GPVI and the not so eager cleaver

From www.bloodjournal.org by guest on July 22, 2018. For personal use only. (see figure, left panel). The fractal dimension, an indication of how completely a fractal appears to fill space as one goes down to finer and finer spatial scales, integrates structural/ mechanical elements as a measure of the network complexity. At the gel point, polymeric clusters establish sufficient connectivity to become sample-spanning, conferring the properties of a solid on the system.9 It is significant that Evans et al1 have studied clotting simply in whole unadulterated blood. Analysis of viscoelastic data from whole blood from healthy subjects revealed that the fractal dimension has a clearly defined value, within a narrow range, 1.74 (⫾ 0.07), which the authors refer to as a “Healthy Index.” The addition of unfractionated heparin to whole blood caused the expected changes in laboratory markers of coagulation and a progressive decease in fractal dimension as well as an increase in clotting time. In fact, the fractal dimension, together with activated partial thromboplastin time, was the most significant predictor of anti-factor Xa activity. These results also indicate that heparin affects clot structure in addition to clotting time. In conclusion, this study introduces a novel method that allows the measurement of a single parameter, the fractal dimension, which can be used to characterize the complex relationship between the clotting system and the microstructure of incipient clots in healthy uncoagulated blood. Because many diseases affect hemostasis and hence clot properties, the next step will be extending these studies to thrombotic and hemorrhagic states to determine whether this functional biomarker remains consistently valid and can therefore provide insights into the diagnosis and management of these conditions. Conflict-of-interest disclosure: The author declares no competing financial interests. ■ tecture is associated with hypofibrinolysis and premature coronary artery atherothrombosis. Arteroscler Thromb Vasc Biol. 2006;26(11):2567-2573. 8. Mills JD, Ariens RA, Mansfield MW, Grant PJ. Altered fibrin clot structure in the healthy relatives of patients with premature coronary artery disease. Circulation. 2002; 106(15):1938-1942. 7. Undas A, Zawilska K, Ciesla-Dul M, et al. Altered fibrin clot structure/function in patients with idiopathic venous thromboembolism and in their relatives. Blood. 2009;114(19):4272-4278. 9. Chernysh IN, Weisel JW. Dynamic imaging of fibrin network formation correlated with other measures of polymerization. Blood. 2008;111(10):4854-4861. ● ● ● THROMBOSIS & HEMOSTASIS Comment on Bender et al, page 3347 GPVI and the not so eager cleaver ---------------------------------------------------------------------------------------------------------------Peter J. Newman BLOODCENTER OF WISCONSIN Platelets control their responsiveness, in part, by shedding adhesion and signaling receptors from their surface. The molecular mechanism by which this occurs, however, is incompletely understood. In this issue of Blood, Bender and colleagues make judicious use of mice genetically deficient in selected candidate proteases to shed new light on the unexpected complexity of ectodomain shedding.1 ematopoietic cells control their adhesive phenotype through several mechanisms, including de novo synthesis of adhesion receptors (eg, VCAM-1, ICAM-1), compartmental- H ization within the cell (eg, P-selectin, LAMP-3), conformational change (eg, integrins), receptor clustering (eg, growth factor and cytokine receptors), and proteolytic removal of the REFERENCES 1. Evans PA, Hawkins K, Morris RHK, et al. Gel point and fractal microstructure of incipent blood clots are significant new markers of hemostasis for healthy and anticoagulated blood. Blood. 2010;116(17):3341-3346. 2. Weisel JW. The mechanical properties of fibrin for basic scientists and clinicians. Biophys Chem. 2004;112(2-3):267-276. 3. Brown AE, Litvinov RI, Discher DE, Purohit PK, Weisel JW. Multiscale mechanics of fibrin polymer: gel stretching with protein unfolding and loss of water. Science. 2009;325(5941):741-744. 4. Weisel JW. Structure of fibrin: impact on clot stability. J Thromb Haemost. 2007;5(suppl 1):116-124. 5. Evans PA, Hawkins K, Lawrence M, et al. Rheometry and associated techniques for blood coagulation studies. Med Eng Phys. 2008;30(6):671-679. 6. Collet J-P, Allali Y, Lesty C, et al. Altered fibrin archi- 3124 Who’s shedding now? The mechanism by which ectodomain shedding of GPVI occurs depends on how the platelets are treated. Addition of calmodulin inhibitors like W7 result in activation of ADAM10, while agents like CCCP that influence mitochondrial metabolism and the generation of reactive oxygen species initiate ADAM17-mediated cleavage of the GPVI extracellular domain. Surprisingly, antibody binding–mediated shedding of GPVI, a contemplated therapeutic approach, appears to take place via an entirely different mechanism that involves a still-to-be-defined protease. (Professional illustration by Paulette Dennis). 28 OCTOBER 2010 I VOLUME 116, NUMBER 17 blood From www.bloodjournal.org by guest on July 22, 2018. For personal use only. extracellular domain of transmembrane adhesion and signaling receptors (eg, L-Selectin, TNF-␣). The latter, commonly referred to as ectodomain shedding, has been shown to regulate such diverse processes as cell adhesion and migration, development, cellular signaling, and apoptosis.2 Ectodomain shedding plays an important role in platelet biology as well. An array of adhesion and signaling receptors reside on the surface of platelets that, upon exposure to their ligands, initiate the activation of a complex network of signaling pathways leading to platelet activation, adhesion, and thrombus formation. Several of these receptors are known to be removed from the plasma membrane after platelet activation as part of a naturally occurring negative feedback loop intended to limit platelet accumulation and thrombus growth. The first example of a shed platelet plasma membrane– derived adhesion receptor was provided by the laboratory of the recently deceased Graham Jamieson, who published a series of studies in the 1970s characterizing glycocalicin3: a soluble, circulating plasma protein that was later found to be derived from glycoprotein (GP) Ib␣, the major subunit of the platelet receptor for von Willebrand factor. The importance of ectodomain shedding in passivating platelet responsiveness has been extended by findings that the extracellular domains of GPV and GPVI are also rapidly lost from the platelet surface after various, seemingly unrelated, forms of cellular activation. GPVI is a 62-kDa platelet-specific type I transmembrane glycoprotein expressed on the surface of human and murine platelets in a noncovalent complex with the immunoreceptor tyrosine-based activation motif (ITAM)– containing subunit, the FcR␥ chain.4,5 GPVI is composed of 2 extracellular immunoglobulin (Ig)– homology domains, a transmembrane domain, and a 51-amino acid cytoplasmic domain,6,7 and serves as the major platelet-activating receptor for collagen, signaling via the Syk/SLP-76/PLC␥2 pathway to activate the integrins ␣2␤1 and ␣IIb␤3 leading to platelet activation and thrombus formation.8,9 Studies performed by Nieswandt and colleagues nearly a decade ago revealed the surprising finding that injection into mice of rat anti–mouse GPVI monoclonal antibodies results in loss of GPVI from the surface of circulating murine platelets,10 with a corresponding reduction in platelet responsiveness blood 2 8 O C T O B E R 2 0 1 0 I V O L U M E 1 1 6 , N U M B E R 1 7 to collagen.11 Antibody-induced shedding (sometimes termed immunodepletion) of GPVI appears to be operable in humans as well, as platelets from a patient with a circulating autoantibody specific for GPVI also become devoid of cell-surface GPVI and fail to form thrombi over collagen-coated surfaces, while remaining responsive to other platelet agonists.12 Taken together with the observation that GPVI can be down-regulated in human platelets in vivo using monoclonal antibody therapy,13 there is growing interest in the development of GPVI-based therapeutics, in part because the use of more broadly acting antiplatelet agents like ADP receptor or antifibrinogen receptor antagonists is associated with a low, but still problematic, incidence of bleeding. Understanding how potential antiGPVI– based agents lead to removal of this adhesion and signaling receptor from the platelet surface, and to corresponding platelet passivation, is therefore of paramount importance before widespread adoption of this novel therapeutic modality can be considered. Ectodomain shedding of transmembrane receptors is largely carried out by members of 2 large families of zinc-dependent metalloproteinases: MMPs (matrix metalloproteinases) and ADAMs (metalloproteinases containing a disintegrin and metalloprotease domain). ADAMs are type I transmembrane glycoproteins, and are thought to cleave their substrates in cis: that is, they eat their neighbors (see figure)! There are 21 distinct genes in humans that encode ADAM proteases, though only 12 (ADAMs 8, 9, 10, 12, 15, 17, 19, 20, 21, 28, 30, and 33) are thought to have actual protease activity.2 Of these, there is compelling evidence that ADAM17 (also known as tumor necrosis factor ␣ converting enzyme, or TACE) is responsible for the cleavage and release of the extracellular domains of GPIb␣14 and GPV.15 The identity of the ADAM that sheds GPVI from the platelet surface, however, is less clear. The extracellular domain of GPVI has been shown to be shed from the platelet surface in response to a wide variety of physiologic and nonphysiologic agents. These include binding of GPVI-specific antibodies,10,12,13,16 binding of GPVI-specific ligands such as collagen, collagen-related peptide (CRP), and convulxin,16,17 engagement of the ITAM-linked Fc receptor, Fc␥RIIa,18 dissociation of calmodulin from the cytoplasmic domain of GPVI induced by the calmodulin inhibitor, W7,17 and treatment of platelets with phorbol 12-myristate-13-acetate, Nethylmaleimide (NEM), or the mitochondrial poison, carbonyl cyanide m-chlorophenyl hydrazone (CCCP).19,20 Regardless of the type of stimulation, GPVI shedding can in all cases be blocked by addition of ethylenediaminetetraacetic acid (EDTA) or the broad-range metalloproteinase inhibitor, GM6001, demonstrating that removal of the GPVI ectodomain is a cation-dependent MMP- or an ADAMmediated event. Although no direct evidence exists, each of these stimuli presumably induce minor conformational changes in GPVI that expose cryptic sequences on the receptor that make it vulnerable to proteolytic cleavage. This process appears to require subthreshold levels of cellular activation, as inhibition anywhere along the GPVI/FcR␥-chain ITAM3 Src3 Syk3 LAT3 PLC␥2 signal transduction circuit blocks GPVI shedding.17,21 In this issue, Bender and colleagues1 use newly generated megakaryocyte-specific ADAM10-deficient mice, mice lacking ADAM17 protease activity, and mice whose platelets lack both ADAM10 and ADAM17 functional activity to examine their relative roles in mediating GPVI ectodomain shedding. The authors make the fascinating and unexpected finding that the ADAM used very much depends on the way that the platelets are stimulated. Thus, although ADAM10 functions as the predominant GPVI sheddase when platelets are stimulated in vitro with the calmodulin inhibitor W7, ADAM17 serves as the predominant GPVI sheddase when platelets are activated, also in vitro, using CCCP (see figure). Perhaps most relevant to the contemplated development of large or small molecule therapeutic agents that target GPVI directly, neither ADAM10 nor ADAM17 appears to function as GPVI sheddases when anti-GPVI antibodies are used to down-regulate the receptor in vivo. The identity of the protease that becomes activated in response to antibody-induced shedding is unknown. What might account for the differences seen in GPVI ectodomain shedding induced by various agonists? It is possible that chemical agents like NEM, W7, and CCCP act indirectly to activate ADAM proteases, perhaps in addition to their effects on GPVI itself or its downstream signaling components. This would explain why agents, like antibodies, that bind GPVI directly, are unable to initiate 3125 From www.bloodjournal.org by guest on July 22, 2018. For personal use only. ADAM10- or ADAM17-mediated cleavage. Solving the interesting and important puzzle of ADAM10 and ADAM17 activation, however, will not address the mechanism by which molecules that bind GPVI directly induce shedding in vivo. Because targeting the GPVI/FcR␥-chain complex represents a promising therapeutic approach—not only for its potential to selectively limit platelet reactivity to collagen while preserving other plateletactivation pathways,13 but also for its potential to dampen inflammatory disease22—the intriguing observations of Bender et al that GPVI is shed in vivo in an ADAM10/ ADAM17-independent manner warrant further investigation. Identification of the protease responsible for mediating in vivo cleavage of this unique platelet adhesion and signaling receptor may very well reveal additional molecular targets that extend beyond GPVI biology for treating thrombotic and inflammatory human disease. Conflict-of-interest disclosure: The author declares no competing financial interests. ■ REFERENCES 1. Bender M, Hofmann S, Stegner D, et al. Differentially regulated GPVI ectodomain shedding by multiple plateletexpressed proteinases. Blood. 2010;116(17):3347-3355. 2. Reiss K, Saftig P. The “a disintegrin and metalloprotease” (ADAM) family of sheddases: physiological and cellular functions. Semin Cell Dev Biol. 2009;20(2):126-137. 3. Jamieson GA, Okumura T, Hasitz M. Structure and function of platelet glycocalicin. Thromb Haemost. 1980; 42(5):1673-1678. 4. Gibbins JM, Okuma M, Farndale R, Barnes M, Watson SP. Glycoprotein VI is the collagen receptor in platelets which underlies tyrosine phosphorylation of the Fc receptor gamma-chain. FEBS Lett. 1997;413(2):255-259. 5. Tsuji M, Ezumi Y, Arai M, Takayama H. A novel association of Fc receptor gamma-chain with glycoprotein VI and their co-expression as a collagen receptor in human platelets. J Biol Chem. 1997;272(38):23528-23531. 6. Clemetson JM, Polgar J, Magnenat E, Wells TN, Clemetson KJ. The platelet collagen receptor glycoprotein VI is a member of the immunoglobulin superfamily closely related to FcalphaR and the natural killer receptors. J Biol Chem. 1999;274(41):29019-29024. 7. Jandrot-Perrus M, Busfield S, Lagrue AH, et al. Cloning, characterization, and functional studies of human and mouse glycoprotein VI: a platelet-specific collagen receptor from the immunoglobulin superfamily. Blood. 2000;96(5): 1798-1807. 8. Nieswandt B, Watson SP. Platelet-collagen interaction: is GPVI the central receptor? Blood. 2003;102(2):449-461. 9. Chen H, Kahn ML. Reciprocal signaling by integrin and nonintegrin receptors during collagen activation of platelets. Mol Cell Biol. 2003;23(14):4764-4777. 10. Nieswandt B, Schulte V, Bergmeier W, et al. Longterm antithrombotic protection by in vivo depletion of platelet glycoprotein VI in mice. J Exp Med. 2001;193(4): 459-469. 11. 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(1):41-49. 3126 12. Boylan B, Chen H, Rathore V, et al. Anti-GPVIassociated ITP: An acquired platelet disorder caused by autoantibody-mediated clearance of the GPVI/FcRgamma-chain complex from the human platelet surface. Blood. 2004;104(5):1350-1355. 17. Gardiner EE, Arthur JF, Kahn ML, Berndt MC, Andrews RK. Regulation of platelet membrane levels of glycoprotein VI by a platelet-derived metalloproteinase. Blood. 2004;104(12):3611-3617. 13. Boylan B, Berndt MC, Kahn ML, Newman PJ. Activation-independent, antibody-mediated removal of GPVI from circulating human platelets: development of a novel NOD/SCID mouse model to evaluate the in vivo effectiveness of anti-human platelet agents. Blood. 2006;108(3): 908-914. 14. Bergmeier W, Piffath CL, Cheng G, et al. Tumor necrosis factor-alpha-converting enzyme (ADAM17) mediates GPIbalpha shedding from platelets in vitro and in vivo. Circ Res. 2004;95(7):677-683. 15. Rabie T, Strehl A, Ludwig A, Nieswandt B. Evidence for a role of ADAM17 (TACE) in the regulation of platelet glycoprotein V. J Biol Chem. 2005;280(15):14462-14468. 18. Gardiner EE, Karunakaran D, Arthur JF, et al. Dual ITAM-mediated proteolytic pathways for irreversible inactivation of platelet receptors: De-ITAM-ising FcgammaRIIa. Blood. 2008;111(1):165-174. 19. Bergmeier W, Rabie T, Strehl A, et al. GPVI downregulation in murine platelets through metalloproteinasedependent shedding. Thromb Haemost. 2004;91(5):951-958. 20. Gardiner EE, Karunakaran D, Shen Y, et al. Controlled shedding of platelet glycoprotein (GP)VI and GPIbIX-V by ADAM family metalloproteinases. J Thromb Haemost. 2007;5(7):1530-1537. 21. Rabie T, Varga-Szabo D, Bender M, et al. Diverging signaling events control the pathway of GPVI downregulation in vivo. Blood. 2007;110(2):529-535. 16. Stephens G, Yan Y, Jandrot-Perrus M, et al. Platelet activation induces metalloproteinase-dependent GP VI cleavage to down-regulate platelet reactivity to collagen. Blood. 2005;105(1):186-191. 22. Boilard E, Nigrovic PA, Larabee K, et al. Platelets amplify inflammation in arthritis via collagen-dependent microparticle production. Science. 2010;327(5965):580-583. ● ● ● TRANSPLANTATION Comment on Sun et al, page 3129 Transplant survivorship: a call to arms ---------------------------------------------------------------------------------------------------------------Corey Cutler DANA-FARBER CANCER INSTITUTE In this issue of Blood, Sun and colleagues from the Bone Marrow Transplant Survivors Study report the chronic health outcomes of more than 1000 survivors of stem cell transplantation. Their results demonstrate a significant burden of chronic conditions among survivors. here has been a consistent increase in the number of hematopoietic stem cell transplantations performed annually for the past 2 decades. Enabling this expansion of transplantation have been refinements in HLA matching, infectious disease therapy, and general supportive care of the transplant patient. A welcome consequence has been an increased number of long-term survivors. While early health-related outcomes and quality of life have been studied, until recently, the longterm health outcomes of these long-term survivors have not been adequately addressed. In this issue, Sun et al describe the burden of chronic health conditions among long-term transplantation survivors, and compare the incidence of these chronic health conditions with their siblings.1 Compared with siblings, transplantation survivors were twice as likely to have any chronic health condition and 3.5-fold more likely to have a serious chronic health condition. Likewise, multiple chronic health conditions were more common among transplantation recipients. The frequency of these health conditions was staggering (cumulative T incidence at 10 years: 59%) and was related to the type of transplantation received, with recipients of allogeneic grafts having a higher incidence of chronic health conditions compared with autologous graft recipients. Chronic graft-versus-host disease and the use of an unrelated donor or a total body irradiation– containing conditioning regimen were significant risks for chronic health conditions among allogeneic transplantation recipients. The cumulative incidence of the chronic health conditions noted in this study increased steadily over time, without an apparent plateau at any time point. Of course, with an aging population this is expected to occur, however, this relationship was noted even for individuals under the age of 40 at the time of transplantation. The implications of this study are obvious, and demonstrate that while transplantation may be curative of the underlying tumor it is not a panacea for the overall health of the patient with a hematologic malignancy. Rather, as many of us have often told our patients, with transplantation we simply trade one set of problems for another. As a 28 OCTOBER 2010 I VOLUME 116, NUMBER 17 blood From www.bloodjournal.org by guest on July 22, 2018. For personal use only. 2010 116: 3124-3126 doi:10.1182/blood-2010-08-299255 GPVI and the not so eager cleaver Peter J. Newman Updated information and services can be found at: http://www.bloodjournal.org/content/116/17/3124.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved.