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Am J Physiol Cell Physiol 295: C1045-C1054, 2008. First published August 20, 2008; doi:10.1152/ajpcell.90644.2007
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RECEPTORS AND SIGNAL TRANSDUCTION

Platelets undergo phosphorylation of Syk at Y525/526 and Y352 in response to pathophysiological shear stress

Henry E. Speich,1,2 Svetozar Grgurevich,2 Teddi J. Kueter,2 Angela D. Earhart,2 Steven M. Slack,1 and Lisa K. Jennings1,2

1Department of Biomedical Engineering, The University of Memphis; and 2Vascular Biology Center of Excellence, College of Medicine, University of Tennessee Health Science Center, Memphis, Tennessee

Submitted 21 December 2007 ; accepted in final form 12 August 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Atherosclerotic plaques can lead to partial vascular occlusions that produce abnormally high levels of arterial wall shear stress. Such pathophysiological shear stress can promote shear-induced platelet aggregation (SIPA), which has been linked to acute myocardial infarction, unstable angina, and stroke. This study investigated the role of the tyrosine kinase Syk in shear-induced human platelet signaling. The extent of Syk tyrosine phosphorylation induced by pathophysiological levels of shear stress (100 dyn/cm2) was significantly greater than that resulting from physiological shear stress (10 dyn/cm2). With the use of phospho-Syk specific antibodies, these data are the first to show that key regulatory sites of Syk at tyrosines 525/526 (Y525/526) and tyrosine 352 (Y352) were phosphorylated in response to pathophysiological shear stress. Increased phosphorylation at both sites was attenuated by pharmacological inhibition of Syk using two different Syk inhibitors, piceatannol and 3-(1-methyl-1H-indol-3-yl-methylene)-2-oxo-2,3-dihydro-1H-indole-5-sulfonamide (OXSI-2), and by inhibition of upstream Src-family kinases (SFKs). Shear-induced response at the Syk 525/526 site was ADP dependent but not contingent on glycoprotein (GP) IIb-IIIa ligation or the generation of thromboxane (Tx) A2. Pretreatment with Syk inhibitors not only reduced SIPA and Syk phosphorylation in isolated platelets, but also diminished, up to 50%, the platelet-mediated thrombus formation when whole blood was perfused over type-III collagen. In summary, this study demonstrated that Syk is a key molecule in both SIPA and thrombus formation under flow. Pharmacological regulation of Syk may prove efficacious in treating occlusive vascular disease.

GPIb; GPIIb-IIIa; signal transduction; thrombosis; collagen

NUMEROUS STUDIES have shown that platelets can be activated, in the absence of exogenous agonists, by both physiological and pathophysiological levels of shear stress (27). Such activation can lead to shear-induced platelet aggregation (SIPA), which has been linked to acute myocardial infarction, unstable angina, and stroke (18, 25, 27). Pathologically elevated levels of shear stress are often attributed to atherosclerosis. Blood flow is constricted at a stenosis, rapidly increasing the velocities and shear forces within the vessel (27, 60). These disruptions in the fluid dynamic environment correlate with increased platelet reactivity. Signaling pathways involved in shear-induced platelet activation are attractive targets for pharmacological intervention because they offer the potential for preferential treatment of pathological thrombosis with a seemingly negligible impact on normal hemostasis (26).

Spleen tyrosine kinase, commonly referred to as Syk, has been implicated in numerous signaling cascades in platelets. Syk is a 72-kDa nonreceptor tyrosine kinase, the activity of which is correlated to its autophosphorylation at specific tyrosine residues (53). Inside-out signaling phenomena involving Syk include, but are not limited to, collagen-induced activation, through glycoprotein VI (GPVI) and the FcR {gamma}-chain (GPVI-FcR{gamma} complex), and thrombin-induced activation (15, 49, 65). Syk also participates in outside-in signaling following ligand occupancy of activated GPIIb-IIIa, where it is downstream of Src kinase (16, 31, 44). Syk has been indirectly linked to shear-induced activation through the GPIb-V-IX complex, primarily through studies using the snake venom probe botrocetin and the fungal derivative ristocetin. These agents allow soluble von Willebrand factor (vWF) to interact with, and thus initiate, signaling through the platelet GPIb-IX-V complex. Ligation of vWF to GPIb ordinarily requires elevated levels of shear stress. Stimulation of platelets with either botrocetin-vWF or ristocetin-vWF induces Syk phosphorylation (3, 33, 64).

Relatively few studies have more directly linked Syk to shear-induced signaling by using shear stress as an experimental component. Prior work in our laboratory established that platelets exhibited a unique pattern of tyrosine phosphorylation when exposed to 1 min of shear stresses up to the pathophysiological level of 100 dyn/cm2 (20). These data tentatively identified the tyrosine kinase Syk as one of the key platelet proteins activated by shear. More substantial evidence of shear-induced Syk phosphorylation was introduced in a report that in platelet phosphatidylinositol 3-kinase (PI 3-kinase) immunoprecipitates, a coprecipitating 72-kDa band at the migration locale of Syk had increased tyrosine phosphorylation in response to 1 min of pathophysiological shear stress (51). The observed increase in phosphorylation was inhibited by high concentrations of piceatannol (25 µg/ml), a commonly used pharmacological inhibitor of Syk. The investigators proposed that shear-induced vWF binding to GPIb prompted ADP secretion, which in turn stimulated the P2Y12 receptor and led to the phosphorylation of PI 3-kinase-associated Syk. More recently, shear stress has been reported to cause dissociation of Syk from the β3-integrin in platelets (14).

The current study was designed to more clearly define the role of Syk and the contributions of its specific tyrosine residues in platelet activation induced by pathophysiological levels of shear stress. We investigated the mechanism(s) associated with Syk phosphorylation by looking at the roles of Src family kinases (SFKs) and various platelet surface receptors. We concluded by examining the ability of pharmacological inhibitors of Syk to prevent thrombosis in an ex vivo flow model. The results from this study support that Syk may be a novel anti-thrombotic target that will diminish detrimental platelet response to pathophysiological levels of shear stress.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Antibodies and reagents. The monoclonal anti-Syk 4D10, anti-c-Src, and normal murine IgG2a were from Santa Cruz Biotechnologies (Santa Cruz, CA). The goat anti-mouse and goat anti-rabbit antibodies used for Western blot analysis were from Pierce Biotechnology (Rockford, IL). The RC20-horseradish persoixdase (HRP) and anti-FAK (no. 610088) were from BD Biosciences Pharmigen (San Diego, CA). The anti-phosphotyrosine clone 4G10 antibody was from Upstate Biotechnology (Charlottesville, VA). The anti-phospho-Syk (Y352), anti-phospho-Syk (Y525/526), and anti-phospho-Src (Y416) antibodies were from Cell Signaling Technology (Beverly, MA). The anti-phospho-FAK (Y397) antibody was from Invitrogen BioSource (Carlsbad, CA). AK2, an anti-GPIb antibody, was from Serotec (Oxford, UK). The anti-GPIb antibodies 6D1 and IB-23 were generously donated by Dr. Barry Coller and Dr. J. H. Beer, respectively. Dr. Michael Berndt kindly provided 5D2, an anti-vWF antibody. VCL, a peptide that prevents vWF binding to GPIb, was obtained from Bio-Technology General (Rehovot, Israel). Purified vWF and the Src inhibitors PP2 and SU6656 were from Calbiochem (San Diego, CA). The {gamma}-thrombin was a kind gift from Dr. John Fenton II. FITC-murine IgG (no. 2012) and anti-mouse secondary antibody (no. 9670) used for flow cytometery were obtained from Sigma-Aldrich (St. Louis, MO). All other flow cytometry antibodies and FACSCaliber station were from BD Biosciences (San Jose, CA). Eptifibatide was obtained from Schering-Plough (Kenilworth, NJ). Portola Pharmaceuticals (San Francisco, CA) provided Factor Xa Inhibitor 034. The Syk inhibitors piceatannol and 3-(1-methyl-1H-indol-3-yl-methylene)- 2-oxo-2,3-dihydro-1H-indole-5-sulfonamide (OXSI-2, catalog no. 574711) and the Protein G Plus/Protein A Agarose Suspension were from EMD Biosciences (San Diego, CA). Protein G Sepharose was from GE Healthcare (Piscataway, NJ). All other chemical reagents, unless otherwise specified, were purchased from Sigma-Aldrich.

Washed platelet preparation. Studies utilizing human blood were approved by a University of Tennessee Health Science Center Institutional Review Board for human subject research. Blood was drawn from healthy, consenting adult volunteers into ACD anticoagulant (0.05 M sodium citrate, 0.1 M dextrose, 0.07 M citric acid, pH 4.5). Donors denied taking medications known to affect platelet function. Blood was centrifuged at 135 g for 20 min before platelet-rich plasma (PRP) was carefully removed and centrifuged at 800 g for 10 min. The platelet pellet was resuspended and washed three times in CGS (0.12 M sodium citrate, 0.1 M dextrose, and 0.1 M NaCl, pH 6.5) with centrifugation steps at 800 g for 10 min. The final resuspension was in Tyrode buffer (in mM: 138 NaCl, 2.9 KCl, 12 NaHCO3, 0.4 MgCl2, 5.5 dextrose, 0.36 NaH2PO4, and 1.8 CaCl2, pH 7.4). The platelet count, conducted on a Coulter Z2 Particle Counter (Coulter, Healeah, FL), was adjusted to 2.5 x 108/ml. Platelet activation inhibitors were not used during platelet isolation due to their potential effects on signaling pathways involved in shear-induced platelet activation. Washed platelet suspensions were allowed to rest for 30 min at 37°C before conducting experiments.

Analysis of platelet function status and shear-induced vWF binding. To ensure that platelets isolated for these studies were minimally activated and functional, the activation state of the final platelet suspensions was evaluated. Flow cytometric analysis was used to examine expression of platelet membrane glycoproteins CD62 and CD63. Elevated expression of these glycoproteins on the platelet surface correlates with platelet activation (37, 71). Washed platelet suspensions were incubated with FITC-conjugated anti-CD62P, anti-CD63, or murine IgG for 15 min at 37°C and then analyzed with a FACSCalibur station for bound chromaphore. The FITC-conjugated murine IgG served as the control for nonspecific antibody binding. The surface expression of each the respective marker was quantified in the form of mean fluorescent intensity (MFI). As a positive control, platelets were activated with {gamma}-thrombin (0.06 U/ml). This agonist was selected based on its ability to activate via the PAR-1 receptor, with minimal fibrinogen cleaving activity, and induce a robust aggregation response (72). This concentration achieved maximum surface expression of CD62 (data not shown).

Viscometer experiments with washed platelets (see Shear system) were conducted without the addition of exogenous vWF. However, the absence of exogenous vWF should not be limiting because elevated shear stress can induce platelet activation and secretion, even in platelets from patients with severe vWF disease (22, 40). Furthermore, in normal patients, platelet-released vWF multimers are sufficient to support aggregation (40). Our lab has previously documented shear-induced platelet activation, procoagulant activity, microparticle formation, and aggregation using the cone-and-plate viscometer approach without the readdition of exogenous vWF to washed platelets (20, 21). As an ancillary confirmation that our shear system was inducing endogenous vWF secretion from washed platelets, allowing for subsequent shear-dependent vWF ligation and signaling through GPIb, we performed flow cytometric assays on washed platelet and PRP suspensions exposed to either static or elevated shear conditions (100 dyn/cm2, 1 min) in the viscometer.

After centrifugation, washed platelet suspension or PRP was adjusted to a platelet count of 2.5 x 108/ml with Tyrode buffer or platelet-poor plasma, respectively. Suspensions were allowed to rest at 37°C for 30 min and then pretreated with eptifibatide (4,000 nM, 2 min, 37°C) to prevent subsequent aggregation, thereby ensuring that data were collected from single platelets and not aggregates. Aliquots were then exposed to static conditions or pathophysiological shear stress (100 dyn/cm2, 1 min, 37°C) in the viscometer.

Platelets were incubated with 6D1 (5 µg/ml, 30 min, room temperature) immediately after exposure to shear stress or static conditions, followed by incubation with a phycoerythrin-conjugated anti-mouse secondary antibody (15 min, room temperature). Binding of the anti-GPIb antibody 6D1 is directly correlated to vWF binding potential because 6D1 binds to GPIb at the same site as vWF, competing with vWF for that site (13, 38). Therefore, increases in the amount of GPIb-bound vWF decrease the extent of 6D1 binding to the platelet surface. The surface expression of 6D1 was quantified as MFI. In some experiments, washed platelet suspensions were supplemented with 5 µg/ml of exogenous purified human vWF immediately before shear stress exposure.

Light transmission aggregometry. To further ensure that platelet reactivity was maintained, all washed platelet suspensions were required to demonstrate a typical aggregation response. Aggregations were performed using a modest concentration of collagen (2 µg/ml) on a Payton Lumi-Aggregation Modules (Series 1000B) (71). PRP adjusted to 2.5 x 108 platelets/ml was used. Experiments were aborted when suspensions produced atypical tracings.

A dose-response curve for the newly described Syk inhibitor OXSI-2 was also established. Aggregations in adjusted PRP, induced by collagen concentrations of 0.5 and 2 µg/ml, were assayed. Platelets were pretreated with vehicle or inhibitor for 5 min before aggregation.

Shear system. A modified Hercules Hi-Shear Viscometer (Kaltec Scientific Instrument, Novi, MI) was used to provide exact levels of wall shear stress. The viscometer had both Couette and cone-plate style viscometer regions. Matching the rotational velocity of both regions creates identical levels of shear stress (61). This system has been described in depth elsewhere (59). The technique, in general, is a well understood and widely used method for subjecting cells in suspension to fluid dynamic parameters (27, 40, 41, 56, 61).

The viscometer's cup and bob were exposed to 50% human serum for at least 30 min at 37°C before experiments to prevent surface-induced platelet activation (20). The temperature of the cup and bob was maintained at ~37°C. Vehicle or inhibitor pretreatments were performed after the platelet resting period and immediately before sample exposure to shear stress. Unless otherwise specified, pretreatments were for 5 min at 37°C. Between shear runs, the cup and bob were thoroughly rinsed with PBS and gently blotted dry. After exposure, sample aliquots were immediately placed into lysis buffer (see Immunoprecipitation).

Quantification of SIPA. Platelet aggregation was quantified by comparing single platelet counts, conducted with an ICHOR hematology analyzer (Helena Laboratories, Beaumont, TX), before and immediately after platelet exposure to shear stress. The extent of SIPA was defined as the drop in single platelet count following exposure. Percent aggregation (%PA) was therefore calculated as %PA = [(count before shear – count after shear)/count before shear] x 100.

Immunoprecipitation. Platelet suspensions were transferred directly from the viscometer into an equal amount of 2x lysis buffer on ice and gently mixed. For experiments involving the RC20-HRP antibody, the final (1x) composition was as follows: 20 mM Tris·HCl, 1% (vol/vol) Triton X-100, 10 mM Na pyrophosphate, 10 mM NaF, 1.2 mM Na3VO4, 5 mM EDTA, and 150 mM NaCl. The buffer was supplemented with one Roche Protease Inhibitor tablet (Roche Pharmaceuticals, Nutley, NJ) per 15 ml and adjusted to pH 7.4. For experiments involving the 4G10 and phospho-Syk antibodies, the following final composition was used: 20 mM Tris·HCl, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 5 mM EDTA, 150 mM NaCl, 1% protease inhibitor cocktail (Sigma), 1% phosphatase inhibitor cocktail-I, and 1% phosphatase inhibitor cocktail-II. Samples were allowed to lyse on ice for 1 h and then clarified by centrifugation at 15,000 g for 10 min at 4°C. The supernatant was subjected to immunoprecipitation.

In experiments using the RC20 antibody, clarified lysates were precleared with an isotype-matched control antibody for 1 h at 4°C. After addition of a 100-µl aliquot of protein A/G agarose bead suspension, the lysates were incubated for 1 h at 4°C, and the control antibody was precipitated by centrifugation at 1,000 g for 1 min. This preclearing step was eliminated in subsequent experiments, as immunoprecipitation with the isotype-matched control antibody was incorporated into the Western blot analysis as a negative control.

Clarified lysates or precleared supernatants were incubated with 2 µg/ml of control or 4D10 antibody overnight at 4°C. Protein A/G beads or Protein G beads (anti-phospho-Syk experiments only) were then added for 1 h with rotation at 4°C. After precipitation of beads (1,000 g for 1 min), the supernatant was removed, and the beads were washed with fresh 1x lysis buffer. The beads were resuspended in modified reducing sample buffer (mRSB, 2% SDS, 10% glycerol, 0.01% bromophenol blue, 1 mM Na3VO4, 5 mM EDTA, 50 mM Tris·HCl, and 2% β-mercaptoethanol, pH 6.8) and boiled for 10 min. The entire content of each sample tube was loaded for electrophoresis.

Gel electrophoresis and immunoblotting. Samples in mRSB were electrophoresed through 5%-20% exponential SDS-polyacrylamide gradient gels at 55 volts. Proteins were then transferred to a polyvinylidenedifluoride membrane (Millipore, Billerica, MA). Membranes were blocked with immune stain buffer (10 mM Tris·HCl, 0.9% NaCl, 5% bovine serum albumin, and 0.1% Tween-20, pH 7.4) for at least 1 h at 4°C. Blots were incubated with appropriate primary and secondary antibodies in immune stain buffer, typically for 90 min each at room temperature. Phospho-Syk primary antibodies required overnight incubations. Next, membranes were washed with Tris-buffered saline with Tween-20 (TBSt: 10 mM Tris, 100 mM NaCl, and 0.1% Tween-20, pH 7.4). Blots were developed in Supersignal Dura chemiluminescent substrate solution per manufacturer's instruction (Pierce, Rockford, IL) and images captured by X-ray film. Antibody stripping of the blots, if necessary, was performed with Restore Western Blot Stripping Buffer (Pierce) per manufacturer's instruction.

Capillary perfusion experiments. The effect(s) of Syk inhibitors on thrombus formation were documented using a capillary perfusion aggregometer provided by Millennium Pharmaceuticals (Cambridge, MA). This apparatus is an evolved rendition of capillary perfusion devices described elsewhere (1, 22). Experiments were conducted on whole blood from healthy adult donors. The first 2 ml of blood drawn were discarded to minimize the risk of including platelets activated during the initial stages of the phlebotomy. The experimental sample was then drawn directly into a syringe containing Factor Xa Inhibitor 034 (10 µM final concentration) and gently mixed. Experiments were completed within 1 h of blood collection.

Cellular constituents of blood were labeled with rhodamine 6G (4 µl/ml) at 37°C for 10 min before perfusion. The dimensions of type III human collagen-coated capillaries were 0.2 x 2.0 mm. A buffer (in mM: 130 NaCl, 2 KCl, 12 NaHCO3, 2.5 CaCl2·2H2O, and 0.9 MgCl2·6H2O, pH 7.4) was briefly perfused through each new capillary at 2,600 1/s to rehydrate adherent collagen and flush away any unbound collagen. Drug pretreatments, if any, were timed to finish simultaneously with rhodamine incubations. After pretreatment, the tube containing the blood was placed in a 37°C sleeve while a syringe pump pulled blood through the capillary at a shear rate of 1,100 1/s. The aggregometer's fluorescent microscope recorded images at a set rate to visualize thrombus dynamics. Instrument software analyzed the captured two-dimensional images to calculate thrombus volume, area, and perimeter for each image per time point.

Statistical methods. t-Tests, one-factor analysis of variance (ANOVA), or two-factor ANOVA were used to determine statistically significant differences between experimental data collected for various treatments. When significant differences were detected with ANOVA, the Student-Newman-Keuls multiple comparison procedure was applied to identify statistically different treatment pairs (17). Microsoft Excel (Version X, Microsoft, Seattle, WA) and SigmaStat (Version 2.03, Systat, Point Richmond, CA) software packages were employed.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Throughout this study we designed several experiments to validate our model system and to monitor platelet reactivity status. Flow cytometric analysis demonstrated that both the CD62 and CD63 activation markers exhibited an approximate 10-fold difference (P < 0.001) in surface expression between untreated and thrombin-activated washed platelets (Fig. 1A), whereas no difference was seen with the control antibody between treatment groups. These results indicated that there was minimal activation of the rested, washed platelet suspensions, and that the platelets were responsive and susceptible to physical and pharmacological perturbation.


Figure 1
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  		Fig. 1. Flow cytometric analysis of washed platelet activation and shear-induced von Willebrand (vWF) binding. A: flow cytometry was used to compare the surface expression of platelet activation markers for rested washed platelets (open bars) versus rested washed platelets activated with 0.06 U/ml {gamma}-thrombin (closed bars). Mean fluorescent intensity (MFI) values + standard deviations are reported for IgG (control for nonspecific binding), CD62 (P-selectin), and CD63 antibodies (n ≥ 4). B: binding levels for anti-GPIb antibody 6D1, which competes with vWF for GPIb on the platelet surface, are shown for suspensions of washed platelets (WP), washed platelets supplemented with 5 µg/ml vWF (WP + vWF), and platelet-rich plasma (PRP). Each suspension was exposed to either static conditions (open bars) or pathophysiological levels of wall shear stress (closed bars), specifically 100 dyn/cm2, for 1 min at 37°C, before incubation with 6D1. MFI values +SD are reported (n = 5 for all groups).

 
We also evaluated whether our shear system was inducing platelet secretion of endogenous vWF, allowing for subsequent shear-dependent vWF ligation and signaling through GPIb. Shear-induced GPIb/vWF binding was assessed by exploiting the ability of anti-GPIb monoclonal antibody 6D1 to compete for the vWF binding site on GPIb. Platelet suspensions in the presence or absence of supplemented vWF (5 µg/ml) or PRP suspensions containing endogenous plasma vWF were exposed to either static or elevated shear conditions (100 dyn/cm2, 1 min) in the viscometer and then incubated with 6D1. The extent of 6D1 binding was analyzed by flow cytometry (Fig. 1B). No significant difference in 6D1 binding was detected between any of the static suspensions. However, exposure to shear stress caused a significant decrease in 6D1 binding in all suspensions, indicating that shear-induced vWF binding had occurred. This decrease was significantly greater in PRP, presumably due to a much higher vWF concentration in plasma than that in the washed and supplemented suspensions, allowing for more effective competition by the ligand with 6D1 for the vWF binding site. Supplementing washed platelets with vWF did not significantly influence 6D1 shear-induced vWF binding.

OXSI-2, a new pharmacological inhibitor of Syk, was recently shown to potently inhibit Syk activity in in vitro kinase assays, in assays of IgE/Fc{epsilon}RI-triggered basophil cell degranulation and in other applications involving Syk-dependent phenomena (29, 43, 52, 75). OXSI-2, which is structurally unique from the classic Syk inhibitor piceatannol, was used in many of our experiments to corroborate our results. In light transmission aggregometry experiments, approximate EC50 values of 5.5 and 30 µM OXSI-2 were determined for 0.5 and 2.0 mg/ml collagen stimulations, respectively (Fig. 2). Based on these data and the results of a thrombosis assay, which will be described subsequently, we chose to use 3.13 µM OXSI-2 to inhibit Syk signaling in our platelet studies. This concentration is 10 times the EC50 for basophil degranulation noted earlier (29).


Figure 2
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  		Fig. 2. Dose-response curves for OXSI-2 inhibition of collagen-induced aggregation. Platelets were pretreated with vehicle or OXSI-2 at various concentrations for 5 min at room temperature. Aggregation was then induced with either 0.5 µg/ml (open squares) or 2 µg/ml collagen (diamonds). Values shown are percent inhibition with respect to maximum vehicle aggregation ±SD (n ≥ 3 for all points of 0.5 µg/ml, n ≥ 5 for all points of 2 µg/ml).

 
To confirm that Syk is tyrosine phosphorylated in response to pathophysiological shear stress, Syk was immunoprecipitated with the 4D10 anti-Syk antibody. As predicted, a band at an apparent molecular mass of 72 kDa exhibited increased tyrosine phosphorylation in response to shear stress (1 min, 100 dyn/cm2) (Fig. 3A). Pretreatment with vehicle 0.1% DMSO resulted in no deviation of the phosphotyrosine level compared with the corresponding lanes of untreated platelets. However, pretreatment with the Syk inhibitor piceatannol (5 µg/ml) almost completely abolished the 72-kDa phosphorylation response to elevated shear stress. The 72-kDa band was confirmed as Syk by stripping the immunoblot of bound antibody and reprobing with anti-Syk antibody. Protein loading was equivalent among treatments.


Figure 3
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  		Fig. 3. Inhibition of shear-induced Syk tyrosine phosphorylation and SIPA by piceatannol and OXSI-2. A–C: washed platelets were pretreated with vehicle (0.1% DMSO), the Syk inhibitor piceatannol (piceat, 5 µg/ml), or the Syk inhibitor OXSI-2 (3.13 µM) then exposed to a wall shear stress of either 0, 10, or 100 dyn/cm2 in the viscometer. Syk was immunoprecipitated (IP) from lysates. Western blots (WB) were probed for phosphotyrosine (pY) with RC20 (A) or 4G10 (B and C) antibodies. All blots were stripped and reprobed for total Syk. (n = 3 for each panel). D: platelet aggregation induced by 1-min exposure to a shear stress of 100 dyn/cm2 was significantly inhibited by 3.13 µM OXSI-2 and 5 µg/ml piceatannol (n = 5).

 
Additionally, the level of Syk tyrosine phosphorylation in response to physiological shear (10 dyn/cm2) differed from that seen in response to pathophysiological shear (100 dyn/cm2), as shown in Fig. 3B. In vehicle (0.1% DMSO)-pretreated samples there was a noticeable, but slight, difference in Syk tyrosine phosphorylation between static and physiological shear stress conditions. In contrast, a drastic increase in Syk phosphorylation is apparent at 100 dyn/cm2. Piceatannol pretreatment (5 µg/ml) dramatically inhibited this marked increase. The blot was stripped and reprobed for total Syk, the levels of which were equivalent in all treatment groups. OXSI-2 pretreatment, at a concentration of 3.13 µM, also greatly reduced Syk tyrosine phosphorylation prompted by 1 min of 100 dyn/cm2 shear stress (Fig. 3C). Piceatannol pretreatment of 5 µg/ml functioned as a control in these experiments. Blots were stripped and reprobed for Syk, showing equivalent protein levels.

To determine whether OXSI-2 and piceatannol also prevented SIPA, the ICHOR Hematology Analyzer was used to perform counts of single platelets before and after shear stress exposure. This procedure served as a quantitative mean for assessing platelet aggregate formation. The percent decrease in single platelet count due to shear is analogous to the percent aggregation quantification appearing in Fig. 3D. Both Syk inhibitors caused a significant (P < 0.05) inhibition of SIPA.

Shear-induced Syk activity was further delineated by using site-specific phospho-Syk antibodies as Western blot probes. These antibodies are well-characterized and have been used to detect and/or differentiate between phosphorylated tyrosine residues on Syk at site 352 or at site 525/526 in a variety of cell types, including platelets (8, 39, 50, 52, 64). In vehicle pretreatment samples (0.1% DMSO), tyrosine phosphorylation was increased at SykY525/526 in response to a wall shear stress of 100 dyn/cm2 over levels seen under static conditions (Fig. 4A). This response was inhibited by OXSI-2 (3.13 µM) and piceatannol (5 µg/ml) pretreatments. The same decreases were observed for SykY352 (Fig. 4B). Negative control lanes, which used an isotype-matched IgG for immunoprecipitation, showed no signal for either phospho-Syk probe, demonstrating that the of specificity 4D10 as an immunoprecipitating antibody. As expected, 10 µg/ml collagen stimulation (positive control) resulted in increased phosphorylation of both SykY352 and SykY525/526. Both sites have been shown to be phosphorylated in platelets following stimulation of the collagen receptor GPVI (64).


Figure 4
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  		Fig. 4. Shear-induced Syk phosphorylation occurs at Y525/526 and Y352. Washed platelets were pretreated with vehicle, 3.13 µM OXSI-2, or 5 µg/ml piceatannol (Pic). All lanes, except the positive control (10 µg/ml collagen stimulation), were then exposed to a wall shear stress of 0 or 100 dyn/cm2 in the viscometer. Syk was immunoprecipitated from lysates, except in the negative control lane where isotype-matched mIgG was used. Western blots were probed with site-specific phospho-Syk antibodies anti-pY525/526 or anti-pY352. Blots were stripped and reprobed for total Syk (n = 3 for both probes).

 
SFKs have been shown to be upstream of Syk in a number of platelet-signaling cascades. Therefore, the well-described SFKs inhibitors SU6656 and PP2 were used to determine the effect(s) of inhibiting SFKs on shear-induced phosphorylation of Syk tyrosine residues (4, 5, 7, 23). Pretreatment of platelets with SU6656 (10 µM) or PP2 (5 µM) substantially inhibited shear-induced phosphorylation at SykY525/526 and SykY352 (Fig. 5A). At these concentrations, neither compound should inhibit Syk phosphorylation directly, and any nonspecific effects are diluted by the use of two structurally diverse agents (4, 9).


Figure 5
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  		Fig. 5. Site-specific phosphorylation of Syk is downstream of Src-family kinase and is ADP dependent. A: washed platelets were pretreated with vehicle or Src-family kinase inhibitors PP2 (10 µM) or SU6656 (5 µM) then exposed to 0 or 100 dyn/cm2 wall shear stress. Syk was immunoprecipitated from lysates. Western blots were probed with anti-pY525/526 or anti-pY352 antibody. B: washed platelets were pretreated with vehicle (2.4% saline or 0.1% EtOH), AK2 (10 µg/ml), eptifibatide (Eptif, 4 µM), apyrase (Apy, 5 U/ml), or aspirin (ASA, 100 µM) then exposed to shear stress. Western blots were probed with anti-pY525/526 antibody after Syk immunoprecipitation. C: washed platelets were pretreated with vehicle control or combinations of eptifibatide, apyrase, and/or aspirin (same concentrations as B) then exposed to shear stress. Western blots were probed with anti-pY525/526 antibody after Syk immunoprecipitation. All blots were stripped and reprobed for total Syk (n = 3 for all panels).

 
Further investigation was made regarding which receptors and/or signaling pathways lay upstream of Syk in shear-induced activation. Before exposure to shear stress, platelets were pretreated with anti-GPIb antibody AK2, GPIIb-IIIa antagonist eptifibatide, ADP scavenger apyrase, or cyclooxygenase inhibitor aspirin to inhibit GPIb-, GPIIb-IIIa-, ADP-, and TxA2-dependent signaling, respectively. The effects of each antagonist on shear-induced phosphorylation of SykY525/526 are shown in Fig. 5B. Saline was the vehicle control for AK2, eptifibatide, and apyrase pretreatments. Likewise, ethanol was the vehicle control for aspirin. Unexpectedly, the anti-GPIb antibody AK2 augmented shear-induced phosphorylation upon binding to intact platelets. In subsequent parallel experiments, AK2 was observed to increase phosphorylation of SykY525/526 even under static conditions (n = 3, data not shown). Eptifibatide or aspirin pretreatment had no effect on shear-induced phosphorylation of SykY525/526. Apyrase, on the other hand, caused a minor, but consistent, attenuation of the response. The effect(s) of pretreating platelets with various combinations of eptifibatide, apyrase, and aspirin was also assessed (Fig. 5C). The only pretreatment regimens that attenuated shear-induced phosphorylation of SykY525/526 compared with vehicle controls were the apyrase-aspirin combination and, to a lesser extent, the combination of all three inhibitors.

A final series of experiments were conducted to assess the effect(s) of OXSI-2 and piceatannol on collagen-induced, platelet-mediated thrombus formation under elevated shear stress. This was investigated using a whole blood capillary perfusion system at a shear rate of 1,100 1/s (~40 dyn/cm2) with capillaries coated with Type III human collagen. Because most of our earlier data were collected using higher levels of shear stress, we confirmed, using the viscometer, that a wall shear stress of 40 dyn/cm2 induced phosphorylation at SykY525/526, though the response was not as robust as that seen in response to 100 dyn/cm2 (Fig. 6A). As in Fig. 1B, shear stress magnitude correlates with the extent of Syk phosphorylation. Both OXSI-2 and piceatannol inhibited shear-induced phosphorylation of SykY525/526 at 40 dyn/cm2.


Figure 6
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  		Fig. 6. Phosphorylation of Syk Y525/526 and thrombus formation on Type-III collagen at 40 dyn/cm2 is inhibited by OXSI-2 and piceatannol. A: washed platelets were exposed to 0, 40, or 100 dyn/cm2 shear stress for 1 min in viscometer. Syk was immunoprecipitated from lysates. Western blot analyses were probed with anti-pY525/526 antibody. Blots were stripped and reprobed for total Syk (n = 2). B: whole blood was perfused at 1,100 1/s (~40 dyn/cm2) over type-III collagen in a capillary perfusion aggregometer. Histograms show thrombus volume/area (arbitrary units) at 5 min of perfusion. Thrombus formation was reduced, in a dose-dependent manner, by 5-min pretreatment with either OXSI-2 or piceatannol (n = 5 for OXSI-2, n = 3 for piceatannol). C: platelet thrombus formation (bright areas) at 5 min of perfusion for various pretreatments from a representative capillary perfusion experiment as detailed in B.

 
For thrombosis experiments, whole blood was preincubated with OXSI-2, piceatannol, or vehicle (0.1% DMSO) for 10 min before perfusion. After 300 s of perfusion, OXSI-2 pretreatment at concentrations of 3.13 and 31.3 µM displayed 28% and 50% decreases, respectively, in thrombus formation over vehicle control (Fig. 6B). Similarly, piceatannol at concentrations of 5 and 25 µg/ml inhibited thrombus formation by 35% and 52%, respectively. Each of the inhibitors also elicited a dose response, with greater inhibition of thrombus formation seen at higher doses of the pharmacological agents. OXSI-2 pretreatment at 0.313 µM did not affect thrombus formation (data not shown), as expected, based on the inability of this concentration to inhibit collagen-induced aggregation.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In recent years, significant effort has been directed toward defining the effects of shear stress on platelet physiology. The paradigm for shear-induced platelet aggregation is the shear-dependent interaction of vWF with the platelet GPIb-V-IX adhesion complex (27, 28). Through modulation of this interaction, as well as tethering/adhesion dynamics and diffusion profiles, shear stress and other fluid dynamic parameters are integral factors in platelet-platelet interactions and platelet interactions with constituents of the vessel wall. Because extreme levels of shear stress are found almost exclusively in vascular disease states and linked to negative outcomes in clinical settings, the task of understanding these phenomena is critical, especially in a society where heart disease is the leading cause of morbidity (27).

The first goal of this study was to confirm earlier indications that Syk was involved in shear-induced platelet signaling. This was established by showing phosphorylation of a 72-kDa band in Syk immunoprecipitates from platelets exposed to 100 dyn/cm2 shear stress. Moreover, the response was inhibited by 5 µg/ml piceatannol. This concentration of piceatannol was specifically selected because the inhibitor has been demonstrated to be indiscriminate at higher concentrations. At 5 µg/ml, piceatannol should significantly inhibit Syk while only negligibly inhibiting SFKs Fyn and Lyn (9, 31, 46). Piceatannol has been found to be less discriminate with regards to Src and FAK; however, we detected no shear-induced phosphorylation of Src at Y416 (n = 4, unpublished observation). We did observe FAK phosphorylation at Y397 in response to pathophysiological shear stress that was completely eliminated by pretreating platelets with 4,000 nM eptifibatide (n = 3, unpublished observation). This finding agrees with other studies that indicate FAK is not directly activated through GPIb/vWF interaction, but instead is involved in secondary signaling events following GPIIb-IIIa engagement (2, 11, 14, 45). Since we demonstrate phosphorylation at the Syk activation site, Y525/526, even when GPIIb-IIIa is blocked, the signaling data regarding Syk phosphorylation and the functional aggregation assays presented in this paper should be unaffected by unintentional FAK or Src inhibition.

The conclusions from these studies are futher substantiated by data obtained using a second, structurally distinct inhibitor of Syk, OXSI-2. This inhibitor has been useful for a number of mechanistic studies (29, 43, 47, 52, 66, 75). Recently, Bhavaraju et al. (6) proposed that OXSI-2 may also inhibit SFKs in platelets. This assertion was based, in part, on marginal inhibition by OXSI-2 of PAR4-mediated, "SFK-dependent" ERK phosphorylation. Although the PAR4 signaling pathway may indeed be SFK dependent, the coparticipation of Syk has not been ruled out (55). Furthermore, in the similar botrocetin/vWF-induced platelet signaling pathway, both Lyn, an SFK, and Syk were required for phosphorylation of BTK, which in turn mediated ERK1/2 phosphorylation (32). Evidence that OXSI-2 is unlikely to inhibit SFK exists in reports where serum amyloid P-induced inhibition of monocyte-to-fibrocyte differentiation, as well as CD200-induced phosphorylation of CD200R and Dok1 in mast cells, were inhibited by PP2, but not by OXSI-2 (48, 77). Noting that nonspecific effects for OXSI-2 in platelets are likely to exist in some form, this Syk inhibitor remains an acceptable tool for investigating Syk-dependent phenomenon, especially when piceatannol, a structurally unique inhibitor, is used corroborate results and dilute the possibility of overlapping effects. Interestingly, Bhavaraju et al. did not observe inhibition of convulxin-induced phosphorylation of SykY352, which is unexplained at this time.

After a shear-induced phosphorylation of Syk was established, the phenomenon was further characterized by demonstrating that physiological levels of shear stress (10 dyn/cm2) promoted only a slight increase in Syk tyrosine phosphorylation while pathophysiological levels of shear stress (100 dyn/cm2) induced a substantially more robust phosphorylation. Later, results obtained for stimulation with 40 dyn/cm2 shear stress affirmed that wall shear stress magnitude can influence the extent of Syk phosphorylation. Therefore, wall shear stress, which can differ dramatically between normal and diseased vessels, could affect the degree of Syk-mediated platelet activation by modulating the extent of Syk phosphorylation.

Further experiments were conducted to investigate the site-specific nature of shear-induced Syk phosphorylation. The novel discovery was made, using phospho-Syk specific antibodies, that Syk is phosphorylated at Y525/526 and at Y352 in response to pathophysiological shear stress. SykY525/526 is in the activation loop of Syk and is essential for full Syk function (76). SykY352 is thought to be an important positive regulator of Syk function by influencing Syk binding to PLC{gamma}1, PLC{gamma}2, Vav1, the p85 subunit of PI 3-kinase, Lck, and Grb2 (19, 30, 42, 58). Furthermore, this site can impact Syk phosphorylation of Vav1, ERK, Akt, LAT, and SLP-76 (58). Both SykY352 and SykY525/526 have been shown to be required for Syk binding to the PLC-{gamma}1 SH domain and its subsequent tyrosine phosphorylation of PLC-{gamma}1 in B cells (30).

These data are the first to show site-specific phosphorylation of Syk in response to shear stress. However, site-specific phosphorylation has been described by Suzuki-Inoue et al. (64) in response to stimulation by ristocetin-vWF. The pattern of tyrosine phosphorylation reported in that study differs from that presented in this paper. Ristocetin-mediated stimulation of GPIb-V-IX led to phosphorylation at SykY352 but not at SykY525/526, despite the fact that both sites were phosphorylated in response to convulxin. Therefore, the revelation here that SykY525/526 is phosphorylated by shear stress is of critical significance. The finding allows for the proposal of a model in which Syk actively participates in, via kinase activity, signaling through the GPIb-V-XI complex. These data also dismiss assumptions that shear-related Syk phosphorylation could only be indicative of a scaffolding or adapter role for the protein.

The discrepancy in Syk phosphorylation patterns reported by Suzuki-Inoue et al. (64) and our findings could simply be due to a limitation in the detection system used in the earlier study, possibly with respect to the weak nature of the signal induced by ristocetin or to insufficient affinities of the phospho-Syk antibodies used. More interestingly, the difference could be explained by a report that ristocetin can nonspecifically interact with Syk and p60c-src, abolishing the kinase activities of both molecules, as demonstrated by in vitro kinase assays on immunoprecipitates of the proteins from nonactivated and ristocetin/vWF-activated platelets (3). Moreover, although ristocetin/vWF, botrocetin/vWF, and shear stress/vWF all allow for stimulation of GPIb, each agent may accomplish that feat by a different mechanism. Indeed, Liu et al. (32, 33) contended that although botrocetin/vWF- and shear stress/induced-induced activation are both Syk dependent, they activate GPIIb-IIIa through slightly different mechanisms. Within these contexts, it is not remarkable that differences exist between the site-specific Syk phosphorylation responses induced by the physical agonist (shear stress) and by the biochemical agonists, especially ristocetin.

Mechanisms of shear-induced Syk function were explored by looking at possible upstream affectors. Unfortunately, an adequate means for directly inhibiting GPIb/vWF-dependent signaling was not readily available during this study. The anti-GPIb antibodies AK2, 6D1, and Ib-23, the GPIb/vWF antagonist peptide VCL, and even the anti-vWF antibody 5D2 all initiated phosphorylation of SykY525/526 in the absence of shear stress or augmented shear-induced phosphorylation of SykY525/526 (data not shown). These observations could be due to direct activation via GPIb following binding of the respective antibody or peptide. Conversely, Syk modulation by these agents could stem from cross-linking of GPIb with the platelet Fc-receptor (Fc{gamma}RII), as has been the case with other anti-GPIb and anti-vWF antibodies (12, 24). Limited availability of the inhibitory antibodies rendered the approach of producing and using Fab fragments of the antibodies impractical. Alternatively, the anti-Fc{gamma}RII antibody IV.3 might have been used in conjunction with direct GPIb/vWF blocking agents to negate Fc{gamma}RII signaling. However, suggested interplay between Fc{gamma}RII and GPIb during shear-induced platelet signaling dictated that such a strategy would not have provided a clearer picture of GPIb/vWF involvement (57, 62, 67). In fact, IV.3 by itself has been shown to inhibit both SIPA and ristocetin/vWF-induced Syk phosphorylation (57, 67).

Syk has long been identified as a mediator of outside-in signaling through GPIIb-IIIa (16, 31, 35, 54). Feng et al. reported that Syk associates with the β3-subunit of GPIIb-IIIa in resting platelets and that shear stress induces dissociation of that complex. Such dissociation was prevented by blocking vWF binding to GPIb and by blocking the vWF/fibrinogen binding site of GPIIb-IIIa (14). This observation may explain why shear-induced phosphorylation of SykY525/526 is unaffected by preincubation with the small KGD-containing peptide eptifibatide. GPIIb-IIIa-mediated phosphorylation of Syk is dependent on the interaction of the kinase with the β3 (GPIIIa) cytoplasmic tail (16, 74).

ADP plays and important role in shear-dependent platelet aggregation and thrombus formation (40, 68). The P2Y12 receptor has been directly implicated in Syk-mediated signaling: P2Y12, but not P2Y1, receptor blockade inhibited shear-induced phosphorylation, as well as ADP-induced Syk phosphorylation under unstirred, stirring conditions and in the presence of EGTA (51). Apyrase, an ADP scavenger, was used to prevent signaling induced by secreted ADP through the P2Y1 and P2Y12 receptors. Apyrase inhibited shear-induced phosphorylation of SykY525/526, indicating that ADP-mediated signaling may be important for activation of Syk kinase activity.

The significance of TxA2 signaling to shear-induced platelet signaling is not clear. Aspirin inhibited SIPA in a washed platelet system but not in PRP (63). This phenomenon may be due to sequestration of free arachidonic acid and TxA2 by plasma proteins, principally albumin. Elevated shear stress can also overcome the anti-platelet effects of aspirin in stenosed dog coronary arteries (34). However, Cannobio et al. (10) demonstrated that in ristocetin/vWF-stimulated washed platelets, pleckstrin phosphorylation and serotonin release are aspirin sensitive (10). Conversely, the phosphorylation of Syk, Fc{gamma}RIIa, and PLC{gamma}2 were unaffected by aspirin in the same system. Our data reinforce the conclusion that shear-induced Syk phosphorylation is not aspirin sensitive. Therefore, the initial signals for activation triggered by ligation of GPIb by vWF appear to be independent or upstream of TxA2 production. Indeed, Liu et al. (32, 33), using a system activated by botrocetin-mediated agglutination, suggested that TxA2 production appears to be initiated by Lyn, enhanced by Src, and propagated through Syk, SLP-76, PI 3-kinase, BTK, ERK1/2, PLC{gamma}2, and PKC.

The capillary perfusion experiments demonstrated that both OXSI-2 and piceatannol significantly inhibited thrombus formation on type III collagen at an elevated shear stress of 40 dyn/cm2 in a dose-dependent manner. These results, performed with whole blood, illustrate that Syk plays a prominent, if not critical, role in the formation of stable thrombi under conditions such as might be found at an atherosclerotic lesion. Complementary results have been obtained with piceatannol in a similar, briefly defined system (26). However, we have included additional data indicating that shear stress of the same magnitude (40 dyn/cm2) leads to phosphorylation of the Syk activation site and that this response can be attenuated with Syk inhibitors. These data indicate that shear-induced signaling through Syk may be reinforcing, or perhaps amplifying, the potent GPVI/Syk-dependent activation response to collagen and GPIIb-IIIa/Syk-dependent aggregate stability. To our knowledge, these data are also the first to establish a pharmacological significance for OXSI-2 in platelets, correlating with the inhibition of in vitro Syk kinase activity and B-cell degranulation reported by others (29).

Although, the mechanism(s) of shear-induced platelet signaling are still poorly defined, it is now clear that Syk is involved. Phosphorylation of Syk525/526 is a persuasive indication that Syk activation is a factor in shear-induced platelet signaling. The significance of shear-induced phosphorylation of SykY352 is unknown but may point to direct interactions between Syk and PLC-{gamma}, PI 3-kinase, or Vav1 during shear-induced platelet events. Demonstration of OXSI-2 inhibition of Syk tyrosine phosphorylation in platelets, along with its ability to inhibit platelet function in the form of thrombus formation, warrant further studies to establish this compound as a valuable complement or alternative to piceatannol for platelet function studies involving Syk. Pending additional research, inhibitors of Syk might be efficacious in the treatment of vascular disease due to the importance of Syk in platelet activation and aggregation induced by two key elements of pathological intravascular thrombosis: collagen and shear stress. The use of Syk as a potential therapeutic agent is encouraged by positive outcomes in studies investigating the value of Syk inhibitors for the treatment of allergic and inflammatory conditions (36, 69, 70, 73). In fact, oral administration of Syk inhibitor R406 was recently demonstrated to decrease IgE-mediated basophil activation in humans without remarkable adverse effects (8). Further studies are needed to elucidate the relevance of the shear-induced Syk pathway as a target for the reduction of atherothrombotic events.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was funded by the VBCE and an American Heart Association Southeast Affiliate grant-in-aid to L. K. Jennings.


   ACKNOWLEDGMENTS
 
The authors thank Charlett Golden for work preparing figures presented in this manuscript. Also, we thank Andrew Nishimoto, Shila Cholera, and Melanie White for their help with platelet aggregation experiments.


   FOOTNOTES
 

Address for reprint requests and other correspondence: L. K. Jennings, Vascular Biology Center of Excellence, Univ. of Tennessee Health Science Center, 956 Court Ave., Coleman Bldg., H300, Memphis, TN 38163 (e-mail: ljennings{at}utmem.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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