PLATELETS AND THROMBOPOIESIS

The new tyrosine-kinase inhibitor and anticancer drug dasatinib reversibly affectsplatelet activation in vitro and in vivo*Marie-Pierre Gratacap,1 *Valerie Martin,1 Marie-Cecile Valera,1 Sophie Allart,2 Cedric Garcia,3 Pierre Sie,3

Christian Recher,1,4 and Bernard Payrastre1,3

1Inserm U563, Centre de Physiopathologie de Toulouse Purpan, Departement d’Oncogenese, Signalisation et Innovation therapeutique and Universite ToulouseIII Paul Sabatier, Toulouse; 2Plateforme d’imagerie de l’IFR 30, Toulouse; and 3Laboratoire d’Hematologie and 4Service d’hematologie, Centre HospitalierUniversitaire de Toulouse, Toulouse, France

Dasatinib is an oral potent adenosinetriphosphate (ATP)–competitive inhibitorof BCR-ABL, cKIT, platelet-derived growthfactor receptor, and SRC family kinases(SFKs), which has demonstrated high effi-ciency in patients with imatinib-resistantchronic myelogenous leukemia. Here, weshow that dasatinib weakly affects plate-let activation by thrombin or adenosinediphosphate but is a potent inhibitor ofplatelet signaling and functions initiatedby collagen or Fc�RIIA cross-linking,which require immunoreceptor tyrosine-

based activation motif phosphorylationby SFKs. Accordingly, dasatinib treat-ment rapidly decreases the volume ofthrombi formed under arterial flow condi-tions in whole blood from patients ormice perfused over a matrix of collagen.Moreover, treatment of mice with dasa-tinib increases the tail bleeding time in adose-dependent manner. Interestingly,these effects are rapidly reversible afterinterruption of the treatment. Our dataclearly demonstrate that, in contrast toimatinib, dasatinib affects platelet func-

tions in vitro and in vivo, which hasimportant implications in clinic and couldexplain increased risks of bleeding ob-served in patients. Moreover, dasatinibefficiently prevents platelet activation me-diated by Fc�RIIA cross-linking and bysera from patients with heparin-inducedthrombocytopenia, suggesting that re-versible antiplatelet agents acting asATP-competitive inhibitors of SFKs maybe of therapeutic interest in the treatmentof this pathology. (Blood. 2009;114:1884-1892)

Introduction

Dasatinib is an oral potent adenosine triphosphate (ATP)–competitive inhibitor of tyrosine kinases, which has demon-strated high efficiency in patients with imatinib-resistant chronicmyelogenous leukemia (CML).1-4 BCR-ABL is an oncogenictyrosine kinase with deregulated activity linked to malignanttransformation in CML, which has been first targeted withsuccess in clinic by imatinib.5,6 Dasatinib is a second-generationtyrosine kinase inhibitor, more potent than imatinib on BCR-ABL and efficient on most of the imatinib-resistant ABLmutants.4 It is also effective on cKIT, platelet-derived growthfactor (PDGF)–receptor and, in contrast to imatinib, on SRCfamily kinases (SFKs). Dasatinib is presently approved for allphases of Philadelphia-positive CML with resistance or intoler-ance to prior therapy, including imatinib. This compound isefficient and safe, although side effects, such as myelosuppres-sion, gastrointestinal symptoms, rash, fluid retention syn-dromes, and bleeding episodes, are commonly observed.7 Tocope with these side effects, most patients require reduction ofthe dose administrated or interruption of the treatment. Althoughthe increased risk of bleeding under dasatinib therapy is clearlyestablished with reported fatal brain hemorrhages, subduralhematomas, and gastrointestinal bleeding, it is difficult toascertain to which degree the drug or the disease is responsiblefor the high incidence of bleeding. Moreover, the effect ofdasatinib on platelet functions remains poorly characterized.

Blood platelets are the first line of defense against hemorrhages.On disruption of the vascular integrity, they adhere to exposedsubendothelial matrix proteins and aggregate to stop bleeding.Platelet activation via soluble or immobilized agonists is a highlyregulated process that involves several signaling proteins, includ-ing nonreceptor tyrosine kinases, such as Syk, BTK, FAK, and theSFKs. Six members of the SFK have been shown to be expressed inplatelets, including c-Src, Lyn, Fgr, Fyn, Lck, and Yes.8 Besides itseffect on platelet production and/or turnover leading, in somecases, to thrombocytopenia,7 dasatinib may also affect plateletfunctions through inhibition of SFKs. Consistent with this hypoth-esis, gastrointestinal hemorrhages have been reported in patientswith normal platelet counts.

Under arterial shear rate, interaction of platelets with thedisrupted vessel wall is largely mediated by binding of subendothe-lial von Willebrand factor (VWF) to its membrane receptor onplatelets, the glycoprotein Ib-IX (GPIb-IX). Recent data indicatethat the SFK Lyn mediates VWF/GPIb-IX-V–induced plateletactivation.9 Moreover, maximal platelet responses to vascularinjury require the contribution of integrins such as �2�1 or �IIb�3and of platelet membrane receptors bearing immunoreceptortyrosine-based activation motif (ITAM), including glycoproteinVI-Fc receptor �-chain (FcR�) and Fc�RIIA.10 These receptors andintegrins activate cell signaling via nonreceptor tyrosine kinases. Itis now clearly established that SFKs play a critical role in platelet

Submitted February 12, 2009; accepted May 24, 2009. Prepublished online asBlood First Edition paper, June 3, 2009; DOI 10.1182/blood-2009-02-205328.

*M.-P.G. and V.M. contributed equally to this work.

An Inside Blood analysis of this article appears at the front of this issue.

The online version of this article contains a data supplement.

The publication costs of this article were defrayed in part by page chargepayment. Therefore, and solely to indicate this fact, this article is herebymarked ‘‘advertisement’’ in accordance with 18 USC section 1734.

© 2009 by The American Society of Hematology

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activation downstream of the collagen receptor glycoprotein VI11,12

and Fc�RIIA13,14 and contribute to GPIb9,15 and �IIb�3-dependentsignaling.16 On collagen/glycoprotein VI interaction, SFKs inducephosphorylation of conserved tyrosine residues within the ITAM ofthe FcR�-chain providing a docking site for the tyrosine kinaseSyk. The specific interaction of Syk with the ITAM leads to itsstimulation, an early key step of platelet activation via glycoproteinVI or Fc�RIIA.11,17 During �IIb�3 integrin outside-in signaling,SFKs are also activated and have been proposed to mediatetyrosine phosphorylation of 2 residues of the �3 chain10,18 inaddition to phosphorylate other signaling molecules. Consistentwith these observations, SFKs have been shown to play animportant role in supporting fibrin clot retraction,19 a mechanismrequiring efficient �IIb�3-mediated outside-in signaling.20

In this study, we analyzed the effect of the SFKs inhibitordasatinib on platelet functions in vitro, ex vivo, and in vivo, both inmice and in humans. We demonstrate that, in contrast to imatinib,dasatinib impairs collagen-induced signaling and platelet aggrega-tion, and induces hemostasis defects in mice. The formation ofthrombi under physiologic arterial flow conditions ex vivo is alsoaffected when patients are treated with dasatinib. Interestingly,these effects are rapidly reversible after interruption of the treat-ment. Moreover, we provide evidence that dasatinib efficientlyprevents Fc�RIIA-mediated platelet activation, suggesting that thiscompound or related ones could be of therapeutic interest in thetreatment of heparin-induced thrombocytopenia (HIT). Altogether,these data have potential implications in clinic and may serve as abasis for the development of reversible antiplatelet agents acting asATP-competitive inhibitors of SFKs.

Methods

Materials

The mouse monoclonal antiphosphotyrosine (clone 4G10) was from UpstateBiotechnology; the anti-phospho-Src (Y416) and the peroxidase-conjugatedsecondary antibodies were from Cell Signaling Technology. The anti-Fc�RIIAmonoclonal antibody IV.3 was from Medarex and the anti–mouse IgG F(ab�)2 tocross-link Fc�RIIA/ IV.3 complexes was from Jackson ImmunoResearchLaboratories. All other antibodies and reagents were purchased from Sigma-Aldrich unless otherwise indicated. Dasatinib (Sprycel; Bristol-Myers Squibb)was kindly provided by Bristol-Myers Squibb.

Human blood samples. Samples were obtained from healthy donorsor patients diagnosed at the Department of Hematology, ToulouseUniversity Medical Center (Toulouse, France), after informed consentwas obtained in accordance with the Declaration of Helsinki. TheInstitutional Review Board of the Hopital Purpan (Toulouse, France)

approved the study. The characteristics of patients treated with dasatinibare provided in Table 1. The platelet counts of healthy donors werebetween 156 g/L and 310 g/L.

HIT serum samples. HIT was identified in patients according tostandard criteria,21 including the presence of antiplatelet factor 4/heparinantibodies and a positive platelet aggregation test in the presence of heparin.Sera were stored at �80°C until use. Before processing, they were heated at56°C for 1 hour and centrifuged to remove any residual thrombin activity.

Preparation and activation of human platelets. For in vitro aggrega-tion experiments, blood was collected into acid-citrate-dextrose, andplatelets were isolated by successive centrifugation steps essentially asdescribed previously.22 Briefly, they were washed in a washing buffer(pH 6.5) containing 140 mmol/L NaCl, 5 mmol/L KCl, 5 mmol/L KH2PO4,1 mmol/L MgSO4, 10 mmol/L N-2-hydroxyethylpiperazine-N�-2-ethanesul-fonic acid, 5 mmol/L glucose, and 0.35% bovine serum albumin (wt/vol).The same buffer containing 1 mmol/L CaCl2 was added to the finalsuspension, and pH was adjusted to 7.4. Before stimulation, platelets werepreincubated (or not) for 15 minutes with dasatinib. For adenosine diphos-phate (ADP) stimulation, platelets were activated in platelet-rich plasma.

Cross-linking of the low-affinity receptor for IgG, Fc�RIIA, wasperformed by preincubation of platelets for 1 minute with the monoclonalantibody IV.3 (2 �g/mL) followed by addition of anti–mouse IgG F(ab�)2

(30 �g/mL) at 37°C under gentle shaking as described previously.23 Foractivation of platelets by HIT serum, 180 �L of platelet suspension wasincubated with heparin (0.5 IU) and HIT sera (90 �L).

Platelet aggregation experiments were monitored by a turbidimetricmethod using a dual-channel aggregometer with continuous stirring at 900gat 37°C. P-selectin expression was measured as previously described,24 andATP secretion was recorded by measuring the luminescence from the fireflyluciferin-luciferase reaction using the Chrono-log aggregometer (Kordia).

For the analysis of the whole platelet tyrosine phosphorylation pattern,platelet stimulations were stopped by addition of ristocetin-induced plateletaggregation buffer, protein submitted to sodium dodecyl sulfate–polyacrylamidegel electrophoresis, transferred onto nitrocellulose, and the tyrosine phos-phorylated proteins were detected by immunoblotting with the 4G10antiphosphotyrosine antibody.

Animals. Wild-type mice were of C57Bl/6 genetic background. Theywere housed in the IFR-Toulouse Purpan animal facility according toinstitutional guidelines. For all experiments, 5- to 10-week-old mice wereused. Ethical approval for animal experiments was received from theFrench Ministry of Research in agreement with the European Unionguidelines. Oral administration of dasatinib ranged from 1.25 mg/kg to5 mg/kg as indicated.

In vitro model of thrombosis on immobilized collagen under flowconditions. Glass microcapillaries were coated with 500 �g/mL type Icollagen from equine tendon for 1 hour at 37°C. The flow chamber,mounted on an epifluorescence microscope (Axiovert 200; Carl Zeiss),allowed direct visualization of platelet adhesion and aggregation processes,which were recorded with a CCD camera (Cool Snap HQ, RoperScientific). Human or mouse blood was drawn into lepirudine (200 IU/mL),

Table 1. Characteristics of patients treated with dasatinib or imatinib

Patient no. Age, y Sex Hematologic diseaseDrug treatment,

mg/d Disease status BCR-ABL, % Platelets, g/L

1 73 Male BC-CML D, 100 CHR 2 163

2 41 Female Ph� ALL D, 70 � 2 CHR Undetectable 311

3 78 Male AP-CML D, 100 CHR 28 120

4 58 Female CML D, 70 � 2 CHR 20 219

5 56 Female CML 100 CHR 76 305

6 54 Male Ph� ALL D, 70 � 2 CHR 0.09 132

7 21 Female CML I, 400 CHR Undetectable 160

8 38 Male CML I, 400 CHR 1.2 238

9 68 Female CML I, 600 CHR Undetectable 163

BC indicates blast crisis; Ph�, Philadelphia chromosome–positive; D, dasanitib, I, imatinib; AP, accelerated phase; CML, chronic myeloid leukemia; ALL, acutelymphoblastic leukemia; and CHR, complete hematological response (normalization of the peripheral blood counts and differential, absence of peripheral blasts, anddisappearance of all signs and symptoms of the disease).

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and DiOC6 (2 �M, 30 minutes at 37°C) was used to label platelets in wholeblood. Labeled blood was then perfused through collagen-coated glassmicrocapillaries for 2 minutes at a wall shear rate of 1500 seconds(15 dyne/cm2), followed by washing for 2 minutes at the same shear ratewith phosphate-buffered saline. Thrombus formation was visualized with a40� long working distance objective in real time (acquisition rate, 1 frameevery 5 seconds) for both fluorescent and transmitted light microscopy asdescribed.24 Image sequences of the time lapse recording and analysis ofsurface coverage were performed using the Metamorph software (UniversalImaging Corp). After deconvolution, a lower intensity threshold wasapplied to distinguish platelets from the background, and the similarthreshold was then used to analyze all Z-stack acquired for a givenexperiment. Thrombus volume was calculated as the summation of partialvolumes measured from the area occupied by platelets in each plane ofZ-stacks.

Lipid extraction and analysis. Platelets were labeled with 0.6 mCi/mL[32P]orthophosphate during 45 minutes in a phosphate-free N-2-hydroxy-ethylpiperazine-N�-2-ethanesulfonic acid-Tyrode buffer (pH 6.5) at 37°C.

32P-Labeled platelets were then washed once in the same buffer andsuspended at a final concentration of 109 platelets/mL in modifiedN-2-hydroxyethylpiperazine-N�-2-ethanesulfonic acid-Tyrode buffer(pH 7.38). After stimulation, reactions were stopped by addition ofchloroform/methanol (vol/vol) containing 0.6 N HCl, and lipids wereimmediately extracted and analyzed by a combination of thin-layerchromatography and high-performance liquid chromatography as de-scribed previously.23

Tail bleeding time

We measured bleeding time by 3-mm tail-tip transection in mice anesthe-tized by an intraperitoneal injection of a mixture of ketamine (25 mg/kg)and xylazine (10 mg/kg). A stopwatch was started immediately on transec-tion to determine the time required for the bleeding to stop. Blood dropswere removed every 15 seconds with the use of a paper filter. If bleeding didnot resume within 30 seconds of cessation, it was considered stopped.

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Figure 1. Effect of dasatinib on human platelet aggre-gation and tyrosine phosphorylation on thrombin orcollagen stimulation. Platelets from healthy donorswere treated or not with increasing concentrations ofdasatinib for 15 minutes and stimulated with (A) throm-bin, (B) ADP, or (C) collagen. Platelet aggregation wasassessed using a Chrono-log dual-channel aggregome-ter under stirring at 900g. The aggregation profiles shownare representative of 3 independent experiments. Rightpanels show the effect of dasatinib on whole platelettyrosine phosphorylation pattern in response to thrombinor collagen. Results are representative of 3 independentexperiments. (D) Western blot analysis of phospho-Srcfamily (Tyr416) in human platelets incubated with increas-ing doses of dasatinib and stimulated by 2.5 �g/mLcollagen for 3 minutes. The Western blots shown arerepresentative of 3 independent experiments.

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Results

Dasatinib inhibits collagen-induced platelet activation

To address the effect of dasatinib on human platelet responses,washed platelets were treated with increasing doses of dasatiniband stimulated by different concentrations of thrombin, ADP, orcollagen. Dasatinib significantly inhibited platelet aggregation inresponse to low concentrations of thrombin but only weaklyaffected aggregation at higher concentrations of agonist (Figure1A). P-selectin expression and ATP secretion induced by thrombinwere not significantly reduced (12.2% 2% and 0.5% 10%inhibition at 50 nM dasatinib in response to 0.1 IU/mL thrombin,respectively). Dasatinib, even at 200 nM, had hardly any detectableeffect on platelet aggregation induced by 0.1 IU/mL thrombin (notshown), whereas the pattern of tyrosine phosphorylation wasstrongly affected in a dose-dependent manner by the drug (Figure1A right panel). These data suggest that dasatinib affects �IIb�3outside-in signaling, which is known to involve SFKs10,16 andaccounts for a major part of tyrosine phosphorylation in response tothrombin in aggregating conditions. ADP-induced platelet aggrega-tion in platelet-rich plasma was not significantly affected bydasatinib (Figure 1B). Interestingly, dasatinib treatment veryefficiently inhibited platelet aggregation in response to collagen.Indeed, 50 nM of the drug abolished platelet shapes change andaggregation in response to all collagen concentrations tested(Figure 1C). P-selectin expression and ATP secretion were alsoclearly decreased (86.5% 2% and 60% 5% inhibition at50 nM dasatinib in response to 5 �g/mL collagen, respectively).Under these conditions, tyrosine phosphorylations were stronglyinhibited (Figure 1C right panel). Moreover, using a phosphospe-cific antibody recognizing the active form of SFKs, we show thatdasatinib efficiently inhibited its targets (Figure 1D). To furtherinvestigate the effect of dasatinib on signaling events initiated bycollagen, we analyzed 2 critical pathways downstream of glycopro-tein VI: the phospholipase C� (PLC�) and the phosphoinositide3-kinase (PI 3-kinase) pathways. Phosphatidic acid (PtdOH) produc-tion, which reflects the activity of PLC and diacylgycerol kinase(DAG-kinase) in platelets, was strongly inhibited by 50 nMdasatinib (Figure 2A). Conversely, thrombin-induced PtdOH syn-thesis, which is known to use the Gq/PLC�/DAG-kinase pathway,was not affected (105% 10% of control, n 3). The productionof D3-phosphoinositides by PI 3-kinase was also very efficientlyinhibited in response to collagen (Figure 2B-C). Altogether, theseresults indicate that dasatinib has a relatively modest impact onthrombin or ADP-induced washed platelet aggregation, whereascollagen-induced signaling and platelet responses are stronglyinhibited by clinically relevant doses of the drug. Indeed, after 2 to5 hours of treatment, the concentration of dasatinib can reachapproximately 150 nM and 100 nM in plasma of patients receivingthe 140-mg or the 70-mg regimen, respectively.25 Interestingly,imatinib acting on PDGF-R, cKit, and ABL, but not on SFKs, hadno detectable effects on washed platelet aggregation in response tothrombin or collagen (supplemental Figure 1, available on theBlood website; see the Supplemental Materials link at the top ofthe online article). Even at high concentrations, imatinib did notaffect the tyrosine phosphorylation pattern of collagen-activatedplatelets (supplemental Figure 1), whereas at similar doses itblocked tyrosine phosphorylation in K562 cells expressing BCR-ABL (not shown).

Dasatinib treatment affects thrombus formation under flow

To further investigate the effect of dasatinib on human platelets, wecompared thrombus formation under physiologic flow conditionsin whole blood from healthy donors and from patients treated withdasatinib or imatinib. Normal blood was used as a control in theseexperiments because the patients tested were all under completehematologic response (Table 1). Moreover, because imatinib hadno significant effect on washed platelet activation (supplementalFigure 1), we compared the effect of dasatinib treatment of CMLpatients to that of imatinib. The drugs were administered 4 to5 hours before blood collection. Whole blood containing fluores-cent labeled platelets was perfused over a matrix of collagen at anarterial shear rate of 1500 seconds (15 dyne/cm2) during 2 minutes.Blood from the healthy donors reproducibly formed denselypacked platelet thrombi on collagen fibers, whereas blood fromdasatinib-treated patients formed much smaller thrombi (Figure 3;Table 1). Both the surface covered by platelet aggregates (notshown) and the thrombi volume were significantly reduced (Fig-

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Figure 2. Effect of dasatinib on collagen-induced PLC/DAG-kinase and PI3-kinase activation. 32P-labeled platelets were treated or not with 50 nM dasatinibfor 15 minutes and stimulated by 5 �g/mL collagen for 2 minutes under stirring at900g, and the levels of (A) 32P-PtdOH, (B) 32P-PtdIns(3,4)P2, and (C) 32P-PtdIns(3,4,5)P3 were analyzed as indicated in “Methods.” Results are mean SEMof 3 independent experiments.

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ure 3C). Patients treated with imatinib showed a reduction ofthrombus growth compared with healthy donors, but the effect ofdasatinib was much more pronounced (Figure 3C).

To check whether the effect of dasatinib was dose dependent,we treated mice with increasing doses (1.25-5 mg/kg) ofdasatinib. Figure 3D shows that the thrombus volume obtained

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Figure 3. Rapid and dose-dependent effect of dasatinib treatment on platelet thrombus formation on collagen under arterial flow conditions in humans and mice.DiOC6-labeled platelets in whole blood from healthy donor or patients 1 to 4 treated with dasatinib 4 hours earlier were perfused through a collagen-coated microcapillary at ashear rate of 1500 seconds for 2 minutes. Thrombus formation was visualized with a 40� long working distance objective in real time and then imaged using transmitted lightmicroscopy. Representative images are shown (A). After a washing step with phosphate-buffered saline for 2 minutes at the same shear rate to remove nonadherent cells,slides were visualized using differential interference contrast microscopy, and representative images are shown (B). Images were visualized using an epifluorescencemicroscope (Axiovert 200; Carl Zeiss Inc); 40�/1.3 NA oil objective, no solution, with DiOC6. Images were acquired using a charge-coupled device camera (Cool Snap HQ;Roper Scientific) and Metamorph Version 6.21.6 software. Thrombus volume (C) was measured at 2 surface locations in each of the 4 independent experiments performedfrom healthy donors, dasatinib-treated patients, and imatinib-treated patients (mean SEM). **Significant difference (P � .005), according to Student t test. Scale barrepresents 20 �m. The same experiments were performed using whole blood from mice treated with 1.25, 2.5, or 5 mg/kg dasatinib (5 mice per group) 4 hours before bloodcollection, and the thrombus volumes were measured (D). Results shown are representative of 2 independent experiments.

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under physiologic shear rate conditions decreased progressivelyaccording to the dose of dasatinib ingested.

Overall, these results indicate that, under physiologic arterialshear rate, dasatinib treatment rapidly affects thrombus formationon collagen fibers both in mice and humans.

Dasatinib treatment reversibly increases mouse bleeding time

To investigate the effect of dasatinib on primary hemostasis in vivo,we analyzed the tail bleeding time of mice treated by increasingconcentrations of the drug. Four hours after ingestion of dasatinib,bleeding induced by amputation of the tail tip increased in adose-dependent manner (Figure 4A). Twenty-four hours afteringestion, the effect of dasatinib decreased, especially when lowdoses were administered (Figure 4B). Interestingly, even at highdoses, the effect of dasatinib was no longer detectable 48 hoursafter ingestion (Figure 4C). These results indicate a rapid reversibil-ity of the effect of dasatinib on primary hemostasis in vivo and areconsistent with the pharmacokinetics of the drug described inmice.25 Accordingly, the thrombus surface and volume obtainedunder physiologic shear rate conditions in whole blood wascomparable in control mice and in mice treated with a single doseof dasatinib 48 hours before measurement (Figures 3E, 5). More-over, we could compare ex vivo the thrombus formation in2 patients at 4 and 24 hours after ingestion of dasatinib (supplemen-tal Figure 2). Again, the reversibility of dasatinib action was clearlyobserved. After 24 hours, the thrombi formed on collagen fibers

were much bigger in surface and volume than the thrombi formed4 hours after treatment. The pharmacokinetics of dasatinib inhumans appears consistent with the rapid reversibility of the effect.Indeed, the plasma concentration peaks 2 to 5 hours after drugintake and then decreases progressively to become undetectableafter 24 hours.25 Altogether, these data show clearly that dasatinibrapidly and reversibly affects platelet functions and hemostasis.

Dasatinib prevents platelet activation by serum from HITpatients

To test whether dasatinib may have a potential therapeutic interestin antiplatelet therapy, we analyzed its effects on platelet activationvia clustering of Fc�RIIA by a specific antibody (IV.3) and asecondary IgG F(ab�)2 fragment or by serum from HIT patients.Fc�RIIA is a member of the immunoglobulin gene superfamily andis composed of 2 extracellular Ig homology domains and acytoplasmic tail bearing an ITAM, which becomes tyrosine phos-phorylated by SFKs on receptor clustering. The tyrosine phosphor-ylation of Fc�RIIA by SFKs is a critical step in Fc�RIIA-dependent platelet activation.10 The importance of Fc�RIIA inpathophysiologic disorders such as HIT is well established,26 but itmay also play a role in physiologic platelet activation processes.27,28

Fc�RIIA cross-linking by the specific antibody IV.3 induced anefficient aggregation of washed platelets that was fully inhibited by50 nM dasatinib (Figure 6A). Interestingly, dasatinib also inhibitedplatelet aggregation induced by sera from HIT patients in adose-dependent manner (Figure 6B). At low doses of dasatinib(� 50 nM), platelet aggregation was strongly delayed; whereas athigher concentrations, platelet aggregation was fully abolished.These results are consistent with the well-described role of SFKs inFc�RIIA-dependent platelet activation and show that dasatinib canblock this process at clinically achievable doses.

Discussion

Blood platelets are critical to maintain normal hemostasis and keyplayers in atherothrombosis. On vascular injury, subendothelialmatrix proteins, such as collagen, are exposed and interact withVWF, allowing platelet arrest and subsequent spreading, activation,and secretion of soluble mediators. These soluble agonists recruitcirculating platelets, allowing aggregation and thrombus growthfor the bleeding to stop. Current antiplatelet drugs interfere withimportant steps in their activation process, including thromboxaneA2 synthesis, P2Y12 ADP receptor activation, or �IIb�3 integrinengagement.29,30 So far, intracellular signaling has not been tar-geted by antiplatelet drugs in clinic. Platelet activation is a highlydynamic and regulated process involving many membrane recep-tors and several intracellular signaling cascades. Protein tyrosinekinases are highly expressed in platelets and play a critical role intheir activation process.31 Many of the key platelet membranereceptors recruit and activate nonreceptor tyrosine kinases, particu-larly SFKs, leading to robust tyrosine phosphorylation of severalproteins.32 Some studies have reported the blocking effects oftyrosine kinase inhibitors on different platelet functions.33-35 How-ever, most of the tyrosine kinase inhibitors used so far werenonselective, and it is still difficult to ascertain a role for thedifferent tyrosine kinases present in platelet. Interestingly, theincreasing development of small-molecule inhibitors of kinases,particularly in targeted cancer therapy, allow us now to test moreselective compounds. Imatinib targeting ABL, cKit, and PDGF-Rand more recently dasatinib targeting ABL, cKit, PDGF-R, and

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Figure 4. Reversible effect of dasatinib over the time on tail bleeding time ofmice. The tail bleeding time of control mice and mice treated with dasatinib 4 (A),24 (B), and 48 hours (C) after ingestion of the drug. The single oral administration ofdasatinib was 1.25, 2.5, or 5 mg/kg, as indicated.

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also SFKs have strongly improved the treatment of CML.1

Strikingly, among the adverse effects of dasatinib, bleeding ofdifferent types, including epistaxis, gastrointestinal and brainhemorrhages have been observed.7 Here, we demonstrate that,besides its thrombocytopenic effect reported in some cases, whichcould be explained by the contribution of tyrosine-kinases inmegakaryocytopoiesis,36 dasatinib also strongly affects plateletfunctions in vitro and in vivo. This might explain the gastrointesti-nal hemorrhages observed in patients with normal platelet counts.Our data clearly show that, in contrast to imatinib, which has nodetectable effects on washed platelet responses induced by colla-gen or thrombin, dasatinib strongly inhibits collagen-inducedplatelet activation and affects platelet aggregation in response tolow doses of thrombin. The striking difference between the effectof imatinib and dasatinib on platelet activation in vitro suggests thatthe major effect of dasatinib is mediated by SFK inhibition,although other potential targets of the drug, such as ephrin receptor,may also contribute. Dasatinib is highly efficient in blocking signaltransduction mechanisms in response to collagen, including ty-

rosine phosphorylation, PI 3-kinase activation, and PLC/DAG-kinase activation. This is consistent with the critical role of SFKs,mainly Fyn and Lyn, in the phosphorylation of FcR�-chain, anearly mandatory step in the signaling cascade initiated by glycopro-tein VI triggering.18,37 Fyn and Lyn constitutively interact with theproline-rich domain of the glycoprotein VI cytosolic tail; and oncross-linking, these kinases become active and phosphorylate theFcR�-chain ITAM. This initial step allows Syk recruitment andactivation and the subsequent phosphorylation of several down-stream targets, including LAT and SLP76, 2 adaptor moleculesessential for PLC�2 and PI 3-kinase activation.18,38 Dasatinib alsoaffects thrombus formation measured ex vivo by microscopyreal-time imaging. Whole blood samples from patients treated withdasatinib were perfused under arterial flow over collagen fibers. Inthese conditions, the thrombotic process involves the tethering ofplatelets via GPIb-IX-V to VWF bound to collagen and theinteraction of glycoprotein VI and �2�1 with collagen to mediatestable adhesion.39 Platelet thrombus growth and stabilization arethen linked to secretion and aggregation via �IIb�3. SFKs are

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Figure 5. Reversible effect of dasatinib on plateletthrombus formation under arterial flow conditions.DiOC6-labeled platelets in whole blood from control miceor mice treated with dasatinib (5 mg/kg) 4 or 48 hoursbefore blood collection were perfused through a collagen-coated microcapillary at a shear rate of 1500 seconds for2 minutes. Thrombus formation was visualized and ana-lyzed as in Figure 3. Representative images of plateletadhesion from control and treated mice are shown (A-B).Scale bar represents 20 �m. Area covered by plateletthrombi (C) and thrombus volume (D) were measured at2 surface locations in each of 3 different conditions48 hours after oral administration of dasatinib (1.25, 2.5,or 5 mg/kg as indicated). Results shown are mean valuesof 2 independent experiments.

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important in different phases of this highly dynamic and coordi-nated process.9,27,40 This is consistent with the fact that both thesurface covered by the thrombi and their volume are affected bydasatinib. In washed platelets, high concentrations of dasatinib(100 nM) strongly inhibit tyrosine phosphorylation under aggregat-ing conditions in response to high doses of thrombin, suggestingthat tyrosine kinases activated via �IIb�3 outside-in signaling areaffected. This was not sufficient to reverse aggregation understirring conditions but may contribute to the reduction of thrombusgrowth under arterial flow conditions.

In vivo, 4 hours after ingestion, dasatinib increases the tailbleeding time in a dose-dependent manner in mice, and this effectis rapidly reversible after interruption of the treatment. Thisreversible effect could also be measured ex vivo using bloodsamples from treated mice to follow the thrombus formation underarterial shear rate. Importantly, this reversibility of dasatinib effectcould be measured in 2 patients 24 hours after dasatinib intake.A very recent clinical study also reports a reversible effect ofdasatinib on platelet responses in CML patients.41 Altogether, theseresults demonstrate that dasatinib is affecting platelet functions invitro, ex vivo, and in vivo, which has potential implications inclinic. It is particularly interesting to consider the rapid andreversible effect of dasatinib on platelet functions in vivo, suggest-ing that invasive surgical gesture should be avoided during the firsthours after dasatinib intake.

One important question is the relevance of tyrosine kinaseinhibitors as antiplatelet drugs. Previous studies33-35 have addressedthis question using nonselective tyrosine kinase inhibitors; how-ever, it is difficult to draw clear conclusions. Very recent datasuggest that selective Syk inhibitors may be of interest as antiplate-let agents to prevent thrombosis42 or HIT.43 Here we show that SFKinhibition by dasatinib impairs Fc�RIIA-mediated platelet activa-tion. HIT is a drug-induced immune thrombocytopenia whereantibodies reactive with heparin-PF4 complexes lead to Fc�RIIA-mediated platelet activation. Dasatinib efficiently prevents platelet

activation induced by HIT antibody from patients at clinicallyachievable doses. These results strongly suggest that dasatinib orrelated compounds acting as SFK inhibitors could be of interest asactive agents in HIT treatment. The rapid efficiency of dasatiniband its reversibility of effect could be important advantages toachieve optimal treatment of HIT. The use of the transgenic mousemodel expressing the human Fc�RIIA would allow to test dasatinibin vivo to further validate this drug or related once on an animalmodel of HIT.44 Moreover, defining which SFK member isinvolved in Fc�RIIA ITAM phosphorylation and the degree ofredundancy of these kinases to undergo this key event upstream ofSyk activation would be important to design selective inhibitorsof this pathway. Whether dasatinib or related SFKs inhibitors couldbe of interest as potential new therapy for thrombosis or othervascular obstructive diseases remain to be established usingappropriate animal models, but the increased risk of bleeding mustbe taken into consideration. In this respect, the development ofinhibitors selectively targeting the different SFK members could beuseful to elaborate therapeutic strategies to specifically modulateappropriate platelet functions.

In conclusion, using complementary approaches, we havedemonstrated that inhibition of SFKs by dasatinib treatment rapidlyand reversibly affects platelet activation by ITAM-mediated signal-ing and induces hemostatic defects. We also provide evidence thatthis drug efficiently prevents Fc�RIIA-mediated platelet activation,suggesting that this compound or related ones acting as ATP-competitive inhibitors of SFKs could be of therapeutic interest inthe treatment of HIT.

Acknowledgments

The authors thank M. Plantavid, F. Gaits-Iacovoni, S. Manenti, H.Tronchere, and H. Mohamed for stimulating discussions and forcritical reading of the manuscript.

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Figure 6. Dasatinib efficiently blocks platelet aggre-gation induced by direct Fc�RIIa clustering or bysera from HIT patients. Platelets from healthy donorswere treated or not with increasing concentrations ofdasatinib and stimulated by Fc�RIIA clustering by incuba-tion of platelets with (A) 2 �g/mL of monoclonal antibodyIV.3 for 1 minute followed by addition of 30 �g/mL goatanti–mouse F(ab�)2 or with (B) HIT sera and heparin(0.5 IU). Platelet aggregation was assessed using aChrono-log dual-channel aggregometer under stirring at900g. The aggregation profiles shown are representativeof 3 or 4 independent experiments.

DASATINIB AFFECTS PLATELET FUNCTIONS 1891BLOOD, 27 AUGUST 2009 � VOLUME 114, NUMBER 9

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This work was supported by Inserm, ANR (program JeunesChercheurs, Jeunes Chercheuses; no. ANR-07-JCJC-0093-01),INCa, and Region Midi-Pyrenees.

AuthorshipContribution: M.-P.G, V.M., and M.-C.V. designed and performedmost experiments and analyzed data; S.A. performed microscopy;

C.G. and P.S. selected and prepared HIT sera and performed theexperiments on Fc�RIIA; and C.R. and B.P. designed research,supervised the work, analyzed data, and wrote the paper.

Conflict-of-interest disclosure: The authors declare no compet-ing financial interests.

Correspondence: Bernard Payrastre, Inserm, U563, CentreHospitalier Universitaire Purpan, BP 3028, 31024 Toulouse Cedex03, France; e-mail: bernard.payrastre@inserm.fr.

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online June 3, 2009 originally publisheddoi:10.1182/blood-2009-02-205328

2009 114: 1884-1892  

Christian Recher and Bernard PayrastreMarie-Pierre Gratacap, Valérie Martin, Marie-Cécile Valéra, Sophie Allart, Cédric Garcia, Pierre Sié, reversibly affects platelet activation in vitro and in vivoThe new tyrosine-kinase inhibitor and anticancer drug dasatinib 

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