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GPR56/ADGRG1 Is a Platelet Collagen-Responsive GPCR and Hemostatic Sensor of Shear Force

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GPR56/ADGRG1 Is a Platelet Collagen-Responsive GPCR and Hemostatic Sensor of Shear Force GPR56/ADGRG1 is a platelet collagen-responsive GPCR and hemostatic sensor of shear force Jennifer Yeunga, Reheman Adilia, Emily N. Stringhama, Rong Luob, Alexander Vizurragaa, Luciana K. Rosselli-Muraia, Hannah M. Stovekena, Maiya Yua, Xianhua Piaob,c, Michael Holinstata,d, and Gregory G. Talla,1 aDepartment of Pharmacology, University of Michigan Medical Center, Ann Arbor, MI 48109; bDepartment of Medicine, Boston Children’s Hospital and Harvard Medical School, Boston, MA 02115; cNewborn Brain Research Institute, Department of Pediatrics, Weill Institute for Neuroscience, University of California, San Francisco, CA 94158; and dDepartment of Internal Medicine, Division of Cardiovascular Medicine, University of Michigan Medical Center, Ann Arbor, MI 48109 Edited by Robert J. Lefkowitz, Howard Hughes Medical Institute, Durham, NC, and approved September 14, 2020 (received for review May 6, 2020) Circulating platelets roll along exposed collagen at vessel injury The differences in the experimental approaches used to inves- sites and respond with filipodia protrusion, shape change, and tigate platelet responses to collagen have offered varied inter- surface area expansion to facilitate platelet adhesion and plug pretations of whether GPVI is the key signaling receptor that acts formation. Various glycoproteins were considered to be both to initiate platelet activation (2, 7, 8, 13). For example, exogenous collagen responders and mediators of platelet adhesion, yet the collagen promotes sustained GPVI/Fc Receptor gamma (FcRγ) signaling kinetics emanating from these receptors do not fully signaling through phospholipase Cγ2(PLCγ2), but the Ca+2 mo- account for the rapid platelet cytoskeletal changes that occur in bilization output and kinetics of this multistep signaling pathway blood flow. We found the free N-terminal fragment of the adhe- may not fully account for the quick-onset cytoskeletal changes that sion G protein-coupled receptor (GPCR) GPR56 in human plasma are required for platelet deceleration in rapid blood flow (1, 17). and report that GPR56 is the platelet receptor that transduces sig- Thus, the putative existence of other rapid-acting platelet collagen nals from collagen and blood flow-induced shear force to activate receptors has been considered. These receptor(s) may be en- − − G protein 13 signaling for platelet shape change. Gpr56 / mice gaged prior to or concomitantly with the glycoprotein collagen have prolonged bleeding, defective platelet plug formation, and receptors (7). delayed thrombotic occlusion. Human and mouse blood perfusion Heterotrimeric G protein 13 (G13) signaling is required to MEDICAL SCIENCES studies demonstrated GPR56 and shear-force dependence of plate- stimulate the cytoskeletal-dependent platelet shape changes in let adhesion to immobilized collagen. Our work places GPR56 as an response to collagen (18). Gα13-GTP (guanosine triphosphate) initial collagen responder and shear-force transducer that is essen- rapidly and robustly activates Rho family small G protein sig- tial for platelet shape change during hemostasis. naling to induce actin polymerization (19). Accounting for the means of G13 activation is not readily explained by the actions of hemostasis | platelets | adhesion GPCR | collagen | signal transduction the current repertoire of platelet collagen receptors. Here, we identified the collagen-responsive, G13-coupled, G protein-coupled emostasis is the physiological wound healing process that receptor (GPCR) GPR56 or ADGRG1 on the surface of plate- Hprevents blood loss while maintaining normal blood flow. lets (20–23). GPR56 is an adhesion G protein-coupled receptor Platelets are anucleate cell fragments that serve as major con- tributors to hemostatic plug formation. The process is initiated Significance by platelet surface receptor engagement with subendothelial collagen of the extracellular matrix that becomes exposed during We identified the known collagen receptor GPR56/ADGRG1 on vessel wall injury (1–3). The initiating hemostatic step involves platelets. GPR56 is an adhesion G protein-coupled receptor that platelet glycoprotein Ibα (GPIbα) transiently tethering to collagen- becomes activated following forced dissociation of its N-terminal bound von Willebrand factor (vWF) (2–5). A multistep mechanism fragment and C-terminal fragment or seven-transmembrane ensues in which multiple receptors including glycoprotein VI spanning domain (7TM). Fragment dissociation reveals the (GPVI) and integrin α2β1 participate in platelet spreading, activa- cryptic stalk of the 7TM, which acts as a tethered peptide ago- tion, stable adhesion, and the aggregation events that are necessary nist, and for GPR56, this activates platelet G13 signaling. GPR56 to support thrombus growth (6–8). Within the sequential activating pharmacological probes activated platelets to undergo shape events, tethered platelets undergo rapid reorganization of the cy- change and aggregation, which are critical for the formation of −/− toskeletal structure, expanding from discoid to spherical shapes in hemostatic plugs. Gpr56 mice exhibit prolonged bleeding, order to spread over the damaged surface (9). Actin polymerization defective platelet plug formation in vessel injury assays, and within platelets permits the development and extension of filopodia delayed thrombotic vessel occlusion. Shear-force dependency of protrusions and lamellipodia that enable platelet deceleration in platelet adhesion to immobilizedcollagenwasfoundtobe the blood flow, flattening and stable adhesion. Activated platelets GPR56 dependent. release storage granule contents, including autocrine and paracrine platelet agonists that fulfill the platelet activation process (2, 10, Author contributions: J.Y., A.V., L.K.R.-M., H.M.S., and G.G.T. designed research; J.Y., R.A., E.N.S., A.V., L.K.R.-M., H.M.S., M.Y., and G.G.T. performed research; J.Y., R.L., X.P., and 11). These events are prerequisite to secondary platelet recruitment G.G.T. contributed new reagents/analytic tools; J.Y., E.N.S., A.V., L.K.R.-M., H.M.S., M.H., and formation of stable clots. and G.G.T. analyzed data; and J.Y. and G.G.T. wrote the paper. Dynamic glycoprotein receptor interactions with collagen have The authors declare no competing interest. been viewed as the dominant initiating events leading to platelet This article is a PNAS Direct Submission. plug formation. Arteriole injury and platelet activation experi- This open access article is distributed under Creative Commons Attribution-NonCommercial- ments using a GPVI-deficient mouse model showed defects in NoDerivatives License 4.0 (CC BY-NC-ND). hemostatic plug formation and platelet adhesion to collagen (12, 1To whom correspondence may be addressed. Email: [email protected]. 13). However, additional measures demonstrated that GPVI- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ deficient mice have negligibly prolonged bleeding times and no doi:10.1073/pnas.2008921117/-/DCSupplemental. impairment in induced occlusive thrombus formation (14–16). First published October 23, 2020. www.pnas.org/cgi/doi/10.1073/pnas.2008921117 PNAS | November 10, 2020 | vol. 117 | no. 45 | 28275–28286 Downloaded by guest on October 1, 2021 (AGPCR), a receptor subclass distinguished by the presence of a markers were measured by flow cytometry. Human platelets, but GPCR autoproteolysis-inducing domain that self-cleaves the re- not the RBCs, stained positive for GPR56 and CD41, whereas ceptors at a precise extracellular site proximal to the start of the RBCs stained positive for CD235 but not GPR56 (Fig. 2A). There first transmembrane span (TM1) of the seven-transmembrane was variability of GPR56 abundance among platelets isolated from bundle (24). Self-cleavage is constitutive, and the two resulting the different human donors (SI Appendix,Fig.S2A). Intact plate- receptor pieces, termed the N-terminal fragment (NTF) and lets were costained with the GPR56 NTF antibody and fluorescein C-terminal fragment (CTF), remain noncovalently bound. Recent isothiocyanate (FITC)-phalloidin and visualized by fluorescent biochemical evidence demonstrated that dissociation of the two microscopy. Inclusion of the GPR56 rNTF during the antibody receptor fragments resulted in strong activation of G protein sig- incubation blocked the punctate cell surface GPR56 NTF anti- naling (22, 23). Dissociation of the NTF unveils a stalk peptide body signal (Fig. 2B). Cytosol-free membrane homogenates were emanating from TM1 that acts as a tethered peptide agonist to prepared from human platelets obtained from five donors. The activate the CTF (23, 25). One proposed physiological mechanism membranes were quantitatively immunoblotted with an antibody to of AGPCR activation is through shear force-mediated NTF/CTF detect the GPR56 CTF (Fig. 2C). GPR56 was present in each dissociation. The NTF becomes immobilized to its protein ligand, platelet membrane preparation, and variable abundance was ob- and the force created by cell movement drives the dissociation served, coinciding with the flow cytometry results. The platelet event. Our work shows that GPR56 is a platelet collagen GPCR membrane preparations were then treated with urea to dissociate that has the critical role of providing early-onset G13 activation to the GPR56 CTF and NTF. The majority of platelet GPR56 was facilitate the platelet shape changes and integrin activation that deemed to be self-cleaved as the transmembrane intercalated are necessary for efficient hemostasis
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