Abstract
In contrast to native low density lipoprotein (LDL), mildly oxidized LDL (mox-LDL) induced platelet shape change and stimulated during shape change the tyrosine phosphorylation of specific proteins including Syk; the translocation of Src, Fyn, and Syk to the cytoskeleton; and the increase of cytosolic Ca(2+) due to mainly Ca(2+) entry. The stimulation of these early signal pathways by mox-LDL was inhibited by desensitization of the lysophosphatidic acid (LPA) receptor and specific LPA receptor antagonists, was independent of the alpha(IIb)beta(3)-integrin, and was mimicked by LPA. Stimulation of tyrosine phosphorylation and Syk activation were independent of the increase of cytosolic Ca(2+) and were suppressed by genistein and two specific inhibitors of the Src family tyrosine kinases, PP1 and PD173956. In contrast to PP1 and PD 173956, genistein prevented shape change by mox-LDL. The results indicate that mox-LDL, through activation of the LPA receptor, stimulates two separate early signal pathways, (a) Src family and Syk tyrosine kinases, and (b) Ca(2+) entry. The activation of these early signaling pathways by mox-LDL probably plays a role in platelet responses subsequent to shape change. The inhibition of mox-LDL-induced platelet activation by LPA receptor antagonists or dietary isoflavonoids such as genistein could have implications in the prevention and therapy of cardiovascular diseases.
Highlights
In contrast to native low density lipoprotein (LDL), mildly oxidized LDL induced platelet shape change and stimulated during shape change the tyrosine phosphorylation of specific proteins including Syk; the translocation of Src, Fyn, and Syk to the cytoskeleton; and the increase of cytosolic Ca2؉ due to mainly Ca2؉ entry
Since we have observed recently that mildly oxidized LDL (mox-LDL) contains lysophosphatidic acid (LPA) and stimulates platelet shape change through activation of LPA receptor(s) [18], we investigated whether the early signal transduction mechanisms elicited by mox-LDL were mediated by activation of the LPA receptor
We conclude that shape change induced by LPA and mox-LDL proceeds through a pathway that does not involve an increase of cytosolic Ca2ϩ mox-LDL Stimulates, through Activation of the LPA Receptor, Protein Tyrosine Phosphorylation and the Tyrosine Kinase Syk during Shape Change: Independence of the ␣IIb3-Integrin—We investigated further whether the shape change induced by mox-LDL was associated with an increase of protein tyrosine phosphorylation, since we have previously observed that the Ca2ϩ-independent shape change elicited by thrombin receptor activation of human platelets occurred with the tyrosine kinase-mediated phosphorylation of specific proteins [20]
Summary
Materials—The LPA receptor antagonists N-palmitoyl tyrosine phosphoric acid (NPTyrPA) and N-palmitoyl serine phosphoric acid (NPSerPA) were kindly provided by Dr Gabor Tigyi (University of Tennessee, Memphis, TN) and Dr Robert Bittman (Queens College of City University of New York) [19]. The monoclonal antibodies against phosphotyrosine (4G10), Src (GD11), and Fak (free and covalently bound to protein A-agarose) were from Upstate Biotechnology, Inc. Preparation of Native LDL and mox-LDL—LDL was isolated in the continuous presence of EDTA as described [6]. Immunoprecipitation of Syk, Separation of Platelet Proteins, and Immunoblotting—Syk was immunoprecipitated by using anti-Syk IgG2a bound to protein A-Sepharose or covalently conjugated to agarose as described [22]. Anti-phosphotyrosine immunoblots were stripped by incubating the nitrocellulose membrane in buffer (2% SDS, 62.5 mM Tris1⁄7HCl, and 100 mM mercaptoethanol, pH 6.8) for 30 min at 70 °C and reprobed with specific antiprotein tyrosine kinase antibodies. The range of linearity of the densitometric measurements of tyrosine-phosphorylated Syk was determined by a dilution series of protein samples of mox-LDLstimulated platelets
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