A-Disintegrin-And-Metalloproteinase (ADAM) 10 Activity on Resting

Feb 3, 2016 - The primary platelet collagen receptor, glycoprotein VI (GPVI), plays an important role in platelet activation and thrombosis. The ectod...
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A‑Disintegrin-And-Metalloproteinase (ADAM) 10 Activity on Resting and Activated Platelets Adam Facey,† Isaac Pinar,‡ Jane F. Arthur,† Jianlin Qiao,† Jing Jing,† Belden Mado,† Josie Carberry,‡ Robert K. Andrews,† and Elizabeth E. Gardiner*,† †

Australian Centre for Blood Diseases, Monash University, Melbourne, Victoria, Australia 3004 Department of Mechanical and Aerospace Engineering, Monash University, Clayton, Victoria, Australia 3168

‡

ABSTRACT: The primary platelet collagen receptor, glycoprotein VI (GPVI), plays an important role in platelet activation and thrombosis. The ectodomain of human GPVI (sGPVI) is proteolytically shed from human platelets by adisintegrin-and-metalloproteinase 10 (ADAM10). In this study, we used a novel ADAM10-sensitive fluorescence resonance energy transfer sensor to analyze ADAM10mediated shedding of GPVI from human platelets in response to the exposure of GPVI ligands collagen-related peptide (10 μg/mL), collagen (10 μg/mL), and convulxin (0.1 μg/mL) to shear stress (1000−10000 s−1, 5 min), or a generic activator of metalloproteinases, N-ethylmaleimide (NEM, 5 mM). Elevated shear, NEM, or ligand engagement of GPVI all induced shedding of GPVI, as detected by release of sGPVI; however, only shear or NEM significantly increased ADAM10 enzyme activity. ADAM10 activity was also detectable on the surface of thrombi formed on a collagen-coated surface under flow conditions. Our findings indicate different mechanisms regulate shear- and ligand-induced shedding and shear forces found within the vasculature can regulate ADAM10 activity.

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GPVI shedding can be blocked by inclusion of broad spectrum metalloproteinase inhibitors or a selective ADAM10 inhibitor.8,14 Binding of GPVI by ligand triggers an intracellular signaling pathway that leads to platelet aggregation, degranulation, and secretion and initiates metalloproteolysis of the GPVI ectodomain. Significantly, GPVI shedding initiated by exposure to elevated shear occurs in a manner that is independent of ligand engagement, intracellular signaling pathway activation, and degranulation.8 It is likely that this process regulates the responsiveness of platelets to collagen and may represent a negative feedback loop for controlling platelet activation responses.15 ADAM metalloproteinases are type I transmembrane proteases that proteolytically regulate surface and soluble levels of growth factors, cytokines, adhesion receptors, and ectoenzymes. The ADAMs are closely related to the matrix metalloproteinase (MMP) family of enzymes; however, ADAMs are primarily responsible for regulating “life-essential” molecules, particularly on vascular cells,16 and have a repertoire of substrates tighter than that of MMPs.17 ADAM10 and the related ADAM17 are ubiquitously expressed on many cell types, and their regulation in vivo remains an open question. ADAM10 is expressed in nucleated cells as an ∼85 kDa zymogen consisting of an N-terminal prodomain, a protease domain containing a conserved metal ion coordination motif, a

n response to vascular injury or changes to biorheological conditions, circulating platelets rapidly become activated and adhere to exposed subendothelial matrix proteins such as von Willebrand factor and collagen. These adherent platelets become activated, spread, and release the contents of storage organelles that contain prothrombotic substances, including serotonin and ADP, and the α-granules, which contain a wide range of bioactive proteins such as P-selectin and CD40L, adhesive proteins, coagulation proteins, fibrinolytic proteins, growth factors, cytokines, and chemokines.1 Active platelets also engage with neutrophils, enhancing neutrophil activation and recruitment and contributing to inflammatory responses.2,3 An additional consequence of these activation processes is the triggering of metalloproteinase-mediated ectodomain shedding of platelet surface substrates, an irreversible mechanism releasing receptors and other substrates from the platelet surface.4 Shedding of GPVI from human platelets is regulated primarily by ADAM10, reducing the responsiveness of platelets to collagen and increasing bleeding risk.5,6 Levels of intact (∼65 kDa) GPVI on nonactivated platelets are stable; however, metalloproteolytic loss of the ectodomain portion (∼55 kDa) of GPVI can be rapidly induced (within seconds to minutes) by treating platelets with GPVI ligands, calmodulin inhibitors, or active Factor X (FXa) or exposure to elevated shear rates in the range of 3000−10000 s−1,7−9 which may be encountered in healthy and diseased vessels.10−12 Roles for the related protease, ADAM17, as well as other proteases in the shedding of GPVI from murine platelets have also been described.13 © 2016 American Chemical Society

Received: October 10, 2015 Revised: February 1, 2016 Published: February 3, 2016 1187

DOI: 10.1021/acs.biochem.5b01102 Biochemistry 2016, 55, 1187−1194

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Biochemistry

yl)-L-2,3-diaminopropionyl-RSSSR-NH2] were purchased from R&D Systems (Minneapolis, MN). Lepirudin (rHirudin) was from Bayer HealthCare (Berlin, Germany). The broad spectrum metalloproteinase inhibitor GM6001, arginineglycine-aspartic acid (RGD) peptide, and N-ethylmaleimide (NEM) were purchased from Calbiochem, and GI254023, a selective ADAM10 inhibitor,34 was a kind gift from A. Ludwig (University of Aachen, Aachen, Germany). FRET sensors contained peptides with sequence matching the ADAM10 cleavage site within GPVI (S236PAGPARQYYTKG248) and a Q243K variation (S236PAGPARKYYTKG248) that was not cleaved by ADAM10.14 Each peptide had an Nterminal azidoacetyl alanine and Cy3 fluorophore and a Cterminal quencher BHQ2, similar to those of a recently described thrombin-sensitive FRET reagent.35 FRET sensors were manufactured by Auspep (Melbourne, Australia). Reagents were freshly made as 10 mM stock solutions in dimethyl sulfoxide (DMSO). Human Blood Collection. Blood collection was conducted with the approval of the Monash University Standing Committee on Ethics in Research Involving Humans, and informed consent was obtained from all participants in accordance with the Declaration of Helsinki. Washed platelets were prepared from acid-citrate-dextrose-anticoagulated blood and resuspended in Tyrode’s buffer [0.36 mM NaH2PO4·H2O, 5.5 mM glucose, 138 mM NaCl, 12 mM NaHCO3, 1.8 mM CaCl2, 0.49 mM MgCl2, and 2.6 mM KCl (pH 7.4)] at a platelet count of 3 × 108 mL−1,7 and treated with 1 mM RGD peptide (final concentration) to block αIIbβ3 integrin-mediated platelet aggregation. Some platelet suspensions were exposed to various levels of shear stress for up to 5 min in a cone-and-plate viscometer as previously described.8 ADAM10 Activity Assays. To assess the utility of FRET sensors, 30 nM rADAM10 or rADAM17 was mixed with 5 μM ADAM10 sensor or Q243K sensor in 25 mM Tris-HCl (pH 7.4) containing 2.5 μM ZnCl2. Assays measuring platelet ADAM activity were performed in duplicate in 96-well white plates (Optiplate, PerkinElmer) by continuously monitoring real-time increases in fluorescence intensity, at excitation and emission wavelengths of 500 and 590 nm, respectively, in a FLUOstar Optima fluorescence plate reader (BMG Labtech, Offenburg, Germany) maintained at 37 °C. Enzyme activity measurements used 100 μL of human washed platelets (107 mL−1) in Tyrode’s buffer to which was added 100 μL of 10 mM Tris-HCl/saline buffer (pH 7.4) containing (final concentrations) 2.5 μM ZnCl2 and 1 mM Arg-Gly-Asp (RGD) peptide to block engagement of αIIbβ3 integrin and platelet aggregation. Platelets were exposed to uniform shear ranging from 1000 to 10000 s−1 using a cone-and-plate viscometer8 for up to 5 min or were mixed with buffer containing 10 μg/mL CRP, 5 mM NEM, 100 μM GM6001, or 2 μM GI254023 or vehicle, and background fluorescence levels were obtained from mixtures containing buffer, FRET sensor, and 50 mM ethylenediaminetetraacetic acid (EDTA). Enzyme activity was monitored in all wells immediately after addition of untreated or shear-exposed platelets to wells containing all other reagents. Initial velocities of hydrolysis were determined at various substrate concentrations (0−50 μM), and the Michaelis constant (Km) and maximal velocity (Vmax) were calculated using Prism GraphPad 6.0 (GraphPad Software, La Jolla, CA). To assess association of the ADAM10 sensor with the platelet surface under resting and activated conditions, platelet suspensions were mixed with ADAM10 sensor and Cy3

disintegrin/cysteine-rich domain, a transmembrane domain, and a cytoplasmic tail. In nucleated cells, the prodomain maintains the enzyme in a latent form and assists in intracellular trafficking.18 Exposure to a range of inflammatory stimuli results in the removal of the ADAM10 prodomain and increased levels of active ADAM10 at the cell surface typically over the course of hours to days. Interestingly, the cytoplasmic domain of ADAM10 and prodomain processing are not essential for the rapid activation of ADAM10-dependent shedding events;19 however, a clear role for the ADAM10 transmembrane domain in ADAM10 activity and substrate selectivity, possibly via interaction with one or more tetraspanin membrane proteins,20−23 is emerging. On platelets, ADAM10mediated cleavage of GPVI occurs within seconds of exposure to agonists, implying that ADAM10 is already present on the surface of unactivated circulating platelets. GPVI, however, is not constitutively shed from circulating platelets,14 suggesting that one or more as yet undefined mechanisms protect GPVI from ADAM10-mediated cleavage until shedding is initiated. Antiplatelet reagents such as aspirin, clopidogrel, and prasugrel are key clinical therapeutics used in situations in which individuals are at risk of thrombosis, myocardial infarction, and stroke; however, these reagents carry significant risk of bleeding.24 New antiplatelet reagents would ideally have strong antithrombotic properties but negligible effects on normal hemostasis. GPVI represents an attractive new target as an antiplatelet reagent, because it is expressed only on platelets and platelet precursor cells in bone marrow (megakaryocytes), and GPVI blockade has demonstrated efficient antithrombotic potential in experimental models of thrombosis without enhancing pathological bleeding.25 However, understanding how GPVI is regulated in normal hemostasis and in pathophysiological settings is imperative for the design of new therapeutics that target initiation of thrombus formation.25,26 Platelet GPVI expression is enhanced in patients with acute coronary syndrome,27 and platelet GPVI as well as plasma sGPVI may serve as early markers for an imminent acute coronary event28 or stroke29,30 or other cardiovascular conditions5,31 and may also reflect a bone marrow condition in hematological malignancies.32 Here we use a new GPVI-based FRET sensor with the Cy3BHQ2 fluorophore/quencher pair for direct continuous measurement of platelet-associated ADAM10 activity in live cells. The sensor was first validated in vitro with recombinant (r) enzymes and in assays involving washed platelets. The ADAM10 sensor is useful for real-time assessment of ADAM10 activity on the surface of resting and activated platelets. Using this sensor, we provide evidence of key differences between shear- and ligand-mediated GPVI shedding by ADAM10 and demonstrate the utility of this sensor for monitoring ADAM10 activity during thrombus formation.

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EXPERIMENTAL PROCEDURES Materials. The GPVI-specific agonist, collagen-related peptide (CRP), with amino acid sequence GCO(GPO)10GCOG-NH2 where O represents hydroxyproline, was prepared as described previously.33 Purified convulxin (a GPVI agonist) from the venom of the South American rattlesnake Crotalus durissus terrif icus was a kind gift from K. Clemetson (Berne, Switzerland). Equine collagen (type I) was from Chrono-Log (Havertown, PA). Human rADAM10 and rADAM17 and the ADAM10/ADAM17 FRET biosensor [7(methoxycoumarin-4-yl)acetyl-PLAQAV-N-3-(2,4-dinitrophen1188

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Figure 1. Novel ADAM10 FRET sensor that is cleaved by rADAM10 but not rADAM17. (A) Schematic of the ADAM10 sensor showing a peptide with a sequence matching that of the GPVI ectodomain, a Cy3 fluorophore conjugated at the N-terminus, and a BHQ2 quencher group at the Cterminus (top). The site of cleavage by rADAM1014 is indicated, and an N-terminal azido group is also shown. Cleavage of this sequence releases the quencher group (bottom), and the resultant fluorescence can be quantified. (B) ADAM10 sensor (10 μM) was mixed with 10 μM rADAM10 or rADAM17 in buffer containing ZnCl2, and the increase in fluorescence over time was measured and corrected for background fluorescence, measured in wells containing substrate alone. (C) A commercial ADAM10/17 FRET sensor (10 μM) was mixed with 10 μM rADAM10 or rADAM17 as described for panel B, and fluorescence activity was monitored at excitation and emission wavelengths of 320 and 405 nm, respectively. (D) Utility of the ADAM10 sensor or a control sensor containing a Q243K variation to be cleaved by 10 μM rADAM10 or rADAM17. Cleavage of the ADAM10 sensor was blocked by inclusion of 2 μM GI254023 (GI). Data are representative of at least 10 experiments, showing mean values of triplicate measurements ± the standard deviation.

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fluorescence associated with platelets was quantified by flow cytometry using a FACSCalibur instrument and CellQuest Pro software (BD Biosciences, San Jose, CA) and a platelet gate defined by forward/side scatter and confirmed by positive staining with a PE-labeled anti-αIIb antibody (CD41a, eBioscience, San Diego, CA). Measurement of sGPVI. sGPVI in the supernatant of samples that were double centrifuged after addition of 50 mM EDTA to prevent shedding was measured by an ELISA as described previously.36 sGPVI was calculated as the mean ± the standard deviation (SD) compared with a standard curve using the rGPVI ectodomain in GPVI-depleted plasma. ADAM10 Activity on Thrombi. Blood was collected from healthy donors into a syringe containing (final concentration) 800 units/mL thrombin inhibitor lepirudin (recombinant hirudin) and passed through a microcapillary that was coated with 100 μg/mL bovine type I collagen at a steady input wall shear rate of 1800 s−1. Thrombi were visualized by differential interference contrast (DIC) using a Nikon A1R Plus instrument with a Piezo z-stage and perfect focus system (Nikon, Chiyoda, Tokyo, Japan). Excitation of the ADAM10 sensor was achieved via a 488 nm argon ion laser. Following the formation of thrombi, capillaries were perfused with Tyrode’s buffer containing 5 μM ADAM10 sensor and fluorescence was recorded and analyzed with NIS-Elements (Nikon). The change in the intensity of fluorescence over time was graphed using NIS software.

RESULTS Cogency of the ADAM10 Sensor. The FRET ADAM10 sensor (Figure 1A) was initially tested using rADAM10 and rADAM17 and real-time monitoring of the fluorescent signal using a fluorescent plate reader. Hydrolysis of the ADAM10 sensor by rADAM10 produced a 5−10-fold increase above the fluorescent signal obtained using rADAM17 over 60 min (Figure 1B,D) and was blocked by inclusion of ADAM10specific inhibitor GI254023 at 2 μM. Equivalent concentrations of rADAM17 could cleave a commercial ADAM10/17 FRET sensor (Figure 1C), indicating rADAM17 was active. A linear reaction curve was obtained over 1 h, and the rate of peptide cleavage was essentially constant. No sensor cleavage was detected in the absence of enzyme, and cleavage was completely inhibited by 100 μM GM6001 (data not shown). A kcat/Km of (1.1 ± 0.16) × 104 M−1 s−1 was determined (n = 4), which is in good agreement with a previously published kcat/ K m value of rADAM10 for an EDANS-LAQAVRSSS(DABCYL)-NH2 peptide [(2.5 ± 0.7) × 104 M−1 s−1]37 and within the range of values obtained using a multiplex approach to FRET analysis of metalloproteinase activity.38 As expected, substrate cleavage by rADAM10 was disrupted by inclusion of a Q to K variation at the P′ position (Figure 1D) as previously shown for both the GPVI Q243K peptide and transfected cells expressing GPVI Q243K.14 ADAM10 Activity on Platelets. Levels of ADAM10 activity on resting and activated platelets were assessed by mixing washed platelets with the ADAM10 sensor or Q243K 1189

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Biochemistry sensor and monitoring increases in fluorescence intensity as described above. Figure 2A shows that ADAM10 activity was

100% cleavage of the sensor, and from this value, a sensor specific activity (relative fluorescence units per picomole) was calculated and converted to picomole amounts. Exposure to shear rates at or above 3000 s−1 or to 10000 s−1 for >1 min increased the rate and extent of cleavage of the ADAM10 sensor (data not shown). Exposure of platelets to 10000 s−1 shear induced a 3-fold increase in the level of ADAM10 sensor cleavage. In contrast, a GPVI-specific ligand, CRP, did not increase ADAM10 activity, although CRP did induce GPVI shedding. All ADAM10 activity was significantly inhibited by 2 μM GI254023 (selective ADAM10 inhibitor) or 100 μM GM6001 (Figure 2B). To explore the notion that GPVI ligand-induced shedding of GPVI did not alter platelet ADAM10 activity, we simultaneously compared release of sGPVI and ADAM10 activity on washed platelets treated with GPVI ligands (CRP, collagen, or convulxin) or NEM or exposed to pathophysiological shear. While exposure to shear or treatment with NEM induced a rapid significant increase in the level of ADAM10 sensor cleavage, treatment of platelets with GPVI ligands did not significantly increase ADAM10 activity above levels measured in nontreated platelets. However, treatment with CRP, collagen, or convulxin induced shedding of GPVI (Figure 3). Analysis of platelets from six separate donors showed that NEM treatment or exposure to elevated shear increased Vmax by 7- or 2.5-fold, respectively, whereas for CRP, collagen, and convulxin treatment, the apparent Vmax was equivalent to that of nontreated platelets (Table 1). Levels of sGPVI, however, increased above control levels across all three treatment groups (Table 1). These findings are consistent with the relatively slow rate of release of sGPVI in response to CRP, collagen, or convulxin treatment compared with shear exposure, and with the observation that shear-induced GPVI shedding does not require platelet signaling and activation.8 ADAM10 Activity on a Platelet or Thrombus Surface. To show that the ADAM10 sensor was detecting ADAM10 activity at the platelet surface, suspensions of platelet-rich plasma were treated with CRP or exposed to shear and mixed with ADAM10 sensor, and platelet-associated fluorescence was assessed by flow cytometry. Figure 4A shows detectable Cy3positive events on resting and CRP-treated platelets. An increased number of Cy3-positive events was detected in shear-exposed platelets, indicating the ADAM10 sensor was associated with platelets and cleaved, producing relative fluorescence intensity at levels consistent with data acquired in the fluorescence plate reader. Similarly, the cleavage of the ADAM10 sensor was inhibited by GM6001 (data not shown). The ability of the ADAM10 sensor to detect metalloproteinase activity on the surface of a forming thrombus was assessed by passing buffer containing ADAM10 sensor across thrombi formed from lepirudin-anticoagulated whole blood on collagen-coated microcapillaries. Figure 4B shows a rapid, time-dependent increase in ADAM10 activity at the surface of thrombi within 1−2 min, showing that ADAM10 activity is detectable on the surface of a thrombus. The increased fluorescence was metalloproteinase-specific, because there was minimal cleavage of the ADAM10 sensor in the presence of GM6001-containing buffer under the same conditions (Figure 4C). While the use of fluorescent substrates for thrombin has previously been reported for thrombus formation under flow conditions,35 this is the first demonstration of ADAM10 activity on a developing thrombus.

Figure 2. ADAM10 sensor detects ADAM10 activity on platelets. (A) Washed platelets were mixed with 10 μM ADAM10 sensor or Q243K sensor (Q243K), and fluorescence activity was measured over time and corrected for background fluorescence activity. Data are representative of findings from experiments performed using platelets isolated from six to eight healthy donors. (B) Washed platelets (3 × 108 mL−1) were mixed with 10 μM ADAM10 sensor in buffer alone or containing 5 mM NEM or 10 μg/mL CRP and either 100 μM GM6001 or 2 μM GI254023 as indicated. Some platelet suspensions were exposed to shear in a cone-and-plate viscometer (10000 s−1, 5 min). The fold change in ADAM10 sensor consumption (picomoles per minute) after 60 min was determined; ADAM10 activity in nontreated platelets (NT) was set to a value of 1. Data representative of 4−10 experiments analyzed using an unpaired one-way ANOVA comparing the means of each treatment group to the mean of the nontreated washed platelet group, with correction for multiple comparisons using Dunnett’s test (ns, no significance; ****p < 0.0001).

detectable in suspensions of nontreated platelets and the degree of cleavage of the substrate increased over the 1 h incubation period compared to that of the Q243K sensor. Increased activity of ADAM10 was observed when platelets were treated with NEM, a generic and potent activator of metalloproteinases.39,40 These data indicate that ADAM10 is active on a nonactivated platelet and that the ADAM10 sensor can monitor changes to ADAM10 activity triggered by treatment with NEM. To assess ADAM10 activity under conditions of elevated shear previously shown to induce GPVI shedding,8,41 platelet suspensions were exposed to shear rates of 10000 s−1 for 5 min (Figure 2B). Shear rates of 10000 s −1 approximate pathophysiological shear rates to which platelets and other blood cells are continuously exposed10 and have been measured in partially occluded coronary vessels.12 In these experiments, levels of fluorescence measured after 1 h in wells containing ADAM10 sensor and NEM-treated platelets were set to reflect 1190

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Figure 3. Ligand-induced shedding of GPVI occurs without a change in ADAM10 activity. Washed platelets (3 × 108 mL−1) from six healthy donors were mixed with 10 μM ADAM10 sensor in buffer alone (nontreated) or containing 5 mM NEM, 10 μg/mL CRP, 10 μg/mL collagen, or 0.1 μg/mL convulxin (CVX) or exposed to shear in a cone-and-plate viscometer (10000 s−1, 5 min). After 1 h, an aliquot of a platelet suspension was centrifuged and levels of sGPVI were determined by an ELISA (left). ADAM10 sensor fluorescence activity was monitored for 30 min as described and corrected for background fluorescence measured in control wells (right). A Wilcoxon signed-rank test comparing the means of each treatment group to the mean of the nontreated washed platelet group was performed. Shown are mean values ± the standard deviation for four to eight experiments with different donors. *p < 0.05. ***p < 0.005.

which GPVI is shed that is determined by the type of triggering event. Activation of platelets and of GPVI shedding by ligation of GPVI requires receptor clustering, dissociation of calmodulin from a binding site within the cytoplasmic tail of GPVI,7,47 and activation of an intracellular signaling cascade,48 all of which are likely to increase the time required to activate shedding processes. However, the respective amounts of CRP, collagen, and convulxin used in this study were sufficient to induce platelet aggregation within minutes and induced 2−4-fold increases in the level of sGPVI, confirming that ADAM10mediated shedding of GPVI had occurred (Figure 3 and Table 1). Thus, the data are consistent with different mechanisms governing shear-induced or ligand-induced shedding of GPVI that can be delineated by the ADAM10 sensor. Finally, the utility of the ADAM10 sensor was underscored upon analysis of ADAM10 activity on nontreated and treated platelet suspensions by flow cytometry or by probing the surface of experimental thrombi. Interestingly, ADAM10 sensor fluorescence on the surface of thrombi formed on collagen under shear conditions was not uniform, suggesting local differences in levels of ADAM10 activity that may correlate with sites of increased local shear and enhanced loss of platelet surface proteins such as GPVI. In future studies, it will be of great interest to assess ADAM10 activity within the thrombi both spatially and temporally under varying shear and flow conditions. In summary, the development of a novel ADAM10 sensor will allow assessment of the activity of this enzyme on platelets and other cells. Taken together, data from the enzyme activity assay and sGPVI measurements reveal different regulatory mechanisms of ADAM10-mediated shedding of GPVI on human platelets. The implication of these findings is that shear forces such as those experienced by a membrane surface in flowing blood could potentially regulate endogenous ADAM activity in a manner independent of GPVI ligation. Pathophysiological levels of shear may directly elevate ADAM activity, thereby controlling platelet GPVI expression. Future experiments will explore advantages of therapeutically modulating shear-triggered platelet receptor shedding possibly by targeted delivery of therapeutics to regions of high shear stress.49 Additionally, the ADAM10 sensor can be used to further investigate platelet physiology and may also be applicable to other systems in which ADAM10 activity regulates physiologically relevant processes.

Table 1. Variation in Platelet ADAM10 Activity after Treatment with NEM or GPVI Ligand or Exposure to Elevated Shear Ratesa ADAM10 activity

basal platelet ADAM10 activity 5 mM NEM 10 μg/mL CRP 10 μg/mL collagen 0.1 μM convulxin 10000 s−1 shear (5 min)

Vmax (pmol/s)

Km (μM)

0.73

1.23

1

5.63 0.81 0.85 0.7 1.86

4.10 2.03 ND ND 1.60

7.7 1.1 1.16 0.96 2.5

x-fold increase

cleavage product sGPVI (ng/mL)

x-fold increase

21

1

205 48 56 77 82

10 2.3 2.6 3.6 3.9

a Cleavage product refers to sGPVI. x-fold increase refers to the increase in Vmax over the basal Vmax rate. ND means not determined.

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DISCUSSION In this study, we have (i) developed a novel FRET substrate to measure ADAM10 activity directly and rapidly on human platelets and thrombi and (ii) demonstrated different mechanisms of shear- versus ligand-induced shedding of GPVI. We used this ADAM10 sensor to monitor ADAM10 activity in a cell-free system using purified rADAM10 and on live cells in real time. Under the experimental conditions used here, the ADAM10 sensor was specifically cleaved by both rADAM10 and platelets and blocked by an ADAM10-selective inhibitor. Under the same conditions, cleavage by rADAM17 was minimal. Others have previously designed and applied reagents to study extracellular ADAM activity,37,42,43 as well as a genetically encoded FRET biosensor that monitors both extraand intracellular ADAM activity in transfected cells,44 and these reagents report on ADAMs as well as MMP activity with varying levels of specificity. This is consistent with the broad specificity for substrates exhibited by ADAM enzymes and frequent “substrate sharing” by ADAMs.45,46 One important finding from our analysis was that the ADAM10 sensor detected basal ADAM10 activity on platelets that had not been treated, supporting the concept that platelets express mature ADAM10 on their surface.8 Exposure to shear dramatically increased the level of ADAM10 activity, as did treatment with NEM; however, in contrast, GPVI engagement by CRP, collagen, or convulxin did not alter levels of ADAM10 activity, implying inherent differences in the mechanism by 1191

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Figure 4. ADAM10 sensor that associates with platelets in suspension and with the surface of thrombi. (A) Platelet-rich plasma was left untreated (resting), incubated with 10 μg/mL CRP, or exposed to shear (10000 s−1 for 5 min) in a cone-and-plate viscometer and then mixed with 5 μM ADAM10 sensor. Numbers of platelets with detectable fluorescence activity representing ADAM10 activity were quantified by flow cytometry. Data are representative histograms from one donor (n = 3 individual donors). (B and C) Thrombi were formed by passing lepirudin-treated whole blood over collagen-coated microcapillaries at a rate of 1800 s−1 for 3 min. Fields of thrombi were then perfused with buffer containing (B) 5 μM ADAM10 sensor [fluorescence intensity in the field imaged over time (scale bar of 25 μm)] or (C) 5 μM ADAM10 sensor in the presence or absence of metalloprotease inhibition by 100 μM GM6001 (right panels). The pump was turned off when the buffer reached the microcapillary, so that the monitoring of fluorescence was under no-flow conditions (scale bars are 25 μm). Images were captured after 5 min of no flow. The direction of flow is from right to left in all confocal images.

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AUTHOR INFORMATION

Corresponding Author

REFERENCES

(1) Golebiewska, E. M., and Poole, A. W. (2015) Platelet secretion: From haemostasis to wound healing and beyond. Blood Rev. 29, 153− 162. (2) Jenne, C. N., and Kubes, P. (2015) Platelets in inflammation and infection. Platelets 26, 286−292. (3) Andrews, R. K., Arthur, J. F., and Gardiner, E. E. (2014) Neutrophil extracellular traps (NETs) and the role of platelets in infection. Thromb. Haemostasis 112, 659−665. (4) Fong, K. P., Barry, C., Tran, A. N., Traxler, E. A., Wannemacher, K. M., Tang, H.-Y., Speicher, K. D., Blair, I. A., Speicher, D. W., Grosser, T., and Brass, L. F. (2011) Deciphering the human platelet sheddome. Blood 117, e15−e26. (5) Al-Tamimi, M., Arthur, J. F., Gardiner, E. E., and Andrews, R. K. (2012) Focusing on plasma glycoprotein VI. Thromb. Haemostasis 107, 648−655. (6) Massberg, S., Gawaz, M., Gruner, S., Schulte, V., Konrad, I., Zohlnhofer, D., Heinzmann, U., and Nieswandt, B. (2002) A crucial role of glycoprotein VI for platelet recruitment to the injured arterial wall in vivo. J. Exp. Med. 197, 41−49. (7) Gardiner, E. E., Arthur, J. F., Kahn, M. L., Berndt, M. C., and Andrews, R. K. (2004) Regulation of platelet membrane levels of glycoprotein VI by a platelet-derived metalloproteinase. Blood 104, 3611−3617. (8) Al-Tamimi, M., Tan, C., Qiao, J., Pennings, G. J., Javadzadegan, A., Yong, A. S. C., Arthur, J. F., Davis, A. K., Jing, J., Mu, F. T., Hamilton, J. R., Jackson, S. P., Ludwig, A., Berndt, M. C., Ward, C. M., Kritharides, L., Andrews, R. K., and Gardiner, E. E. (2012)

*Telephone: 61-3-9903-0756. Fax: 61-3-9903-0228. E-mail: [email protected]. Funding

This study was supported by the National Health and Medical Research Council of Australia, the National Heart Foundation of Australia, and the China Scholarship Council. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Mr. H. Qayum and Mrs. C. Berndt for technical assistance and Dr. Andreas Ludwig for the kind gift of GI254023.

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ABBREVIATIONS ACD, acid-citrate dextrose anticoagulant solution; ADAM, a disintegrin and metalloproteinase; ANOVA, analysis of variance; CGS, citrate/glucose/saline solution; CRP, collagenrelated peptide; ELISA, enzyme-linked immunosorbent assay; FRET, fluorescence resonance energy transfer; GP, glycoprotein; NEM, N-ethylmaleimide; PPP, platelet-poor plasma; PRP, platelet-rich plasma; sGPVI, soluble GPVI; TS, Tris saline. 1192

DOI: 10.1021/acs.biochem.5b01102 Biochemistry 2016, 55, 1187−1194

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DOI: 10.1021/acs.biochem.5b01102 Biochemistry 2016, 55, 1187−1194