Kinetic characteristics of fibrinogen and fibrin hydrolysis by plasmin 1 and 2 and miniplasmin

THROMBOSIS RESEARCH41; 681-688, 1986 0049-3848/86 $3.00 + .OO Printed in the USA. Copyright (c) 1986 Pergamon Press Ltd. All rights reserved.

KINETIC CHARACTERISTICS OF FIBRINOGEN FIBRIN HYDROLYSIS BY PLASMIN 1 AND AND MINIPLASMIN

AND 2

T.F. Kastrikina, L.D. Taran, S.A. Kudinov Department of Enzyme Chemistry and Biochemistry The A.V. Palladin Institute of Biochemistry, Ukrainian Academy of Sciences, Kiev 252030, U.S.S.R. (Received 22.4.1985; Accepted in revised form 10.10.1985 by Editor M. Kopec) (Received in final form by the Executive Editorial Office 13.12.1985)

AESTRACT

Fibrinogenand fibrin hydrolysis by native plasmin ‘I aad 2 and by miniplasminwas studied.The degree of hydrolysiswas estimatedby the number of amino groups determinedwith trinitrobenzenesulphonicacid. The processwas shown to obey Michaelisdrlantan kinetics. Kinetic parametersof fibrinogenend fibrin hydrolysis by plasmin forms 1 and 2 were identical(Q = 6.5 x IO+, kcat= 7.1 set") while for hydrolysisby mini= 3.58 sec.'. Thus = 20.0 x lo+, k demonstratedthatCat enzymaticpropertiis of plasmin are to some extant dependenton the presence of lyetie-bindingsites. However, this appearsnot to have a decisive effect on fibrinolyticprocess.

INTRODUCTICN Plasminogenis a glycoproteln,consistingof one polypeptide chain of a known primary structurewith a molecularweight of 92,000 (1). Native plasminogenis availablein the plasma in two forms distinct in their affinity to lysinedepharose (3). The basic structuraldifferenceof these forms, is their relative degree of glycos lation:form I has 2 glycosylationsites, as eragin 288 and tEreonine 345; form II has one, threonine345 P 6). In view of differancesin affinity to lysFneSepharose the authors suggest that the strength of two plasminogenforms binding to _____~

___

__

_

Key words: Plasmin, miniplasmin,Fibrinogen 681

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fibrin can vary. The study of hydrolysisof syntheticsubstrates revealed little enzymaticdifferencesbetween the two plazminogen forms (4). Limitednative plasminogenhydrolysis by elastase allowed us to obtain differentmolecular fragments.The smallest of the known plasminogenfragments,totally preservingpotential enzyme activityis miniplasminogenwith a molecularweight of 38,000 (7,8). The miniplasminogenmolecule preserves the intact light chati and kringle 5 linked to it by disulfide bonds, but lacks a substantialportion of the heavy chain with lysinebinding sites. The existenceof a low-molecularform permits structuraland functionalstudies of plasminogento be made with the attemptsto establishthe role of lysine-bindingsites in exposing enzyme propertiesof plasminogen.Kinetic parameters of syntheticsubstrateshydrolysisby plasmin and by miniplasmin were found to be similar (g,lO),which led the authors to suggest that miniplasminactive centre was nearly identical to that of plasmin. The lack of sensitive quantftationfor fibrinogen hydrolysissearces the evaluationof the catalyticproperties of plasmin structuralforms during the hydrolysisof natural protein substrates.In this very case the role of additional binding sites in plasmtiogenmolecule can sufficiently reveal itself. To establishthe role of lysine-bindingsites and reveal the differencesin carbohydratestructureof plasmin isoforms and their effect on its enzymaticproperties,the kinetic ara. meters KM and kcat of fibrinogenand fibrin hydrolysisby p?asmFn 1 and 2 as well as miniplasminwere compared.The degree of hydrolysiswas assessed by amino groups released,which were determinedusing trinitrobenzenesulphonic acid under condition8 worked out by us.

MATERIAZS AND MRTHODS Native plasminogenwas isolated from donor blood plasma by affinitychromatographyin the presence of contrykal (11). Plasminoganforms 1 and 2 were separatedon lysineaepharose by elutingwith concentrationgradient of &-aminocapronic acid (2). Miniplasminogenwas obtained by plasminogenelastolysis followingwith the separationof the products on lysine-sepharose (8). Preparationhomogeneitywas tested by SIXi-electrophoresis in polyacrylamidegel (12). Plasmlno en was turned into plasmin by activationwith catalytic quantfty of streptokinase in the presence of glycerol.Concentrationof the active centre in plasmin pre arationwas measured using p-nitrophanylp-guanidinebenzoateP13), plasmin activitywas determinedaccording to Robbias (14) and by the micromethod(15). Fibrinogen was isolatedfrom bovine plasma by salting out with sodium sulfate (16) and purified of plasminogentraces (16); thrombinwas obtained from bovine plasma (18). Protein contentwas estimated by$bsorption at 280 nm using extinctioncoefficients (Rag0 = 17.0 for plasminogen,16.0 for miniplasminogen,15.6 for fibrinogen).Fibrinogenhydrolysis by plasminwas performed

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in 0.067 I phosphate buffer, pH 7.4. 0.5 ml of the mixture contained 0.7 - 7yg of the enzyme,with substrateconcentration of 1.5 - 35 x IO4 M. Reaction mixture was incubatedfor 'IO min at 380C. The reaction was stopped by adding 0.2 ml of % trichloraceticacid solution.Supernatantwas supplementedwith !%%sodium tetraboratesolution and 'Iml of 0.86 trinitrobenzene sulphonic acid, kept in the dark for 20 min at 38OC, and absorbance at 420 nm was measured (19). The initialhydrolysisrate was expressedin micromolesof free amino groups of hydrolysed peptide bonds per second. Kinetic studies of the enzymicreaction of plasmin with fibrin where performed Fn the same conditionsas that of the fibrinogen.The fibrin clot was formed by adding thrombin (5 NiH) in 0.5 ml reaction mixture. The clotting time of the fibrinbgenwa6 1 min. The dissolutiontime of the fibrin clot was 15-20 min and depended on plasmin concentration.Clotting of fibrinogenoccured in the presence of plasmin.

RESULQS Separationof native plasminogeninto forms I and 2 was performed in linear E-aminocapronicacid (&-ACA) concentration gradient.Plasminogen1 was eluted at E-BOA concentration of 1.8 x IO-3 Edand plasminogen2 at 2.7 x 'IO% (Fig.1).

*280

E-ACA AI.103

I',6 192 03 094

10 20 30 40 50 ml

FIG. 1 Plasminogenisoform separationon lysine-sepharoseby the linear concentrationgradientof 6-aminocapronicacid. Column size is 2.4 x 15 cm. Starting buffer : 0.1 M of Naphosphate,pH 8.0 (250 ml). Final solution:0.1 M of Naphosphate buffer,pH 8.0, containing0.015 M of ~--aminocapronic acid (250 ml). Flow rate was 60 ml/h.

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The preparationsobtainedwere homogeneouson SIX-electrophoresis in 7.95 polyacrylamidegel. Upon activationthe concentrafor 85-95s of baseline tion of plasmin active centre accounted plasminogenconcentration. Fibrinogenhydrolysis by plasmin was performed in the renge of I:16 to I:350 enzyme/substrate molar ratios. At a given initialrate, hydrolysiscorrespondsto Michaelis-Mentenkinetics. This is confirmed by Unear dependenceof the initial rate on enzyme concentration(Fig. 2A) and hyperbolicdependence of the initial rate on substrateconcentration(Fig. 2B), which is converted into linear dependencewhen plotted on Lineweaver-Burkplots (Fig. 3).

1

2

&m-h7

3

4

5

0 ~o-~~~ITI&s

FIG. 2 A,B Dependenceof fibrinogenhydrolysisrate on plasmin concentration(A) and substrateconcentration(B). To analyse action of plasmin and miniplasminwe also studied the dependenceof reci rocal of the relative substratecon. centrationversus reciproca Y of the relative initial rate. The slope of the dependencenear to 1 (0.98 for miniplasminand 1.05 for plasmin) serve accesory affirm that the enzymic reaction obeyed to Michaelis-Mentenrate equation(20). Thus,_wewere able to determinekinetic parameterswhich appeared identicalfor plasmin forms 1 and 2, 5 = 6.5 x 10 6 Y, kcat = 7.1" sec. The same values were obtainedduring fibrin hydrolysis.The results demonstratethat the structuraldifferences in carbohydrateportion of plasmin molecule appear to have no effect on fibrin binding end the rate of hydrolysis. During fibrjnogenhydrolysisby miniplaamin,alterations of kinetic parameterswere observed,KM increasedthree-fold and kcat reduced two fold as compared to native plasmin. FibrFn hydrolysisyielding the seme results, fibrin as a substratefor plasmin had no advantagesover fihcinogen.

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Plasmln Miniplasmin

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6.5

A 0.75

20.0 f 0.0

71 3.;E+;

On

? 08

0:24

ok

FIG. 3 Effect of fibrinogenconcentrationon the rate of hydrolysis by plasmin (1) end by miniplasmin(2) in LineweaverBurk plots. The data obtained showed that the loss of lysine-binding sites resulted in the chenges of enzymaticpropertiesof plasmFn as regard to the specific substrate.For comparisonVssake we have also studied hydrolysisof non-specificsubstrate,hemoglobin,which, unlike fibrin and fibrinogen,had no regions specific to lyslne-bindingsites of plasminogen.DurFng hemoglobin hydrolysis$ of native plasmin end miniplasminwas found to be practicallyidentical(36.3 x 102 and 39.3 x 'l,o"", whereas kcat values were different(8.3 set and 2.4 set , respectively).Therefore,the reductionof miniplasmincatalytic constant appearedto be typicalnot only of specific substrate hydrolysis but also of hydrolysisof any protein substrates. The G values obtained gave evidence that the heavy chain with lysine-bindingsites was responsiblefor the increase in the strength of plasmin binding to the specificsubstrate.

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DISCUSSION The recent works (2,21) studying fibrin bindingof various fragmants and the whole molecule of plasminogen1 and 2, have shown that the presence of carbohydratechain at asparagin 288 residue reduces considerablyfibrin binding to fragments containingthe lysine-bindingsites of high affinity. However, this has no impact on the binding abilitiesof the whole plasminogenmolecule due, possibly to the existenceof other types of interaction.At the same time plasminogen2 activated by a tissue activatoris 2-4 times more rapid than plasminogen1 in causing fibrin lysis (22). Catalyticproperties of plasmin 1 and 2 being identical,the presence of two glycosylatedregions in plasminogen1 appears to have a certati effect on plasminogenactivationby a physiologicactivator. Kinetic characteristicsof plasmin isoforms 1 and 2 during hydrolysisof low-molecularsyntheticsubstratesbeing available(4,9,10),the questionof interrelationof plasminogen structuralpeculiatiities and-its-functionduring plasminogen interactionwith the specific substrate,fibrinogenand fibrin, remains topical.To reveal the role of lysine-binding sites comparativestudies of fibrinogenhydrolysiswith plasmin and miniplasminhave been performed.However, the methods used permited no kinetic characteristicsof the process to be obtained.Morris et al (10) compared the action of plasmin and miniplasminon fibrinogen,studyingdegradationproducts with high resolution li uid chromatographyand found them similar. Later the authors P23) compared fibrinogenhydrolysis by plasmFn and miniplasminin the presence and absence of E-amiuocapronic acid and observedno differencesin the ro erties of these structuralplasmin forms. Ney and Pizeo PP 24 studied the products of fibrinogenand fibrin hydrolysiswith plasmin and miniplasminusing SIX-electrophoresisin polyacrylamidegel. They have shown that fibrinogenlysis by miniplasminyielded the same products as its lysis with plasmin but at a somewhat reduced speed. Kinetic characteristicsof fibrinogenand fibrin hydrolysis obtained by us demonstratethat the absence of the greater portion of plasmin heavy chain containing lysine-bindingsites has an adverse effect on hydrolysis,while alteringits kinetic parameters,with k,,t/Q decreasing6-fold. However, the changes are not so great to influencesignificanty the fibrinolyticprocess. The basic physiologicalrole of lysine-binding sites appears to reveal itself at the stage of plasminogen activationand plasmin inhibition.

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