Snake venom metalloproteinases, crotarhagin and ...

© 2007 Schattauer GmbH, Stuttgart

Platelets and Blood Cells

Snake venom metalloproteinases, crotarhagin and alborhagin, induce ectodomain shedding of the platelet collagen receptor, glycoprotein VI Lakshmi C. Wijeyewickrema1, Elizabeth E. Gardiner2, Masaaki Moroi3, Michael C. Berndt2, Robert K. Andrews2 1

Department of Biochemistry and Molecular Biology and 2Department of Immunology, Monash University, Alfred Medical Research & Education Precinct, Melbourne, Victoria, Australia; 3Department of Protein Biochemistry, Institute of Life Science, Kurume University, Kurume, Fukuoka, Japan

Summary Glycoprotein (GP)VI, that binds collagen, together with GPIbIX-V which binds vonWillebrand factor, forms an adheso-signalling complex on platelets that initiates thrombus formation in haemostasis and thrombosis. In this study, we show that two snake venom metalloproteinases,crotarhagin and alborhagin,induce ectodomain shedding of GPVI by a mechanism that involves activation of endogenous platelet metalloproteinases.Alborhagin is a viper venom metalloproteinase from Trimeresurus albolabris, while crotarhagin is a previously undescribed toxin from the rattlesnake Crotalus horridus horridus (~60-kDa non-reduced and reduced). Like alborhagin, crotarhagin induces aggregation in human platelet-rich plasma (maximal activity, ~0.3 µg/ ml). Aggregation of washed platelets was inhibited by soluble GPVI ectodomain expressed as an Fc-fusion protein, confirming Keywords ADAMS, adhesion receptors, snake venoms, platelet glycoproteins

Introduction Glycoprotein (GP)VI, which binds collagen, and GPIb-IX-V, which binds von Willebrand factor (VWF), form a unique adheso-signalling complex on platelets. Ligand binding to GPVI or GPIb-IX-V leads to platelet activation, and activation of the integrin, αIIbβ3, that binds fibrinogen or VWF and mediates platelet aggregation (1–4). GPVI and GPIb-IX-V are critical in triggering pathophysiological thrombus formation at arterial shear rates (5–10), and are under investigation as anti-thrombotic targets in human diseases such as heart attack or stroke. Platelet GPVI levels may also be a marker for acute coronary syndrome (11). Snake toxins of the metalloproteinase-disintegrin or C-type lec-

crotarhagin targeted GPVI.Treating washed platelets with crotarhagin or alborhagin resulted in time-dependent loss of surface GPVI and the appearance of an ~55-kDa soluble GPVI fragment in supernatants. Crotarhagin also induced shedding in GPVItransfected RBL-2H3 cells. Crotarhagin-induced shedding was metalloproteinase-dependent (inhibited by EDTA), but also blocked by inhibitors of GPVI signalling (Src kinase inhibitors, PP1 or PP2, or Syk inhibitor, piceatannol), indicating shedding required GPVI-dependent platelet activation.Together, the data suggest that the rattlesnake metalloproteinase, crotarhagin, and the viper toxin alborhagin, induce GPVI shedding by a mechanism involving activation of endogenous platelet metalloproteinases rather than direct cleavage of GPVI.

Thromb Haemost 2007; 98: 1285–1290

tin-like families that target GPVI and/or GPIbα (the major ligand-binding subunit of GPIb-IX-V) have provided valuable selective probes for GPVI- or GPIb-IX-V-dependent platelet aggregation (12–16). GPVI of the immunoglobulin (Ig) receptor superfamily (1, 2), consists of two extracellular Ig domains, a mucin domain, a transmembrane domain, and a cytoplasmic tail, that binds calmodulin (via a juxtamembrane positively-charged sequence) and Src family kinases (via a Pro-rich sequence) (17–19). GPVI is associated with Fc receptor γ-chain (FcRγ), which signals via an ITAM (immunoreceptor-based tyrosine activation motif) in the cytoplasmic tail; ligand-induced cross-linking of GPVI/FcRγ leads to ITAM-dependent activation of Syk (18, 20). Binding of collagen,

Correspondence to: Robert K. Andrews Department of Immunology, Monash University Alfred Medical Research and Education Precinct (AMREP) Commercial Road, Melbourne, Australia Tel.: +61 3 9903 0136, Fax: +61 3 9903 0038 E-mail: [email protected]

Received June 11, 2007 Accepted after resubmission September 5, 2007 Prepublished online November 9, 2007 doi:10.1160/TH07–06–0402

1285

Downloaded from www.thrombosis-online.com on 2015-02-25 | ID: 1000490121 | IP: 130.194.20.173 For personal or educational use only. No other uses without permission. All rights reserved.

Wijeyewickrema et al. Crotarhagin-mediated shedding of platelet GPVI

collagen-related peptide (CRP), or the snake toxin, convulxin, to GPVI induces platelet activation and dissociation of calmodulin from the cytoplasmic tail, resulting in shedding of a soluble ~55-kDa GPVI ectodomain fragment (2, 21–23). Ligand-induced GPVI shedding is metalloproteinase-dependent, and inhibited by EDTA or the metalloproteinase inhibitor, GM6001. Shedding also requires platelet activation, and is blocked by pharmacological inhibitors of GPVI signalling, PP1 or PP2 (Src inhibitors) or piceatannol (Syk inhibitor) (21). GPVI shedding provides a mechanism for down-regulating GPVI function post-adhesion (24, 25). Cleavage of GPVI within an extracellular membrane-proximal sequence, PAR^Q243YY, involves ADAM10, of the ADAM family of mammalian metalloproteinase-disintegrins analogous to reptilian metalloproteinase-disintegrins (26). Immunoblotting with anti-GPVI cytoplasmic tail antibody (that recognizes both intact GPVI and membrane-associated remnant) shows that essentially all GPVI on circulating platelets is intact, and that ligand-induced shedding is tightly controlled (26). We previously showed that a metalloproteinase-disintegrin, alborhagin, from the viper Trimeresurus albolabris targets GPVI, and induces platelet activation and GPVI-dependent aggregation (27). Here, we show that both alborhagin and crotarhagin, a rattlesnake metalloproteinase from Crotalus horridus horridus, induce GPVI proteolysis. However, rather than direct cleavage, this shedding mechanism involves platelet activation, and activation of endogenous platelet sheddases.

Materials and methods Materials Crotalus horridus horridus and Trimeresurus albolabris venom was from Sigma (St Louis, MO, USA). PP1 and PP2 (Src inhibitors), piceatannol (Syk inhibitor), and N-ethylmaleimide (NEM) were from Calbiochem (La Jolla, CA, USA). The GPVIspecific agonist, collagen-related peptide (CRP), GCO(GPO)10GCOG-NH2 (O, hydroxyproline), was prepared as described (21, 28). Ristocetin was from Helena Laboratories (Mount Waverley, Australia). Convulxin from Crotalus durissus terrificus was a gift from Dr. K. Clemetson (Berne, Switzerland). CHH-B, a GPIbα-targeting protein that inhibits VWF binding, has been published (29). GPVI-Fc, consisting of the GPVI ectodomain (residues 21–234, excluding the signal sequence) and human Fc expressed as a secreted fusion protein in HEK-293 cells has been published (30, 31). The monoclonal antibody, 6B12, that immunoblots human platelet GPVI at ~2 µg/ml (100-fold dilution of hybridoma medium), was a gift from Dr. M. Kahn (Philadelphia, PA, USA) (21, 28). Anti-GPIbα antibody, AK2 (32–34), and rabbit anti-mocarhagin IgG that immunoblots mocarhagin and alborhagin (27, 35) have been previously reported. Purification of human fibrinogen, and the assay for metalloproteinase-dependent cleavage were performed using published methods (27, 35).

Figure 1: Effect of crotarhagin on aggregation of human platelets. A) Purified crotarhagin analyzed by SDS-5–20%-polyacrylamide gel electrophoresis under reducing conditions, and stained with Coomassie blue. B) Western blot of crotarhagin with anti-mocarhagin IgG. C) Aggregation of human platelet-rich plasma induced by crotarhagin. The lower trace contained 0.3 µg/ml crotarhagin and 10 mM EDTA. D) Aggregation of platelet-rich plasma induced by 0.3 µg/ml (final concentration) crotarhagin (arrows) or 1.5 mg/ml (final concentration) ristocetin (open arrows), after pre-incubating platelets for 6 minutes at 37oC with the antiGPIbα antibody, AK2 (20 µg/ml) or CHH-B (10 µg/ml). E) Aggregation of washed platelets (5 x 108/ml) in Tyrode’s buffer induced by crotarhagin (0.5 µg/ml), after pre-incubating platelets with TS buffer (upper trace), anti-GPIbα antibody AK2 (20 µg/ml), or Src kinase inhibitor PP2 (10 µM) for 5 minutes at 37oC. Results are representative of three experiments with separate donors.

1286



Purification of alborhagin and crotarhagin Alborhagin was purified from Trimeresurus albolabris venom by heparin-affinity and gel-filtration chromatography (27). Crotarhagin was purified from Crotalus horridus horridus venom using Ni2+-agarose chromatography (35). Lyophilized venom (0.1 µg) in 10 ml TS buffer (0.01 M Tris-HCl, 0.15 M NaCl, pH 7.4) was loaded at ~30 ml/hour(h) onto a 10 x 1-cm Ni2+-agarose column at 22oC. Bound protein was eluted by a linear 100-ml 0–30 mM imidazole gradient in TS buffer. Fractions (5 ml) eluting at ~10 mM imadazole containing crotarhagin, ~60-kDa by SDS-5–20%-PAGE, were concentrated using a YM30 Amicon ultrafiltration device (Danvers, MA, USA), and gel-filtered on a 40 x 2.5-cm Sepharose-CL-6B column in TS buffer at 25 ml/h. Effect of snake venom proteins on platelet aggregation Aggregation in citrated human platelet-rich plasma (PRP) was carried out in a Lumiaggregometer (Chronolog, Havertown, PA,

USA) stirred at 900 rpm at 37oC (26–28, 31, 33). PRP was treated with TS buffer, or (final concentrations) crotarhagin (0.1–0.36 µg/ml), CHH-B (10 µg/ml), or AK2 (20 µg/ml), for 6 minutes (min) at 37oC, prior to adding ristocetin (1.5 mg/ml) or crotarhagin (0.3 µg/ml). In parallel assays, EDTA (10 mM) was added to PRP prior to adding crotarhagin. Aggregation of washed human platelets (108/ml) in Tyrode’s buffer (28) was induced by crotarhagin (0.3 µg/ml), after pre-treating platelets with TS buffer, AK2 (20 µg/ml) or Src inhibitor, PP2 (10 µM), for 5 min at 37oC. In other assays, crotarhagin (0.3 µg/ml) or CRP (5 µg/ml) were pre-incubated with TS buffer or GPVI-Fc (10 µg/ml) for 5 min at 22oC, prior to adding to platelets. Shedding of human platelet GPVI Shedding of GPVI from washed platelets (108/ml) in Tyrode’s buffer was induced by either CRP (2.5 µg/ml), convulxin (1 µg/ ml), alborhagin (1 µg/ml), or crotarhagin (0.1–2.0 µg/ml), and analyzed as previously described (21, 26, 28). After 30 min at

Figure 2: Crotarhagin and alborhagin induce GPVI ectodomain shedding. A) Washed platelets (5 x 108/ml) in Tyrode’s buffer were treated with CRP (2.5 µg/ml), crotarhagin (0.5 µg/ml), or alborhagin (1 µg/ml) for 30–180 minutes at 22 oC. Platelet suspensions were made 10 mM in EDTA, centrifuged, and supernatants immunoblotted with anti-GPVI antibody, 6B12. Platelet lysate is included for molecular weight comparison of the shed fragment with intact GPVI. Data are representative of three separate experiments with different donors. B) Dose response for crotarhagin-induced shedding of GPVI from washed platelets for 30 minutes, in the absence or presence of 10 mM EDTA, and platelet supernatants immunoblotted with anti-GPVI antibody 6B12. C) Washed platelets (5 x 108/ml) in Tyrode’s buffer were treated with TS buffer or crotarhagin (0.5 µg/ml) [top panel], or alborhagin (1 µg/ml) [bottom panel] for 30 minutes at 22oC. Platelets were pre-incubated with TS buffer, Src kinase inhibitors, PP1 or PP2 (10 µM, final concentration), or Syk inhibitor, piceatannol (30 µg/ml, final concentration) for 5 minutes prior to addition of crotarhagin or alborhagin. Platelet suspensions were made 10 mM in EDTA, centrifuged, and supernatants immunoblotted with anti-GPVI antibody, 6B12. GPVIf is the soluble ~55-kDa GPVI fragment.

1287



22oC, samples were made 10 mM in EDTA, platelets were pelleted by centrifugation (14,000 g; 5 min; 22oC), and platelet supernatants immunoblotted with anti-GPVI antibody, 6B12. Parallel assays included EDTA (10 mM), Src inhibitors, PP1 or PP2 (10 µM), or Syk inhibitor, piceatannol (30 µg/ml) (21, 26, 28). GPVI-transfected cells The effect of crotarhagin on GPVI shedding was further evaluated using GPVI-transfected RBL-2H3 cells, rat basophilic leukemic cells that contain no endogenous GPVI, but express FcRγ required for GPVI expression, cultured as previously described (18, 26). GPVI-transfected cells were washed twice in PBS (0.01 M NaH2PO4, 0.15 M NaCl, pH 7.4) and treated with either NEM (5 mM) or crotarhagin (0.5–2.0 µg/ml) for 60 min at 37°C. EDTA (10 mM) was added, and supernatants immunoblotted with anti-GPVI antibody 6B12, as described for GPVI shedding from platelets.

Results

Figure 3: Crotarhagin interaction with GPVI-transfected RBL-2H3 cells and recombinant GPVI. A) GPVI-transfected RBL-2H3 cells were treated with TS buffer, NEM (5 mM), or crotarhagin for 60 minutes at 37°C, cell supernatants were then made 10 mM in EDTA, and immunoblotted with anti-GPVI antibody 6B12. (E, crotarhagin-induced shedding in the presence of 10 mM EDTA). Data are representative of two separate experiments. B) Washed human platelets (5 x 108/ml) in Tyrode’s buffer were treated with CRP (5 µg/ml), which had been preincubated with either TS buffer or GPVI-Fc (10 µg/ml, final concentration) for 5 min at 22oC, prior to addition to the platelets. C) Washed platelets (5 x 108/ml) in Tyrode’s buffer were treated with crotarhagin (0.3 µg/ml), which had been preincubated with either TS buffer or GPVI-Fc (10 µg/ml, final concentration) for 5 minutes at 22oC, prior to addition to the platelets. The bottom trace shows GPVI-Fc (10 µg/ml, final concentration) preincubated with TS buffer for 5 minutes at 22oC (without crotarhagin) before addition.

Effect of crotarhagin on platelet aggregation GPVI is targeted by two snake venom metalloproteinase-disintegrins, alborhagin from the viper Trimeresurus albolabris (27) and crotarhagin from Crotalus horridus horridus (this study). Like alborhagin and other venom metalloproteinase-disintegrins (27, 35), purified crotarhagin is ~60 kDa by SDS-PAGE under reducing (Fig. 1A) or non-reducing conditions (not shown), is immunoblotted by rabbit anti-mocarhagin IgG (Fig. 1B), and exhibits metalloproteinase activity since it partially digested fibrinogen in the presence divalent cation (60 min; 22oC), but not EDTA (data not shown). Crotarhagin also induced platelet aggregation in human PRP in a dose-dependent manner, with maximal activity at ~0.3 µg/ml (Fig. 1C). Aggregation consisted of an initial shape change (increased transmittance) followed by aggregation (decreased transmittance). As for alborhagin (27), EDTA inhibited crotarhagin-induced aggregation, but not shape change (Fig. 1C). This suggests that binding of crotarhagin to platelets is independent of divalent cation or metalloproteinase activity, since EDTA is known to inhibit Ca2+-dependent platelet aggregation, mediated by αIIbβ3, but not platelet shape change (27, 36). Inhibition by EDTA, however, does not discriminate between inhibition of αIIbβ3, Ca2+ signals or endogenous platelet metalloproteinases, or inhibition of intrinsic crotarhagin/alborhagin proteolytic activity which could also potentially contribute to platelet activation. Aggregation was independent of GPIbα, since the venom protein, CHH-B, or the anti-GPIbα antibody AK2, which completely block GPIbα/VWF-dependent aggregation induced by ristocetin (29, 32), had no effect on crotarhagin-induced aggregation (Fig. 1D, E). However, crotarhagin-dependent aggregation of washed platelets is blocked by the Src inhibitor, PP2 (Fig. 1E), which inhibits GPVI-dependent signalling induced by alborhagin or other ligands (21, 24–27). Crotarhagin induces GPVI shedding Crotarhagin and alborhagin induce time-dependent ectodomain shedding of GPVI from human platelets, resulting in a soluble GPVI fragment in the supernatant comparable to that induced by CRP (Fig. 2A). Crotarhagin-induced shedding is dose-depend-

1288



ent, and inhibitable by EDTA (Fig. 2B). Consistent with aggregation studies, crotarhagin-induced GPVI shedding is strongly inhibited by Src inhibitors (PP1 or PP2) and Syk inhibitor (piceatannol) (Fig. 2C). PP1, PP2 and piceatannol also inhibit shedding induced by alborhagin (Fig. 2C) and GPVI ligands, collagen, CRP and convulxin (21). As for human platelets, treating GPVI-transfected RBL-2H3 cells with crotarhagin induced shedding of a soluble ~55-kDa GPVI fragment recognized by the anti-GPVI antibody, 6B12, against the extracellular domain (Fig. 3A). This shed fragment was also induced by NEM, that directly activates ADAM-family sheddases (37, 38), and causes metalloproteinase-dependent shedding of GPVI from human platelets (26, 28). The dose response for crotarhagin-induced shedding from GPVI/RBL-2H3 cells was comparable to platelets (Fig. 2B), and inhibitable by EDTA. Pre-incubating crotarhagin with a soluble GPVI-Fc fusion protein also strongly inhibited crotarhagin-dependent platelet aggregation (Fig. 3C), providing additional evidence for a crotarhagin-GPVI interaction. Confirming specificity, GPVI-Fc inhibited aggregation induced by the GPVI-specific agonist, CRP (Fig. 3B), but not thrombin (1 U/ml) (data not shown). GPVI-Fc alone had no effect on platelets (Fig. 3C).

Discussion GPVI, a major platelet collagen receptor, together with GPIbIX-V that binds VWF, forms an adheso-signalling complex on platelets involved in pathophysiological thrombus formation (1–10, 28). We previously showed that a metalloproteinase-disintegrin, alborhagin, from the viper Trimeresurus albolabris, selectively targets GPVI on platelets or transfected cells, and induces GPVI-dependent signalling and platelet aggregation (27). In this study, we show that both alborhagin and a rattlesnake protein, crotarhagin, from Crotalus horridus horridus, also induce ectodomain shedding of platelet GPVI. Crotarhagin (~60 kDa non-reduced and reduced) is immunoreactive towards anti-mocarhagin IgG, an antibody that recognizes snake venom metalloproteinase-disintegrins (27, 35). Like alborhagin, crotarhagin induces aggregation of human platelets, with a similar dose response in PRP or washed platelets (maximal activity, ~0.3 µg/ml). Several lines of evidence suggest crotarhagin induces platelet aggregation by targeting GPVI. First, crotarhagin-dependent platelet aggregation is independent of GPIbα, since it is unaffected by either CHH-B or AK2, reagents that completely inhibit ristocetin-dependent aggregation involving VWF binding to GPIbα, but have no effect on aggregation involving GPVI (28, 29, 32). Second, like alborhagin and other GPVI ligands, including collagen, CRP and convulxin (21, 26, 28; this study), crotarhagin induced metalloproteinase-mediated shedding of GPVI from platelets. Shedding required platelet activation, because it was strongly blocked by inhibitors of GPVI signalling – the Src inhibitors PP1 and PP2, or the Syk inhibitor, piceatannol – previously shown to block ligand-induced GPVI shedding (21). Third, like CRP-dependent aggregation of washed platelets, crotarhagin-induced platelet aggregation was inhibited by a soluble fusion protein containing the GPVI ectodomain. Finally, crotarhagin induced GPVI shedding from human GPVI-transfected RBL-2H3 cells. Together, these results

Figure 4: Shedding of GPVI. Binding of snake venom metalloproteinase-disintegrins, crotarhagin or alborhagin, to GPVI induces signalling, and activation of endogenous platelet sheddase (ADAM10), generating an ~55-kDa soluble ectodomain fragment of GPVI (GPVIf) and an ~10-kDa membrane-associated remnant. Shedding is blocked by inhibitors of Src (PP1 or PP2) or Syk kinase (piceatannol).

suggest that, rather than direct cleavage, crotarhagin and alborhagin induce GPVI shedding by a mechanism involving platelet activation, and activation of endogenous platelet sheddases (Fig. 4). This would be a unique mechanism for regulating GPVI expression by this class of proteins, consistent with the abrogation of shedding by inhibitors of GPVI signalling, and the size of the shed soluble fragment being indistinguishable from that induced by the GPVI-selective ligand, CRP. The metalloproteinase involved in shedding human platelet GPVI induced by GPVI ligands has been identified asADAM10, a member of the ADAM (a disintegrin and metalloproteinase) family; ADAM10 or ADAM17 are also implicated in regulating levels of not only GPVI, but also GPIbα or GPV in murine or human platelets (21, 24–26, 39–41). Mammalian ADAMs are analogous to reptilian metalloproteinase-disintegrins, although mechanisms underlying their effects on surface expression of GPVI or GPIb-IX-V are not fully understood. Cleaving GPVI at an extracellular membrane-proximal site by exogenous metalloproteinases would presumably be limited by the platelet glycocalyx, a highly glycosylated dense barrier comprised of receptor sialomucins including GPIbα which extends 37.2 ± 3.3 nm beyond the membrane surface (42). However, the present work suggests an alternative mechanism by which metalloproteinasedisintegrins can shed GPVI, that is, by acting as ligands and inducing activation of endogenous platelet metalloproteinases. Selective recognition of Ig domains in the extracellular region of GPVI by metalloproteinases such as crotarhagin and alborhagin (27; this study), may be compared to Ig domain-containing proteins in marsupial serum that inhibit snake venom metalloproteinases (43–45). The interaction of immunoreceptors such as GPVI with metalloproteinase-disintegrins warrants further investigation. Acknowledgements We thank Carmen Llerena, Cheryl Berndt and Jing Jing for technical assistance, and the NHMRC of Australia for financial support.

1289



References 1. Moroi M, Jung SM. Platelet glycoprotein VI: its structure and function. Thromb Res 2004; 114: 221–233. 2. Nieswandt B, Watson SP. Platelet-collagen interaction: is GPVI the central receptor? Blood 2003; 102: 449–461. 3. Andrews RK, Berndt MC, López JA. The glycoprotein Ib-IX-V complex. In: Platelets, 2nd ed, Academic Press, San Diego; 2006: 145–163. 4. Andrews RK, Gardiner EE, Shen Y, et al. Molecules in focus: Glycoprotein Ib-IX-V. Int J Biochem Cell Biol 2003; 35: 1170–1174. 5. Kroll MH, Hellums JD, McIntire LV, et al. Platelets and shear stress. Blood 1996; 88: 1525–1541. 6. Goto S, Ikeda Y, Saldivar E, Ruggeri ZM. Distinct mechanisms of platelet aggregation as a consequence of different shearing flow conditions. J Clin Invest 1998; 101: 479–486. 7. Goto S, Tamura N, Handa S, et al. Involvement of glycoprotein VI in platelet thrombus formation on both collagen and von Willebrand factor surfaces under flow conditions. Circulation 2002; 106: 266–272. 8. Massberg S, Gawaz M, Gruner S, et al. A crucial role of glycoprotein VI for platelet recruitment to the injured arterial wall in vivo. J Exp Med 2003; 197: 41–49. 9. Bergmeier W, Piffath CL, Goerge T, et al. The role of platelet adhesion receptor GPIb? far exceeds that of its main ligand, von Willebrand factor, in arterial thrombosis. Proc Natl Acad Sci USA 2006; 103: 16900–16905. 10. Kleinschnitz C, Pozgajova M, Pham M, et al. Targeting platelets in acute experimental stroke. Impact of glycoprotein Ib, VI, and IIb/IIIa blockade on infarct size, functional outcome, and intracranial bleeding. Circulation 2007; 115: 2323-2330. 11. Bigalke B, Lindemann S, Ehlers R, et al. Expression of platelet collagen receptor glycoprotein VI is associated with acute coronary syndrome. Eur Heart J 2006; 27: 2165–2169. 12. Andrews RK, Berndt MC. Snake venom modulators of platelet adhesion receptors and their ligands. Toxicon 2000; 38: 775–791. 13. Andrews RK, Gardiner EE, ShenY, et al. Structureactivity relationships of snake toxins targeting platelet receptors, glycoprotein Ib-IX-V and glycoprotein VI. Curr Med Chem Cardiovasc Hematolog Agents 2003; 1: 143–149. 14. Lu Q, Clemetson JM, Clemetson KJ. Snake venoms and hemostasis. J Thromb Haemost 2005; 3: 1791–1799. 15. Kamiguti AS. Platelets as targets of snake venom metalloproteinases. Toxicon 2005; 45: 1041–1049. 16. Wijeyewickrema LC, Berndt MC, Andrews RK. Snake venom probes of platelet adhesion receptors and their ligands. Toxicon 2005; 45: 1051–1061. 17. Andrews RK, Suzuki-Inoue K, Shen Y, et al. Interaction of calmodulin with the cytoplasmic domain of platelet glycoprotein VI. Blood 2002; 99: 4219–4221.

18. Locke D, Liu C, Peng X, Chen H, et al. FcRγ-independent signaling by the platelet collagen receptor glycoprotein VI. J Biol Chem 2003; 278: 15441–15448. 19. Suzuki-Inoue K, Tulasne D, Bori-Sanz T, et al. Association of fyn and lyn with the proline rich domain of GPVI regulates intracellular signalling. J Biol Chem 2002; 277: 21561–21566. 20. Bori-Sanz T, Inoue KS, Berndt MC, et al. Delineation of the region in the glycoprotein VI tail required for association with the Fc receptor γ-chain. J Biol Chem 2003; 278: 35914–35922. 21. Gardiner EE, Arthur JF, Kahn ML, et al. Regulation of platelet membrane levels of glycoprotein VI by a platelet-derived metalloproteinase. Blood 2004; 104: 3611–3617. 22. Stephens G, Yan Y, Jandrot-Perrus M, et al. Platelet activation induces metalloproteinase-dependent GPVI cleavage to down-regulate platelet reactivity to collagen. Blood 2005; 105: 186–191. 23. Bergmeier W, Rabie T, Strehl A, et al. GPVI downregulation in murine platelets through metalloproteinase-dependent shedding. Thromb Haemost 2004; 91: 951–958. 24. Andrews RK, Karunakaran D, Gardiner EE, et al. Platelet receptor proteolysis: a mechanism for down regulating platelet reactivity. Arterioscler Thrombos Vasc Biol 2007; 7: 1511-1520. 25. Berndt MC, Karunakaran D, Gardiner EE, et al. Programmed autologous cleavage of platelet receptors. J Thromb Haemost 2007; Suppl. 1: 212-219. 26. Gardiner EE, Karunakaran D, Shen Y, et al. Controlled shedding of platelet glycoprotein (GP)VI and GPIb-IX-V by ADAM family metalloproteinases. J Thromb Haemost 2007; 5: 1530-1537. 27. Andrews RK, Gardiner EE, Asazuma N, et al. A novel viper venom metalloproteinase, alborhagin, is an agonist at the platelet collagen receptor GPVI. J Biol Chem 2001; 276: 28092–28097. 28. Arthur JF, Matzaris M, Gardiner EE, et al. Glycoprotein (GP)VI is associated with GPIb-IX-V on the membrane of resting and activated platelets. Thromb Haemost 2005; 93: 716–723. 29. Andrews RK, Kroll MH, Ward CM, et al. Binding of a novel 50-kilodalton alboaggregin from Trimeresurus albolabris and related viper venom proteins to the platelet membrane glycoprotein Ib-IX-V complex. Effect on platelet aggregation and glycoprotein Ib-mediated platelet activation. Biochemistry 1996; 35: 12629–12639. 30. Miura Y, Takahashi T, Jung SM, et al. Analysis of the interaction of platelet collagen receptor glycoprotein VI (GPVI) with collagen. A dimeric form of GPVI, but not the monomeric form, shows affinity to fibrous collagen. J Biol Chem 2002; 277: 46197–46204. 31. Gruner S, Prostredna M, Koch M, et al. Relative antithrombotic effect of soluble GPVI dimer compared with anti-GPVI antibodies in mice. Blood 2005; 105: 1492–1499. 32. Shen Y, Romo GM, Dong J-F, et al. Requirement of leucine-rich repeats of GPIbα for shear-dependent and

static binding of von Willebrand factor to the platelet membrane GPIb-IX-V complex. Blood 2000; 95: 903–910. 33. Shen Y, Cranmer SL, Aprico A, et al. Leucine-rich repeats 2–4 (Leu60-Glu128) of platelet glycoprotein Ibα regulate shear-dependent cell adhesion to von Willebrand factor. J Biol Chem 2006; 281: 26419–26423. 34. Ward CM, Andrews RK, Smith AI, et al. Mocarhagin, a novel cobra venom metalloproteinase, cleaves the platelet von Willebrand factor receptor glycoprotein Ibα. Identification of the sulfated tyrosine/anionic sequence Tyr276-Glu282 of glycoprotein Ibα as a binding site for von Willebrand factor and α-thrombin. Biochemistry 1996; 35: 4929–4938. 35. Wijeyewickrema LC, Gardiner EE, Berndt MC, et al. Fractionation of snake venom metalloproteinases by metal ion affinity: a purified cobra metalloproteinase, Nk, from Naja kaouthia binds Ni2+-agarose. Toxicon 2007; 50: 1064-1072. 36. Holme S, Murphy S. Quantitative measurement of platelet shape by light transmission studies: application to storage of platelets for transfusion. J Lab Clin Med 1978; 92: 53–64. 37. Amit T, Amit T, Hochberg Z, et al. Shedding of growth hormone-binding protein is inhibited by hydroxamic acid-based protease inhibitors: proposed mechanism of activation of growth hormone-binding protein secretase. J Endocrinol 2001; 169: 397–407. 38. Allinson TM, Parkin ET, Turner AJ, et al. ADAMs family members as amyloid precursor protein α-secretases. J Neurosc Res 2003; 74: 342–352. 39. Rabie T, Strehl A, Ludwig A, et al. Evidence for a role of ADAM17 (TACE) in the regulation of platelet glycoprotein V. J Biol Chem 2005; 280: 14462–14468. 40. Bergmeier W, Piffath CL, Cheng G, et al. Tumor necrosis factor-α-converting enzyme (ADAM17) mediates GPIbα shedding from platelets in vitro and in vivo. Circ Res 2004; 95: 677–683. 41. Aktas B, Pozgajova M, Bergmeier W, et al. Aspirin induces platelet receptor shedding via ADAM17 (TACE). J Biol Chem 2005; 280: 39716–39722. 42. Erlandsen SL, Greet Bittermann A, White J, et al. High-resolution CryoFESEM of individual cell adhesion molecules (CAMs) in the glycocalyx of human platelets: detection of P-selectin (CD62P), GPI-IX complex (CD42A/CD42bα, bβ), and integrin GPIIbIIIa (CD41/CD61) by immunogold labeling and stereo imaging. J Histochem Cytochem 2001; 49: 809–819. 43. Neves-Ferreira AG, Perales J, Fox JW, et al. Structural and functional analyses of DM43, a snake venom metalloproteinase inhibitor from Didelphis marsupialis serum. J Biol Chem 2002; 277: 13129–13137. 44. Rocha SL, Lomonte B, Neves-Ferreira AG, et al. Functional analysis of DM64, an antimyotoxic protein with immunoglobulin-like structure from Didelphis marsupialis serum. Eur J Biochem 2002; 269: 6052–6062. 45. Perales J, Neves-Ferreira AG, Valente RH, et al. Natural inhibitors of snake venom hemorrhagic metalloproteinases. Toxicon 2005; 45: 1013–1020.

1290
