention iβ not aββociated with any particular problems but can preferably be made by first digesting the fibrinogen with enzyme and then diββolving the resulting fibrinogel by the addition of peptide. If required, the enzyme can then be re¬ moved, e.g. with matrix-bound antibodieβ againβt the same. Aβ was mentioned above, the invention also refers to certain particular applications or uses of the composition, which have been made possible or have been substantially im¬ proved as compared to the prior art by the suprior properties of the soluble fibrin preparation that iβ the result of the invention. These uses can be aββigned to the determination of important fibrinolytic parameters, the background being as foil we . Fibrin deposits in the circulatory tract are degraded to soluble components by the βerine protease plasmin. Thiβ enzyme iβ formed from the plasma protein plasminogen by act¬ ion of plasminogen activators. The known plasminogen activa¬ tors are serine proteases, which split an Arg-Val-peptide bond in the plasminogen molecule to form the active two chain enzyme plasmin. Plasmin βhowβ βome substrata specificity and preferably splits peptide bonds in the fibrin matrix and in the fibrin precursor fibrinogen. This specificity is far from absolute, and plasmin sensitive bonds can be found in moβtβ proteinβ (many of the numerouβ trypβin βenβitβivβ bonds are also split by plasmin) . Plasmin formed during fibrinolysis may therefore damage plasma proteins and cell surface proteins. In order .to avoid this organism haβ developed mechanisms that localize the plasmin formation and restrict its effect. Two such mechanisms are commonly recognized, namely: 1. The tissue plasminogen activator (t-PA), an important plasminogen activator in the blood, is effective only in the presence of fibrin. In the absence of fibrin the rate of the t-PA-catalyzed plasminogen activation iβ low, but in the preβence of fibrin the reaction rate ia increaβed up to 1000 timeβ . The generation of plasmin iβ, thuβ, localized to fibrin, which iβ then degraded. When the fibrin iβ once degraded, the rate of plasmin formation returns to low values. 2. Generated plaβmin not involved in fibrin degradation iβ rapidly inhibited by the plaβmin inhibitor α-2-antiplaβ- min. The effect of generated plaβmin iβ, thuβ, limited to the vicinity of the clot. Normally, fibrin clotβ have a useful • function, and con¬ sequently they should not be exposed to premature lysis. By this reason nature haβ developed mechanisms to avoid thiβ, and these mechanisms are at present beginning to be better understood. The now discovered fast acting plasminogen acti- vator inhibitor can constitute the base for such a mechanism. Alterations, during fibrinolysis, of the stimulatory proper¬ ties of the fibrin can constitute another one. The present invention relates to certain uses, which are related to the above-mentioned. One first use of the com- position more specifically implies that it is used in con¬ nection with the detection or determination or quantification of the enzyme tissue plasminogen activator (fcΦA), the fibrin potentiating the activity of the enzyme towards the physiolo¬ gical substrate plasminogen or towards some synthetic sub- βtrate. The method per se . i.e. analysis with respect to tissue plasminogen activator, is previously disclosed by Ranby et al in Thrombosis Research (1982) 27: 743-749. However, according to the invention it has been shown that the fibrin composi- tion according to the invention is considerably more effect¬ ive than the composition deβcribed in said publication as regards the ability of stimulating the tissue plasminogen activator in the activation of plasminogen. Furthermore, when diluting fibrin I βolubilizβd with the peptide referred to, fibrin gel iβ formed more βlowly than when diluting fibrin I solubilized with urea. This iβ of a βubβtantial practical significance when solubilized fibrin I iβ uβed aβ a "βtimula- tor" in the determination of t-PA according to the abovβ- -mentioned method. The effectβ of the compoβition claimed will be further elucidated below. An alternative application or use according to the in- vention of the compoβition meanβ that it iβ utilized to de¬ tect trace amounts of fibrin diββolved in biological fluidβ, e.g. blood plasma, the composition being uβed aβ a βtandard. Thiβ uβe is made poββiblβ inter alia by the favourable gel forming kinetic shown by the fibrin compoβition according to the invention, and the compoβition according to the invention will most likely be of a great practical importance. Thus, the concentration of fibrin i blood or in blood plasma is expected to become an important diagnostic parameter inter alia for the condition D.I.C, (diβseminatded intravascular coagulation) . j A second alternative application or use according to the invention is the use of the compoβition in measuring or studying fibrinolytic activity i_x vitro or .in ivivo through the addition of the compoβition and regisβtration or recordal of the degradation of the fibrin contained in the composit¬ ion. In connection with thiβ uβe, the unique fact should be emphasized that the fibrin composition according to the in¬ vention is most likely non-toxic and iβ presented in a phy¬ siological buffer, so that the compoβition can be adminiβter- ed without any inconvenience into the circulatory tract, e.g. by intravenous injection. The fibrin will, inter alia owing to itβ gel formation kineticβ, be diβtributed over the entire circulatory tract, where it iβ most likely complexβd to fib¬ rinogen. Due to fibrinolytical activity in the blood and in the vascular bed this fibrin will be degraded and disappear out of circulation. The rate at which thiβ occurs iβ a measu- re of the total fibrinolytical capacity of the organism. This parameter, which can also be measured j__ vitro. may come to be of a great diagnostic importance, whereby the composition according to the invention will be an extremely valuable in¬ strument in such a diagnoβiβ. The compoβition according to the invention iβ for inβtance an extraordinarily well suited fibrin derivative for uβe ill vivo, aβ it iβ preβented in a phyβiological buffer and aβ the peptide iβ likely to be harm¬ less to the organism. To facilitate the analysis the fibrin can be labelled, e.g. with radio labelled iodine or with biotin. EXAMPLES The invention will now be illustrated further by some working examples, which are preβented merely to illustrate the invention and not in any reβtricting βence. In the exampleβ highly purified preparations of prote¬ ins and some less known chemicalβ are uβed. These are descri¬ bed below together with information on where they can be ob¬ tained. The compositions of buffers uβed are also indicated. Glu-Plas inoσen from human plasma; lyophilized prepara¬ tion from BioPool AB, Umea, which according to βpecificationβ contains less than IX of Lyβ-plaβminogen, less than 10 ppm of plasmin and less than 0.0001 IU of t-PA/mg. At use the prepa¬ ration was reconstituted in sterile PBS (see below), to a final concentration of 5 mg/ml and centrifuged 2 min at 11.000 x g. Reconstituted material was kept at +4°C for no more than 48 hours . Tissue plasminogen activat-or. (t-PA), in its single- chain form from human melanoma sells; lyophilized preparation without any carrier substance from BioPool AB, Umea, Accord¬ ing to the manufacturer; specific activity 500,000 IU/mg, more than 95% active, containing less than 2% of twochain t-PA and less then IX of non t-PA protein. The preparation waβ reconβtituted in 1M KHCO3 to a concentration of 50,000 IU/ml and kept at +4°C for no more than 48 hours. Further dilution waβ made with Triβ/Tween buffer, (βee below). Aβ a reference for t-PA activity the WHO firβt international stan¬ dard lot 83/517 from NIBSAC, Holly Hill, London, was used. Fibrinogen from human plaβma; lyophilized preparation from Imco Corporation Ltd AB, Stockholm, according to speci¬ fications 97% coagulability and free from plaβminogen. The preparation waβ reconβtituted with H2O to a concentration of 20 mg/ml and waβ kept a +4°C for at moβt 4 hours. Batroxobin from βnakβ venom from Bothropβ atrox maran- hao; lyophilized preparation from Pβntapharm AG, Basel, Switzerland. According to specificationβ at leasts 100 BU/ml . The preparation was reconstituted in PBS to a concentration of 20 BU/ml and kept aβ 0.2 ml aliquoteβ in liquid nitrogen (-196°C) . Thrombin from bovine plaβma; βterilβ βolution Topoβta- βin from Hoffman-La Roche, Basel, Switzerland. Glv-Pro-Arg-Pro. (glycyl-L-prolyl-L-arginyl-L-prolin) , aβ the acetate βalt from BioPool AB, Umea. The salt waβ dis¬ solved in H2O to a concentration of 100 mg/ml and kept at -20°C. Plasmin substrate Spectrozyme PL. (H-D-norleucyl-L- hexahydrotyrosyl-L-lysinyl-paranitro-anilide) ; lyophilized preparation from American Diagnostics Inc., Greenwich, USA. Buffers: PBS ("phosphate buffered saline"), with the compoβition 0.1 mole/1 of NaCl, 0.02 mole/1 of sodium phos¬ phate buffer pH 7.3. Tris/Tween has the composition 0.1 mol/1 of Tris/acetic acid buffer pH 8.3 (at 37°C) , containing 0.1 g/1 of Tween 80. EXAMPLE 1 Fibrinogen, 5 mg/ml, diββolved .in PBS waβ aliquoted aβ 2 ml aliquoteβ in φ 9 mm polystyrene ^ test tubes. To these either 0.01 ml of bovine thrombin, 20 NIH/ml, or 0.01 ml of batroxobin (from B A Maranhao) , 20 BU/ml waβ added. Hereby, in all teβt tubββ, a coagulate waβ formed within 5 minuteβ. To theβe small fibrin coagulates 0.02 ml of glycyl- L-prolyl-L-arginyl-prolin, (Gly-Pro-Arg-Pro), with a protein concentration between 0 and 115 mg/ml, was added. The final concentration of peptide in the cogulateβ waβ, thuβ, between 0 and 10 mg/ml. Every tventy minute the test tubββ were βhaken, and after 3 hours the appearance of the coagulates were register¬ ed. The experiment waβ performed at room temperature (about 20°C). The reβultβ are preβented in Table 1 below. TABLE 1 Solub il ity for fibrin I and fibrin I I , respectively Cone. rag/ml 0 0.1 0.2 0.4 0.6 1 .0 2.0 4.0 10.0 fibrin I (obtained rigid soft soft weakly clear clear clear clear clea with gel gel gel opaque sol . sol . sol . sol . sol. batroxobin) sol . fibrin II rigid rigid rigid rigid rigid rigid rigid rigid loos obtained gel gel gel gel gel gel opaque opaque gel with gel gel thrombin) From Table 1 above it can be seen that solubilization of f ibrin I was achieved already at a concentration of about 0 . 4 mg/ml and that full solubilizat ion of the f ibrin ref erred to was achieved at a peptide concentration of 0 .6 mg/ml or more . On the contrary no solubilization of f ibrin I I could be obtained within the concentration range tested . Aβ 10 mg/ml may be a pract icable upper limit f or certain applicat ions , as suggested above , and as will be further illustrated below, it is clear that merely the combination of peptide with f ibrin I is practically useful . EXAMPLE 2 Preparation of peptide solubilized fibrin I (used in Examples 3, 4 and 5). 1 volume 20 mg/ml of fibrinogen was diluted with 1 volume of PBS. To thiβ one eightieth volume 20DU/ιal of batroxobin (end concentration 0.25 BU/ml) waβ add¬ ed, and incubation waβ performed at room temperature (lβ°C). A βoft gel waβ formed within 15 minutes. After 90 minutes the gel was solubilized by the addition of one fifthieth volume of 100 mg/ml of Gly-Pro-Arg-Pro (final concentration in the coagulate 2 mg/ml). Gentle agitation for 3 hours. The βolution waβ βterilβ filtered (0.45 μm membrane filter), and aliquoted aβ 100 μl aliquoteβ (1 g of fibrin I, 0.2 mg of peptide). Some of these were kept at -20°C, (lot 12A) , and some were lyophilized and kept under a nitrogen atmosphere at +4°C (lot 12B) . The t-PA stimulating property of these preparations were tested and compared with an earlier lyophilized prepa¬ ration (lot 11B, see Table 1). The lyophilized preparationβ, (lot 11B and lot 12B) , were reconβtituted by the addition of 100 μl of Triβ/Tween. Lot 12B2d iβ a reconstituted material Table 2. 0.5 ml aliquoteβ of 0.5 mg/ml of Glu-plasmi- nogen, 0.5 mM Spectrozymβ and 0.2 ΣU/ml of one chain t-PA diββolved in Triβ/Tweβn were diβpenβated in 9 mm polyβtyrene tubeβ . To theβe tubeβ 1, 3, 5 or 7 μl of solubilized fibrin I derivative were added, and immediately after the additions the tubes were agitated and placed in a water bath held at 37°C. After i h of incubation the reaction was stopped by addition of 50 μl of 10% acetic acid and the absorbance at 405 n was measured. The increase in absorbance was calculat¬ ed by subtracting the absorbance in solution where the react¬ ion was stopped immediately after addition of fibrin. This absorbance was 0.083. In the absence of stimulator an incu¬ bation at 37°C for 4 hours resulted in an absorbance increase of 0.017. TABLE 2 Added amount of Absorbance increase at 405 nm obtained fibrin I prep. with different prepa .rations of solubilizβd fibrin I (Ul) lot 11B lot 12A lot 12B lot 12B2d 1 0.635 0.621 0.572 0.562 3 0.755 0.714 0.705 0.688 5 0.800 0.787 0.776 0.750 7 0.809 0.804 0.795 0,778 The reβultβ in Table 2 βhow that βoluble fibrin compo¬ sitions, prepared according to the invention, can be prepared with reproducible t-PA βtimulatory effects. Furthermore it iβ demonstrated that thiβ βtimulatorγ effect iβ on the whole unaffected by lyophilization with subsequent reconβtitution. Finally, it iβ shown that reconβtituted preparations have practically acceptable stabilities (48 h) . Taken to¬ gether the experiment βhowβ that soluble fibrin, prepared according to the invention, haβ propertieβ that make it extremely well βuited for the determination of t-PA activity. EXAMPLE 3 In order to determine to what extent the peptide Gly— Pro-Arg-Pro inhibits the fibrin stimulated t-PA activation of plasminogen the following experiment waβ performed. To ali- quotes of 0.49 ml of Tris/Tween, containing 0.025 mg of Glu- -plasminogen, 0.25 μmole of Spectrozyme and 0.1 IU of t-PA, there was added 0.01 ml of peptide, at a concentration of 5, 2.5, 1.25, 0.63, 0.32 or 0 mg/ml. The reaction waβ started by addition of 5 μl of soluble fibrin according to the invention (see Example 2) and aβ the fibrin preparation containβ 2 mg/ml of the peptide the final peptide concentrations were 0.12, 0.07, 0.045, 0.0325, 0.0263, and 0.2 mg/ml. 60 minutes at 37°C after the fibrin addition the reaction was stopped by the addition of 50 μl of 10% acetic acid and the absorbance at 405 nm waβ measured. At each peptide concentration 4 experiments were performed and the mean values of the absorbance increase and its standard deviations are accounted for in Fig. 1. Thus, Fig. I shows the t-PA stimulating effect of 0.1 mg/ml of soluble iibrin according to the invention, dependant on the final concentra¬ tion of peptide. More specifically the ordinate denotes the absorbance increase at 405 nm, while the abβciββa denoteβ the final concentration of Gly-Pro-Arg-Pro in mg/ml. In the ab- sence of fibrin the absorbance increase waβ about 0.006 at all examined peptide concentrations. From the experiment it iβ clear that a final concentra- tion of 0.033 mg/ml of peptide reβultβ in a βignificant dβ- creaβe in t-PA mediated activation of Glu-plaβminogen. In thiβ way an upper limit for a uββful final concentration of peptide in the preparation of soluble fibrin according to the invention iβ provided. Thiβ iβ 10 mg/ml and iβ motivated by the activation reaction being inhibited at peptide concentra¬ tions exceeding 0.033 mg/ml and by the fact that at least one threβhundredth volume of fibrin preparation haβ to be added at the beginning of the reaction (0.033 x 300 = 10). g Mpkg 4 , The determination of t-PA activity while utilizing so¬ luble fibrin according to the invention can be made in the following way: 500 μl of TAR (tissue plasminogen activator reagent) consisting of 0.05 mg/ml of Glu-plasminogen and 0.5 mM of Spectrozyme PL dissolved in Tris/Tween are aliquoted in 0 9 mm polystyrene test tubeβ. To these 50 μl samples consis¬ ting of the WHO t-PA standard diluted in Tris/Tween (t-PA concentrations between 0 and 3 IU/ml) are added. To this (immediate mixing) 5 μl soluble fibrin I is added, aβ is de¬ scribed in Example 2. After 60 minutes at 37°C the reaction is stopped by 50 μl of 10% acetic acid. The absorbance in¬ crease at 405 nm iβ determined as in Example 2 and is plotted along the ordinata against the t-PA content in the sample (IU/ml) , Figure 2. To demonstrate the determination of t-PA in plasma samples 10 μl of t-PA dissolved in Tris/Tween were added to 200 μl of t-PA depleted plasma to yield final concentrations between 0 and 20 IU/ml. To destroy the plasmin inhibitor con- tent of the plaβma 200 μl of 1 M acetate buffer pH 3.9 were- added, followed by a 15 minuteβ incubation at 37°C. After that 200 μl of Triβ/Tween were added, whereby the t-PA con-- centration became 0-2.5 IU/ml. 50 μl samples thereof were analyzed for t-PA activity aβ described above. The result is shown in Fig. 2. The figure shows the analysis of t-PA dis¬ solved in buffer (•) and of t-PA dissolved in blood plasma (*> . From Figure 2 it can be seen that soluble fibrin ac¬ cording to the invention is extraordinarily useful in the determination of t-PA activity with application to biological βampleβ . The content of inhibitors for t-PA iβ determined by adding a known amount of t-PA to the sample, whereafter the residual exceββ of t-PA iβ determined. Fibrin preparationβ according to the invention are, thuβ, very uβeful alβo for the determination of the content of t-PA inhibitor in e.g. blood or blood plaβma. In the t-PA measurements in Examples 2 and 3 a solution containing 0.05 mg/ml of plasminogen was uβed. During the measurement leββ than 1% thereof iβ activated. Thuβ the high concentration of plasminogen iβ not justified by the consump- tion but by the fact that a high activation rate, meaning sensitivity, demands such concentrations. However, during the development of the invention it waβ found that partially degraded fibrin (fibrin with C-terminal parts of the α-chains split off) can stimulate t-PA to ef¬ fectively activate plasminogen at plasminogen concentrations of 0.005 mg/ml. Soluble fibrin according to the invention, where the jC-terminal parts of the α-chains have been removed from the fibrin by enzymatic digestion, e.g. with matrix bound plasmin, would allow t-PA measurement with considerably lower plasminogen consumption. Plasminogen of the necessary high quality is an expensive component in tPa measurements, which means that the possibility of reducing the concentrat¬ ion is of ! great importance. EXAMPLE 5 Fibrin may be found in circulating blood, where it most likely occurs in the form of complexes with other plasma pro¬ teins. The presence of such fibrin indicates proceeding coagulation procesβeβ and a quantification of circulating fibrin iβ likely to be of a great clinical interest. The ability of the tisβue plasminogen activator of activating plasminogen is greatly potentiated by fibrin aβ was described above. If the concentration of fibrin is low, the activation rate depends directly thereupon, as waβ shown by Ranby M, 1982, Biochimica and Biophysics Acta 704: 461- -469. In connection with work on the present invention it was noted that also fibrin in plaβma stimulated the capacity of t-PA to activate plaβminogen and that thiβ could conβtitute a principle on which analytical methodβ for the determination of fibrin could be baβed. However, for βuch methodβ there will ariββ a need for a standard and soluble fibrin according to the invention iβ here demonβtrated to be a good, perhapβ the only practicable one, βtandard for thiβ purpose. To 1 ml of human blood plaβma 10 μl of soluble fibrin according to the invention were added (see Example 2). Thiβ plaβma (containing 0.09 mg/ml of added fibrin) waβ diluted with plaβma, plaβma samples containing 0.09, 0.045, 0.023, 0.011, and 0.0056 mg/ml being made. From these samples 50 μl were mixed with 500 VI of 0.05 mg/ml of plasminogen, 0.5 mM Spectrozyme and 50 IU/ml of t-PA. After 25 minutes at 37°C the reactions were stopped by the addition of 50 μl of 10% acetic acid. The absorbance increase at 405 nm was determined and plotted along the ordinata aβ a function of the concen¬ tration of added fibrin, Figure 3. The results here accounted for demonstrate that soluble fibrin according to the invention is well suited as a stan¬ dard in analyeee aiming at the determination of the fibrin content in circulating blood or blood plasma. EXAMPLE 6 It seems probable that an important function of the fibrinolysis system is to free circulating blood from low amounts of fibrin (see Example 5). Probably the enzyme tissue plasminogen activator (t-PA) is of significance in this re¬ spect, but also other enzymes maybe involved. Reasonably, what is of importance in clinical connections is not which enzyme or enzymeβ iβ (are) active, but the results of their action. Therefore, the soluble fibrin according to the inven¬ tion may become of a great diagnostic importance aβ this makes it possible to add to blood, in. vivo or .in vitro. or to plood plaβma a controlled amount of fibrin. The efficiency of the fibrinolytic system can then be established by recording how thiβ added fibrin iβ degraded. The key to thiβ type of determination is a handleable fibrin derivative. In the present example there iβ added to normal blood plaβma a certain amount of soluble fibrin (70 mg/1 or 200 nmole/1). At regular intervals βampleβ are taken and the con¬ centration of remaining fibrin iβ determined by the method diβcloβed in Example 5. The experiment iβ alβo performed after an increase of the t-PA activity in plasma of 10 IU/ml. To two 600 μl βampleβ of normal blood plaβma there were added 10 μl of Triβ/Tween (βample A) or 10 μl of 600 IU/ml of one-chain t-PA (βample B) . To theβe two βampleβ there were added 4 μl of peptide solubilized fibrin I (see Example 2), the plaβma samples containing 70 mg/1 of added fibrin. Imme¬ diately after the addition of fibrin (time = 0) and at 5, 10, 20 and 30 minutes aliquotes were taken from the plaβma samp¬ les and the fibrin concentrations were determined as in Example 2. The results are presented in Figure 4, where the ordi- nata shows the remaining fibrin concentration in mg/1 and the abscissa shows the incubation time in minutes. Thuβ, soluble fibrin according to the invention was added to normal human plasma (•) and to normal human plasma enriched with 10 IU/ml of t-PA (x) . After the additions the concentration of fibrin was in each case 70 mg/1. The plasma samples were incubated at 25°C and aliquotes were analyzed for fibrin at 0, 5, 10, 20 and 30 minutes. From the results (see Fig. 4) it is clear that the fib- rin concentration of sample A fell from 70 mg/1 at the time 0 to 48 mg/1 after 20 minutes. The corresponding figures for sample B were 70 mg/ml (time 0) and 20 mg/ml (20 minutes). Thuβ, soluble fibrin disappears rather rapidly when added to normal plaβma. Thiβ procesβ will be even more rapid if the fibriolytic activity in plaβma iβ increased by the addition of t-PA. The ability of blood to make itself free from circu¬ lating soluble fibrin can be expected to be reduced for pati- ents with a tendency to thromboβiβ and/or infarct. The solub¬ le fibrin according to the invention makes possible a quanti¬ fication of said ability.

Claims: CLAIMS 1. A βolubilizable, fibrin based composition, characterized in that the fibrin is desAA-fibrin or deβAA- fibrin from which the C-terminal portions of the α-chains have been removed by enzymatic digestion, and that it contains a βolubilizing amount of a tetrapeptide containing the amino acid sequence -L-prolyl-L-arginyl- and having the ability of βolubilizing said desAA-fibrin, and preferably also a physiological buffer. 2. A compoβition according to claim 1, characterized in that the peptide iβ glycyl-L-prolyl-L-arginyl-L-prolin. 3. A compoβition according to claim 1 or 2, characterized in that the deβAA-fibrin haβ been obtained by a selective " removal of fibrinopeptide A from fibrinogen with an enzyme, preferably batroxobin. 4. A compoβition according to any one of the preceding claims, characterized in that the peptide concentration is at least 0.4, preferably 0.4 - 10, mg/ml of a ready to use-preparation. 5. A compoβition according to claim 4, characterized in that the peptide concentration is 0.6 - 4, preferably about 2, mg/ml of a ready to use-preparation. 6. Use of a compoβition according to any one of claims 1 - 5 in connection with the detection or quantification of the enzyme tissue plasminogen activator, the fibrin potentiating the activity of the enzyme towards the physiological substrate plasminogen .or some synthetic substrate. 7. Use of a composition according to any one of claims 1 - 5 for the detection of soluble fibrin in biological fluids, e.g. blood plasma, the composition being used as a standard. 8. Use of a composition according to any one of claims 1 - 5 for measuring or studying fibrinolytic activity in vitro or .in vivo through addition of the composition and registration of the degradation of the fibrin contained therein.

Inventors: RAONBY MATS GUSTAF, RAONBY, MATS, GUSTAF
Applicants: BIOPOOL AB
Application year: 1986
Main classification: C12Q1
IPC codes: C07K5, C12Q1