Participation of Tyrosine Phosphorylation in Cytoskeletal Reorganization, αIIbβ3 Integrin Receptor Activation, and Aspirin-Insensitive Mechanisms of Thrombin-Stimulated Human Platelets
Background—Fibrinogen binding to the active conformation of the αIIbβ3 integrin receptor (glycoprotein IIb/IIIa) and cytoskeletal reorganization are important events in platelet function. Tyrosine phosphorylation of platelet proteins plays an essential role in platelet signal transduction pathways. We studied the participation of tyrosine kinases on these aspects of platelet reactivity and their importance in cyclooxygenase (COX)-1–independent mechanisms in thrombin-stimulated human platelets.
Methods and Results—Using washed platelets from normal donors and tyrphostin-A47 and aspirin as tyrosine kinase and COX-1 inhibitors, respectively, we found that tyrphostin-A47 downregulated (1) the thrombin-activated conformational change of αIIbβ3, (2) actin polymerization and cytoskeletal reorganization, and (3) the quantity of tyrosine-phospho-rylated proteins associated with the reorganized cytoskeleton. The latter are important components of multimolecular signaling complexes. Concomitantly, platelet aggregation and secretion were significantly reduced. Aspirin did not affect receptor activation or tyrosine phosphorylation but did decrease the initial (30-second) burst of actin polymerization. Importantly, aspirin significantly amplified the inhibitory effect of tyrphostin-A47 on all aspects of platelet reactivity that we evaluated.
Conclusions—Tyrosine protein phosphorylation is a regulatory control system of the inside-out mechanism of αIIbβ3 activation and cytoskeletal assembly in thrombin-stimulated human platelets. Inhibition of these aspects of platelet function with tyrphostin-A47 is amplified when platelets are treated with aspirin. Therefore, tyrosine phosphorylation is a major component of early signaling events and of COX-1–independent mechanisms of thrombin-induced platelet reactivity. The study results may indicate a novel target for therapeutic intervention.
The regulation of agonist-induced platelet reactivity requires coordination and control of multiple biochemical signaling pathways that culminate in platelet functional responses. These pathways include activation of GTP-binding proteins, inositol phospholipid turnover, eicosanoid metabolism, calcium movement, and protein phosphorylation processes.1 2 With regard to phosphorylation, various serine/threonine and tyrosine kinases and phosphatases are involved in biochemical events that lead to platelet activation.3 4 5
The precise role of protein tyrosine phosphorylation in signal transduction in platelets is not fully understood. However, it has functional implications. The stimulation of platelets with physiologically relevant agonists, such as collagen or thrombin, induces the phosphorylation of tyrosine residues of several platelet proteins.3 6 Conversely, the treatment of platelets with inhibitors of tyrosine kinases results in inhibition of platelet aggregation and secretion.3 4 7 8 9
Agonist-induced platelet aggregation requires fibrinogen binding to the αIIbβ3 integrin receptor and cell–cell contact to achieve ligand-mediated bridging of αIIbβ3 complexes between platelets.5 In resting platelets, the αIIbβ3 receptor is present in an inactive, nonadhesive conformation. On platelet stimulation, a conformational change occurs, enabling binding of adhesive proteins to the integrin. This results from an intracellular signal transduction process known as “inside-out” receptor signaling.5 Fibrinogen binding to the active conformation of the receptor initiates another biochemical pathway known as “outside-in” signaling that reinforces the platelet aggregation process.5 The latter has been consistently associated with tyrosine phosphorylation of platelet proteins and is altered in Glanzman’s thrombasthenic platelets.4 5 Less information is available concerning the role of tyrosine kinases in the regulation of “inside-out” mechanism or mechanisms of αIIbβ3 receptor signaling.5
Another early event after platelet activation is cytoskeletal reorganization.10 11 12 This results in a rapid increase in actin polymerization and the association of αIIbβ3 with actin-binding proteins (ABPs) and signaling molecules into the platelet actin-based cytoskeleton.11 The anchorage of αIIbβ3 to cytoskeletal structures regulates adhesive properties of the receptor and stabilizes fibrinogen binding via mechanisms that are not completely elucidated.5 10 13 Tyrosine-phospho-rylated proteins may play an important role in the formation of cytoskeletal multimolecular protein complexes.14 15
Eicosanoid synthesis via cyclooxygenase-1 (COX-1) is an important pathway by which platelets respond to agonists. The inhibition of COX-1 by aspirin (ASA) is clinically beneficial for patients with vascular diseases.16 Nevertheless, platelets readily respond to stimuli by mechanisms independent of COX-1.2 17 Platelet reactivity is also modulated by cell–cell interactions that involve other blood cells, via both COX-1–dependent and –independent mechanisms.18 19 20 21 22 Mechanisms that regulate COX-1–independent pathways are of pathophysiological importance but are incompletely understood.
We evaluated the participation of tyrosine kinases in inside-out αIIbβ3 receptor activation and cytoskeletal reorganization in thrombin-stimulated platelets. Furthermore, we determined the role of tyrosine phosphorylation in COX-1–independent mechanisms of platelet activation by thrombin. We used ASA for COX-1 inhibition and tyrphostin-A47 (AG213; RG-50864) as an inhibitor of tyrosine kinases.7 8 Tyrphostin-A47 inhibits Src, Fyn, Yes, and Lyn tyrosine kinases and platelet aggregation and secretion.7 8
Our data demonstrate that protein tyrosine phosphorylation plays an important regulatory role in the early events of stimulus–response coupling in thrombin-stimulated platelets. This includes both inside-out mechanisms of αIIbβ3 receptor activation and cytoskeletal reorganization. Moreover, protein tyrosine phosphorylation is a major component of ASA-insensitive mechanisms of platelet reactivity, suggesting the possibility of a novel pharmacological approach for the treatment of thrombotic diatheses.
Equipment and Reagents
Platelet aggregometer model 540 was obtained from Chronolog. Cell counter Microdiff 18, Epics XL flowcytometer, and Phoenix software were obtained from Coulter. The blotting system was obtained from Hoefer. Collagen was obtained from Hormon. Aprotinin was obtained from Bayer. Leupeptin, pepstatin, and phenylmethylsulfonyl fluoride (PMSF) were obtained from Boehringer-Mannheim. Tyrphostin-A47 (AG 213, RG-50864) and tyrphostin-1 (tyrphostin-A1, AG9) were obtained from Sigma-Aldrich. Metrizamide was obtained from Nycomed. FITC-PAC-1 monoclonal antibody was obtained from Becton Dickinson. PAGE reagents and nitrocellulose membranes were obtained from Bio-Rad. [14C]5-Hydroxytryptamine (5-HT [serotonin]) creatinine sulfate, anti-mouse IgG peroxidase-linked antibody, and Hyperfilm-ECL Western blotting detection reagents were obtained from Amersham. Anti-phosphotyrosine mouse monoclonal 4G10 was obtained from Upstate Biotechnology. Protein standards were obtained from Novex. All other reagents were obtained from Sigma-Aldrich or Merck.
Blood Collection, Platelet Processing, and Labeling
Venous blood (160 mL) was obtained via free flow (after an overnight fast) from normal volunteers who had been medication free for ≥15 days.18 The blood was collected into 50-mL polypropylene tubes containing 6 mL acid-citrate-dextrose as anticoagulant (total 46 mL)18 23 after informed consent was obtained, as approved by the institutional review board. Platelets were isolated, washed, and adjusted to 109 cells/mL.18 23 PRP from acid-citrate-dextrose–anticoagulated blood was also used to prepare 14C-5-HT–labeled platelets.21 23 For flow cytometry, platelets were obtained before and 2 hours after the ingestion of 500 mg ASA, isolated in a metrizamide gradient,24 and suspended in calcium-free HEPES buffer A (5 mmol/L HEPES, 140 mmol/L NaCl, 5 mmol/L KCl, 1.2 mmol/L MgCl2, pH 7.4).
Platelet aggregation was assessed with optical aggregometry in 500 μL washed platelet suspension (2×108 platelets/mL) in HEPES buffer A containing 1 mmol/L CaCl2 and 0.38 mg/mL fibrinogen at 37° with constant stirring (1200 rpm). Thrombin-induced platelet aggregation was monitored for up to 5 minutes. Tyrphostin-A47 and tyrphostin-1 (analog with similar structure but lacking tyrosine kinase inhibitory activity) were prepared as DMSO stock solutions (−70°C); working dilutions of inhibitors and ASA were prepared daily in buffer. Inhibitors were added to platelets, mixed, and maintained at 37°C for 10 minutes without stirring before platelet stimulation. Solvent controls (DMSO <0.1%) were run in parallel.
Imipramine (2.5 μmol/L) was added 1 minute before stimulation to 14C-5-HT–labeled washed platelets (2×10 platelets/mL). Aggregation was monitored for 3 minutes, and 125 μL 5× ice-cold stop solution (0.63 mol/L formaldehyde, 0.05 mol/L EDTA) was added.25 The sample was immediately transferred to an Eppendorf tube (0°C) and centrifuged (13 000g, 1 minute) within 1 hour. Supernatant [14C]5-HT was quantified through scintillation counting.21 23
Protein Tyrosine Phosphorylation
Protein tyrosine phosphorylation was examined as described.8 Thrombin-induced platelet aggregation was terminated by the addition of 125 μL 5× stop solution (2% SDS, 1 mmol/L EDTA, 1 mmol/L EGTA, 0.5 mmol/L Na3VO4, 10 mmol/L NaF, 10 mmol/L HEPES, 2 mg/mL aprotinin, 0.5 mg/mL leupeptin, 0.7 mg/mL pepstatin, 170 mg/mL PMSF, pH 7.4) and stored at −20°C. Samples were diluted 1:1 with 2× Laemmli’s buffer, reduced with 5% β-mercaptoethanol, and heated (30 minutes, 60°C). Proteins were separated with SDS-PAGE (7.5% gels).
Separated proteins were transferred onto nitrocellulose.26 Membranes were stained with Ponceau S to determine protein transfer efficiency. Nitrocellulose was soaked (1 hour) in blocking buffer TBS (20 mmol/L Tris base, 137 mmol/L NaCl, pH 7.4) that contained 5% low-fat powdered milk and 0.05% NaN3. Membranes were washed twice (5 minutes) in TBS and incubated (2 hours, 20°C) with anti-phosphotyrosine antibody 4G10 (diluted 1:1000 in blocking buffer).8 Membranes were washed 3 times (5 minutes) with TBS and incubated with peroxidase-conjugated secondary antibody (diluted 1:3000 in blocking buffer without NaN3). After 5 washes (5 minutes) with TBS, blots were visualized with ECL chemiluminescence. Molecular weights of phosphorylated substrates were compared with known standards.
Cytoskeleton isolation was performed essentially as described.11 Thrombin-induced aggregation (0.5 mL; 2×108 platelets/mL) was terminated with 125 μL 5× ice-cold lysis buffer (1% Triton X-100, 5 mmol/L EGTA, 1 mg/mL leupeptin, 1 mmol/L PMSF, 1 mmol/L Na3VO4, 50 mmol/L Tris-HCl, pH 7.4). Detergent-insoluble fractions were pelleted (15 600g, 4 minutes), rinsed twice with 1× lysis buffer, resuspended in 200 μL Laemmli’s buffer, and boiled for 3 minutes. Proteins were separated with SDS-PAGE and detected with Coomassie blue. The presence of phosphorylated tyrosine residues in cytoskeletal proteins was determined with immunoblotting as described above. In these experiments, 0.4 U/mL thrombin was used to enhance the degree of cytoskeletal reorganization.
Aspirin-free or ASA-treated platelets (2×108 platelets/mL) in HEPES buffer (10 mmol/L HEPES, 150 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L MgSO4, 10 mmol/L glucose, pH 7.4) plus 1 mmol/L CaCl2 were incubated without stirring (10 minutes, 37°C) with tyrphostin-A47, tyrphostin-1 (negative control), or solvent. Thrombin was added, and incubation was continued for 5 minutes without stirring. Duplicate 5-μL aliquots of thrombin-stimulated platelets were transferred to polypropylene tubes that contained 50 μL HEPES buffer without calcium.27 To each sample, 4 μL FITC-PAC-1 monoclonal antibody (25 μg/mL) was added, kept undisturbed (30 minutes, 20°C, dark), quench-diluted with 500 μL ice-cold HEPES, and maintained at 0°C in the dark. To quantify the percentage of platelets binding FITC-PAC-1, 5000 platelets per sample were analyzed. Because 0.1 U thrombin/mL yielded PAC-1 binding in only 4.25% of platelets, 1 and 0.4 U/mL concentrations were used.
Effects of Tyrphostin-A47 on Platelet Function and Tyrosine Phosphorylation of Proteins
Platelet aggregation and release were monitored 3 minutes after the addition of thrombin (0.1 U/mL) to ASA-free or ASA-treated platelets (same donor). Aspirin alone yielded the expected inhibition of platelet aggregation and release compared with ASA-free platelets (Table 1⇓). Tyrphostin-A47 significantly inhibited platelet aggregation and 5-HT release in ASA-free platelets (Table 1⇓), as reported for tyrosine kinase inhibitors.3 4 8 9 When ASA and tyrphostin-A47 were combined, a further reduction in platelet aggregation and secretion occurred, exceeding the effect of either ASA or tyrphostin-A47 alone (Table 1⇓). The inactive analog (tyrphostin-1) had no significant inhibitory effect (Table 1⇓). Functional effects of tyrphostin-A47 occurred concomitant with a decrease in tyrosine-phosphorylated substrates in thrombin-stimulated platelets (Figure 1⇓). The effect was greater when ASA and tyrphostin-A47 were combined (Figure 1⇓). In contrast, the tyrphostin-1 analog had no effect on protein tyrosine phosphorylation of ASA-free or ASA-treated platelets (Figure 1⇓).
Time course studies of platelet aggregation in response to thrombin demonstrated a time-dependent increase in amplitude (Table 2⇓, control). This was inhibited at all time points with either ASA or tyrphostin-A47 alone. Interestingly, inhibition with ASA at 30 seconds (53%) was much greater than that at 3 minutes (21%). Platelet aggregation was inhibited even more strongly when tyrphostin-A47 was added to ASA-treated platelets, particularly at an early time point (30 seconds, 97%) (Table 2⇓).
The results of kinetic studies of the effect of tyrphostin-A47 on protein tyrosine phosphorylation in whole cell extracts of ASA-free and ASA-treated platelets are shown in Figure 2⇓. Tyrphostin-A47 (90 μmol/L) markedly inhibited tyrosine phosphorylation of the 75/80-, 64-, and 50- to 60-kDa substrates and virtually blocked phosphorylation of the 95/97-kDa proteins (Figure 2⇓).
Aspirin treatment alone had no effect on tyrosine phosphorylation at any time point (Figure 2⇑). Importantly, tyrphostin-A47 had a much greater inhibitory effect on tyrosine phosphorylation when added to ASA-treated platelets (Figure 2⇑). Tyrphostin-A47 consistently induced phosphorylation of a 90-kDa protein. This was strongly reduced with ASA in tyrphostin-A47–treated platelets (Figure 2⇑).
To confirm the greater effect of tyrosine kinase inhibition in ASA-treated platelets, we also used a different tyrosine kinase inhibitor, genistein. The treatment of platelets with 100 μmol/L genistein had a greater inhibitory effect on tyrosine phosphorylation and platelet function when platelets were treated with ASA (not shown). Thus, tyrosine phosphorylation of platelet proteins is an important regulatory mechanism in COX-1–independent platelet reactivity to thrombin.
Effect of Tyrphostin-A47 on Inside-Out Activation of the αIIbβ3 Integrin Receptor: Modulation by Aspirin
The binding of FITC-PAC-1, a monoclonal antibody specific for neoepitopes exposed on the activated form of the αIIbβ3, was determined in ASA-free and ASA-treated platelets through flow cytometry.27 28 Tyrphostin-A47 (90 μmol/L) significantly reduced thrombin (1 U/mL)-induced PAC-1 binding, whereas, as reported,29 ASA alone had no effect (Figure 3⇓). The reduction in PAC-1 binding was significantly enhanced when ASA-treated platelets were exposed to tyrphostin-A47. This trend was also observed with 0.4 U/mL thrombin (Figure 3⇓). In contrast, tyrphostin-1 did not modify PAC-1 binding.
Thus, tyrphostin-sensitive tyrosine kinases modulate inside-out αIIbβ3 receptor activation. Moreover, ASA-sensitive events converge with tyrosine kinase–modulated effects to downregulate the initial integrin activation.
Effect of Tyrphostin-A47 on Organization of the Platelet Cytoskeleton: Role of Aspirin
We stimulated ASA-free or ASA-treated platelets with thrombin (0.4 U/mL) in the presence or absence of tyrphostin-A47. Aspirin treatment decreased the quantities of ABP, talin, and polymerized actin at 30 seconds compared with control platelets (Figure 4⇓). However, the ASA inhibition was overcome if platelet aggregation was allowed to proceed. Tyrphostin-A47 (90 μmol/L) significantly reduced the quantity of polymerized actin and associated proteins in the cytoskeleton of thrombin-stimulated platelets, particularly at early time points (Figure 4⇓). When platelets were simultaneously treated with ASA and tyrphostin-A47, a greater reduction was observed in ABP and talin in the cytoskeleton (Figure 4⇓). Thus, tyrosine kinase activities modulate platelet cytoskeletal reorganization, whereas ASA downregulates the initial burst of actin polymerization and augments the inhibitory effects of tyrphostin-A47.
Effect of Aspirin and Tyrphostin-A47 on Tyrosine Phosphorylated Proteins on the Cytoskeleton
Shortly after thrombin stimulation, phosphorylation of 130-, 75/80-, 64-, and 50/60-kDa proteins was detected in the cytoskeleton. The 95/97-kDa substrates appeared later (3 minutes) (Figure 5⇓). The pattern of tyrosine-phosphorylated substrates in the cytoskeleton was similar, although not identical, to that observed in whole cell extracts (Figure 2⇑ versus Figure 5⇓). Tyrosine-phosphorylated proteins in the cytoskeleton were minimally detectable in unstimulated platelets (not shown).
Aspirin treatment of platelets significantly reduced the quantity of tyrosine-phosphorylated proteins in the cytoskeleton at 30 seconds (Figure 5⇑). This was in sharp contrast with the lack of effect of ASA on tyrosine phosphorylation in whole cell extracts (Figure 2⇑). No differences were observed between control and ASA-treated platelets at later time points (Figure 5⇑).
Tyrphostin-treatment (90 μmol/L) of ASA-free platelets markedly decreased tyrosine-phosphorylated proteins in the cytoskeleton at 30 seconds and 3 minutes. However, at 5 minutes, the differences observed between tyrphostin-treated and control platelets were minimal (Figure 5⇑). Importantly, tyrphostin-A47 virtually abolished the presence of tyrosine-phosphorylated proteins in the cytoskeleton at all time points in ASA-treated platelets (Figure 5⇑).
Because tyrosine-phosphorylated motifs are important for the assembly of multimolecular signaling complexes in the cytoskeleton,14 15 this suggests that ASA plus tyrphostin-A47 is a potent inhibitor of this process.
Fibrinogen binding to the activated αIIbβ3 integrin receptor and cytoskeletal reorganization are important events in platelet function. The reorganized cytoskeleton serves as a scaffold for association of cytoskeletal proteins, integrins, and signaling molecules.14 The resulting multimolecular complexes participate in signal transduction and platelet functional responses.3 10 Tyrosine phosphorylation of platelet proteins is an essential component of platelet signal transduction pathways.4 5 We studied the participation of tyrosine kinases on these aspects of platelet reactivity and investigated their importance in COX-1–independent mechanisms in thrombin-stimulated human platelets. COX-1–independent mechanisms are of importance because clinical trials indicate that only 25% to 30% of patients with vascular disease benefit from ASA therapy.16 We hypothesize that the lack of benefit in the remaining 70% of patients is due, at least in part, to platelet activation by mechanisms independent of COX-1. This also includes stimulatory effects elicited by cell–cell interactions with other blood cells as we and others have demonstrated.18 19 20 21 22
Our results extend previous data on the inhibition of platelet aggregation and secretion with tyrosine kinase inhibitors.3 4 8 9 Importantly, this inhibitory effect is significantly increased in ASA-treated platelets (Tables 1⇑ and 2⇑). Thus, tyrosine phosphorylation constitutes a major COX-1–independent mechanism whereby platelets respond to thrombin. The effects of tyrphostin-A47 are due to tyrosine kinase inhibition specifically, because tyrphostin-1 (negative control) is inactive (Table 1⇑, Figure 1⇑). Moreover, the amplified inhibitory effect with ASA was also observed with genistein (another kinase inhibitor).
Regarding the biochemical mechanisms that underlie these functional effects, our results show that tyrosine kinases modulate (directly and/or indirectly) inside-out signaling of αIIbβ3 in thrombin-stimulated platelets. This follows from our observation that tyrphostin-A47 significantly reduces thrombin-induced PAC-1 binding (Figure 3⇑). This is in agreement with data with genistein-treated permeabilized platelets,28 platelets stimulated with the thrombin receptor agonist peptide SFLLRN,29 piceatannol-treated ADP-stimulated platelets,30 and platelets treated with herbimycin A.9 The specific kinases involved have not been identified. Law et al30 reported the participation of Syk tyrosine kinase, because receptor activation is inhibited (32%) in Syk-null murine platelets. However, this partial inhibition suggests that tyrosine kinases other than Syk are also involved in this step of receptor activation.
Although ASA alone did not modify receptor activation, as reported,29 the inhibition of tyrosine kinase in ASA-treated platelets resulted in further reduction in receptor activation as shown by reduced PAC-1 binding (Figure 3⇑). This demonstrates ASA amplification of tyrosine kinase inhibition of this important aspect of platelet reactivity and suggests that αIIbβ3 downregulation is at least 1 mechanism whereby tyrosine kinases regulate COX-1–dependent and –independent mechanisms of platelet reactivity.
Our results show that tyrosine kinase inhibition with tyrphostin-A47 reduced actin polymerization and decreased the quantity of actin-associated proteins in cytoskeletons of thrombin-stimulated platelets, particularly at early time points (Figure 4⇑). This demonstrates that tyrosine kinases regulate cytoskeletal reorganization. Unexpectedly, we consistently observed an ASA-sensitive, but reversible, decrease in cytoskeletal reorganization shortly after thrombin addition (30 seconds) (Figure 4⇑). Thus, an ASA-sensitive mechanism regulates the early burst of actin polymerization. When ASA- and tyrphostin-sensitive mechanisms were inhibited simultaneously, greater inhibition of cytoskeletal reorganization resulted. This represents a novel, COX-1–independent mechanism of tyrosine kinase regulation of platelet function.
In our experiments, tyrphostin-A47 effects on αIIbβ3 activation and cytoskeletal reorganization paralleled a reduction in tyrosine-phosphorylated proteins in the actin-based cytoskeleton (Figure 5⇑). Interestingly, ASA also reduced tyrosine phosphorylation of proteins in the cytoskeleton shortly after thrombin addition (30 seconds) (Figure 5⇑). This coincides temporally with decreased actin polymerization (Figure 4⇑) and the greatest degree of inhibition of platelet aggregation (Table 2⇑). It is unrelated to an effect of ASA on tyrosine kinase activity, because ASA had no effect on tyrosine-phosphorylated proteins in whole cell extracts (Figure 2⇑). Importantly, tyrphostin-A47 induced a dramatic reduction of tyrosine-phosphorylated proteins in the cytoskeleton of ASA-treated platelets (Figure 5⇑). This is of interest if we consider that protein phosphotyrosine domains and SH2 and SH3 homology regions are essential components of protein–protein assembly for multimolecular complex formation in the cytoskeleton.14 These protein complexes participate in focal adhesion, signal transduction, and platelet functional responses.3 10 Therefore, the inhibition demonstrated in our study of tyrosine-phosphorylated proteins in the cytoskeleton might represent a reduction in signaling complex formation in the cytoskeleton of thrombin-stimulated human platelets. This would contribute to the significant inhibition of platelet aggregation (Tables 1⇑ and 2⇑), αIIbβ3 receptor activation (Figure 3⇑), and cytoskeletal reorganization (Figure 4⇑) that we observed.
In conclusion, our results support the concept that tyrosine kinases participate in inside-out mechanisms of αIIbβ3 receptor activation and extend it to the amplifying effect of ASA on downregulation that results from tyrosine kinase inhibition. In addition, we present novel evidence of regulation by tyrosine kinases of both cytoskeletal organization of structural proteins and the accumulation of tyrosine-phosphorylated proteins in actin-rich cytoskeleton of thrombin-stimulated platelets. These are required for multimolecular signaling complex formation in the cytoskeleton. Furthermore, our ASA experiments demonstrate that tyrosine phosphorylation is a major component of COX-1–independent mechanisms of platelet activation with thrombin. This may constitute a novel target for future antithrombotic drug development.
This work was supported in part by grants 95/0639 and 98/0906 from the Fondo de Investigaciones Sanitarias (FIS) (Drs Santos, Vallés, and Aznar); a Merit Review grant from the Department of Veterans Affairs (Dr Marcus); and NIH grants HL-47073, HL-46403, and HL-07423 (Drs Marcus and Broekman). The authors gratefully acknowledge the technical assistance of M. Carmen Insa, Amparo Garrido, and Pilar Ferriz.
- Received February 25, 2000.
- Revision received May 23, 2000.
- Accepted May 31, 2000.
- Copyright © 2000 by American Heart Association
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