Plasmin enzymatic activity in the presence of actin

E. I. Yusova © 2015 E. I. Yusova; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited ISSN 0233-7657 Biopolymers and Cell. 2015. Vol. 31. N 5. P. 345–350 doi: http://dx.doi.org/10.7124/bc.0008F6


Introduction
Plasminogen/plasmin system takes part in a wide spectrum of physiological and pathophysiological processes in human organism, such as reproduction, embryogenesis, tissue remodeling, allergic and inflammatory reactions, carcinogenesis and metastasis formation.The main role of this proteolytic system is fibrinolysis that prevents the formation of fibrin deposits or provides the degradation of the formed blood clots.A key reaction of the fibrinolytic process is the conversion of plasminogen (zymogen) to the active serine protease plasmin (E.C.3.21.7).Human plasma plasmin hydrolyzes fibrinogen, fibrin, coagulation factors V, VIII, XII, converts single chain forms of the tissue plasminogen activator (tPA) and urokinase to more active double-chained forms, activates metalloproteases and lipoxygenases, growth factors [1].Multifunctional properties of plasmin are realized due to its high specificity towards different high molecular weight substrates, and, on the other hand, due to the changing of its specificity during the interaction with modulator proteins, cellular receptors.Specific binding of modulator proteins to plasmin influences its functional activity.One of the most studied regulators of the plasmin substrate specificity is streptokinase, a plasminogen activator of bacterial origin.Plasmin in a complex with streptokinase (unlike free protease) is able to activate plasminogen by cleavage of the Arg561-Val562 activation peptide bond in its molecule.Streptokinase increases the amidase activity of plasmin and de-E.I. Yusova creases its protease activity towards fibrin, its physiological substrate.The complex streptokinase-plasmin makes plasmin resistant to α 2 -antiplasmin, its physiological inhibitor [2][3][4][5].
Wang H et al. showed [6] that an extracellular membrane of the carcinoma PC-3, HT 1080 and MDA-MB231 cellular lines contains β-actin that mediates both the plasmin interaction with cellular surface and the enzyme autoproteolysis leading to the formation of angiostatins 4.5.The similar effect of β-actin was observed in vitro in the cell-free system.Actin has been found in plasma of healthy subjects, and under different diseases associated with various types of the tissue and cell injury its concentration in the blood achieves a few micromoles.That is why the damaged cells are considered as the main source of circulating actin [7].The actin concentration may be significantly increased due to the cell damage in the micro medium of the inflammation and clot formation sites, i.e. in the places where plasmin is localized and expresses its functional activity.To clarify the actin ability to modulate the specificity of action of free circulating plasmin, in the present work the actin influence on the changes in the plasmin activity towards substrates with high and low molecular mass was investigated.

Materials and Methods
Glu-plasminogen was purified from fresh citrated human plasma by affinity chromatography on lysine-Sepharose at 4°C in the presence of aprotinin [8].
Plasmin was obtained by activation of plasminogen on urokinase-Sepharose (1 mg of zymogen/1250 IU urokinase) at 25 о C in the presence of 25 % glycerol.The enzyme was stored in the presence of 50 % glycerol at -20 о C.
For the estimation of fibrinolytic activity of plasmin or plasminogen activated by tissue activator, the turbidimetric method was applied as described in [11].The final concentration of desAB-fibrin was 6.2·10 -7 M, plasmin -3.5·10 -8 M, Glu-plasminogen -3.2·10 -8 M, tissue activator -100 IU, actin -35·10 -8 M or 32 •10 -8 M. The rate of fibrin clot lysis by plasmin was calculated as V=1/t 1/2.Time t 1/2 was measured as the time period from the initiation step of the clot formation till the step characterized by 50 % fall in absorbance from maximum.
To study the effects of actin on the rate of plasmin-mediated fibrin clot lysis, 35·10 -8 M or 32 •10 -8 M actin was added to the reaction medium.
To detect the plasmin autolysis in the presence or in the absence of actine the amidolytic activity of enzyme preincubated during different time was studied.Plasmin (1·10 -6 M) was incubated in 0.6 ml 0.05 M tris-HCl buffer with 0.13 M NaCl (pH 7.4) with/ without actine (1·10 -5 M). 0.05 M tris-HCl buffer with 0.13 M NaCl, pH 7.4, plasmin -3.5·10 -8 M, actin -35·10 -8 M, S2251 -3·10 -3 M were added to the thermostatic cuvette of SF 26.Total volume of the mixture was 1 ml.The absorbance of p-nitroaniline was monitored at certain time intervals at 405 nm.The amount of hydrolysed substrate was calculated using following formula: V=∆A 405 /∆t, where ∆A 405 is a change of absorbance at 405 nm and ∆t is a time of absorbance measuring.
Biotinylation of Glu-plasminogen and its fragments was carried out as described in [12].
Binding assay was performed using avidin-biotin system.Biotin labeling of Glu-plasminogen and tPA was carried out according to manufacturer recommendation.The wells of high binding polysterene microtitrate plates (Nunc, MaxiSorp) were coated with 1µg of actin in 0.1 ml 0.01 M sodium phosphate buffer (pH 7.4) and 0.15 M NaCl at 4°C overnight.After binding an excess of protein were removed by washing buffer with 0.1 % Tween 20.To avoid nonspecific sorption, 2 % BSA was pipetted into the plate wells, incubated for 2 h at 37 °C and washed out.After washing procedures, bound actin in each well was incubated with 0.1 ml biotinylated Glu-plasminogen or its fragments for 4 h at 37°C and washed out.Then 0.1 ml of avidin-phosphatase solved, as manufacturer recommends, were added into the wells and washed out after 60 min incubation.0.1 ml of alkaline phosphatase substrate (1 mg/ml p-nitrophenylphosphate in 10 % diethanolamine, pH 9.8) was pipetted in the wells and incubated for 45 min at 37 °C.The binding of biotin labeled proteins with actin immobilized on the well surface was estimated by measurement of p-nitrophenol absorbance at 405 nm using the reader (Titertek Multiscan, Finland).
The obtained proteins were homogeneous according to the electrophoresis data in PAAG [14].
The data w analysed by ORIGIN 8.6 (Originlab Corporation, USA).The permissible error did not exceed 5 % (P < 0.05).The presented curves are typical for a set of repeated experiments (at least three repeats for each experiment).

Results and Discussion
In circulating blood plasminogen, zymogen of plasmin, is present as a Glu-form, a glycoprotein composed of 791 amino acids.The conversion of plasminogen in plasmin is the key process of fibrinolysis.The plasminogen activation by physiological activators, such as tPA, urokinase, is mainly based on the enzymatic cleavage of a single peptide bond Arg561-Val562 [15,16].The cleavage results in the formation of the enzyme active center as well as in the appearance of a two-chain plasmin molecule, composed of an N-terminal heavy-chain (A) with molecular mass of 56 kDa and a C-terminal light one (B) with molecular mass of 26 kDa.The light chain has two disulfide bonds Cys 548-Cys 666 and Cys 558-Cys 566.The desAB fibrin is known as a potent stimulator of the activation reaction [17].In solutions in the absence of fibrin, Glu-plasminogen (a native form of zymogen) is activated very slowly by t-PA.The reaction is characterized by a low value of k cat (0.06 s -1 ), and Km is approximately 60 µM.In the presence of fibrin, Кm decreases till 0.16 µM, and k cat value increases till 0.1 s -1 .Besides fibrin, the tPA-dependent plasmin formation can be stimulated by the products of fibrin(ogen) hydrolysis, membrane-bound proteins, intercellular matrix proteins such as thrombospondin, gelatin, and collagen IV [18].
Nowadays, there are many data confirming physiological importance of the plasmin(ogen) interaction with actin.They considered the regulation activity of fibrinolytic system and the processing of biologically active molecules as well as the modulating action of plasminogen/plasmin system on the cells [19].It was established that actin has a high affinity towards plasmin and tPA (K d 70-140 nM and 0.55 µM, correspondingly) [20].The actin influence on the rate of fibrin clot degradation by the free enzyme and by plasmin formed due to the Glu-plasminogen activation by tPA was studied with G-actin (the protein monomeric soluble form) using turbidimetric assay.As it is shown in Fig. 1, actin inhibits both the plasmin activity and the enzyme formed as a result of the Glu-plasminogen activation by tPA.At 10 fold molar excess of actin, the rate of hydrolysis of polymeric desAB fibrin reduced by approximately 50 % in both cases.

E. I. Yusova
The observed effect of actin can be a result of the protein interaction with the enzyme active center or with the lysine-binding sites of plasmin(ogen) kringle domains, that are responsible for the plasmin interaction with its physiological substrate, polymeric fibrin.To clarify this problem we investigated the amidase activity and enzyme autolysis in the presence of actin.It was shown that actin taken in equimolar amount or in 10 fold molar excess makes no influence on the plasmin amidase activity.The rate of hydrolysis of plasmin chromogenic substrate in the absence and in the presence of actin does not change during 60 minutes and is within 0.0068 optical units per minute.(Fig. 2).
To determine the possible influence of actin on the enzyme autolysis, we incubated plasmin or plasmin mixture with two-molar excess of actin for 3 h at 37 ° C. The aliquots of reaction mixture were taken at certain intervals and added to the medium to determine the amidase activity.The presented data (see Fig. 3) do not show any effect of actin.A decrease in the enzyme amidolytical activity was observed when the enzyme was preincubated in the absence or in the presence of actin.After three-hour incubation, the residual amidase activity was approximately 30 % of the original in both cases (Fig. 3).
According to the presented results we can conclude that during the actin interaction with plasmin, the plasmin active center is open and available for the substrates with high and low molecular mass.Actin inhibition of the plasmin fibrinolytic activity is probably due to the actin interaction with lysinebinding sites of enzyme kringle domains.To prove this suggestion we compared actin binding to Gluplasminogen and its fragments: first three kringles (K1-3), fourth kringle (K4) and Val442-plasminogen (mini-plasminogen).The investigations were carried out using linked immunosorbent assay and a biotinavidin phosphatase system.Actin was deposited onto the surface of microtiter plate wells.Then, prebiotinylated plasminogen and its fragments (1-50 µg/ml) were added to wells.After that phosphataselabeled avidin was added.Adsorption of the proteins was measured according to the amount of p-nitrophenylphosphate converted by the phosphatase.The protein concentrations corresponding to 50 % saturation of binding sites for actin were determined using the obtained saturation curves.As it is shown in Table 1, actin has equally high affinity towards the native zymogen molecule and its fragment K 1-3.C 50 values are 113 and 117 nM, correspondingly.At the same time, there is no binding of actin to isolated kringle 4 or mini-plasminogen.Similar affinity of Glu-plasminogen and kringle 1-3 to actin gives us the reason to believe that lysine-binding sites of first three kringle domains of the proenzyme are involved into interaction with actin.
So, we have shown that actin can modulate specificity of the plasmin action towards substrates with high molecular mass through its interaction with lysine-binding sites of enzyme kringle domains.The actin inhibition of the fibrinolytic activity of plasmin is due to its competition with fibrin for lysine binding sites of plasminogen/plasmin.Thus, actin can cause a reduction of fibrinolytic system potential and can be involved in the hemostasis disorders related to sepsis, inflammation and tissue damages.