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Involvement of Src kinases and PLCgamma2 in clot retraction.

Suzuki-Inoue K, Hughes CE, Inoue O, Kaneko M, Cuyun-Lira O, Takafuta T, Watson SP, Ozaki Y - Thromb. Res. (2006)

Bottom Line: In the present study, we demonstrate that the structurally distinct Src kinase inhibitors, PP2 and PD173952, significantly reduced the rate of clot retraction, but did not prevent it reaching completion.This effect was accompanied by abolition of alpha(IIb)beta(3)-dependent protein tyrosine phosphorylation, including PLCgamma2.Furthermore, platelet adhesion to fibrinogen leads to MLC phosphorylation through a pathway that is inhibited by PP2 and by the PLC inhibitor, U73122.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical and Laboratory Medicine, Faculty of Medicine, Yamanashi University, 1110 Shimokato, Chuo, Yamanashi, 409-3898, Japan. katsuei@yamanashi.ac.jp

ABSTRACT
The integrin alpha(IIb)beta(3) plays a critical role in mediating clot retraction by platelets which is important in vivo in consolidating thrombus formation. Actin-myosin interaction is essential for clot retraction. In the present study, we demonstrate that the structurally distinct Src kinase inhibitors, PP2 and PD173952, significantly reduced the rate of clot retraction, but did not prevent it reaching completion. This effect was accompanied by abolition of alpha(IIb)beta(3)-dependent protein tyrosine phosphorylation, including PLCgamma2. A role for PLCgamma2 in mediating clot retraction was demonstrated using PLCgamma2-deficient murine platelets. Furthermore, platelet adhesion to fibrinogen leads to MLC phosphorylation through a pathway that is inhibited by PP2 and by the PLC inhibitor, U73122. These results demonstrate a partial role for Src kinase-dependent activation of PLCgamma2 and MLC phosphorylation in mediating clot retraction downstream of integrin alpha(IIb)beta(3).

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αIIbβ3 outside-in regulation of MLC phosphorylation was inhibited by Src inhibitor or PLC inhibitor. 0.4 ml of human washed platelets at 3 × 108/ml pretreated with 10 μM indomethacin and 3 U/ml of apyrase followed by being incubated without (A) or with 50 μM PP2, PP3 (B), 10 μM U73122, or U73323 (C), and then they were seeded on the surfaces coated with BSA or fibrinogen (fib) for indicated times. Bound and unbound platelets were solubilized by 4× Laemmli sample buffer, followed by immediate sonication for three periods of 5 s. Platelet proteins were separated by SDS-PAGE on 15% gels, electrotransferred, and then phospho-MLC or total MLC were blotted using anti-phospho-MLC mAb or anti-MLC pAb. (D) 0.4 ml of washed platelets at 3 × 108/ml from PLCγ2+/+ and PLCγ2-/- mice were pretreated with 10 μM indomethacin and 3 U/ml of apyrase and then they were seeded on the surfaces coated with BSA or fibrinogen (fib) for indicated times. The following procedure is the same as that for human platelets. The data are representatives of at least two experiments.
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fig4: αIIbβ3 outside-in regulation of MLC phosphorylation was inhibited by Src inhibitor or PLC inhibitor. 0.4 ml of human washed platelets at 3 × 108/ml pretreated with 10 μM indomethacin and 3 U/ml of apyrase followed by being incubated without (A) or with 50 μM PP2, PP3 (B), 10 μM U73122, or U73323 (C), and then they were seeded on the surfaces coated with BSA or fibrinogen (fib) for indicated times. Bound and unbound platelets were solubilized by 4× Laemmli sample buffer, followed by immediate sonication for three periods of 5 s. Platelet proteins were separated by SDS-PAGE on 15% gels, electrotransferred, and then phospho-MLC or total MLC were blotted using anti-phospho-MLC mAb or anti-MLC pAb. (D) 0.4 ml of washed platelets at 3 × 108/ml from PLCγ2+/+ and PLCγ2-/- mice were pretreated with 10 μM indomethacin and 3 U/ml of apyrase and then they were seeded on the surfaces coated with BSA or fibrinogen (fib) for indicated times. The following procedure is the same as that for human platelets. The data are representatives of at least two experiments.

Mentions: Potentially, clot retraction could also be regulated through activation of PLCβ by thrombin and/or PLCγ2 by αIIbβ3, thereby leading to Ca2+ elevation and activation of MLC kinase. In consideration of this, we investigated whether outside-in signals by αIIbβ3 activate MLC phosphorylation in the absence of thrombin. To address this, we seeded washed, human platelets on a fibrinogen-coated surface in the absence of thrombin. Indomethacin and apyrase were included in these studies to prevent stimulation of phosphorylation through release of thromboxane A2 and ADP. Immobilized fibrinogen stimulated weak MLC phosphorylation (Fig. 4A), which was inhibited in the presence of the Src kinase inhibitors, PP2 (Fig. 4B) and PD173952 (not shown), and by the phospholipase inhibitor, U73122 (Fig. 4C). The inactive enantiomers of PP2 and U73122, namely PP3 and U73323 respectively, had no effect (Fig. 4B and C). Moreover, MLC phosphorylation in platelets seeded on immobilized fibrinogen is inhibited in PLCγ2-deficient mice (Fig. 4D). These findings confirm that αIIbβ3 stimulates MLC phosphorylation through a pathway that is partially dependent on Src kinases and PLCγ2.


Involvement of Src kinases and PLCgamma2 in clot retraction.

Suzuki-Inoue K, Hughes CE, Inoue O, Kaneko M, Cuyun-Lira O, Takafuta T, Watson SP, Ozaki Y - Thromb. Res. (2006)

αIIbβ3 outside-in regulation of MLC phosphorylation was inhibited by Src inhibitor or PLC inhibitor. 0.4 ml of human washed platelets at 3 × 108/ml pretreated with 10 μM indomethacin and 3 U/ml of apyrase followed by being incubated without (A) or with 50 μM PP2, PP3 (B), 10 μM U73122, or U73323 (C), and then they were seeded on the surfaces coated with BSA or fibrinogen (fib) for indicated times. Bound and unbound platelets were solubilized by 4× Laemmli sample buffer, followed by immediate sonication for three periods of 5 s. Platelet proteins were separated by SDS-PAGE on 15% gels, electrotransferred, and then phospho-MLC or total MLC were blotted using anti-phospho-MLC mAb or anti-MLC pAb. (D) 0.4 ml of washed platelets at 3 × 108/ml from PLCγ2+/+ and PLCγ2-/- mice were pretreated with 10 μM indomethacin and 3 U/ml of apyrase and then they were seeded on the surfaces coated with BSA or fibrinogen (fib) for indicated times. The following procedure is the same as that for human platelets. The data are representatives of at least two experiments.
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Related In: Results  -  Collection

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fig4: αIIbβ3 outside-in regulation of MLC phosphorylation was inhibited by Src inhibitor or PLC inhibitor. 0.4 ml of human washed platelets at 3 × 108/ml pretreated with 10 μM indomethacin and 3 U/ml of apyrase followed by being incubated without (A) or with 50 μM PP2, PP3 (B), 10 μM U73122, or U73323 (C), and then they were seeded on the surfaces coated with BSA or fibrinogen (fib) for indicated times. Bound and unbound platelets were solubilized by 4× Laemmli sample buffer, followed by immediate sonication for three periods of 5 s. Platelet proteins were separated by SDS-PAGE on 15% gels, electrotransferred, and then phospho-MLC or total MLC were blotted using anti-phospho-MLC mAb or anti-MLC pAb. (D) 0.4 ml of washed platelets at 3 × 108/ml from PLCγ2+/+ and PLCγ2-/- mice were pretreated with 10 μM indomethacin and 3 U/ml of apyrase and then they were seeded on the surfaces coated with BSA or fibrinogen (fib) for indicated times. The following procedure is the same as that for human platelets. The data are representatives of at least two experiments.
Mentions: Potentially, clot retraction could also be regulated through activation of PLCβ by thrombin and/or PLCγ2 by αIIbβ3, thereby leading to Ca2+ elevation and activation of MLC kinase. In consideration of this, we investigated whether outside-in signals by αIIbβ3 activate MLC phosphorylation in the absence of thrombin. To address this, we seeded washed, human platelets on a fibrinogen-coated surface in the absence of thrombin. Indomethacin and apyrase were included in these studies to prevent stimulation of phosphorylation through release of thromboxane A2 and ADP. Immobilized fibrinogen stimulated weak MLC phosphorylation (Fig. 4A), which was inhibited in the presence of the Src kinase inhibitors, PP2 (Fig. 4B) and PD173952 (not shown), and by the phospholipase inhibitor, U73122 (Fig. 4C). The inactive enantiomers of PP2 and U73122, namely PP3 and U73323 respectively, had no effect (Fig. 4B and C). Moreover, MLC phosphorylation in platelets seeded on immobilized fibrinogen is inhibited in PLCγ2-deficient mice (Fig. 4D). These findings confirm that αIIbβ3 stimulates MLC phosphorylation through a pathway that is partially dependent on Src kinases and PLCγ2.

Bottom Line: In the present study, we demonstrate that the structurally distinct Src kinase inhibitors, PP2 and PD173952, significantly reduced the rate of clot retraction, but did not prevent it reaching completion.This effect was accompanied by abolition of alpha(IIb)beta(3)-dependent protein tyrosine phosphorylation, including PLCgamma2.Furthermore, platelet adhesion to fibrinogen leads to MLC phosphorylation through a pathway that is inhibited by PP2 and by the PLC inhibitor, U73122.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical and Laboratory Medicine, Faculty of Medicine, Yamanashi University, 1110 Shimokato, Chuo, Yamanashi, 409-3898, Japan. katsuei@yamanashi.ac.jp

ABSTRACT
The integrin alpha(IIb)beta(3) plays a critical role in mediating clot retraction by platelets which is important in vivo in consolidating thrombus formation. Actin-myosin interaction is essential for clot retraction. In the present study, we demonstrate that the structurally distinct Src kinase inhibitors, PP2 and PD173952, significantly reduced the rate of clot retraction, but did not prevent it reaching completion. This effect was accompanied by abolition of alpha(IIb)beta(3)-dependent protein tyrosine phosphorylation, including PLCgamma2. A role for PLCgamma2 in mediating clot retraction was demonstrated using PLCgamma2-deficient murine platelets. Furthermore, platelet adhesion to fibrinogen leads to MLC phosphorylation through a pathway that is inhibited by PP2 and by the PLC inhibitor, U73122. These results demonstrate a partial role for Src kinase-dependent activation of PLCgamma2 and MLC phosphorylation in mediating clot retraction downstream of integrin alpha(IIb)beta(3).

Show MeSH
Related in: MedlinePlus