Regulation of platelet membrane protein shedding in health and disease

Abstract A monoclonal antiplatelet antibody (MA-13G8E1) is described that dose- dependently induces platelet aggregation and serotonin release in an Fc- independent fashion. Whereas platelets were equally aggregated by F(ab')2 fragments of this monoclonal antibody (MoAb), its Fab fragments, on the other hand, were inactive, indicating that divalent interaction is an essential requirement to induce platelet activation by MA-13G8E1. In addition, we could show that platelet epitope cross- linking by MA-13G8E1 occurred on the same platelet. MA-13G8E1 stimulated platelet phospholipase C (PLC) and induced activation of protein kinase C (PKC), both of which were almost unaffected by aspirin pretreatment. Furthermore, PLC activation appeared to be a direct antibody-mediated effect, since intracellular Ca2+ rises were not inhibited by EGTA, cytochalasin B, or aggregation-blocking MA-16N7C2 (antiglycoprotein [anti-GP]IIb/IIa). The MA-13G8E1 antigen is constitutively expressed on resting platelets of different species (7,100 +/- 800 molecules per human platelet), but not on other cell types tested. Both immunoprecipitation and affinity isolation by MA- 13G8E1 showed two low-molecular weight proteins (45 and 36 kD), having slightly acidic isoelectric pH levels (4.5 to 5.5) and forming multimolecular complexes. In conclusion, we found an MoAb that is able to induce platelet activation in an Fc-independent fashion. The mechanism involves cross-linking of a hitherto undescribed platelet membrane protein, leading to PLC and PKC stimulation.

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Platelet Agglutinating Protein p37 Causes Platelet Agglutination through its Binding to Membrane Glycoprotein IV

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647462 ◽

1991 ◽

Vol 65 (01) ◽

pp. 102-106 ◽

Cited By ~ 22

Author(s):

Eric C-Y Lian ◽

Farooq A Siddiqui ◽

G A Jamieson ◽

Narendra N Tandon

Keyword(s):

Monoclonal Antibodies ◽

Membrane Proteins ◽

Membrane Protein ◽

Receptor Site ◽

Platelet Membrane ◽

Membrane Glycoprotein ◽

Thrombocytopenic Purpura ◽

Normal Platelet ◽

Reducing Conditions ◽

Rabbit Igg

SummaryA 37 kDa platelet agglutinating protein (PAP p37) has previously been shown to be present in a subset of patients with thrombotic thrombocytopenic purpura and has been purified from their plasma. Using solubilized platelet membrane proteins from normal donors, it was shown by Western blotting that r2sI-p37 bound to a membrane protein of 97 kDa (red/unred). Furthernore, the same protein was identified by reverse immunoblotting in which purified p37 was electrophoresed, transferred to the nitrocellulose sheet and incubated with solubilized normal platelet membrane proteins. The complex formed between p37 and the membrane protein was identified by autoradiography using polyclonal and monoclonal (OKM5) anti- GPIV antibodies, but was not detected by polyclonal antibody to GPIIIa. Similar studies with purified platelet GPIV under both reducing and non-reducing conditions demonstrated the binding of 125I-p37. Polyclonal and monoclonal antibodies to GPIV completely inhibited the platelet agglutination induced by TTP plasma containing p37, however, normal rabbit IgG, rabbit anti- GPIIIa IgG, and murine monoclonal anti-GPIIb/IIIa (10E5) antibodies had no effect. These data indicate that platelet GPIV is the receptor site for PAP p37.

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Structural analysis of the Na+/H+ exchanger isoform 1 (NHE1) using the divide and conquer approachThis paper is one of a selection of papers published in a Special Issue entitled CSBMCB 53rd Annual Meeting — Membrane Proteins in Health and Disease, and has undergone the Journal’s usual peer review process.

Biochemistry and Cell Biology ◽

10.1139/o10-140 ◽

2011 ◽

Vol 89 (2) ◽

pp. 189-199 ◽

Cited By ~ 16

Author(s):

Brian L. Lee ◽

Brian D. Sykes ◽

Larry Fliegel

Keyword(s):

Crystal Structure ◽

Membrane Protein ◽

Peer Review Process ◽

Divide And Conquer ◽

Transmembrane Helices ◽

Transmembrane Segments ◽

Sodium Proton Exchanger ◽

Attractive Therapeutic Target ◽

Health And Disease ◽

Selection Of

The sodium/proton exchanger isoform 1 (NHE1) is an ubiquitous plasma membrane protein that regulates intracellular pH by removing excess intracellular acid. NHE1 is important in heart disease and cancer, making it an attractive therapeutic target. Although much is known about the function of NHE1, current structural knowledge of NHE1 is limited to two conflicting topology models: a low-resolution molecular envelope from electron microscopy, and comparison with a crystal structure of a bacterial homologue, NhaA. Our laboratory has used high-resolution nuclear magnetic resonance (NMR) spectroscopy to investigate the structures of individual transmembrane helices of NHE1 — a divide and conquer approach to the study of this membrane protein. In this review, we discuss the structural and functional insights obtained from this approach in combination with functional data obtained from mutagenesis experiments on the protein. We also compare the known structure of NHE1 transmembrane segments with the structural and functional insights obtained from a bacterial sodium/proton exchanger homologue, NhaA. The structures of regions of the NHE1 protein that have been determined have both similarities and specific differences to the crystal structure of the NhaA protein. These have allowed insights into both the topology and the function of the NHE1 protein.

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Human platelet membrane protein

Biochemistry ◽

10.1021/bi00804a002 ◽

1970 ◽

Vol 9 (2) ◽

pp. 200-205 ◽

Cited By ~ 16

Author(s):

Ralph L. Nachman ◽

Barbara Ferris

Keyword(s):

Membrane Protein ◽

Human Platelet ◽

Platelet Membrane

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Report on the 53rd Annual Meeting of the Canadian Society of Biochemistry, Molecular and Cellular Biology: “Membrane Proteins in Health and Disease”

Biochemistry and Cell Biology ◽

10.1139/o10-157 ◽

2011 ◽

Vol 89 (2) ◽

pp. 79-81

Author(s):

Reinhart A.F. Reithmeier ◽

Joseph R. Casey

Keyword(s):

Protein Structure ◽

Membrane Proteins ◽

Membrane Protein ◽

Protein Trafficking ◽

Membrane Protein Structure ◽

Full Spectrum ◽

Membrane Protein Trafficking ◽

Bicarbonate Transporters ◽

Health And Disease ◽

And Function

The meeting “Membrane Proteins in Health and Disease” featured 6 sessions and 2 satellite meetings. At the opening session, Gunnar von Heijne delivered a plenary lecture entitled Insertion of Membrane Proteins into the Endoplasmic Reticulum. The following session topics were Membrane Protein Trafficking and Folding, Regulation of Membrane Proteins, Membrane Protein Structure, Membrane Proteins in Diverse Species, and Membrane Proteins and Diseases. The satellite meetings discussed bicarbonate transporters and Na+/H+ exchangers. Together the 21 lectures and 106 posters presented at the meeting spanned the full spectrum of current research into membrane protein structure and function.

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Release of Ca2+ by inositol 1,4,5-trisphosphate in platelet membrane vesicles is not dependent on cyclic AMP-dependent protein kinase

Biochemical Journal ◽

10.1042/bj2570715 ◽

1989 ◽

Vol 257 (3) ◽

pp. 715-721 ◽

Cited By ~ 18

Author(s):

F O'Rourke ◽

G B Zavoico ◽

M B Feinstein

Keyword(s):

Protein Kinase ◽

Cyclic Amp ◽

Membrane Protein ◽

Protein Phosphorylation ◽

Kinase Inhibitor ◽

Membrane Vesicles ◽

Platelet Membrane ◽

Dependent Protein Kinase ◽

Inositol 1,4,5 Trisphosphate ◽

Dependent Protein

In contrast with previous reports, it was found that membrane-protein phosphorylation by the catalytic subunit (CS) of cyclic AMP-dependent protein kinase had no effect on Ca2+ uptake into platelet membrane vesicles or on subsequent Ca2+ release by inositol 1,4,5-trisphosphate (IP3). Furthermore, IP-20, a highly potent synthetic peptide inhibitor of CS, which totally abolished membrane protein phosphorylation by endogenous or exogenous CS, also had no effect on either Ca2+ uptake or release by IP3. Commercial preparations of protein kinase inhibitor protein (PKI) usually had no effect, but one preparation partially inhibited Ca2+ uptake, which is attributable to the gross impurity of the commercial PKI preparation. IP3-induced release of Ca2+ was also unaffected by the absence of ATP from the medium, supporting the conclusion that Ca2+ release by IP3 does not require the phosphorylation of membrane protein.

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