Jane F. Arthur

Controlled shedding of platelet glycoprotein (GP)VI and GPIb-IX-V by ADAM family metalloproteinases.

Background: Platelet glycoprotein (GP)VI that binds collagen, and GPIb–IX–V that binds von Willebrand factor, initiate thrombus formation.Objectives: In this study, we investigated the mechanisms of metalloproteinase‐mediated ectodomain shedding that regulate the surface expression of GPVI, GPIbα (the major ligand‐binding subunit) and GPV (that regulates thrombin‐dependent activation via GPIbα).Methods and results: Immunoblotting human platelet lysates using affinity‐purified antibodies against cytoplasmic domains of GPVI, GPIbα or GPV allowed simultaneous analysis of intact and cleaved receptor, and revealed (i) that a significant fraction of GPIbα, but not GPVI, exists in a cleaved state on platelets, even when isolated in the presence of metalloproteinase inhibitor (GM6001) or EDTA; (ii) the same‐sized membrane‐associated fragments of GPVI or GPIbα are generated by phorbol‐ester (PMA), the mitochondrial‐targeting reagent CCCP, the calmodulin inhibitor W7, or the thiol‐modifying reagent, N‐ethylmaleimide, that directly activates ADAM10/ADAM17; and (iii) GPV is shed by both metalloproteinase‐ and thrombin‐dependent mechanisms, depending on the concentration of thrombin. Based on the predicted cleavage area defined by these studies, ADAM10, but not ADAM17, cleaved a GPVI‐based synthetic peptide within the extracellular membrane‐proximal sequence (PAR^Q243YY) as analyzed by MALDI‐TOF‐MS. In contrast, ADAM17, but not ADAM10, cleaved within the GPIbα‐based peptide (LRG^V465LQ). Both ADAM10 and ADAM17 cleaved within a GPV‐based peptide (AQP^V494TT). Metalloproteinase‐mediated shedding of GPIbα from GPIb‐IX‐transfected or GPVI‐transfected cells induced by W7 or N‐ethylmaleimide was inhibited by mutagenesis of sequences identified from peptide analysis.Conclusions: These findings suggest surface levels of GPVI, GPIbα and GPV may be controlled by distinct mechanisms involving ADAM10 and/or ADAM17.

Glycoprotein VI is associated with GPIb-IX-V on the membrane of resting and activated platelets

The platelet collagen receptor, glycoprotein (GP)VI, initiates platelet aggregation at low shear stress while GPIb-IX-V, which binds von Willebrand factor, elicits platelet aggregation under high shear conditions. To investigate the possibility that GPIb-IX-V and GPVI are associated on the platelet surface, we first ascertained that aggregation induced by a GPVI-specific agonist, collagen-related peptide, like collagen, is markedly cross-blocked by a GPIb alpha-specific monoclonal antibody, SZ2. Immunoprecipitation of GPIb-IX with anti-GPIb alpha from the 1% (v/v) Triton-soluble fraction of unstimulated platelets and immunoblotting with anti-GPVI demonstrated association between GPIb-IX and GPVI. This association was maintained when platelets were activated by thrombin. Pre-treatment of platelets with methyl-beta-cyclodextrin to disrupt lipid rafts did not affect association in resting platelets under these conditions of detergent lysis. The association is also independent of cytoskeletal attachment, since it was unaffected by treatment with N-ethylmaleimide or DNaseI, which dissociate GPIb-IX from filamin and the actin-containing cytoskeleton, respectively. Finally, the association involves an interaction between the ectodomains of GPIb alpha and GPVI, since soluble fragments of GPIb alpha (glycocalicin) and GPVI are co-precipitated from the platelet supernatant under conditions where GPVI is shed. A contribution of GPIb-IX-V to GPVI-induced platelet responses, and vice versa, therefore warrants further investigation.

Platelet glycoprotein VI‐related clinical defects

Human patients with defects associated with the platelet collagen receptor, glycoprotein (GP)VI, are rare and usually described as having a mild bleeding disorder. However, here we review clinical profiles of patients with familial or acquired GPVI defects, revealing the bleeding defect is often severe and associated with immune dysfunction. GPVI is a member of the immunoreceptor family, and co‐expressed on platelets with Fc receptor γ‐chain (FcRγ). Ligand binding to GPVI leads to activation of platelet integrins, in particular αIIbβ3 that mediates platelet aggregation; and activation of endogenous platelet metalloproteinases resulting in ectodomain shedding and release of a soluble GPVI fragment. Increasing evidence supports the functional importance of GPVI/FcRγ in thrombus formation at arterial shear rates, and expression levels of platelet GPVI may be a marker of thrombotic risk. Over the past 20 years, patients have been reported with GPVI‐related defects involving: (i) an acquired deficiency, resulting from (a) anti‐GPVI autoantibodies or (b) other causes; or (ii) a congenital deficiency, where (c) GPVI is not expressed or (d) is expressed in a dysfunctional form with defective signalling to αIIbβ3. Clinical consequences of GPVI‐related defects may be uniquely informative about the role of platelet GPVI in health and disease.

Pathologic shear triggers shedding of vascular receptors: a novel mechanism for down-regulation of platelet glycoprotein VI in stenosed coronary vessels.

Ligand-induced ectodomain shedding of glycoprotein VI (GPVI) is a metalloproteinase-dependent event. We examined whether shear force, in the absence of GPVI ligand, was sufficient to induce shedding of GPVI. Human-citrated platelet-rich plasma or washed platelets were subjected to increasing shear rates in a cone-plate viscometer, and levels of intact and cleaved GPVI were examined by Western blot and ELISA. Pathophysiologic shear rates (3000-10 000 seconds(-1)) induced platelet aggregation and metalloproteinase-dependent appearance of soluble GPVI ectodomain, and GPVI platelet remnant. Shedding of GPVI continued after transient exposure to shear. Blockade of α(IIb)β(3), GPIbα, or intracellular signaling inhibited shear-induced platelet aggregation but minimally affected shear-induced shedding of GPVI. Shear-induced GPVI shedding also occurred in platelet-rich plasma or washed platelets isolated from a von Willebrand disease type 3 patient with no detectable VWF, implying that shear-induced activation of platelet metalloproteinases can occur in the absence of GPVI and GPIbα ligands. Significantly elevated levels of sGPVI were observed in 10 patients with stable angina pectoris, with well-defined single vessel coronary artery disease and mean intracoronary shear estimates at 2935 seconds(-1) (peak shear, 19 224 seconds(-1)). Loss of GPVI in platelets exposed to shear has potential implications for the stability of a forming thrombus at arterial shear rates.

Akt-mediated Cardiomyocyte Survival Pathways Are Compromised by Gαq-induced Phosphoinositide 4,5-Bisphosphate Depletion

Expression of the wild type α subunit of Gq (GqWT) in cardiomyocytes induces hypertrophy, whereas a constitutively active Gαq subunit (GqQ209L) induces apoptosis. Akt phosphorylation increases with GqWT expression but is markedly attenuated in cardiomyocytes expressing GqQ209L or in those expressing GqWT and treated with agonist. A membrane-targeted Akt rescues GqQ209L-expressing cardiomyocytes from apoptotic cell death. In contrast, leukemia inhibitory factor fails to activate Akt or promote cell survival in these cells. Association of Akt and PDK-1 with the membrane is also diminished in GqQ209L-expressing cardiomyocytes. Phosphatidylinositol 3,4,5-trisphosphate (PIP3), the primary regulator of Akt, increases significantly in GqWT-expressing cells but not in cardiomyocytes expressing GqQ209L. Levels of phosphatidylinositol 4,5-bisphosphate (PIP2), the immediate precursor of PIP3, are also markedly lower in GqQ209L-expressing compared to control cells. Expression of a GqQ209L mutant that has diminished capacity to activate phospholipase C does not decrease PIP2 or Akt or induce apoptosis. In transgenic mice with cardiac Gαq overexpression, heart failure and increased cardiomyocyte apoptosis develop during the peripartal period. Akt phosphorylation and PIP2 levels decrease concomitantly. Our findings suggest that an Akt-mediated cell survival pathway is compromised by the diminished availability of PIP2 elicited by pathological levels of Gq activity.

Ligand binding rapidly induces disulfide-dependent dimerization of glycoprotein VI on the platelet plasma membrane.

Thrombus formation in hemostasis or thrombotic disease is initiated by adhesion of circulating platelets to damaged blood vessel walls. Exposed subendothelial collagen interacting with platelet glycoprotein (GP) VI leads to platelet activation and integrin αIIbβ3-mediated aggregation. We previously showed that ligand binding to GPVI also induces metalloproteinase-dependent shedding, generating an ∼55-kDa soluble ectodomain fragment and an ∼10-kDa membrane-associated remnant. Here, treatment of platelets with collagen or the GPVI-targeting rattlesnake toxin convulxin also induces rapid (10–30 s) formation of a high molecular weight GPVI complex (GPVIc) under nonreducing conditions, as detected by immunoblotting with anti-GPVI antibodies. The appearance of an ∼20-kDa remnant detectable using a polyclonal antibody against the GPVI cytoplasmic tail under nonreducing, but not reducing, conditions after ectodomain shedding and nonreduced/reduced two-dimensional SDS-polyacrylamide gel analysis of biotinylated platelets confirmed that that GPVIc was a homodimer. Formation of disulfide-linked GPVIc was prolonged in the presence of metalloproteinase inhibitor GM6001 and was independent of GPVI signaling because it was unaffected by inhibitors of Src kinases, Syk, or phosphoinositide 3-kinase. To identify the thiol involved in disulfide bond formation, wild-type or mutant GPVI, where two available sulfhydryls (Cys-274 and Cys-338) were individually mutated to serine, was expressed in rat basophilic leukemia cells. Dimerization of wild-type and C274S GPVI, but not the C338S mutant, was observed after treating cells with convulxin. We conclude that (i) a subpopulation of GPVI forms a constitutive dimer on the platelet surface, facilitating rapid disulfide cross-linking, (ii) convulxin or other GPVI agonists induce disulfide-linked GPVI dimerization independent of GPVI signaling, and (iii) the penultimate residue of the GPVI cytoplasmic tail, Cys-338, mediates disulfide-dependent dimer formation.

ITAM receptor-mediated generation of reactive oxygen species in human platelets occurs via Syk-dependent and Syk-independent pathways

Background: Ligation of the platelet‐specific collagen receptor, GPVI/FcRγ, causes rapid, transient disulfide‐dependent homodimerization, and the production of intracellular reactive oxygen species (ROS) generated by the NADPH oxidase, linked to GPVI via TRAF4. Objectives: The aim of this study was to evaluate the role of early signaling events in ROS generation following engagement of either GPVI/FcRγ or a second immunoreceptor tyrosine‐based activation motif (ITAM)‐containing receptor on platelets, FcγRIIa. Methods and Results: Using an H2DCF‐DA‐based flow cytometric assay to measure intracellular ROS, we show that treatment of platelets with either the GPVI agonists, collagen‐related peptide (CRP) or convulxin (Cvx), or the FcγRIIa agonist 14A2, increased intraplatelet ROS; other platelet agonists such as ADP and TRAP did not. Basal ROS in platelet‐rich plasma from 14 healthy donors displayed little inter‐individual variability. CRP, Cvx or 14A2 induced an initial burst of ROS within 2 min followed by additional ROS reaching a plateau after 15–20 min. The Syk inhibitor BAY61‐3606, which blocks ITAM‐dependent signaling, had no effect on the initial ROS burst, but completely inhibited the second phase. Conclusions: Together, these results show for the first time that ROS generation downstream of GPVI or FcγRIIa consists of two distinct phases: an initial Syk‐independent burst followed by additional Syk‐dependent generation.

Phosphorothioate backbone modifications of nucleotide-based drugs are potent platelet activators.

Flierl et al. show that phosphorothioate (PS) oligonucleotides activate platelets via interacting with the collagen receptor GPVI. As PS backbone modification is currently used for nucleotide-based drug candidates, the findings suggest that this widely used method may present a risk to patients in the form of arterial thrombosis.

TNF receptor-associated factor 4 (TRAF4) is a novel binding partner of glycoprotein Ib and glycoprotein VI in human platelets

Summary.  Background: Reactive oxygen species generation is one consequence of ligand engagement of platelet glycoprotein (GP) receptors GPIb‐IX‐V and GPVI, which bind VWF/collagen and initiate thrombosis at arterial shear; however, the precise molecular mechanism coupling redox pathway activation to engagement of these receptors is unknown. Objective: The objective of this study was to identify novel binding partners for GPIb‐IX‐V and GPVI that could provide a potential link between redox pathways and early platelet signaling events. Methods and Results: Using protein array analysis and affinity‐binding assays, we demonstrated that the orphan TNF receptor‐associated factor (TRAF) family member, TRAF4, selectively binds cytoplasmic sequences of GPIbβ and GPVI. TRAF4, p47phox [of the NADPH oxidase (Nox2) enzyme complex] and other redox relevant signaling proteins such as Hic‐5, co‐immunoprecipitate with GPIb/GPVI from human platelet lysates whilst MBP‐TRAF4 or MBP‐p47phox fusion proteins specifically pull‐down GPIb/GPVI. GPIb‐ or GPVI‐selective agonists induce phosphorylation of the TRAF4‐associated proteins, Hic‐5 and Pyk2, with phosphorylation attenuated by Nox2 inhibition. Conclusion: These results describe the first direct association of TRAF4 with a receptor, and identify a novel binding partner for GPIb‐IX‐V and GPVI, providing a potential link between these platelet receptors and downstream TRAF4/Nox2‐dependent redox pathways.

Focusing on plasma glycoprotein VI

New methods for analysing both platelet and plasma forms of the platelet-specific collagen receptor, glycoprotein VI (GPVI) in experimental models or human clinical samples, and the development of the first therapeutic compounds based on dimeric soluble GPVI-Fc or anti-GPVI antibody-based constructs, coincide with increased understanding of the potential pathophysiological role of GPVI ligand binding and shedding. Platelet GPVI not only mediates platelet activation at the site of vascular injury where collagen is exposed, but is also implicated in the pathogenesis of other diseases, such as atherosclerosis and coagulopathy, rheumatoid arthritis and tumour metastasis. Here, we describe some of the critical mechanisms for generating soluble GPVI from platelets, and future avenues for exploiting this unique platelet-specific receptor for diagnosis and/or disease prevention.