SLAP/SLAP2 prevent excessive platelet (hem)ITAM signaling in thrombosis and ischemic stroke in mice

Regular Article

THROMBOSIS AND HEMOSTASIS

SLAP/SLAP2 prevent excessive platelet (hem)ITAM signaling inthrombosis and ischemic stroke in miceDeya Cherpokova,1 Markus Bender,1 Martina Morowski,1 Peter Kraft,2 Michael K. Schuhmann,2 Sarah M. Akbar,3

Cheryl S. Sultan,3 Craig E. Hughes,4 Christoph Kleinschnitz,2 Guido Stoll,2 Leonard L. Dragone,5 Steve P. Watson,4

Michael G. Tomlinson,3 and Bernhard Nieswandt1

1Department of Experimental Medicine, University Hospital Wurzburg and Rudolf Virchow Center for Experimental Biomedicine, Wurzburg, Germany;2Department of Neurology, University Hospital Wurzburg, Wurzburg, Germany; 3School of Biosciences, College of Life and Environmental Sciences, and4Centre for Cardiovascular Sciences, School of Clinical and Experimental Medicine, Institute of Biomedical Research, College of Medical and Dental

Sciences, University of Birmingham, Birmingham, United Kingdom; and 5Departments of Pediatrics and Immunology, University of Colorado, School of

Medicine, Aurora, CO

Key Points

• SLAP and SLAP2 haveredundant functions in theregulation of platelet(hem)ITAM signaling.

• SLAP and SLAP2 in plateletslimit occlusive thrombusformation and ischemic braininfarction.

Glycoprotein VI and C-type lectin-like receptor 2 are essential platelet activating receptors

in hemostasis and thrombo-inflammatory disease, which signal through a (hem)immuno-

receptor tyrosine-basedactivationmotif (ITAM)-dependentpathway.Theadaptermolecules

Src-like adapter proteins (SLAP and SLAP2) are involved in the regulation of immune cell

surface expression and signaling, but their function in platelets is unknown. In this study,

we show that platelets expressed both SLAP isoforms and that overexpression of either

protein in a heterologous cell line almost completely inhibited glycoprotein VI and C-type

lectin-like receptor 2 signaling. In mice, single deficiency of SLAP or SLAP2 had only

moderate effects on platelet function, whereas double deficiency of both adapters

resulted in markedly increased signal transduction, integrin activation, granule release,

aggregation, procoagulant activity, and thrombin generation in response to (hem)ITAM-

coupled,but notGprotein-coupled, receptor activation. In vivo, constitutiveSLAP/SLAP2

knockoutmicedisplayedacceleratedocclusivearterial thrombus formationandadramaticallyworsenedoutcomeafter focal cerebral

ischemia.Thiswasattributedto theabsenceofbothadapterproteins inplatelets,asdemonstratedbyadoptive transferofSlap2/2/Slap22/2

platelets into wild-typemice. Our results establish SLAP and SLAP2 as critical inhibitors of platelet (hem)ITAM signaling in the setting of

arterial thrombosis and ischemic stroke. (Blood. 2015;125(1):185-194)

Introduction

Platelet activation at sites of vascular injury is essential for hemostasis,but is also a major pathomechanism underlying myocardial infarctionand stroke.1,2 The central platelet collagen receptor glycoprotein (GP)VI/FcRg-chain complex3,4 critically contributes to this activation, andits loss or functional inhibition provides profound antithromboticprotection, but only moderately increased bleeding in vivo.4,5

GPVI signals through an immunoreceptor tyrosine-based activa-tion motif (ITAM) pathway in a similar manner to the T- and B-cellantigen receptors (TCR,BCR) and someFc receptors.Ligand-inducedcrosslinking of GPVI leads to phosphorylation of the two tyrosineresidueswithin the ITAMon the FcRg-chain predominantlyby the Srcfamily kinase (SFK) Lyn,6,7 followed by the recruitment, phospho-rylation, and activation of the tyrosine kinase Syk, which initiates adownstream signaling cascade ultimately resulting in the activation ofeffector enzymes, including phosphoinositol-3-kinases and phospho-lipase C (PLC) g2.8 These signaling events downstream of Syk alsooccur upon stimulation of the platelet C-type lectin-like receptor2 (CLEC-2) either by its endogenous ligand, the transmembraneGP podoplanin, or by the snake venom toxin rhodocytin. CLEC-2 is

an ;30 kilodalton (kDa) type II membrane protein that containsa single conserved cytosolic YXXL sequence (hemITAM) whichinitiates signaling upon CLEC-2 dimerization or oligomerization.8

CLEC-2 is highly expressed on megakaryocytes and platelets andat lower levels on a number of leukocytes.9-11 CLEC-2 has beenidentified as a critical player in a plethora of (patho-)physiologicalprocesses, including thrombus formation and stability, lymphaticdevelopment, and tumormetastasis, and similar toGPVI, in themain-tenance of vascular integrity during inflammation.10,12,13

Src-like adapter proteins (SLAP and SLAP2) constitute a familyof adapter molecules of 34 kDa and 25/28 kDa, respectively, thatshare structural similarities with SFKs, characterized by the presenceof a unique N-terminal region, an SH3-, and an SH2-domain.14

Unlike SFKs, SLAP and SLAP2 do not possess a C-terminal kinasedomain.14 Overexpression studies in T- and B-cell lines indicatedthat SLAP and SLAP2 act as negative regulators of TCR and BCRsignaling,15-19 and contribute to TCR and BCR surface expressionlevels.18-21 The latter mechanism involves the interaction of SLAPproteins with phosphorylated components of the TCR or BCR

Submitted June 5, 2014; accepted September 28, 2014. Prepublished online

as Blood First Edition paper, October 9, 2014; DOI 10.1182/blood-2014-06-

580597.

The online version of this article contains a data supplement.

The publication costs of this article were defrayed in part by page charge

payment. Therefore, and solely to indicate this fact, this article is hereby

marked “advertisement” in accordance with 18 USC section 1734.

© 2015 by The American Society of Hematology

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complex, followed by the recruitment of the E3 ubiquitin ligasec-Cbl to the receptor complex which promotes its degradation.19,21,22

In this way, SLAP plays an important role in the regulation ofT- and B-cell maturation and development.18,20

Little is known about the function of SLAP proteins in platelets,other thanuponplatelet stimulation specificallywith theGPVI-activatingsnake venom protein convulxin, SLAP2 co-immunoprecipitates withc-Cbl, Syk, and LAT.23 The functional consequences of these putativeinteractions are unknown and form the aim of this study.

Methods

Animals

Slap2/2, Slap22/2, and Slap2/2/Slap22/2 mice were generated as previouslydescribed,20,24 and these mice were backcrossed for 10 generations onto theBALB/cbackground.Gp62/2mice13were crossedwithSlap2/2/Slap22/2mice.Slap2/2/Slap22/2/Gp61/2 and litter-matched Slap2/2/Slap22/2/Gp61/1 miceon a mixed BALB/c/Sv129/C57BL/6 background were used in this study.Animal studies conducted were approved by the district government ofLower Frankonia (Bezirksregierung Unterfranken, Germany).

Transfections and luciferase assays

TheDT40B-cell linewas transfected by a previously published electroporationmethod,25 which is described in detail in the supplemental Methods on theBloodWeb site.

In vitro platelet studies

Immunoprecipitation is described in the supplemental Methods. Plateletpreparation, western blot analysis, aggregometry, flow cytometry, adeno-sine 59-triphosphate (ATP) release, quantification of phosphatidylserine(PS) exposure, and thrombin generation were performed as describedpreviously.26-28

Tail bleeding time

Micewere anesthetized, 1 mm of the tail tip was removedwith a scalpel, and thetails were immersed in 0.9% isotonic saline (37°C). The time until cessationof bleeding (no blood flow for 1 minute) was determined.

Mechanical injury of the abdominal aorta

Anultrasonicflowprobe (0.5PSB699;TransonicSystems)was placed aroundthe abdominal aorta of anesthetized mice and thrombus formation was in-duced by a single firm compression with a forceps for 10 seconds. Blood flowwas monitored until complete blood vessel occlusion occurred for at least5 minutes, or for a maximum of 30 minutes.

Thrombus formation in FeCl3-injured carotid arteries

Anultrasonicflowprobe (0.5PSB699;TransonicSystems)was placed aroundthe exposed carotid artery of anesthetized mice and thrombosis was inducedby topical application of 2.5% FeCl3 for 90 seconds. For animals subjectedto FeCl3–induced injury of the carotid artery after adoptive platelet transfer,7.5% FeCl3 for 1 minute was used. Blood flowwas monitored until completeblood vessel occlusion occurred for at least 2 minutes, or for a maximum of30 minutes.

Transient middle cerebral artery (MCA) occlusion model

Focal cerebral ischemia was induced in wild-type (WT) and Slap2/2/Slap22/2

mice by a transientMCAocclusion (tMCAO) as described.29 Briefly, a silicon-coated thread was advanced through the carotid artery up to the origin of theMCA causing an MCA infarction. After an occlusion time of 30 minutes,the filament was removed allowing reperfusion of the MCA territory. The

extent of infarction was quantitatively assessed 24 hours after reperfusionon 2,3,5-triphenyltetrazolium chloride stained brain sections. Global neuro-logic function and motor function were evaluated by the Bederson score30 andthe grip test,31 respectively. The percentage of ipsilesional occluded vessels24 hours after tMCAOwas determined on hematoxylin and eosin stained brainsection as previously described.32

Platelet depletion

Thrombocytopenia was induced in BALB/c WT mice by IV injection of ananti-GPIba antibody (0.15mg/g bodyweight). Peripheral platelet countsweredetermined by flow cytometry 12 hours after platelet depletion as previouslydescribed.33

Platelet transfusion

Washed platelets from several donor mice were pooled and 109 platelets in200mLTyrode’s buffer were transferred IV intoWTmice. Purity of the plateletsuspensionwas confirmedbyflowcytometry andmicroscopical inspection, andcontamination by other blood cell types was further excluded using a fullyautomated hematology analyzer (Sysmex KX-21N). Peripheral plateletcounts were determined 30 minutes after platelet transfer, and mice weresubsequently subjected to FeCl3-injury of the carotid artery, or 30 minutes oftMCAO.

Chemicals and data analysis

A list of antibodies and reagents, and statistical data analysis are provided inthe supplemental Methods.

Results

SLAP and SLAP2 independently prevent GPVI and CLEC-2

signaling in a cell line model without affecting surface

expression levels

SLAP proteins have been identified as negative regulators of TCRand BCR expression levels and signaling.14 Platelet GPVI/FcRgsignaling resembles the signaling pathways downstream of the TCRand BCR complexes. Therefore, we hypothesized that SLAP andSLAP2 might have the capacity to regulate GPVI signaling. SLAPproteins were expressed in the DT40 B-cell line model system thatwe have previously used to study GPVI/FcRg signaling.25 In thisassay, an NFAT/AP-1 transcriptional reporter produces luciferasein response to combined Ca21 and mitogen-activated protein kinasesignaling, both of which are downstream of GPVI/FcRg activation.Strikingly, SLAP and SLAP2 almost completely inhibitedGPVI sig-naling in response to collagen (Figure 1A), without affecting theexpression levels of transfected GPVI (Figure 1B). Cells transfectedwith SLAP and SLAP2 responded normally to phorbol myristateacetate and ionomycin stimulation (data not shown), indicating thatthe SLAP proteins were not nonspecifically inhibiting cell signalingin a global manner.

To test the possibility that SLAP or SLAP2might also negativelyregulate signaling via the hemITAM receptor CLEC-2, the NFAT/AP-1-luciferase assay in DT40 B cells was again used, since it is aneffective model system to study hemITAM signaling.34 SLAP andSLAP2 each almost completely inhibited CLEC-2 signaling in re-sponse to rhodocytin (Figure 1C), without significantly reducing theexpression levels of transfected CLEC-2 (Figure 1D). Responses tophorbol-12-myrisate-13-acetate and ionomycin were not affected bythe SLAP proteins (data not shown). Together, these data suggestedthat SLAP andSLAP2 can serve as negative regulators of both ITAMand hemITAM signaling.

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SLAP and SLAP2 are expressed in mouse platelets

We set out to extend our data obtained in a heterologous system andto further investigate the role of SLAP family proteins in plateletbiology by analyzing mice deficient for SLAP or SLAP2.20,24

Western blot analysis confirmed the absence of SLAP in Slap2/2 andof SLAP2 in Slap22/2 platelet lysates (Figure 2A). We observedupregulation of SLAP in Slap22/2 platelets (increase of ;35%compared with WT platelets) (Figure 2A). The two SLAP2 proteinbands of 25 kDa and 28 kDa are in line with previous reports whichindicate that alternative translation leads to the expression of twoSLAP2 isoforms (Figure 2A, arrowheads).16,17 Slap2/2 and Slap22/2

mice had normal platelet counts and size (supplemental Table 1).Flow cytometric measurement of prominent platelet surface re-ceptors and western blot analysis showed slightly increased surfaceexpression of GPVI in Slap2/2 platelets, whereas CLEC-2 levelswere slightly elevated inSlap22/2platelets (supplemental Table 1 andsupplemental Figure 1A). The effect of SLAPor SLAP2 deficiency onplatelet activation was analyzed by aggregation studies and flowcytometry measurements of agonist-induced inside-out activation ofaIIbb3 integrin and degranulation-dependent P-selectin surface

exposure. Stimulation of platelets with GPVI-specific agonists(collagen-related peptide [CRP], the snake venom protein convulxin),or the CLEC-2 agonist rhodocytin, resulted in slightly elevatedintegrin activation anda-granule release, in particular at high agonistconcentrations (supplemental Figure 1B-E). In contrast, activationresponses to low concentrations of (hem)ITAM-specific agonists orto soluble agonists operating via G protein-coupled receptors, suchas adenosine 59-diphosphate (ADP), the thromboxane A2 analogU-46619, and thrombin, were normal. These results suggested apotential negative regulatory function for SLAP family proteinsdownstream of (hem)ITAM receptors in platelets, but this couldbe largely masked by functional compensation in the singleknockouts.

Slap2/2/Slap22/2 platelets are hyperreactive to

(hem)ITAM-specific agonists

To assess a potential functional redundancy of the two adapter proteins,we generated SLAP/SLAP2 double-deficient mice. Slap2/2/Slap22/2

platelets exhibited a significant (P , .001) ;23% increase in surfaceexpression of GPVI and ;15% increase in CLEC-2 compared withWT controls, as determined byflow cytometry (supplemental Table 2)and confirmed by western blot analyses (Figure 2B). Expression ofall other major platelet surface receptors was unaltered (supplemen-tal Table 2).

Slap2/2/Slap22/2 platelets displayed a marked hyperreactivityin response to GPVI or CLEC-2 stimulation (Figure 2C-E). Thiseffect was best evident at subthreshold concentrations of GPVI ag-onists (CRP, convulxin and collagen) as well as the CLEC-2 agonistrhodocytin, which did not induce activation of WT platelets, butresulted in integrin activation, a- and dense-granule secretion, androbust aggregation of the mutant platelets. Importantly, activationresponses to G protein-coupled receptor-specific agonists wereunaltered in Slap2/2/Slap22/2 platelets (Figure 2C-E). Together,these results indicated that SLAP and SLAP2 have redundantfunctions in the regulation of (hem)ITAM signaling in platelets.

To examine whether increased GPVI/ITAM signaling in Slap2/2/Slap22/2 platelets can be explained by the elevated GPVI surfaceexpression levels in SLAP/SLAP2 deficient mice, Slap2/2/Slap22/2

Gp61/2 were generated by crossing Slap2/2/Slap22/2 mice withGp62/2 mice.13 Flow cytometric analysis confirmed that GPVI ex-pression levels in Slap2/2/Slap22/2/Gp61/2 platelets were reducedby approximately 50% compared with platelets of Slap2/2/Slap22/2/Gp61/1 litter-matched mice and were also significantly lower than inWT platelets (Figure 3A). Despite reduced GPVI surface expressionlevels, Slap2/2/Slap22/2/Gp61/2 platelets were markedly hyperre-active to GPVI agonists, as shown by analysis of integrin aIIbb3activation, P-selectin exposure, and aggregation in response to GPVI-specific agonists, most notably at low and intermediate concentrations(Figure 3B-C). These results clearly demonstrated that the enhancedGPVI activation in SLAP/SLAP2 deficient platelets occurred inde-pendently of increased GPVI expression levels in these cells, andindicate a specific defect in the inhibition of GPVI signaling.

To study the function of SLAP/SLAP2 in ITAM signaling inmore detail, we assessed changes in protein tyrosine phospho-rylation in WT, Slap2/2/Slap22/2/Gp61/2, and Slap2/2/Slap22/2/Gp61/1 platelets upon stimulation with convulxin (Figure 3D).We detected enhanced and sustained tyrosine phosphorylation ofkey signaling molecules in the GPVI/ITAM activation pathway,including FcRg-chain, Syk (Y519/520), and PLCg2 (Y759) inSLAP/SLAP2 deficient platelets, which was also seen in Slap2/2/Slap22/2/Gp61/2 platelets, and thus largely independent of GPVI

Figure 1. SLAP and SLAP2 inhibit GPVI/FcRg and CLEC-2 signaling in the

DT40 B-cell line model. (A) The DT40 B-cell line was transfected with a Ca21/

mitogen-activated protein kinase-responsive NFAT/AP-1-luciferase reporter construct,

a b-galactosidase construct, a GPVI, an FcRg, a SLAP or SLAP2 expression construct,

or empty vector control. Cells were either left unstimulated or stimulated with 5 mg/mL

collagen, lysed, and assayed for luciferase and b-galactosidase. Luciferase data

were normalized for b-galactosidase values. (B) The expression of GPVI was

confirmed by flow cytometry with an anti-GPVI mAb. (C) The experiment was con-

ducted as described in (A), with the exception that cells were transfected with

a myc-tagged CLEC-2 expression construct and stimulated with 50 nM rhodocytin.

(D) The experiment was performed as for (B), except that CLEC-2 was detected by

an anti-myc mAb. Results in all panels are expressed as mean 6 standard deviation

(SD) (n 5 3). ***P , .001. ctrl, control; RC, rhodocytin; unstim., unstimulated.

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expression levels (Figure 3D and supplemental Figure 2A). Notably,markedly enhanced tyrosine phosphorylation was observed alsoupon stimulation of SLAP/SLAP2 deficient platelets with rhodocytin(Figure 3E and supplemental Figure 2B).

To address the mechanism by which SLAP proteins inhibitGPVI signaling, we hypothesized that SLAP proteins might com-pete with SFKs for binding to the GPVI/FcRg signaling complex.This is because Lyn and other SFKs are known to interact withGPVI,6,35 and because of the sequence similarity between Lynand SLAP/SLAP2 (33% to 34% identity for SH3 and 51% identity forSH2 domains). Indeed, we detected strongly increased binding ofLyn to activatedGPVI in the absence of SLAP/SLAP2 comparedwithcontrol (Figure 3F), consistent with the increased tyrosine phos-phorylation of downstream signaling proteins (Figure 3D).

Markedly enhanced GPVI-dependent procoagulant activity in

Slap2/2/Slap22/2 platelets

We next investigated consequences of SLAP/SLAP2 deficiency onplatelet adhesion to collagen and aggregate formation under flowusing a whole blood perfusion system. Unexpectedly, WT andSlap2/2/Slap22/2 platelets formed large aggregates to the sameextent and with the same kinetics at intermediate shear rates of1000 s21 (supplemental Figure 3A-B).

GPVI signaling leads to a marked increase in cytosolic Ca21

concentrations, subsequent surface exposure of procoagulant PS,and resultant thrombin generation.4,36 To test whether SLAP/SLAP2regulate coagulant activity of platelets, blood fromWT and Slap2/2/Slap22/2 mice was anticoagulated, perfused over immobilized

Figure 2. Increased integrin activation, a2 and dense-granule release, and aggregation in Slap2/2/Slap22/2 platelets upon GPVI or CLEC-2 stimulation.

(A) Analysis of SLAP and SLAP2 expression in WT, Slap2 /2, and Slap22 /2 platelets by western blot. Actin expression was used as loading control and for

quantification. Arrowheads indicate both SLAP2 isoforms (25 kDa and 28 kDa) in WT platelets. (B) Western blot analysis of GPVI and CLEC-2 expression in

Slap2 /2 /Slap22 /2 platelets. GPIIIa expression was used as loading control and for quantification. (A-B) Equal protein amounts were loaded and expression levels

were quantified by densitometry with ImageJ software (National Institutes of Health) on 10 (A) or 12 (B) samples per genotype. Results represent mean 6 SD.

Expression levels were adjusted such that SLAP-to-actin, GPVI-to-GPIIIa, or CLEC-2-to-GPIIIa ratio in WT platelets was set to 1. (C) Flow cytometric analysis of

activated integrin aIIbb3 (binding of JON/A-PE) (left) and degranulation-dependent P-selectin exposure (right) upon stimulation with the indicated agonists in

WT and Slap2 /2 /Slap22 /2 platelets. Results are expressed as mean fluorescence intensity 6 SD (n 5 4 mice per group) and are representative of 6 independent

experiments. Dividing lines indicate separate measurements. (D) Washed platelets were activated with the indicated agonists and light transmission was recorded

on a Fibrintimer 4-channel aggregometer. ADP measurements were performed in platelet-rich plasma (PRP). Aggregation traces representative of 3 independent

experiments with n 5 4 are depicted. Arrows indicate addition of the respective agonist. (E) Washed platelets were incubated with Luciferase-Luciferin reagent

and ATP release was measured in a Lumi-aggregometer upon stimulation with thrombin, CRP, or rhodocytin. Results are representative of 2 individual

experiments with n5 4. Results represent mean6 SD. *P, .05; **P, .01; ***P, .001. A/U, ADP1 U-46619; CRP, collagen-related peptide, CVX, convulxin; FITC, fluorescein

isothiocyanate; PE, phycoerythrin; PRP, platelet-rich plasma; RC, rhodocytin; thr., thrombin; U46, U-46619.

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collagen at a shear rate of 1000 s21, and PS exposure was determinedby Annexin-A5 staining. The surface covered by collagen-adherentplatelets was similar inWT and mutant samples, whereas PS exposurewas markedly increased in Slap2/2/Slap22/2 blood (Figure 4A-B).EnhancedGPVI-dependent procoagulant activity ofSlap2/2/Slap22/2

platelets was confirmed by flow cytometric analysis of Annexin-A5-positive cells upon stimulation with different agonists (Figure 4C andsupplemental Figure 3C). In agreement with enhanced PS-exposure,lack of SLAP/SLAP2 resulted in altered maximal amount of newlygenerated thrombin and significantly accelerated response upon stim-ulationwith (hem)ITAM-specific agonists,whereas the overall amount

of thrombin produced was comparable to the values in WT controlsamples (Figure 4D). Importantly, SLAP/SLAP2 deficiency did notaffect plasmacoagulation, as confirmedbyunaltered activated plasmathromboplastin time and prothrombin time in Slap2/2/Slap22/2micecompared with the WT control (supplemental Figure 4).

SLAP/SLAP2 limit pathological thrombus formation

We next studied the role of SLAP and SLAP2 in hemostasis andthrombosis. Combined deficiency of SLAP and SLAP2 did not affectplatelet hemostatic functions (mean tail bleeding time: 54622 seconds

Figure 3. Increased GPVI/ITAM signaling in Slap2/2/Slap22/2

platelets occurs independently of elevated GPVI expression

levels. (A) Analysis of GPVI expression levels in WT, Slap2/2/Slap22/2/

Gp61/2, and Slap2/2/Slap22/2/Gp61/1 platelets. Diluted whole

blood was stained with fluorescein isothiocyanate-labeled JAQ1

antibody and platelets were analyzed by flow cytometry (n 5 4

mice per group). (B) Detection of integrin aIIbb3 activation (binding of

JON/A-PE) (top) and a-granule release (bottom) in response to the

indicated agonists in WT, Slap2/2/Slap22/2/Gp61/2, and Slap2/2/

Slap22/2/Gp61/1 platelets. Results are expressed as mean fluores-

cence intensity6SD (n5 4 mice per group). (C) Washed platelets were

activatedwith CRP and light transmission wasmonitored on a Fibrintimer

4-channel aggregometer. (D) WT, Slap2/2/Slap22/2/Gp61/2 (HET), and

Slap2/2/Slap22/2/Gp61/1 (HOM) platelets were stimulated with

0.5 mg/mL convulxin for the indicated time points. Whole-cell

lysates were western-blotted and probed with the anti-phosphotyro-

sine antibody 4G10 or with phospho-specific antibodies. Staining

of the respective nonphosphorylated proteins and actin served

as loading control. (E) WT and Slap2/2/Slap22/2 platelets were

stimulated with 1 mg/mL rhodocytin and analysis of tyrosine phos-

phorylation patterns was performed as described for (D). (F)

SLAP/SLAP2 attenuate Lyn binding to activated GPVI. Washed

WT and Slap2/2/Slap22/2 (DKO) platelets were left unstimulated or

stimulated with 0.5 mg/mL convulxin for 20 seconds, lysed, and

proteins immunoprecipitated (IP) with Lyn were resolved by sodium

dodecyl sulfate-polyacrylamide gel electrophoresis under nonreduc-

ing conditions and immunoblotted (WB) for GPVI or Lyn. An im-

munoblot of whole-cell lysate (WCL) loaded at 0.4% of Lyn

immunoprecipitation input is also shown. Note that GPVI is detected

as an ;65 kDa monomer and an ;120 kDa dimer, respectively.

Results in all panels are representative of 3 independent experiments.

*P , .05; **P , .01; ***P , .001. A/U, ADP 1 U-46619; CVX,

convulxin; CRP, collagen-related peptide; DKO, double knockout;

FITC, fluorescein isothiocyanate; HOM, Slap2/2Slap22/2Gp61/1

platelets; PE, phycoerythrin; RC, rhodocytin; thr., thrombin; WB,

western blot; WCL, whole-cell lysate.

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inWTmice, and 496 14 seconds in Slap2/2/Slap22/2mice; P5 .6)(Figure 5A).

To assess the extent to which the above-described defects affectthrombotic events in vivo, Slap2/2/Slap22/2 and WT mice weresubjected to two models of occlusive arterial thrombus. In the firstmodel, the abdominal aorta is mechanically injured and thrombusformation occurs mainly through collagen-dependent mechanisms.The severity of injury was adjusted to achieve stable vessel occlusionin only 20% of the WT mice. In marked contrast, the majority (89%)of Slap2/2/Slap22/2mice formed stable vessel occlusion, which alsooccurred considerably faster (2826 151 seconds in Slap2/2/Slap22/2

mice vs 713 6 131 seconds in WT mice) (Figure 5B). In a secondmodel, the carotid artery was injured by topical application of a lowconcentration of FeCl3. Stable vessel occlusion occurred in 93% of alltested Slap2/2/Slap22/2mice, whereas 60% of theWTmice (9 out of15) were not able to form occlusive thrombi within the observationperiod of 30 minutes (P , .01); mean time to occlusion: 607 6 146seconds inWTmice vs 4836 149 seconds in Slap2/2/Slap22/2mice(Figure 5C). Importantly, similar results were obtained upon adoptiveplatelet transfer of Slap2/2/Slap22/2 platelets into platelet-depletedWT mice, thus excluding a contribution of other cell types thanplatelets to the observed phenotype (stable vessel occlusion in 30%of mice transfused with WT platelets, vessel occlusion in 92% ofmice transfused with Slap2/2/Slap22/2 platelets; P, .01) (Figure 5D).Notably, application of a very low concentration of FeCl3 resulted inthe formation of occlusive platelet-rich thrombi in Slap2/2/Slap22/2

mice, which did not differ morphologically from those observed inWT control mice treated with a higher concentration of FeCl3,demonstrating that thrombus formation in Slap2/2/Slap22/2 micefollows the samemechanisms as inWTmice, but occurs at amarkedlylower threshold stimulus (supplemental Figure 5). Together, thesefindings demonstrated that SLAP/SLAP2 deficiency in platelets

resulted in a prothrombotic phenotype, confirming the notion thatthese adapter proteins limit platelet activation in vivo, and therebyefficiently prevent occlusive thrombus formation and infarction.

SLAP/SLAP2 deficiency in platelets dramatically aggravates

neurologic damage after focal cerebral ischemia

Ischemic stroke is a complex diseasewithmultiple cellular interactionsinvolved in the progression of thromboembolic vessel occlusion toinfarct development.2 Although the exact mechanisms by whichplatelets contribute to the development of ischemic brain infarction arestill poorly understood, there is increasing evidence that GPVI (andGPIb) plays amajor role in this process.29,37 To determine the effect ofSLAP/SLAP2 deficiency on cerebral infarct development, Slap2/2/Slap22/2 andWTmice were subjected to the tMCAOmodel of acutestroke where the MCA is occluded by a filament for 30 minutes,thereby reducing cerebral flow by .90%.29 In agreement withprevious reports,38 WT mice developed only small infarctions afterthis short ischemia. In sharp contrast, infarct volumes in Slap2/2/Slap22/2 mice were increased by;92% compared with WT mice(58.8 6 33.5 mm3 in WT mice vs 113.1 6 14.3 mm3 in Slap2/2/Slap22/2 mice; P , .001) (Figure 6A). The increase in infarct sizewas functionally relevant, as it was associated with an overallimpairment of neurologic and motor function, demonstrated by thesignificantly worsened outcome in the Bederson score and grip test,respectively (Figure 6B-C). The dramatic increase in infarct volumewas accompanied by enhanced intracerebral thrombosis, as dem-onstrated by the markedly elevated number of occlusive vessels(Figure 6D). In agreement with the higher proportion of thromboticvessels, immunohistochemical and western blot analyses revealedincreased accumulation of fibrin(ogen) in the infarcted basal gangliaand cortices of Slap2/2/Slap22/2 mice (data not shown).

Figure 4. SLAP/SLAP2 deficiency enhances procoagulant activity and (hem)ITAM-dependent thrombin generation in platelets. (A) Unaltered surface coverage by

collagen-adherent platelets in WT and Slap2/2/Slap22/2 blood. Representative phase-contrast images are depicted. (B) Increased PS exposure of Slap2/2/Slap22/2

platelets, as demonstrated by Annexin-A5-DyLight 488 staining of platelets. Procoagulant index indicates the ratio of surface coverage of PS-exposing platelets

(Annexin-A5-DyLight 488 staining of platelets) to the total surface covered by platelets. Results in (A) and (B) are representative of 2 independent experiments with

n $ 6. Images were obtained at 340. Bars represent 50 mm. (C) Washed platelets were stimulated with 0.1 U/mL thrombin, 20 mg/mL CRP, a combination of both

thrombin and CRP, 1 mg/mL convulxin, or 0.12 mg/mL rhodocytin, stained with saturating amounts of Annexin-A5-DyLight 488, and directly analyzed by flow cytometry.

Results are representative of 2 independent experiments with n 5 5. (D) Citrate-anticoagulated PRP was left unstimulated, or platelets were activated by incubation

with convulxin (1 mg/mL), CRP (20 mg/mL), rhodocytin (1 mg/mL), or the Ca21 ionophore A23187 (A23) (10 mM) for 10 minutes at 37°C. Thrombin generation was

triggered with tissue factor/CaCl2. Endogenous thrombin potential (left); quantification of thrombin peak height (middle); and quantification of time to peak (right). n 5 3

for A23187; n 5 7 for CRP and RC; and n 5 10 for PRP and CVX. *P , .05; **P , .01; ***P , .001. A23, A23187; CRP, collagen-related peptide; CVX, convulxin; ETP,

endogenous thrombin potential; PRP, platelet-rich plasma; RC, rhodocytin; thr., thrombin.

190 CHERPOKOVA et al BLOOD, 1 JANUARY 2015 x VOLUME 125, NUMBER 1

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To specifically investigate the role of platelet SLAP and SLAP2in the progression of ischemic stroke, we performed adoptive platelettransfer into platelet-depleted WT mice. Importantly, the trans-fused platelet populations were free of leukocytes, as tested by flowcytometry, microscopical inspection, and automated blood cellanalysis. Furthermore, flow cytometric and transmission electronmicroscopy analyses confirmed that the platelet suspensions werealso free of (leukocyte- or platelet-derived) microparticles. Strikingly,infarct volumes inWTmice transfusedwithSlap2/2/Slap22/2plateletswere dramatically increased by ;97% compared with WT micetransfusedwithWTplatelets (123.06 23.4mm3 vs 62.66 31.8mm3;P, .001) (Figure 7A), and this was accompanied by severe neu-rologic deficits (Figure 7B-C). These findings demonstrated thatSLAP/SLAP2 activity in platelets is critically involved in limitinginfarct growth following focal cerebral ischemia.

In a control experiment, WT mice received leukocytes isolatedfrom blood of Slap2/2/Slap22/2 or WT mice, and were then sub-jected to 30minutes of tMCAO.Both groups ofmice developed onlysmall infarcts, clearly demonstrating that white blood cells are notresponsible for the increased susceptibility of Slap2/2/Slap22/2

mice toward ischemic stroke (supplemental Figure 6). Furthermore,induction of local cytokine production, infiltration by T cells (whichhave been shown to contribute to infarct growth in this model),39,40

complement activation destabilization of the blood-brain barrier

were comparable inWT and Slap2/2/Slap22/2 mice for 24 hoursafter 30minutes of tMCAO (data not shown). Together, these resultsfurther corroborated the notion that enhanced thrombotic, rather thanproinflammatory activity, is a major determinant of the increasedsusceptibility of the mutant mice toward ischemic brain infarction.

Figure 5. SLAP/SLAP2 limit pathological thrombus formation. (A) Unaltered

hemostasis in Slap2/2/Slap22/2 mice. Tail bleeding times of WT and Slap2/2/

Slap22/2 mice in saline at 37°C. Each symbol represents 1 animal. (B) The

abdominal aorta was mechanically injured using a forceps (compression for 10

seconds) and blood flow was monitored by an ultrasonic flow probe until complete

vessel occlusion for at least 5 minutes, or for a maximum of 30 minutes. Each symbol

represents 1 animal. (C) The right carotid artery of WT and Slap2/2/Slap22/2 mice

was injured by topical application of 2.5% FeCl3 for 90 seconds and blood flow was

monitored with a Doppler flow probe until complete vessel occlusion for at least

2 minutes, or for a maximum of 30 minutes. Each symbol represents 1 animal. (D) WT

mice were treated with a platelet-depleting anti-GPIba antibody. Platelet-depleted

WT mice were transfused with WT or Slap2/2/Slap22/2 (DKO) platelets and 7.5%

FeCl3 were topically applied to the right carotid arteries of these mice for 1 minute.

Time to complete vessel occlusion was determined as described for (C). Each symbol

represents 1 animal. **P , .01. DKO, double knockout; NS, not significant.

Figure 6. SLAP/SLAP2 deficiency dramatically aggravates neurologic damage

after focal cerebral ischemia. Infarct volumes (A) and functional outcome (B-C)

24 hours after focal cerebral ischemia in WT and Slap2/2/Slap22/2 mice were in-

vestigated in a murine model of ischemic stroke. Mice were subjected to 30 minutes

of tMCAO. (A) Brain infarct volumes in WT (n 5 9) and Slap2/2/Slap22/2 (n 5 12)

mice were measured by planimetry (left). Results represent mean 6 SD. Rep-

resentative images of 3 coronal brain sections stained with 2,3,5-triphenyltetrazolium

chloride for 24 hours after 30 minutes of tMCAO (right). Arrows indicate infarcted

areas in Slap2/2/Slap22/2 mice. Bederson score (B) and grip test (C) were

determined 24 hours after tMCAO. Each symbol represents 1 animal. (D) SLAP/

SLAP2 deficiency dramatically increases microvascular thrombosis after 30 minutes

of tMCAO. Representative hematoxylin and eosin stains from the ipsilesional

hemispheres of WT (n 5 6) and Slap2/2/Slap22 /2 (n 5 8) mice (left), and

determination of the percentage of occluded vessels (right). Number of thrombotic

vessels was significantly increased in Slap2/2/Slap22/2 mice (arrows), whereas the

microvascular patency was largely preserved in WT mice (arrowheads). Images

were obtained at 340. Bar represents 50 mm. *P , .05; **P , .01; ***P , .001.

BLOOD, 1 JANUARY 2015 x VOLUME 125, NUMBER 1 SLAP/SLAP2 IN PLATELETS 191

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Discussion

In this study, we have shown that SLAP and SLAP2 negatively regu-late surface expression levels and signaling of the ITAM receptorGPVI and the hemITAM receptor CLEC-2 in platelets. However,single deficiency of either adapter protein had only a minor effecton platelet (hem)ITAM signaling, whereas the combined loss ofSLAP and SLAP2 resulted in a marked platelet hyperreactivityto (hem)ITAM-coupled agonists in vitro, which translated into asignificant prothrombotic phenotype and dramatically increasedsusceptibility to focal cerebral ischemia in vivo. Our results clearlydemonstrate a functional redundancy of the two adapter proteins inthe regulation of platelet (hem)ITAM signaling and reveal a crucialfunction of SLAP and SLAP2 in the control of thrombotic andthrombo-inflammatory events.

Involvement of SLAP in the regulation of surface expression levelsof ITAM-coupled immune cell receptors, such as the TCR and BCR,is well-established.14 In vivo studies have demonstrated an importantrole for SLAP in regulating TCR expression during a specificT lymphocyte developmental stage at which repertoire selectiontakes place.20 Similarly, SLAP is required for the fine tuning of BCRlevels and signal strength during B-cell development.18 Importantly,our data clearly demonstrate that increased GPVI receptor densitywas not responsible for the marked hyperreactivity, as this was alsoseen in SLAP/SLAP2 deficient Gp61/2 platelets, which expressedlower GPVI surface levels than WT control platelets (Figure 3A-D).

This was supported by observations in a model cell line, in which ex-pression of either SLAP or SLAP2 almost completely inhibited GPVIsignaling without affecting expression levels (Figure 1A-B). Wepropose that the mechanism by which GPVI signaling is inhibitedby SLAP proteins involves, at least in part, competition with SFKsfor binding to the GPVI/FcRg signaling complex. In support of thismodel, immunoprecipitation of the SFK Lyn contained substan-tially more GPVI in the absence of SLAP/SLAP2 (Figure 3F), andtyrosine phosphorylation of downstream signaling proteinswas alsoelevated (Figure 3D). Collectively, these results establish SLAP/SLAP2 as negative regulators of ITAM signaling in platelets,presumably through competition with Lyn for binding to the GPVIcytosolic tail, and a similar mechanism may also apply to CLEC-2signaling.

Our data further demonstrated that SLAP/SLAP2 significantlycontributed to dampening collagen-induced procoagulant activity inplatelets and ensuing thrombin generation (Figure 4). These resultsare in line with earlier studies indicating that key components ofthe GPVI/ITAM signaling cascade, including the FcRg-chain, LAT,Syk, and PLCg2, as well as GPVI-induced increase in cytosolicCa21 concentration, are critically involved in collagen-dependent PSexposure.28,36,41 Increasing evidence suggests that platelet procoagu-lant activity, in particular by propagation of the contact phase ofcoagulation, plays an important role in initiating arterial thrombusformation.1 Our results revealed that SLAP and SLAP2 are essentialto prevent overshooting thrombotic and thrombo-inflammatory pro-cesses in vivo (Figures 5B-D, 6, and 7). Remarkably, usingadoptive platelet transfer, we were able to unambiguously attributethis function to SLAP/SLAP2 expression in platelets and thecontribution of both adapter proteins to attenuation of (hem)ITAMsignaling.

Compelling evidence suggests that platelets are essentially in-volved in the progression of ischemic stroke, presumably byorchestrating a complex thrombo-inflammatory process, in whichinteractions between platelets, immune cells, and the contact ac-tivation system result in the disruption of the blood-brain barrierand neurologic damage.37,38 Although the underlying mecha-nisms of platelet contribution to infarct development are notcompletely understood, integrin aIIbb3-dependent platelet ag-gregation was shown to be dispensable.29 By contrast, the importanceof initial platelet adhesion and activation steps involving GPIb-vonWillebrand factor interactions is now well-established.29,37,42-44

Likewise, GPVI also appears to play a crucial role in the patho-physiology of acute stroke, as shown by profound protection ofGPVI-depleted mice in the tMCAO model of ischemic stroke,29

and indirect evidence indicating increased GPVI activity in acutestroke patients.45,46 Our findings further corroborate the hypoth-esis that GPVI/ITAM-mediated activation processes are of centralimportance during stroke progression. Significantly, GPVI hasemerged as an attractive potential target for antithrombotic ther-apy, as its blockade or antibody-induced depletion provided pow-erful protection from experimental arterial thrombosis withoutaffecting platelet hemostatic functions.4,5,47 Together, these ex-perimental and clinical data emphasize a central role of GPVI inthe course of acute ischemic disease states, and thereby establishmodulators of GPVI activity, such as SLAP/SLAP2, as centralfactors in the pathology of atherothrombosis and ischemic stroke.

Taken together, our studies establish two distinct roles for the ad-apter proteins SLAP and SLAP2 in platelets. Firstly, SLAP/SLAP2negatively regulate expression levels of two major platelet-activatingreceptors: the ITAM-containing collagen receptor GPVI/FcRg-chain,which parallels SLAP/SLAP2 regulation of BCR and TCR levels, and

Figure 7. SLAP/SLAP2 deficiency in platelets induces a dramatic increase in

infarct volume in a model of ischemic stroke. Infarct volumes (A) and functional

outcome (B-C) 24 hours after focal cerebral ischemia in WT mice reconstituted with

WT or Slap2/2/Slap22/2 (DKO) platelets after depletion of endogenous platelets

were investigated in a murine model of ischemic stroke. Mice were subjected to

30 minutes of tMCAO. (A) Brain infarct volumes in platelet-depleted WT mice

transfused with WT (n5 12) or Slap2/2/Slap22/2 (n5 8) platelets were measured by

planimetry (left). Results represent mean 6 SD. Representative images of 3 coronal

brain sections stained with 2,3,5-triphenyltetrazolium chloride for 24 hours after

30 minutes of tMCAO (right). Arrows indicate infarcted areas in WT mice recipients

of Slap2/2/Slap22/2 platelets. Bederson score (B) and grip test (C) were determined

24 hours after tMCAO. Each symbol represents 1 animal. *P , .05; ***P , .001. DKO,

double knockout; plts, platelets.

192 CHERPOKOVA et al BLOOD, 1 JANUARY 2015 x VOLUME 125, NUMBER 1

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the hemITAM-containing CLEC-2 receptor. Secondly, SLAP/SLAP2are central regulators of (hem)ITAM signaling in platelets that areessential to limit arterial thrombus growth and thrombo-inflammatoryprocesses.

Acknowledgments

The authors thank Sarah Schiebl for help with thrombin generationassays, and Jonas Muller, Andrea Sauer, and Daniela Urlaub forexcellent technical assistance.

This study was supported by the Deutsche Forschungsgemein-schaft (Sonderforschungsbereich 688) (B.N., G.S., and C.K.), theRudolf Virchow Center, the Wellcome Trust (088410), a BritishHeart Foundation Senior Research Fellowship (FS/08/062/25797)(M.G.T.), and a British Society for Haemostasis and Thrombosistravel grant (M.G.T.). S.P.W. holds a British Heart Foundation chairposition.

Authorship

Contribution:D.C. performedexperiments, analyzeddata, andwrote themanuscript; M.B., M.M., P.K., M.K.S., S.M.A., C.S.S., and C.E.H.performed experiments and analyzed data; C.K., G.S., L.L.D., andS.P.W. assisted with experimental design and contributed to thewriting of the manuscript; and M.G.T. and B.N. designed andsupervised research, interpreted data, and wrote the manuscript.

Conflict-of-interest disclosure: The authors declare no competingfinancial interests.

Correspondence: Bernhard Nieswandt, University HospitalWurzburg andRudolfVirchowCenter for Experimental Biomedicine,University ofWurzburg; Josef-Schneider-Strasse 2, 97080Wurzburg,Germany; e-mail: [email protected];and Michael G. Tomlinson, School of Biosciences, College of Lifeand Environmental Sciences, University of Birmingham, Edgbaston,Birmingham B15 2TT, United Kingdom; e-mail: [email protected].

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online October 9, 2014 originally publisheddoi:10.1182/blood-2014-06-580597

2015 125: 185-194  

Steve P. Watson, Michael G. Tomlinson and Bernhard NieswandtAkbar, Cheryl S. Sultan, Craig E. Hughes, Christoph Kleinschnitz, Guido Stoll, Leonard L. Dragone, Deya Cherpokova, Markus Bender, Martina Morowski, Peter Kraft, Michael K. Schuhmann, Sarah M. thrombosis and ischemic stroke in miceSLAP/SLAP2 prevent excessive platelet (hem)ITAM signaling in 

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