Lys-plasminogen stimulates vitronectin exposure on the platelet surface

Aim. To study the vitronectin exposure on the platelet surface in the presence of two forms of the plasminogen molecule: Lysand Glu-plasminogens. Methods. Research was conducted on human platelets. Aggregometry was used to check the platelet vitality and cell response towards thrombin. To evaluate the infl uence of different plasminogen forms on the platelet vitronectin exposure we used the method of fl ow cytometry. Results. It was found that incubation of resting platelets with Lys-plasminogen increased the amount of vitronectin-positive cells, but did not affect signifi cantly their fl uorescent intensity. Thrombin stimulation led to an increase in both: the number of vitronectin-positive platelets and the signal of fl uorescence at least by two times. The Lys-plasminogen adding to the suspension of washed platelets followed by the thrombin stimulation enhanced the vitronectin exposure on the platelet surface and increased the amount of vitronectin-positive cells as compared to the isolated thrombin stimulation. Gluplasminogen had no effect on the vitronectin exposure in case of resting or stimulated platelets. Conclusions. Lys-plasminogen but not its Glu-form enhances the exposure of vitronectin on the platelet surface. We suggest that the binding of Lys-plasminogen to the surface platelet receptors may generate plasmin that leads to the activation of intracellular signaling cascade.


Introduction
The platelet activation and following thrombus formation involve many adhesive proteins which are secreted from the platelet α-granules including fibrinogen, von Willebrand factor, fi bronectin and vitronectin (Vn) [1].However, it has been recently demonstrated that thrombus formation still occurs in the mice lacking both von Willebrand factor and fibrinogen [2] suggesting that other proteins such as fi bronectin, thrombospondin and Vn may be able to mediate the platelet adhesion and aggregation [3].Among these proteins, a special attention has been paid to Vn, which plays a signifi cant role in platelet physiology.It may regulate thrombosis and fi brinolysis through the interaction with integrins, plasminogen activator inhibitor-1 (PAI-1), collagen, and the urokinase receptor.In plasma, this protein circu-lates in an inactive folded form, in which most of its ligand-binding domains are hidden.Conversely, in the extracellular matrix and in blood platelets Vn is present in the activated multimeric form [4,5].It was suggested that this adhesive protein is a thrombus stabilizer as it has a site (Arg-Gly-Asp) with the potential for binding to integrins [6,7].However, the ideas about its exact role in thrombus formation are controversial.It was shown that the platelet-released Vn enhances the platelet aggregation, whereas the plasma Vn inhibits this process.It was reported that Vn has binding site to plasminogen, as monoclonal antibodies against the C-terminal portion of Vn inhibited plasminogen binding [8].Recently we have shown that Lys-plasminogen but not its Glu-form inhibits the thrombin-and collagen-induced aggregation in the preparations of washed human platelets.In this study, we investigated the effects of two forms

Materials and Methods
Human plasma and platelet preparations were obtained from blood of healthy volunteers (n = 5) as it was described before [9].Research protocols were approved by the Ethical Committee of Palladin Institute of Biochemistry of NASU (from 3 rd of November 2014, protocol N 10).
Aggregometry was used to check platelet vitality and cell response towards thrombin.Platelet aggregation was measured by optical aggregometry (aggrego meter «Solar AT-02», Belorussia).All assays were per formed within 60-180 minutes after platelet collection.Experimental data were statistically analyzed using software «MS Excel» and «Agregometr 2.01».Thrombin (Sigma Aldrich, USA) at fi nal concentration 1 NIH unit/ml was used to stimulate platelets.
Glu-plasminogen was purifi ed from fresh donor plasma and Lys-plasminogen was isolated from Cohn fraction II-III.Lysine-sepharose chromatography was used in both cases [10].All preparations of plasminogen had no spontaneous plasmin activity.
To evaluate the infl uence of different plasminogen forms on Vn exposure by platelets the method of fl ow cytometry was used.The levels of exposed Vn were detected with use of respective antibodies to Vn (Sigma Aldrich, USA).The washed platelets (10 6 cells) were transferred into 100 μl of 20 mM HEPES buffer, pH 6.8 containing 137 mM NaCl, 4mM KCl, 0.2 mM MgCl 2 , 0.2 % glucose and 0.2 % bovine serum albumin (BSA).To perform investigations three groups of cells were used: resting platelets (control); platelets incubated with Glu-or Lys-plasminogen (ti me of exposure 3 min); platelets treated with thro mbin (5min), and platelets treated with thrombin after their preliminary incubation with Lys-plasminogen (3min).All procedures with platelets were carried out at 22-25 C to avoid platelet hyperactivation.Platelets shortly thereafter incubation with plas minogen or resting platelets (control group) were treated with anti-Vn antibodies.To carry out immunochemical detection of Vn we prepared the mixture of antibodies in 0.05 M sodium phosphate buffer (PBS), pH 7.4 with 0.13 M NaCl and 1 % BSA.Antibodies to Vn (2 μg) we re mixed with 100μl of PBS and secondary FITC-conjugated antibodies (Sigma Aldrich, USA) (fi nal dilution 1:100).The mixture was incubated in the darkness at 4 C for 15 min.Platelets were incubated with the obtained mixture in the darkness at room temperature for 30 min.As a control of non-specifi c antibody binding we used platelets which were incubated only with secondary FITC-conjugated antibodies.Non-bound antibodies were removed by double washing in PBS by centrifugation at 3,000 g for 3 min at 20 C.The obtained suspension of washed platelets was resuspended in 1 ml of PBS and used for the investigation of Vn exposure at COULTER EPICS XL (Beckman Coulter, USA) equipped with argon laser, excitation λ = 488nm.Evaluation of Vn exposure on the platelet surface was done using two parameters: 1) percentage of Vn-positive platelets of the whole amount of investigated cells in the sample; 2) intensity of fl uorescence, which was monitored by the channel FL1 (515-535 nm).To sort the Fig. 1.Effects of Lys-and Glu-plasminogen on vitronectin exposure by intact and thrombin-stimulated platelets: 1 -resting platelets, 2 -resting platelets incubated with Glu-plasminogen, 3resting platelets incubated with Lys-plasminogen, 4 -thrombinstimulated platelets, 5 -thrombin-stimulated platelets preliminary incubated with Glu-plasminogen, 6 -thrombin-stimulated platelets preliminary incubated with Lys-plasminogen.Blue columnsresting platelets, red columns -thrombin-stimulated platelets.The concentrations of plasminogen forms and thrombin were 1.2 μM and 1.0 NIH units/ml respectively in all experiments.Difference in the compared groups is statistically signifi cant (р < 0.05): *compared to the group of 'resting platelets», # -compared to the group of «thrombin-stimulated platelets» platelets the corresponding gates were established.The chan ges in signal of fl uorescence, which were observed in case of the impact of the investigated molecules, were defi ned according to the displacement of fl uorescence cur ves relative to the marker, which limits the fl u o rescence curve of the intact platelets.The quantitative changes of the fl uorescence were expressed in arbitrary units (AU) that mean the common logarithm according to scale Log FL1.To obtain statistically signifi cant results at least 10 thousand events were analyzed.
The measurement of cytometric parameters in each group of cells was fulfi lled in two parallels.Then, a mean value for the corresponding data obtained from all donors was calculated.The results were graphically presented using «FCS Express V3» (De Novo Software, USA).The data were expressed as mean ± SD.Statistical treatment of the results was made with Student's t-test, the difference was considered as signifi cant with p < 0.05.

Results and Discussion
Using the fl ow cytometric analysis we determined the amount of Vn-positive cells and their fl uorescence intensity.The presented data characterize the vitro nectin exposure on the surface of resting and throm binstimulated washed platelets (Fig. 1).The appearance of Vn-positive cells in the population of resting platelets can be due to the spontaneous activation of a part of cells taken in the experiment.It is recommended to use the population of platelets, the level of spontaneous activation of which does not exceed 50-55 % [11] Fig. 2. Infl uence of Lys-plasminogen (А) and Glu-plasminogen (В) on vitronectin exposure by resting human platelets.Count -the amount of events, FL1 LOG -logarithm of fl uorescence intensity with FITC.Black colour-resting platelets, green colour-platelets incubated with Lys-plasminogen, blue colour-platelets incubated with Glu-plasminogen Fig. 3. Infl uence of Lys-plasminogen (А) and Glu-plasminogen (В) on vitronectin exposure by stimulated human platelets.Count -the amount of events, FL1 LOG -logarithm of fl uorescence intensity with FITC.Black colour-resting platelets, blue colour-thrombin stimulated platelets, red colour-thrombin-stimulated platelets preliminary incubated with Lys-plaminogen (A) or Glu-plasminogen (B) that is in accordance with our data.When Lys-plasminogen was introduced into the incubation medium with intact platelets, an increase of vitronectin-positive cells was noted.The introduction of Glu-plasminogen had no effect (Fig. 1).It is known that the platelet affi nity for Lys-plasminogen is higher as compared to Glu-plasminogen [12].In its turn, the bound Lys-plasminogen may be considered as the preferable adhesive lygand for Vn, since the affi nity of Lys-plasminogen towards vitronectin is signifi cantly higher (K d = 100 nM and K d = 1 mM respectively) [8].
We established that thrombin activation caused an increase of Vn-positive cells at least by 2 times as compared to the population of resting platelets.It is known that thrombin activation leads to secretion of alpha-granules, which contain vitronectin.According to the previous data, the activated platelets secreted highly adhesive multimeric Vn, half of which remained the platelet bound [13].If Lys-plasminogen was added to the incubation medium prior to the thrombin activation, we noticed a signifi cant increase of Vn-positive cells.In case of Glu-plasminogen, signifi cant changes in the amount of Vn-positive cells were observed in comparison with the case of thrombin stimulation without prior plasminogen incubation.This difference may be also explained by different affi nity of Glu-and Lys-plasminogens towards vitronectin.
The changes in intensity of the fl uorescence signal of intact and thrombin-activated platelets have the same tendency as it was demonstrated for the quantitative rearrangement of the cells.The resting platelets incubated with Glu-or Lys-plasminogen do not reveal any signifi cant changes of the fl uorescence signal as compared to the control curve (Fig. 2).In case of the thrombin-activated platelets it was an essential shift of the peak of the fl uorescence curve to the right of the control curve (Fig. 3), which agrees with the appearance of vitronectin-binding sites on the surface of thrombin-activated platelets.The preliminary incubation of stimulated platelets with Lys-plasminogen leads to the shift of the peak of fl uorescence to the right (Fig. 3, A), whereas the incubation with Glu-plasminogen did not cause any changes as compared to the curve obtained for thrombin-stimulated platelets (Fig. 3, B).
The obtained results may be interesting consi-dering the role of plasminogen in cellular signaling.As it has been recently shown on peripheral monocytes and macrophages, the plasminogen binding to the annexin A2 S100 A10 may induce the plasmin generation, which results in the activation of intracellular signaling cascade and leads to a pro-infl ammatory response [14].It is quite pos-sible that the binding of Lys-plasminogen (which has open conformation and therefore is more readily converted into plasmin) to the platelet membrane may serve as an initial step of the cellular response.However, the further investigations should be done to prove this suggestion.