Articles |
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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Key Words: calpain tyrosine phosphorylation tyrosine dephosphorylation platelets
| Introduction |
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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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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% SDSpolyacrylamide
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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| Discussion |
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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 |
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Received September 28, 1994; accepted February 15, 1995.
| References |
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