Michael C. Berndt

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.

Molecular mechanisms of platelet adhesion and activation

When a blood vessel is injured, control of bleeding starts with the rapid adhesion of circulating platelets to the site of damage. Within seconds, the adhered platelets are activated, secrete the contents of storage organelles, spread out over the damaged area and recruit more platelets to the developing thrombus. However, if this same process occurs in a diseased, sclerotic or occluded vessel, the resulting platelet thrombus may break away and block the coronary artery, causing a heart attack, or restrict blood supply to the brain, causing a stroke. The glycoprotein (GP) Ib-IX-V complex, a member of the leucine-rich protein family, is a constitutive platelet membrane receptor for von Willebrand Factor (vWF), a multimeric adhesive glycoprotein found in the matrix underlying the endothelial cell lining of the blood vessel wall and in the plasma. Binding of vWF to the GP. Ib-IX-V complex regulates adhesion of platelets to the subendothelium at high shear flow, and initiates signal transduction leading to platelet activation. The GP Ib-IX-V complex also constitutes a binding site for alpha-thrombin, an interaction that facilitates thrombin-dependent platelet activation. This review will focus on recent detailed analysis of the GP Ib-IX-V complex and vWF that has identified discrete amino acid sequences that mediate their interaction. An anionic/sulfated tyrosine sequence of the GP Ib alpha-chain that is critical for binding of the GP Ib-IX-V complex to both vWF and alpha-thrombin is analogous to sulfated anionic amino acid sequences mediating interactions of other adhesive proteins, including P-selectin binding to PSGL-1 and Factor VIII binding to vWF.

Mechanism of Activation of the GM-CSF, IL-3, and IL-5 Family of Receptors

The process of ligand binding leading to receptor activation is an ordered and sequential one. High‐affinity binding of GM‐CSF, interleukin 3 (IL‐3), and IL‐5 to their receptors induces a number of key events at the cell surface and within the cytoplasm that are necessary for receptor activation. These include receptor oligomerization, activation of tyrosine kinase activity, phosphorylation of the receptor, and the recruitment of SH2 (src‐homology) and PTB (phosphotyrosine binding) domain proteins to the receptor. Such a sequence of events represents a recurrent theme among cytokine, growth factor, and hormone receptors; however, a number of very recent and interesting findings have identified unique features in this receptor system in terms of: A) how GM‐CSF/IL‐3/IL‐5 bind, oligomerize, and activate their cognate receptors; B) how multiple biological responses such as proliferation, survival, and differentiation can be transduced from activated GM‐CSF, IL‐3, or IL‐5 receptors, and C) how the presence of novel phosphotyrosine‐independent signaling motifs within a specific cytoplasmic domain of βC may be important for mediating survival and differentiation by these cytokines. This review does not attempt to be all‐encompassing but rather to focus on the most recent and significant discoveries that distinguish the GM‐CSF/IL‐3/IL‐5 receptor subfamily from other cytokine receptors.

P-selectin Glycoprotein Ligand-1 Is the Major Counter-receptor for P-selectin on Stimulated T Cells and Is Widely Distributed in Non-functional Form on Many Lymphocytic Cells

P-selectin glycoprotein ligand-1 (PSGL-1) is the high affinity counter-receptor for P-selectin on myeloid cells (Sako, D., Chang, X. J., Barone, K. M., Vachino, G., White, H. M., Shaw, G., Veldman, G. M., Bean, K. M., Ahern, T. J., Furie, B., Cumming, D. A., and Larsen, G. R.(1993) Cell 75, 1179-1186). Here we demonstrate that PSGL-1 is also widely distributed on T- and B-lymphocytic tumor cell lines, resting peripheral blood T and B cells, and on stimulated peripheral blood T cell and intestinal intraepithelial lymphocyte (IEL) lines. However, the majority of PSGL-1-positive resting peripheral blood lymphocytic cells and lymphoid tumor cell lines do not display significant P-selectin binding. In contrast, in vitro stimulated peripheral blood T cell and IEL lines avidly bind P-selectin, and PSGL-1 is the sole high affinity counter-receptor mediating this binding. During the course of in vitro stimulation, cell surface expression levels of PSGL-1 do not change as P-selectin binding increases. Rather, the activities of two glycosyltransferases reportedly involved in the production of functional PSGL-1 in myeloid cells are substantially higher in the stimulated T-lymphocytic lines than in resting T lymphocytes, consistent with the hypothesis that activation-dependent post-translational events contribute to the expression of functional PSGL-1 on lymphocytes.

Glycoprotein Ib-IX-V

Glycoprotein (GP) Ib-IX-V is a remarkable platelet adhesion receptor of the leucine-rich repeat family. It has evolved to fulfil its major function of initiating platelet aggregation (thrombus formation) at high-shear stress in flowing blood. In addition to binding von Willebrand factor (vWF) in subendothelial matrix or plasma to trigger platelet aggregation, GPIb-IX-V also binds counter-receptors, alphaMbeta2 (Mac-1) on neutrophils or P-selectin on activated platelets or endothelial cells. GPIb-IX-V ligands also include alpha-thrombin, clotting factors XI/XIIa, and high-molecular-weight kininogen. Interactions involving GPIb-IX-V are therefore central to vascular processes of thrombosis and inflammation, and the receptor is under intense scrutiny as a potential therapeutic target.

The SH2-containing inositol polyphosphate 5-phosphatase, SHIP-2, binds filamin and regulates submembraneous actin.

SHIP-2 is a phosphoinositidylinositol 3,4,5 trisphosphate (PtdIns[3,4,5]P3) 5-phosphatase that contains an NH2-terminal SH2 domain, a central 5-phosphatase domain, and a COOH-terminal proline-rich domain. SHIP-2 negatively regulates insulin signaling. In unstimulated cells, SHIP-2 localized in a perinuclear cytosolic distribution and at the leading edge of the cell. Endogenous and recombinant SHIP-2 localized to membrane ruffles, which were mediated by the COOH-terminal proline–rich domain. To identify proteins that bind to the SHIP-2 proline–rich domain, yeast two-hybrid screening was performed, which isolated actin-binding protein filamin C. In addition, both filamin A and B specifically interacted with SHIP-2 in this assay. SHIP-2 coimmunoprecipitated with filamin from COS-7 cells, and association between these species did not change after epidermal growth factor stimulation. SHIP-2 colocalized with filamin at Z-lines and the sarcolemma in striated muscle sections and at membrane ruffles in COS-7 cells, although the membrane ruffling response was reduced in cells overexpressing SHIP-2. SHIP-2 membrane ruffle localization was dependent on filamin binding, as SHIP-2 was expressed exclusively in the cytosol of filamin-deficient cells. Recombinant SHIP-2 regulated PtdIns(3,4,5)P3 levels and submembraneous actin at membrane ruffles after growth factor stimulation, dependent on SHIP-2 catalytic activity. Collectively these studies demonstrate that filamin-dependent SHIP-2 localization critically regulates phosphatidylinositol 3 kinase signaling to the actin cytoskeleton.

Transforming Growth Factor β Suppresses Human Telomerase Reverse Transcriptase (hTERT) by Smad3 Interactions with c-Myc and the hTERT Gene

Telomerase underpins stem cell renewal and proliferation and is required for most neoplasia. Recent studies suggest that hormones and growth factors play physiological roles in regulating telomerase activity. In this report we show a rapid repression of the telomerase reverse transcriptase (TERT) gene by transforming growth factor β (TGF-β) in normal and neoplastic cells by a mechanism depending on the intracellular signaling protein Smad3. In human breast cancer cells TGF-β induces rapid entry of Smad3 into the nucleus where it binds to the TERT gene promoter and represses TERT gene transcription. Silencing Smad3 gene expression or genetically deleting the Smad3 gene disrupts TGF-β repression of TERT gene expression. Expression of the Smad3 antagonist, Smad7, also interrupts TGF-β-mediated Smad3-induced repression of the TERT gene. Mutational analysis identified the Smad3 site on the TERT gene promoter, mediating TERT repression. In response to TGF-β, Smad3 binds to c-Myc; knocking down c-Myc, Smad3 does not bind to the TERT gene, suggesting that c-Myc recruits Smad3 to the TERT promoter. Thus, TGF-β negatively regulates telomerase activity via Smad3 interactions with c-Myc and the TERT gene promoter. Modifying the interaction between Smad3 and TERT gene may, thus, lead to novel strategies to regulate telomerase.

A Novel Cobra Venom Metalloproteinase, Mocarhagin, Cleaves a 10-Amino Acid Peptide from the Mature N Terminus of P-selectin Glycoprotein Ligand Receptor, PSGL-1, and Abolishes P-selectin Binding

Initial rolling of circulating neutrophils on a blood vessel wall prior to adhesion and transmigration to damaged tissue is dependent upon P-selectin expressed on endothelial cells and its specific neutrophil receptor, the P-selectin glycoprotein ligand-1 (PSGL-1). Pretreatment of neutrophils, HL60 cells, or a recombinant fucosylated soluble form of PSGL-1 (sPSGL-1.T7) with the cobra venom metalloproteinase, mocarhagin, completely abolished binding to purified P-selectin in a time-dependent and EDTA- and diisopropyl fluorophosphate-inhibitable manner consistent with mocarhagin selectively cleaving PSGL-1. A polyclonal antibody against the N-terminal peptide Gln-1-Glu-15 of mature PSGL-1 immunoprecipitated sPSGL-1.T7 but not sPSGL-1.T7 treated with mocarhagin, indicating that the mocarhagin cleavage site was near the N terminus. A single mocarhagin cleavage site between Tyr-10 and Asp-11 of mature PSGL-1 was determined by N-terminal sequencing of mocarhagin fragments of sPSGL-1.T7 and is within a highly negatively charged amino acid sequence 1-QATEYEYLDYDFLPETEPPE, containing three tyrosine residues that are consensus sulfation sites. Consistent with a functional role of this region of PSGL-1 in binding P-selectin, an affinity-purified polyclonal antibody against residues Gln-1-Glu-15 of PSGL-1 strongly inhibited P-selectin binding to neutrophils, whereas an antibody against residues Asp-9-Arg-23 was noninhibitory. These combined data strongly suggest that the N-terminal anionic/sulfated tyrosine motif of PSGL-1 as well as downstream sialylated carbohydrate is essential for binding of P-selectin by neutrophils.

Platelet GPIb‐IX‐V‐dependent signaling

Summary.  Although the signaling pathways related to GPIb‐IX‐V have not been fully elucidated, an accumulating body of evidence suggests that phospholipase C (PLC)γ2 activation, subsequent Ca++ release and oscillations constitute an essential signal transduction pathway related to GPIb‐IX‐V. Src family kinases are required for PLCγ2 activation, while FcRγ‐chain/FcγRIIA may be dispensable for PLCγ2 activation. Although PI‐3K serves to potentiate various signaling events culminating in αIIbβ3 activation, PI‐3K activity may be dispensable for Src‐PLCγ2 activation in GPIb‐IX‐V‐mediated signaling. Glycosphingolipid‐enriched microdomains (GEMs) appear to provide platforms for the signal transduction pathway related to GIb‐IX‐V, as the interaction between GPIb‐IX‐V and Src or PLCγ2 tyrosine phosphorylation occurs exclusively in GEMs.

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.