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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:511-514.)
© 1995 American Heart Association, Inc.

Articles

Participation of Calpain in Protein-Tyrosine Phosphorylation and Dephosphorylation in Human Blood Platelets

Hideo Ariyoshi; Atsushi Oda; Edwin W. Salzman

From the Department of Surgery, Beth Israel Hospital, Harvard Medical School, Boston, Mass.

Correspondence to Edwin W. Salzman, MD, Beth Israel Hospital, 330 Brookline Ave, Boston, MA 02215.

	Abstract

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Abstract The possible role of calpains in protein-tyrosine phosphorylation in platelets was examined by the use of the cell-permeant calpain inhibitor calpeptin. In platelets stimulated by 1 U/mL thrombin, protein-tyrosine phosphorylation was maximal after 2 minutes and was followed by protein-tyrosine dephosphorylation. Calpeptin (30 µmol/L) or vanadate (2 mmol/L) enhanced protein-tyrosine phosphorylation and delayed protein-tyrosine dephosphorylation. The effects of these two compounds were not additive. We also observed proteolysis of pp60src and autoproteolysis of µ-calpain. Cleavage of the former was significantly slower than that of the latter and slower than protein-tyrosine dephosphorylation. The activity of protein-tyrosine phosphatase in the platelet lysate was transiently increased to 190% by addition of Ca²⁺. Ca²⁺-dependent activation of protein-tyrosine phosphatase was not observed in the presence of leupeptin. Those observations suggest that platelet calpains may be involved in modulation of protein-tyrosine phosphorylation through activation of protein-tyrosine phosphatase rather than through the inactivation of pp60src, a mechanism that was previously suggested.

Key Words: calpain • tyrosine phosphorylation • tyrosine dephosphorylation • platelets

	Introduction

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Calpains (EC 3.4.22.17) are calcium-activated cysteine proteases that are ubiquitous in mammalian and avian tissue. Their activities are regulated by calcium ions and the endogenous inhibitor calpastatin. There are at least two types of calpain: a protease that requires micromolar amounts of calcium (µ-calpain) and one that requires millimolar amounts of calcium (m-calpain).¹ Although several platelet proteins have been reported to be substrates of calpain, the physiological role of these platelet proteins is still obscure.² As we reported previously, the cell-permeant calpain inhibitor calpeptin did not inhibit thrombin-induced platelet aggregation, generation of inositol-1,4,5 trisphosphate, and cytoplasmic Ca²⁺ elevation, suggesting that calpain activity is not essential for the early steps of platelet activation.³ However, autoproteolysis of µ-calpain⁴ and calpain-mediated microparticle formation⁵ were observed during platelet activation. It is likely that platelet calpains, if they are involved at all, are involved in a late phase of platelet aggregation.

Platelets contain high levels of pp60src and related kinases, which can be activated by thrombin, collagen, U46619 (a thromboxane A₂ analogue), and platelet-activating factor.^{6 7} Some aspects of tyrosine phosphorylation are mediated by platelet aggregation; cell-to-cell interaction through an integrin, glycoprotein (Gp) IIb/IIIa; and adhesive proteins.⁸ There is evidence that pp60src is cleaved by calpains.⁹ The activation of calpains was also reported to be related to an integrin, GpIIb/IIIa, in aggregating platelets.¹⁰ It appears that at least two signal transduction systems, one employing calpain and the other, protein-tyrosine phosphorylation, may interact during platelet activation. In this study, we report on the possible further participation of calpains during protein-tyrosine phosphorylation in human blood platelets.

	Methods

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Materials
Prostaglandin E₁ was obtained from Biomol. Sodium orthovanadate and PT 60 monoclonal antibody, which recognizes phosphotyrosine, were from Sigma Chemical Co. Monoclonal antibody 327, specific for pp60src, was kindly donated by Dr Joan S. Brugge (University of Pennsylvania).¹¹ Monoclonal antibody 1A8A2, specific for the large subunit of µ-calpain, was kindly provided by Dr Sei-ichi Kawashima (Tokyo Metropolitan Institute of Gerontology).¹² Other chemicals were of the highest analytical grade available.

Platelet Preparation
Human blood was drawn into 0.1 volume of 3.8% (wt/vol) trisodium citrate. Platelet-rich plasma, prepared by centrifugation at 200g for 20 minutes at room temperature, was mixed with prostaglandin E₁ (1 µmol/L). The platelet-rich plasma was spun at 800g for 20 minutes. The platelet pellet was resuspended in 1 mL of a modified HEPES-Tyrode's buffer (129 mmol/L NaCl, 8.9 mmol/L NaHCO₃, 0.8 mmol/L KH₂PO₄, 0.8 mmol/L MgCl₂, 5.6 mmol/L dextrose, and 10 mmol/L HEPES, pH 7.4). To make gel-filtered platelets, the platelet suspension was then layered onto a Sepharose 2B gel column (9 mL) preequilibrated with a modified HEPES-Tyrode's buffer.

Platelet Stimulation and Immunoblot Analysis
Gel-filtered platelets (4.0x10⁸/mL) suspended in a modified HEPES-Tyrode's buffer containing 1 mmol/L CaCl₂ were stimulated by thrombin (1 U/mL) at 37°C at a continuous stirring rate of 1000 rpm in a Lumiaggregometer (Chronolog). Reactions were terminated by boiling for 3 minutes with a Laemmli sample buffer containing 5 mmol/L EDTA and 1 mmol/L sodium orthovanadate, and proteins were separated by 10% SDS–polyacrylamide gel electrophoresis (PAGE).¹³ Immunoblot analysis was carried out as described previously.¹⁴

Protein-Tyrosine Phosphatase Assay in Platelet Lysate
Gel-filtered platelets (5x10⁸/mL) were lysed by the addition of 0.1% Triton X-100 in the presence of 2 mmol/L EDTA and 2 mmol/L EGTA in an ice bath. After addition of 2 mmol/L Ca²⁺, the platelet lysate was incubated at 25°C for several seconds in the presence or absence of 100 µmol/L leupeptin. Reactions were terminated by the addition of 2 mmol/L EDTA and 2 mmol/L EGTA, and samples were stored on ice until the protein-tyrosine phosphatase assay. The activity of protein-tyrosine phosphatase was assayed by using L-phosphotyrosine as a substrate as described by Zhao et al.¹⁵ The reaction mixture (1 mL) contained 25 mmol/L sodium acetate (pH 4.75), 1 mmol/L EDTA, 1 mmol/L DTT, 0.5 mmol/L phosphotyrosine, and 25 µL of platelet lysate. Reactions were initiated by adding platelet lysates, and the changes in optical density at 280 nm due to the conversion of phosphotyrosine to tyrosine were monitored at 25°C for 5 minutes.

	Results

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Fig 1A shows time-dependent protein-tyrosine phosphorylation in calpeptin-treated (right panel) or untreated (left panel) gel-filtered platelets stimulated by thrombin (1 U/mL). As shown in the left panel, thrombin induced a transient protein-tyrosine phosphorylation in several platelet proteins. Maximal phosphorylation was observed approximately 3 minutes after stimulation, which was then followed by disappearance of protein-tyrosine phosphorylation. Although preincubation of platelets with 30 µmol/L calpeptin did not affect platelet aggregation, cytoplasmic Ca²⁺ elevation, or [¹⁴C]serotonin release (data not shown), it did substantially inhibit protein-tyrosine dephosphorylation, as shown in the right panel. As illustrated in Fig 2B, calpeptin enhanced protein-tyrosine phosphorylation in a dose-dependent manner. Because pp60src is the major protein-tyrosine kinase in platelets^{6 7} and is also thought to be a substrate for calpains,⁹ we examined the cleavage of pp60src and autoproteolysis of µ-calpain in the same samples that were used in the studies shown in Fig 1A. We observed cleavage of pp60src (Fig 1B) and autoproteolysis of µ-calpain (Fig 1C), and these processes were completely inhibited by the specific calpain inhibitor calpeptin. The possibility that cleavage of pp60src and µ-calpain was an artifact due to postlytic proteolysis was negligible, because 5 mmol/L EDTA had been added to the sample buffer to inactivate µ-calpain in the platelet lysate. The time course of pp60src cleavage was slower than that of the autoproteolytic activation of calpain. Less than 10% of pp60src was cleaved after a 10-minute incubation, by which time almost all of the tyrosine-phosphorylated protein had disappeared. It is unlikely that proteolytic inactivation of pp60src was responsible for the decrease of protein-tyrosine phosphorylation after the initial peak effect in thrombin-stimulated platelets. Because dephosphorylation of protein could result from a combination of protein-kinase inactivation and protein-phosphatase activation, we examined the effect of the phosphatase inhibitor sodium vanadate^{15 16} on protein-tyrosine phosphorylation in thrombin-stimulated platelets. As shown in Figs 2 and 3 sustained protein-tyrosine phosphorylation was observed in vanadate-pretreated platelets. Calpeptin did not increase the effect of vanadate on protein-tyrosine phosphorylation. To confirm the involvement of calpain in protein-tyrosine phosphorylation, protein-tyrosine phosphatase activity was assayed in a platelet lysate. As shown in Fig 4, addition of Ca²⁺ resulted in a transient increase of protein-tyrosine phosphatase activity (by approximately 1.9-fold) and in autoproteolysis of µ-calpain, both of which were completely inhibited by the cell-impermeable calpain inhibitor leupeptin, suggesting that calpains were responsible for the Ca²⁺-dependent activation of protein-tyrosine phosphatase in the platelet lysate. These observations suggest that calpain may exert its effect by enhancing the activity of a protein-tyrosine phosphatase.

View larger version (66K): [in this window] [in a new window]   Figure 1. Effect of calpeptin on (A) protein-tyrosine phosphorylation, (B) cleavage of pp60src, and (C) proteolysis of µ-calpain in thrombin-stimulated platelets. Platelets preincubated with 30 µmol/L calpeptin or control DMSO were stimulated with 1 U/mL thrombin at time zero in the presence of 1 mmol/L extracellular Ca2+. Reactions were terminated by SDS sample buffer at the times indicated. Samples were subjected to SDS–polyacrylamide gel electrophoresis, and separated proteins were transferred onto nitrocellulose membranes. Bound proteins were detected by anti-phosphotyrosine monoclonal antibody PT60 (A), anti-pp60src monoclonal antibody 327 (B), or anti–µ-calpain large-subunit monoclonal antibody 1A8A2 (C). Figures are typical and representative of five different experiments.

View larger version (77K): [in this window] [in a new window]   Figure 2. Dose-dependent effects of calpeptin on protein-tyrosine phosphorylation in the presence (A) or absence (B) of vanadate in resting and thrombin-stimulated platelets. Platelets were preincubated with several concentrations of calpeptin in the presence or absence of 2 mmol/L sodium vanadate at 37°C for 5 minutes before addition of 1 U/mL thrombin. Reactions were terminated by SDS sample buffer at 5 minutes after stimulation. Samples were subjected to SDS–polyacrylamide gel electrophoresis, and immunoblot analysis was carried out as described in "Methods." Figures are typical and representative of five different experiments.

View larger version (17K): [in this window] [in a new window]   Figure 3. Time-dependent protein-tyrosine phosphorylation of 100-kD platelet protein in thrombin-stimulated platelets pretreated with calpeptin and/or vanadate. Time-dependent protein-tyrosine phosphorylation was detected as described in the legend to Fig 1A. Platelets were stimulated with 1 U/mL thrombin in the presence of 1 mmol/L extracellular Ca2+ after preincubation of platelets with control saline + DMSO (), 30 µmol/L calpeptin (), 2 mmol/L vanadate (), or 30 µmol/L calpeptin +2 mmol/L vanadate (). Tyrosine phosphorylation in 100-kD protein was quantified by densitometry. Each value was expressed as a fraction of the protein-tyrosine phosphorylation in unstimulated platelets. Similar results were obtained from two other experiments.

View larger version (24K): [in this window] [in a new window]   Figure 4. Activation of protein-tyrosine phosphatase by calpains in platelet lysate. Gel-filtered platelets (5.0x108/mL) were lysed in HEPES-Tyrode's buffer containing 0.1% Triton X-100 in the presence of 2 mmol/L EDTA and 2 mmol/L EGTA in an ice bath. After addition of 1 mmol/L Ca2+, the platelet lysate was incubated in the absence () or presence () of 100 µmol/L leupeptin at 25°C for several seconds as indicated. Reactions were terminated by addition of 2 mmol/L EDTA and 2 mmol/L EGTA. Protein-tyrosine phosphatase activity was assayed as described in "Methods" (n=4, mean±SD). Protein-tyrosine phosphatase activity in the samples at time zero in the presence of leupeptin was defined as 100%. Typical Western blot analysis of the proteolysis of µ-calpain at 45 seconds in the presence (a) or absence (b) of leupeptin is also shown.

	Discussion

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Although platelets are known to contain protein-tyrosine kinases and µ-calpain in their cytosol, the physiological functions of these enzymes have remained obscure, at least in part because of the lack of knowledge of how these enzymes are regulated in resting platelets or during platelet activation under physiological conditions. In response to thrombin, platelets rapidly undergo shape change, secretion, and aggregation. These reactions are accompanied by a transient increase in protein-tyrosine phosphorylation⁶ and the activation of µ-calpain.¹⁷ Several reports have suggested that the major platelet protein-tyrosine kinase, pp60src, and the major calcium-dependent protease, µ-calpain, are associated with the membrane fraction in aggregating platelets.^{17 18} There is evidence that pp60src is cleaved by calpains.⁹ Therefore, we suspected that µ-calpain might be involved in protein-tyrosine phosphorylation in aggregating platelets. To examine this hypothesis in intact platelets, we used the cell-permeant calpain inhibitor calpeptin. This study presents evidence that activation of µ-calpain, which is abolished by calpeptin, is involved in protein-tyrosine dephosphorylation in aggregating platelets.

Calpeptin extended the duration of protein-tyrosine phosphorylation (by 10 minutes) in thrombin-stimulated platelets. We also observed calpain-mediated cleavage of pp60src and the activation of calpain, which was demonstrated by the proteolysis of calpain itself (autoproteolysis). However, the time course of pp60src cleavage was slower than that of µ-calpain autoproteolysis, and less than 10% of total pp60src was cleaved, even after 10 minutes of incubation. These findings indicate that protein-tyrosine dephosphorylation is unlikely to be mediated through the inactivation of tyrosine kinases and suggest that other reactions, such as activation of protein-tyrosine phosphatase, are more likely to exert a regulatory effect. This hypothesis is supported by published observations on protein-phosphatase activity in a platelet lysate.¹⁹ Addition of Ca²⁺ to a platelet lysate caused transient activation of protein-tyrosine phosphatase, which was completely abolished by the addition of leupeptin, suggesting that Ca²⁺-dependent activation of protein-tyrosine phosphatase experiments was mediated by calpains.

Although the physiological implications of protein-tyrosine phosphorylation are still controversial, several reports have suggested that protein-tyrosine phosphorylation is involved in postaggregation events in platelets, such as clot retraction, ADP-induced granule secretion, and thromboxane production (reviewed in Reference 20²⁰ ). In addition, Yano et al⁵ reported that calpain participates in the formation of microparticles in aggregating platelets. Perhaps the effects of calpain might be exerted on the phenomena that occur relatively late during platelet activation, possibly through the regulation of protein-tyrosine phosphorylation. In aggregating platelets, this could result from their capacity for proteolytic stimulation of protein-tyrosine phosphatase.

	Acknowledgments

 
This work was supported by grants 37610 and 33014 from the National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Md.

Received September 28, 1994; accepted February 15, 1995.

	References

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