Glu- and lys-forms of plasminoGen differentially affect phosphatidylserine exposure on the platelet surface

Plasminogen/plasmin system is known for its ability to support hemostatic balance of blood. however, plasminogen may be considered as an adhesive ligand and in this way could affect the functioning of blood cells. We showed that exogenous Lys-plasminogen, but not its Glu-form, inhibited platelet aggregation and suppressed platelet α-granule secretion. The aim of this work was to investigate the influence of Gluand Lysform of plasminogen on the formation of platelet procoagulant surface using phosphatidylserine exposure as a marker. human platelets were obtained from human platelet-rich plasma (donors were healthy volunteers, men aged 30-40 years) by gel-filtration on Sepharose 2B. Phosphatidylserine exposure on the platelet surface was evaluated by flow cytometry with FITC-conjugated annexin A5. Gluand Lys-plasminogen have different impact on the platelet functioning. Exogenous Lys-plasminogen has no significant effect on phosphatidylserine exposure, while Glu-plasminogen increases phosphatidylserine exposure on the surface of thrombinand collagen-activated human platelets. Glu-plasminogen can be considered as a co-stimulator of agonist-induced platelet secretion and procoagulant surface formation. Meanwhile effects of Lys-plasminogen are probably directed at platelet-platelet interactions and not related to agonist-stimulated pro-apoptotic changes. The observed different effects of Gluand Lys-plasminogen on phosphatidylserine exposure can be explained by their structural peculiarities.

Plasminogen/plasmin system is known for its ability to support hemostatic balance of blood.however, plasminogen may be considered as an adhesive ligand and in this way could affect the functioning of blood cells.We showed that exogenous Lys-plasminogen, but not its Glu-form, inhibited platelet aggregation and suppressed platelet α-granule secretion.The aim of this work was to investigate the influence of Glu-and Lysform of plasminogen on the formation of platelet procoagulant surface using phosphatidylserine exposure as a marker.human platelets were obtained from human platelet-rich plasma (donors were healthy volunteers, men aged 30-40 years) by gel-filtration on Sepharose 2B.Phosphatidylserine exposure on the platelet surface was evaluated by flow cytometry with FITC-conjugated annexin A5.Glu-and Lys-plasminogen have different impact on the platelet functioning.Exogenous Lys-plasminogen has no significant effect on phosphatidylserine exposure, while Glu-plasminogen increases phosphatidylserine exposure on the surface of thrombin-and collagen-activated human platelets.Glu-plasminogen can be considered as a co-stimulator of agonist-induced platelet secretion and procoagulant surface formation.Meanwhile effects of Lys-plasminogen are probably directed at platelet-platelet interactions and not related to agonist-stimulated pro-apoptotic changes.The observed different effects of Glu-and Lys-plasminogen on phosphatidylserine exposure can be explained by their structural peculiarities.k e y w o r d s: Glu-and Lys-plasminogen, phosphatidylserine exposure, platelets.P lasminogen/plasmin system plays the key role in degradation of fibrin clots and supports hemostatic balance of blood.However, it was reported that plasminogen can be considered as an adhesive ligand for integrins and modulate functioning of blood cells [1].There are two forms of plasminogen, which differ according to their structure and functional role in hemostasis system.The native form of the molecule, Glu-plasminogen, possesses closed conformation and it is the main form of plasminogen circulating in plasma.Lys-plasminogen is a partially degraded form, which is formed due to catalytic action of plasmin on Glu-plasminogen.Lysplasminogen is not present in blood under physiological conditions but a low amount of this form was detected in blood of patients undergoing thrombolytic therapy with tissue plasminogen activator [2].It was shown that Lys-plasminogen has more open conformation and possesses more affinity towards fibrin and membrane receptors [3].It is known that both forms of plasminogen are able to bind to platelet surface, however, Lys-plasminogen binds to platelets with higher affinity [4].In our previous investigations, we showed that exogenous Lys-plasminogen inhibits platelet aggregation and suppresses platelet α-granule secretion, meanwhile the native form of plasminogen has no significant influence on the above-mentioned processes [5,6].We suggested that plasminogen effects, which were shown during platelet aggregation and secretion, can be observed at the step of procoagulant phospholipid membrane formation, i.e. phosphatidylserine exposure on the platelet surface.The present work is aimed to study plasminogen influence on phosphatidylserine exposure on the surface of platelets activated by thrombin or collagen.

materials and methods
Plasminogen preparation.Glu-plasminogen was purified from fresh blood plasma of healthy volunteers and Lys-plasminogen was isolated from Cohn fraction III2,3.Research protocols were ap-doi: https://doi.org/10.15407/ubj89.si01.102proved by the Ethical Committee of Palladin Institute of Biochemistry of NASU (from 3 rd of November 2014, protocol N 10).
Lysine-sepharose chromatography was used in both cases [7].All preparations of plasminogen had no spontaneous plasmin activity.Plasminogen preparations in all experiments were used at final concentration 1.2 µM.
Platelet aggregation.Intact platelets were obtained from human platelet-rich plasma by gel-filtration on Sepharose 2B [8].Platelet aggregation was measured by optical aggregometry (aggregometer Solar AT-02, Belorussia).All assays were performed within 60-80 min after platelet collection.Thrombin (Sigma Aldrich, USA) at final concentration 1 unit NIH/ml and collagen (Tekhnologia Standard , Russia) at final concentration 1.25 mg/ml were used to stimulate platelets.Experimental data were statistically analyzed using software MS Office Excel 2007 and Agregometr 2.01.
Flow cytometry.To investigate plasminogen effects on phosphatidylserine exposure flow cytometry with FITC-conjugated annexin A5 (Sigma Aldrich, USA) was used.Washed platelets (2.5×10 6 ) were transferred into 100 µl of 20 mM HEPES buffer , pH 6.8, containing 137 mM NaCl, 4 mM KCl, 0.2 mM MgCl 2 , 0.2% glucose and 0.2% BSA.The platelets were incubated with Glu-or Lys-plasminogen for 3 min at 22-25 ºC with or without further incubation with thrombin or collagen for 5 min at 22-25 ºC.Intact platelets were served as control.After incubation, platelets were washed by 0.05 M PBS, pH 7.4, with 0.13 M NaCl by centrifugation at 1,000 g at 25 ºC.Platelets were resuspended in 1.5 ml of 0.05 M PBS, pH 7.4, with 0.13 M NaCl, according with manufacturer's instruction.FITCconjugated annexin A5 was added to all samples and suspension was incubated in the darkness at room temperature for 30 min.Fluorescence intensity was monitored by Coulter Epics flow cytometer (Beckman Coulter, USA) via FL1 channel (515-535 nm).Annexin A5 exposure on the platelet surface was estimated using two parameters: 1) the percentage of annexin A5-positive cells of total cell number in the sample 2) the fluorescence intensity which was detected via FL1 channel (515-535 nm).To obtain statistically significant results no less than 15,000 events were analyzed for each sample.The measurement of cytometric parameters in each group of cells was carried out in duplicate, and the mean value was calculated based on the values obtained from all donors.
The results were graphically presented using "FCS Express V3" (De Novo Software, USA).The data were expressed as mean ± SD.Statistical analysis of the results was performed with using of Student's t-test, the difference was considered as significant at P < 0.05.

results and discussion
For better presentation of flow cytometry results two gated regions were created.Gated region M1 was set to estimate the population of annexin A5-positive platelets and M2 marker was created around the clearly identifiable sub-population showing high level of annexin A5 binding.(Fig. 1).We found that among intact platelets there was only small amount of cells exposing phosphatidylserine on their surface (3.6 ± 1.3%), see Fig. 2. Thrombin stimulation (1 unit NIH/ml) increased the amount of annexin A5 positive cells in 8.6 times (31 ± 3.9%).It has to be noted that large proportion of the platelets (2/3) displayed high level of annexin A5 binding .Framing such sub-population during thrombin activation was shown at the dot graph (see Addendum 1): dot density was significantly shifted on the right by axis FL1 LOG, indicating the high fluorescence intensity of thrombin-stimulated platelets.The average fluorescence of this sub-population was 1.5-2 times higher than in the whole population of annexin A5-positive cells.Thrombin stimulation led to phosphatidylserine exposure and as a result annexin A5 binding on the platelet surface.The proportion of annexin A5-positive platelets became larger and it was noticed the appearance of sub-population of platelets with intensive fluorescence.
Incubation with Glu-plasminogen followed by thrombin stimulation resulted in the increase of population of annexin A5-positive platelets (in 1.6 times).Under these conditions, the effect of Lysplasminogen was more moderate.In case of Lysplasminogen effect, the changes were related with the sub-population of platelets showing low level of annexin A5 binding (Addendum 1, group "Lys-Pg + Thr").It has to be noted that plasminogen itself, in the absence of stimulation, had no influence on platelet functions.
Collagen was a potent stimulator of phosphatidylserine exposure as the amount of annexin A5binding platelets increased on 55.3 ± 8.8% (Fig. 3,  a).Most annexin A5-positive cells possessed high fluorescence.The fluorescence intensity of these cells was 6 times higher as compared to this one The relationship between plasminogen binding to cells and phosphatidylserine exposure has been earlier described in the literature [9][10][11].O'Mullane et al. showed that in the presence of apoptotic factors (thrombin is considered as one of them) exogenous Glu-plasminogen considerably increases phosphatidylserine exposure on the surface of cells of monocyte culture U 937 [12].According to our data, thrombin is not only stimulates platelet activa- tion and secretion of α-granules, but also potentiates the formation of procoagulant surface.Thrombin stimulation led to activation of surface receptors GP Ib and IIbIIa and triggers the chain of events which leads to the formation of procoagulant surface (see Fig. 4).As it was previously shown the inhibition of these receptors on the platelet surface by specific antibodies blocked the exposure of procoagulant activity on platelets [13].

Fig. 3. Effects of Glu-and Lys-plasminogen on annexin A5 exposure on collagen-stimulated platelets (n = 3): А -amount of annexin A5 positive cells, B -fluorescence intensity. M1 -population of annexin A5-positive platelets; М2 -sub-population of platelets showing high level of annexin A5 binding. Difference in the com
According to our data, Glu-plasminogen can be considered as a co-stimulator of agonist-induced platelet secretion and procoagulant surface formation.Effects of Lys-plasminogen are probably directed at platelet-platelet interactions and not related to agonist-stimulated pro-apoptotic changes.We showed that the stimulating effect of Glu-plasminogen in exposure of phosphatidylserine was more pronounced under thrombin stimulation.It can be explained by the fact that these agonists realize their stimulating effects through different ways.Dormann et al. suggested that in case of thrombin, platelet gly-coprotein GP Ib plays an essential role in the formation of platelet procoagulant surface [13].In case of collagen, this glycoprotein is not involved.
The obtained results highlight the possibili ty of the practical use of two forms of plasminogen in therapeutic practice.As it was noted before, Lysplasminogen in complex with streptokinase can be considered as a tool for the treatment of patients with deep vein thrombosis [14] and administration of Glu-plasminogen improves the healing of chronic diabetes wounds in a mouse model of diabetes [15].Besides, it was shown that platelets in type 2 diabetic individuals adhere to vascular endothelium and aggregate more readily than those in healthy people [16].Lys-plasminogen, which shows its anti-aggregation effect, seems to be a good candidate for correlation of such pathological state.
In conclusion, the obtained results indicate the different effects of Glu-and Lys-plasminogen on platelet functions.The precise mechanisms of these effects need further investigation and will be clari- Glu-Pg + Thr Glu-Pg fied in the future studies.However, we suggest that the conformational differences between native and truncated forms of plasminogen molecules could play the principal role for realization of above-mentioned effects.