| | Effect of homocysteine on platelet activation induced by collagenReceived 30 December 2004; accepted 19 April 2005. Abstract ObjectiveThe present study investigated the effects of homocysteine on platelet activation induced by collagen and the downstream signaling pathways potentially involved in these effects. MethodsWashed human platelets were incubated with homocysteine and collagen type I. The effects of homocysteine on platelet aggregation and adhesion and the tyrosine phosphorylation of total platelet proteins, Src kinase, and phospholipase-Cγ2 (PLCγ2) were studied. ResultsHomocysteine (10 to 100 μM) increased collagen-induced aggregation of washed platelets. Upon homocysteine (50 to 100 μM) treatment, platelet deposition to collagen-coated surface was significantly augmented under the low shear rate model (100/s) but not under the high shear rate model (1600/s). Collagen-stimulated total protein tyrosine phosphorylation in platelets was further enhanced by incubation with homocysteine. This effect was almost abrogated by genistein. Homocysteine potentiated collagen-stimulated tyrosine phosphorylation of the Src kinase and PLCγ2, which was partly decreased by integrin β1 blocking antibody. ConclusionHomocysteine (at 10 to 100 μM) potentiates collagen type I induced-platelet activation through signaling components of glycoprotein VI and integrin α2β1 pathway. Our results suggested that upregulation of tyrosine phosphorylation of proteins such as Src and PLCγ2 is involved in the downstream signaling events of homocysteine stimulation in human platelets.
Introduction  Hyperhomocysteinemia is one risk factor for the development of atherosclerosis, thrombosis, and other cardiovascular diseases. The presence of even mildly increased plasma homocysteine could increase thrombotic risk [1]. Plausible mechanisms by which homocysteine might contribute to atherogenesis include promotion of platelet activation and enhanced coagulability, increased smooth muscle cell proliferation, cytotoxicity, induction of endothelial dysfunction, and stimulation of low-density lipoprotein oxidation [2], [3], [4]. However, the molecular mechanisms of how increased circulating homocysteine causes vascular injury and promote thrombosis remain elusive. Platelets play an important role in physiologic homeostasis and pathologic thrombosis that complicate the course of vascular disorders. Increased platelet activation may be one mechanism through which hyperhomocysteinanemia confers an increased thrombotic risk in atherosclerosis [5], [6], [7]. Experimental data suggest that oxidative stress and lipid peroxidation might be enhanced in atherosclerosis or thrombosis, which contributes to platelet activation. Homocysteine is one inducer of oxidative damage. Homocysteine induces oxidative stress in human platelets in vitro. The imbalance in platelet redox state and increased thromboxane A2 and B2 formation may generate hyperactivation of platelets, which contributes to a thrombogenic state that leads to cardiovascular diseases [8], [9]. Many platelet functions, including adhesion, aggregation, and recruitment, are controlled by nitric oxide (NO) that is generated by platelets and endothelial cells. Homocysteine has been shown to have many effects, such as to inactivate NO, to increase intracellular Ca2+ concentration, to cause endothelial cell injury, and to induce the progressive loss of NO-mediated inhibition of platelet aggregation [10], [11]. There are reports that homocysteine potentiates platelet response to thrombin and adenosine diphosphate (ADP), and this effect is associated with changes of the l-arginine/NO pathway [11], [12]. However, the mechanisms of how homocysteine affects collagen-induced platelet aggregation have not been defined. The present study investigated 1) the effects of homocysteine on collagen-induced platelet activation and 2) the possible signaling pathways involved in these effects. Materials and methods Materials The study protocol was approved by the medical ethical committee of the local hospital. Informed consent was obtained from all subjects who participated in the study. Collagen type I, genistein, cysteine, and dl-homocysteine were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Antiphosphotyrosine monoclonal antibody (PY-20), fluorescein isothiocyanate (FITC)-labeled antiphosphotyrosine monoclonal antibody (FITC-P-Tyr/PY-20), anti-Src, and anti–phospholipase-Cγ2 (PLCγ2) monoclonal antibodies, and integrin β1 blocking antibody were purchased from Santa Cruz Biotech (Santa Cruz, CA, USA). Protein-A Sepharose CL-4 beads, horseradish peroxidase–conjugated secondary antibodies, and the enhanced chemiluminescence detection system was from Amersham Biosciences Biotech (Piscataway, NJ, USA). Preparation of washed platelets Human blood from 12 normal healthy volunteers, who declared that they had not taken drugs that were known to interfere with platelet function 2 wk before blood was drawn, was collected in a 130-mM aqueous trisodium citrate anticoagulant solution (9:1). Washed platelets were prepared by differential centrifugation, as previously described [11] . Briefly, platelet-rich plasma (PRP) was obtained by centrifugation at 100g for 25 min. Platelets, isolated from PRP centrifugation at 1000g for 20 min, were washed twice with RCD-PGE1 buffer (108 mM of sodium chloride, 3.8 mM of potassium chloride, 1.67 mM of sodium bicarbonate, 21.2 mM of sodium citrate, 27.7 mM of glucose, 1.1 mM of magnesium chloride, and 50 ng/mL prostaglandin E1, pH 6.5). Then platelets were resuspended in modified Tyrode’s HEPES buffer (134 mM of NaCl, 0.34 mM of Na2HPO4, 2.9 mM of KCl, 12 mM of NaHCO3, 20 mM of HEPES, 5 mM of glucose, and 1 mM of MgCl, pH7.4) and maintained at 37°C for all subsequent experiments. Platelet aggregation study Platelet aggregation experiments were carried out at 37°C under conditions of continuous stirring (1000 rpm) in a PAP-4 aggregometer (Bio-Data) and quantified by light transmission for 3 min according to a previous report [12]. Aggregation was expressed as the maximal percent change in light transmittance from baseline, using platelet-poor plasma as a reference. Washed platelets (2.0 × 108) were preincubated for 5 min with saline, four different concentrations (10, 25, 50, and 100 μM) of cysteine (as a negative control), or homocysteine at 37°C before the addition of 5 μg/mL of collagen type I. No changes in pH after homocysteine or collagen treatment were detected in platelet suspensions. Measurement of platelet adhesion Platelet-rich plasma was preincubated for 5 min with saline, four different concentrations (10, 25, 50, and 100 μM) of cysteine (as a negative control), or homocysteine at 37°C and then subjected to adhesion study. Platelet adhesion to collagen was evaluated in a parallel plate flow chamber (GlycoTech Co., Rockville, MD, USA) at a shear rate of 1600/s or 100/s. The characteristics of the flow chamber and the experimental setup have been described previously [13]. PRP treated with saline, cysteine, or homocysteine was perfused through the flow chamber at 37°C for 4 min. After washing with phosphate buffered saline, adherent platelets on collagen-coated coverslips were stained with Giemsa and platelet adhesion was quantified with a light microscope at 1000× magnification. Measurement of protein tyrosine phosphorylation For flow cytometric or protein precipitation experiments, platelets were suspended in buffer containing 1 mM of ethyleneglycotetracetic acid (EGTA) to prevent aggregation. Protein tyrosine phosphorylation of the entire platelet was determined by flow cytometry, as previously described [14]. Washed platelets (2 × 108) were treated with saline or homocysteine (10, 25, 50, or 100 μM) at 37°C for 5 min and then stimulated with collagen (25 μg/mL) for 2 min. Platelet suspensions were incubated with 0.1% Triton X-100 for 20 min at room temperature and then incubated with FITC-P-Tyr/PY-20 for 1 h at room temperature. Parallel immunostaining with a FITC-labeled mouse anti-human immunoglobulin G1 (IgG1) served as a negative control. After being washed and resuspended in phosphate buffered saline (pH 7.4), immunostained platelets were analyzed in a flow cytometer (EPICS XL-MCL, Beckman Coulter Inc., Fullerton, CA, USA). Immunoprecipitation and western blotting Previously, tyrosine phosphorylation of Src kinase and PLCγ2 in platelet was observed by using immunoprecipitation and western blotting [15], [16]. In a typical experiment, washed platelets (2 × 108) were incubated for 5 min with different concentrations of homocysteine at 37°C, followed by stimulation with collagen (25 μg/mL) for 2 min. Platelet treatment was terminated by adding an equal volume of ice-cold lysis buffer (25 mmol/L of Tris-buffered saline, 1% Triton X-100, pH 7.8, containing a protease inhibitor cocktail; Roche, Indianapolis, IN, USA) and the supernatant was obtained by centrifugation at 16000g for 15 min at 4°C and the detergent-insoluble debris was removed. Lysates were precleared by mixing protein-A Sepharose CL-4 beads for 1 h at 4°C. Beads were removed from the lysates, and the antibody to Src kinase or PLCγ2 was added. Immunoprecipitation of lysates with a mouse anti-human IgG1 served as a negative control. After rotation for 2 h at 4°C, immune complexes were recovered with protein-A Sepharose CL-4 beads. Beads were washed four times with lysis buffer and resuspended in Laemmli’s sample buffer. Samples were electrophoresized on 10% sodium dodecylsulfate polyacrylamide gel and transferred to a polyvinyl diflouride membrane (Millipore, Bedford, MA, USA) using a MiniProtein II system (Bio-Rad, Hercules, CA, USA). The membrane was blocked with 5% non-fat milk in Tris-buffered saline/0.05% Tween-20 at 4°C overnight and then washed three times with Tris-buffered saline/0.05% Tween-20. After washing, the membrane was incubated with primary antibody, the monoclonal antibody specific for phosphotyrosine (PY-20), at room temperature for 1 h. After washing, the membrane was incubated with 1:2000 diluted horseradish peroxidase–labeled, goat ant-mouse IgG (Amersham Pharmacia, Piscataway, NJ, USA) for 1 h at room temperature, washed again, and detected by enhanced chemiluminescence reagents. Immunoblots were stripped by using mild antibody stripping solution and reprobed with another antibody (anti-Src or anti-PLCγ2). Tyrosine phosphorylation of Src kinase and PLCγ2 was quantified by densitometric measurement after western blotting using Quantity One for Windows (Bio-Rad). Statistical analysis Experimental data of platelet aggregation, adhesion, and flow cytometry are presented as mean ± standard deviation. Group means were compared by one-way analysis of variance using SPSS 10.0 for Windows (Chicago, IL, USA), and P < 0.05 was considered statistically significant. Results Effect of homocysteine on platelet aggregation induced by collagen Homocysteine has been shown to increase platelet aggregation in washed platelets and PRP by a number of different agonists including thrombin and ADP. However, because the data were conflicting on how homocysteine affected platelet aggregation induced by collagen, it was necessary to confirm its effect in our experimental system. To verify whether homocysteine could influence platelet function, we used the washed platelets in the aggregation study. Platelets were incubated for 5 min with homocysteine, cysteine, or saline and then stimulated with collagen type I for 2 min. In the presence of collagen type I, homocysteine (10 to 100 μM) caused a concentration-dependent increase of platelet aggregation, whereas homocysteine alone did not induce platelets aggregation (Fig. 1). In contrast, cysteine did not affect platelet aggregation at any tested concentration, even in the presence of collagen type I (data not shown). Effect of homocysteine on platelet adhesion to collagen-coated surfaces After treatment with homocysteine, platelet adhesion to collagen-coated coverslip was examined at two different shear rates. At low shear rate (100/s), higher concentrations of homocysteine (50 or 100 μM) significantly increased platelet adhesion to collagen, but this effect was not observed with lower concentrations of homocysteine (10 or 25 μM; Fig. 2). At high shear rate (1600/s), fewer platelets adhered to the collagen-coated surfaces compared with low shear rate adhesion, and homocysteine (10 to 100 μM) had no effect on platelet adhesion to collagen (Fig. 2). In contrast, cysteine had no effect on platelet adhesion to collagen under either flow condition (data not shown). Effect of homocysteine on collagen-stimulated platelet protein tyrosine phosphorylation In this and the following experiments, platelets were stimulated with collagen under non-aggregation conditions with EGTA (1 mM). In this procedure, calcium was chelated, and the primary signaling events were presumably by collagen, rather than a secondary effect after paracrine secretion or aggregation. However, EGTA treatment also decreased levels of positive feedback signaling. To observe a substantial increase in protein tyrosine phosphorylation in such a condition, it is necessary to increase collagen concentration to 25 μg/mL [17]. The effect of homocysteine on platelet protein tyrosine phosphorylation was examined using flow cytometric analysis. Consistent with a previous report [17], collagen (25 μg/mL) induced an increase in total platelet protein tyrosine phosphorylation compared with basal levels. Pretreatment of platelets with homocysteine for 5 min before stimulation with collagen (25 μg/mL) resulted in a further enhancement of protein tyrosine phosphorylation compared with stimulation with collagen alone. The effect of homocysteine on collagen-induced platelet protein tyrosine phosphorylation was confirmed by using a tyrosine kinase inhibitor, genistein. When genistein, a selective tyrosine kinase inhibitor, was incubated with homocysteine, the homocysteine-elicited increase in total protein tyrosine phosphorylation was almost abrogated (Fig. 3). Effect of homocysteine on tyrosine phosphorylation of Src kinase and PLCγ2 To explore the mechanism of how homocysteine enhanced downstream signaling of collagen-induced total platelet protein tyrosine phosphorylation, we examined whether homocysteine affected the tyrosine phosphorylation of two important components downstream of the collagen-signaling pathway, i.e., Src kinase and PLCγ2. Before collagen stimulation, incubating platelets with homocysteine for 5 min increased tyrosine phosphorylation of Src kinase and PLCγ2 (Fig. 4). Addition of the integrin β1 blocking antibody with homocysteine during incubation resulted in a decrease of collagen-stimulated Src tyrosine phosphorylation by 68% and a decrease of PLCγ2 tyrosine phosphorylation by 66% (Fig. 4). However, this partial inhibition on Src and PLCγ2 tyrosine phosphorylation was not observed in the same experimental condition when the integrin β1 blocking antibody was replaced by a mouse anti-human IgG1. Discussion Homocysteine is a sulfhydryl amino acid metabolite of dietary methionine. Increased plasma concentration of homocysteine is associated with increased risks of arteriosclerosis and arterial thrombosis. Hyperhomocysteinemia can be caused by a deficiency in enzymes such as cystathionine β-synthase or vitamins or by pharmaceutical agents. Increased levels of homocysteine promote endothelial cell damage, platelet hyperactivity, and the production of abnormal clotting factors that lead to the development of thromboembolic plaques in the coronary, carotid, and peripheral vascular systems [2], [3], [4]. The molecular mechanisms of how homocysteine exerts these effects have not been fully elucidated. Different possible mechanisms have been proposed, including endothelial dysfunction or hemostatic abnormalities. Oxidative stress, as a result of the production of intracellular and extracellular reactive oxygen species, plays a major player in the pathogenesis of cardiovascular and other diseases. Because homocysteine and other thiols have prooxidant activity, the oxidant stress hypothesis is frequently invoked to explain the damaging effects of homocysteine on vascular cells and tissues. Homocysteine contains a reactive sulfhydryl group (-SH). Like most thiols (RSH), it can undergo oxidation in the presence of O2 to form a disulfide bond (RSSR) in physiologic pH [8], [9]. There are reports associating hyperhomocysteinemia with enhanced platelet aggregation and macrophage-derived tissue factor, and homocysteine-induced oxidative stress might account for this hypercoagulation state [18]. In contrast, other thiol-containing amino acids, such as cysteine and N-acetylcysteine, have no such an effect on platelet aggregation and adhesion, even though they also can undergo oxidation. The concentration of total cysteine in normal plasma is 25 to 30 times higher than that of total homocysteine, and about 94% to 95% of total cysteines are oxidized forms. Therefore, oxidative stress generated through the daily flux of cysteine appears to be much greater than that of homocysteine, but cysteine usually is not considered to be a significant risk factor for cardiovascular diseases [19]. Thus, the mechanisms of homocysteine-induced platelet hyperactivity need to be further investigated. Previous studies have found that homocysteine significantly decreases l-arginine uptake and NO intracellular formation, increases [Ca2+] from intracellular stores, and potentiates platelets response to thrombin or ADP in vitro [10], [11]. Homocysteine has also been shown to enhance the aggregation of platelets induced by collagen [18], although a specific mechanism for this effect has not been established. Consistent with previous reports, our experiments with washed platelets showed that homocysteine at 10 to 100 μM increased collagen-induced platelet aggregation in vitro in a dose-dependent manner. A normal plasma level of homocysteine is 5 to 12 μM, the level for mild hyperhomocysteinemia is 12 to 25 μM, and the level for severe hyperhomocysteinemia is 50 to 500 μM [19]. Therefore, we propose a hypothesis that, through the stimulating effect of homocysteine on platelet aggregation induced by collagen, increased homocysteine level in hyperhomocysteinemia may correlate with more severe levels of cardiovascular diseases such as thrombosis and atherosclerosis. This study represents the first detailed investigation on the effect of homocysteine on platelet adhesion to collagen under different flow conditions. Our results showed a significant increase in adhesion to collagen-coated surface under a low shear rate of 100/s flow conditions, when platelets were treated with a high concentration of homocysteine (50 or 100 μM), but not with low concentration of homocysteine (10 or 25 μM). In contrast, platelet adhesion to collagen remained unchanged under a high shear rate model (1600/s) after homocysteine treatment. We observed that 10 and 25 μM of homocysteine increased collagen-induced platelet aggregation but had no effect on platelet adhesion to collagen surface under either flow condition. Platelet aggregation and adhesion are different functional assays, and the signaling pathways that lead to aggregation and adhesion probably are also different. Collagen receptor α2β1 responds only to an immobilized ligand such as collagen-coated coverslips, and this activation of this pathway is not detectable in platelet when in suspension [14]. Aggregation of platelets is mainly mediated by another collagen receptor, glycoprotein (GP) VI [20]. GP VI signaling may be more sensitive to homocysteine stimulation than α2β1 receptor. Such a possibility needs further investigation. Another possibility for the inconsistency in the dose responses in aggregation and adhesion to homocysteine is that the two assays have different sensitivities. One study showed that, under flow conditions of a shear rate of 350/s, treatment of endothelial cells with homocysteine resulted in a two- to three-fold enhancement of platelet adhesion to endothelial cells [21]. Low shear stress plays an important role in atherogenesis. For example, one study has shown that progressive atherosclerosis is more likely to develop in regions of a vessel that are subjected to low shear stress [22]. Our data support the idea that atherosclerosis-prone sites are regions where shear stress is low. Platelet activation is an integrated process that involves subendothelial matrix proteins and soluble agonists that support adhesion and activation. Platelet activation in response to vessel wall injury is an early event in atherothrombotic diseases including stroke and myocardial infarction. Collagen is a vessel wall protein that directly activates platelets, and platelet activation by exposed collagen after vessel wall injury is believed to be an early and important step in cardiopathogenetic diseases [23]. Platelets interact with collagen through two major surface receptors, the integrin α2β1 and the non-integrin receptor GP VI. GP VI is a platelet membrane protein with a molecular weight of 62 kDa that was identified as a physiologic collagen receptor. The GP VI–induced activation mechanism is initiated by tyrosine phosphorylation of FcRγ, an immunoreceptor tyrosine-based activation motif. Then this signal is transduced to many downstream signaling proteins (e.g., Src family kinases) primarily through tyrosine phosphorylation [20]. Src tyrosine kinases such as Src, Lyn, and Fyn are critical for GP VI–mediated signaling that leads to platelet aggregation [24]. In platelets, integrin α2β1 is another major receptor for collagen and it can also be activated by G protein–coupled receptor agonists that are independent of GP VI. Inoue et al. [14] recently provided evidence that α2β1 mediates spreading through a Src kinase-dependent pathway that lies upstream of PLCγ2 and Ca2+. Signal through α2β1 may also have a significant role in pathologic thrombosis [25]. The intracellular signaling cascade used by α2β1 shares many features of the GP VI signaling cascade, including participation of Src kinases and PLCγ2 [15], [26]. The Src kinases and PLCγ2 have also been reported to play a critical role in the platelet activation induced by thrombin or ADP [26], [27]. Our present study on the effect of homocysteine on the signaling pathway involved in platelet activation induced by collagen is helpful in understanding the pathogenesis of atherothrombotic diseases and for the development of new therapies to treat these diseases. Because of the marked effect of homocysteine on platelet functions, we investigated the effect of homocysteine on collagen-stimulated signal transduction. Stimulation of platelets with collagen results in the activation of a tyrosine kinase-dependent signaling pathway. As a consequence, platelet activation with collagen is accompanied by tyrosine phosphorylation of multiple platelet proteins. Protein tyrosine phosphorylation is essential for platelet activation and plays a major role in platelet aggregation [15], [17], [24], [28]. Homocysteine increased collagen-stimulated protein tyrosine phosphorylation in platelets. In addition, we found that genistein, a selective tyrosine kinase inhibitor, when incubated with homocysteine, abolished the homocysteine-elicited increase of total platelet protein tyrosine phosphorylation. We therefore investigated the effect of homocysteine on specific signal molecules in the collagen-stimulated platelet activation pathway. Src kinase activation is a key intermediate step in the activation of platelets by the physiologic agonist collagen [15], [24]. Another signal-transduction cascade that accompanies collagen-induced platelet activation is activation of the phosphoinositide pathway. Phosphoinositide-specific PLC cleaves to phosphatidylinositol 4,5-bisphosphate to form the second messengers inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. PLCγ2 lies downstream of the integrins α2β1 and αIIbβ3 and the major receptor for collagen, GP VI [15]. Src and PLCγ2 are required for downstream signaling of both collagen receptors in platelets, so we investigated the effect of homocysteine on tyrosine phosphorylation of Src kinase and PLCγ2 using immunoprecipitation and western blotting. Homocysteine increased the tyrosine phosphorylation of Src kinase and PLCγ2 stimulated by collagen. Our experimental data also showed that enhancement of homocysteine on tyrosine phosphorylation of Src kinase and PLCγ2 was partly decreased by a blockade of integrin α2β1 activity by a polyclonal blocking antibody to integrin β1. This result suggests that homocysteine affects collagen-induced platelet activation through interaction with both major collagen receptor signals. Homocysteine is a potential stimulation that interferes with a variety of collagen-platelet interactions, and it can be recognized not only by the primary platelet adhesion receptor α2β1 but also by other collagen receptors such as GP VI. In conclusion, our study has shown that, by enhancing tyrosine phosphorylation in whole cellular protein, especially tyrosine phosphorylation of signaling molecules in the GP VI and integrin α2β1 pathway, such as Src kinase and PLCγ2, homocysteine might potentiate platelet response to collagen and might contribute to the thrombogenic effects described in hyperhomocystinemia. 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a Laboratory of Cardiovascular Diseases, West China Hospital, Sichuan University, Chengdu, Sichuan, Peoples Republic of China b Department of Pathophysiology, West China Medical Center, Sichuan University, Chengdu, Sichuan, Peoples Republic of China  Corresponding author. Tel.: +86-28-8550-3300; fax: +86-28-8516-0219.
This work was supported by grants 30470437, 39800054, and 39700068 from the Natural Science Foundation of China (Xiaojing Liu), grant 04GY029-083-1 from the Research Foundation of Science and Technology Bureau of Sichuan Province (Fengming Luo), and grant 02951181 from the Research Foundation of West China Hospital of Sichuan University (Fengming Luo). PII: S0899-9007(05)00235-2 doi:10.1016/j.nut.2005.04.012 © 2006 Elsevier Inc. All rights reserved. | |
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