MONOCLONAL ANTIBODIES RECOGNISING PROTEINS OF THE OUTER AND INNER SURFACE OF THE PLATELET PLASMA MEMBRANE

Platelet membrane preparations can be fractionated into two major subpopulations by free flow electrophoresis and these have been shown to correspond to the plasma membrane and the endoplasmic reticulum of the platelet. The plasma membrane fraction can be shown, by two-dimensional electrophoresis, to contain the major surface glycoproteins together with considerable amounts of actin and actin-associated proteins such as the 250 kDa actin-binding protein (filamin), P235 (talin), myosin, α-actinin and tropomyosin (Hack, N. … Crawford, N., Biochem. J. 222, 235 (1984). These cytoskeletal proteins are associated with the cytoplasmic face of the plasma membrane and probably interact with transmembrane glycoproteins. We have raised monoclonal antibodies to the purified plasma membrane preparation in order to investigate the nature of these glycoprotein-cytoskeletal interactions. In two fusion experiments, out of 804 tested, 104 hybrids secreted antibody to the membrane preparation and of these 24 were selected for further study. Initial assays were by ELISA using either the membrane preparation or whole fixed platelets as the target antigen. The specificity of the antibodies was investigated further by immunoblotting of SDS gels of total platelet proteins prepared under reducing and nonreducing conditions, by immunofluorescence, by immunohisto-chemistry and by crossed immunoelectrophoresis. The majority of the antibodies recognise major surface glycoproteins; of these, four bind to glycoprotein Ib under all conditions examined while another seven recognise the glycoprotein IIb/IIIa complex as detected by crossed immunoelectrophoresis. Three antibodies recognise the actin binding protein and these cross-react with the smooth muscle protein filamin in a number of different species. Further characterisation of these antibodies in both structural and functional terms will be presented.We are grateful to the Smith and Nephew Foundation for financial support for these studies

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Binding of Human Platelet Glycoprotein lb and Actin to Fragments of Actin-Binding Protein

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648421 ◽

1992 ◽

Vol 67 (02) ◽

pp. 252-257 ◽

Cited By ~ 9

Author(s):

Anne M Aakhus ◽

J Michael Wilkinson ◽

Nils Olav Solum

Keyword(s):

Actin Filaments ◽

High Speed ◽

Binding Protein ◽

Protein A ◽

Actin Binding ◽

Platelet Glycoprotein ◽

Actin Binding Protein ◽

Crossed Immunoelectrophoresis ◽

Triton X ◽

Calpain Inhibitors

SummaryActin-binding protein (ABP) is degraded into fragments of 190 and 90 kDa by calpain. A monoclonal antibody (MAb TI10) against the 90 kDa fragment of ABP coprecipitated with the glycoprotein lb (GP lb) peak observed on crossed immunoelectrophoresis of Triton X-100 extracts of platelets prepared without calpain inhibitors. MAb PM6/317 against the 190 kDa fragment was not coprecipitated with the GP lb peak under such conditions. The 90 kDa fragment was adsorbed on protein A agarose from extracts that had been preincubated with antibodies to GP lb. This supports the idea that the GP Ib-ABP interaction resides in the 90 kDa region of ABP. GP lb was sedimented with the Triton-insoluble actin filaments in trace amounts only, and only after high speed centrifugation (100,000 × g, 3 h). Both the 190 kDa and the 90 kDa fragments of ABP were sedimented with the Triton-insoluble actin filaments.

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EFFECTS OF SOME ORGANIC SOLVENTS ON GP lb AND ACTIN-BINDING PROTEIN IN BLOOD PLATELETS

10.1055/s-0038-1643511 ◽

1987 ◽

Author(s):

A M Aakhus ◽

N O Solum ◽

I Hagen

Keyword(s):

Organic Solvents ◽

Von Willebrand Factor ◽

Early Stage ◽

Binding Protein ◽

Blood Platelets ◽

Cytoskeletal Proteins ◽

Actin Binding ◽

Actin Binding Protein ◽

Von Willebrand ◽

Willebrand Factor

Effects on filamentous proteins appear to be a central phenomenon in the neuronal toxic effects of organic solvents. We have therefore compared the effects of some organic solvents (particularly isopropylalcohol, IPA) to the previously observed effects of dibucaine (DBC) on platelet cytoskeletal proteins. Incubation of platelets with 6% IPA at 37° C, like DBC, initiates a degradation of actin-binding protein (ABP) as substrate for a calcium activated protease (CAP), shown by SDS-PAGE. IPA leads to an increase followed by a decrease in bovine von Willebrand factor-induced agglutination. The decrease is accompanied by a release of glycocali-cin from the GP lb α-chain. The process was also studied using CIE of Triton X-100 extracts of platelets against antiserum to glycocalicin. Incubation of platelets with IPA before extraction in the presence of 4.2 mM leupeptin leads to a time-dependent transformation of GP Ib-related immunoprecipitates from that of the slow-migrating peak III complex (probably between ABP and GP lb) to the faster migrating GP Ib-precipitate. Our working hypothesis is that IPA induces an activation of the CAP by mobilizing calcium. This leads to degradation of ABP and liberation of GP lb from the cytoskeleton accompanied by an increased tendency for agglutination. The following decrease is explained by degradation of the glycocalicin part of the GP lb enchain which contains the binding-site for von Willebrand factor. We conclude that IPA has a similar effect on GP lb and ABP as DBC. Preliminary studies with 1% DMSO and 0,005% toluene at 37° C revealed that these organic solvents have some similar effects on platelets as described for IPA. Possibly the described effects are characteristic of certain cells at an early stage in a process ultimately leading to cell lysis.

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Identification of membrane proteins mediating the interaction of human platelets

The Journal of Cell Biology ◽

10.1083/jcb.86.1.77 ◽

1980 ◽

Vol 86 (1) ◽

pp. 77-86 ◽

Cited By ~ 234

Author(s):

D Phillips ◽

L Jennings ◽

H Edwards

Keyword(s):

Actin Filaments ◽

Binding Protein ◽

Membrane Surface ◽

Direct Interaction ◽

Cytoskeletal Proteins ◽

Actin Binding ◽

Insoluble Residue ◽

Membrane Glycoproteins ◽

Actin Binding Protein ◽

Activated Platelets

Membrane glycoproteins that mediate platelet-platelet interactions were investigated by identifying those associated with the cytoskeletal structures from aggregated platelets. The cytoskeletal structures from washed platelets, thrombin-activated platelets (platelets incubated with thrombin in the presence of mM EDTA to prevent aggregation) and thrombin- aggregated platelets (platelets activated in the presence of mM Ca(++) were prepared by first treating platelet suspensions with 1 percent Triton X-100 and 5 mM EGTA and then isolating the insoluble residue by centrifugation. The readily identifiable structures in electron micrographs of the residue from washed platelets had the shape and dimensions of actin filaments. Analysis of this residue from washed platelets had the shape and dimensions of actin filaments. Analysis of this residue by SDS gel electrophoresis showed that it consisted primarily of three proteins: actin (mol wt = 43,000), myosin (mol wt = 200,000) and a high molecular weight polypeptide (mol wt = 255,000) which had properties indentical to actin-binding protein (filamin). When platelets are activated with thrombin in the presence of EDTA to prevent aggregation, there was a marked increase in the amount of insoluble precipitate in the subsequent Triton extraction. Transmission electron microscopy showed that this residue not only contained the random array of actin filaments as seen above, but also organized structures from individual platelets which appeared as balls of electron-dense filamentous material approximately 1mum in diameter. SDS polyacrylamide gel analysis of the Triton residue of activated platelets showed that this preparation contained more actin, myosin and actin-binding protein than that from washed platelets plus polypeptides with mol wt of 56,000 and 90,000 and other minor polypeptides. Thus, thrombin activation appeared to increase polymerization of actin in association with other cytoskeletal proteins into structures that are observable after Triton extraction. The cytoskeletal structures from thrombin-aggregated platelets were similar to those from thrombin-activated platelets, except that the structural elements from individual platelets remained aggregated rather than randomly dispersed in the actin filaments. This suggested that the membrane components that mediate the direct interaction of platelets were in Triton residue from aggregated platelets. Only a small percentage of the membrane surface proteins and glycoproteins were found in the cytoskeletal structures from either washed platelets or thrombin-activated platelets. In contrast, the aggregated cytoskeletal structures from thrombin-aggregated platelets contained membrane glycoproteins IIb (26 percent of the total in pre-extracted platelets) and III (14 percent), suggesting that one or both of these glycoproteins participate in the direct interaction of platelets during aggregation.

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Isoform specific expression of the neuronal F-actin binding protein, drebrin, in specialized cells of stomach and kidney epithelia

Journal of Cell Science ◽

10.1242/jcs.113.2.325 ◽

2000 ◽

Vol 113 (2) ◽

pp. 325-336 ◽

Cited By ~ 4

Author(s):

B.H. Keon ◽

P.T. Jedrzejewski ◽

D.L. Paul ◽

D.A. Goodenough

Keyword(s):

Plasma Membrane ◽

Cell Lines ◽

Cultured Cells ◽

Binding Protein ◽

Specific Pattern ◽

Actin Binding ◽

Apical Plasma Membrane ◽

Specific Expression ◽

Actin Binding Protein ◽

Acid Secreting

To further understand the functional role that the F-actin binding protein, drebrin (developmentally regulated brain protein), plays in the regulation of F-actin, we characterized its expression in non-neuronal cells. Using nanoelectrospray mass spectrometry methods, we initially identified drebrin in non-neuronal cultured cells. Using a drebrin-specific monoclonal antibody, we were able to detect drebrin protein in several different cell lines derived from fibroblasts, astrocytomas, and simple epithelia, but not in cell lines derived from stratified epithelia. Double-label immunofluorescence experiments of cultured cell monolayers revealed the localization of drebrin at the apical plasma membrane together with a pool of submembranous F-actin. Immunoblot analysis of mouse organs revealed that, in addition to its high levels of expression in brain, drebrin was present in stomach and to a lesser degree in kidney, colon, and urinary bladder. Drebrin protein detected in the non-brain organs migrated faster through SDS-PAGE gels, indicating that the lower molecular weight embryonic brain isoform (E2) may be the prominent isoform in these organs. RT-PCR experiments confirmed the specific expression of the E2 isoform in adult stomach, kidney, and cultured cells. In situ immunofluorescence experiments revealed a cell-type specific pattern in both stomach and kidney. In stomach, drebrin was specifically expressed in the acid-secreting parietal cells of the fundic glands, where it accumulated at the extended apical membrane of the canaliculi. In kidney, drebrin was expressed in acid-secreting type A intercalated cells, where it localized specifically to the apical plasma membrane. Drebrin was expressed as well in the distal tubule epithelial cells where the protein was concentrated at the luminal surface and present at the interdigitations of the basolateral membranes.

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Role of the membrane skeleton in preventing the shedding of procoagulant-rich microvesicles from the platelet plasma membrane.

The Journal of Cell Biology ◽

10.1083/jcb.111.2.483 ◽

1990 ◽

Vol 111 (2) ◽

pp. 483-493 ◽

Cited By ~ 97

Author(s):

J E Fox ◽

C D Austin ◽

J K Boyles ◽

P K Steffen

Keyword(s):

Plasma Membrane ◽

Binding Protein ◽

Membrane Skeleton ◽

Membrane Properties ◽

Actin Binding ◽

Ionophore A23187 ◽

Membrane Interactions ◽

Membrane Glycoproteins ◽

Actin Binding Protein ◽

Hydrolysis Of

The platelet plasma membrane is lined by a membrane skeleton that appears to contain short actin filaments cross-linked by actin-binding protein. Actin-binding protein is in turn associated with specific plasma membrane glycoproteins. The aim of this study was to determine whether the membrane skeleton regulates properties of the plasma membrane. Platelets were incubated with agents that disrupted the association of the membrane skeleton with membrane glycoproteins. The consequences of this change on plasma membrane properties were examined. The agents that were used were ionophore A23187 and dibucaine. Both agents activated calpain (the Ca2(+)-dependent protease), resulting in the hydrolysis of actin-binding protein and decreased association of actin with membrane glycoproteins. Disruption of actin-membrane interactions was accompanied by the shedding of procoagulant-rich microvesicles from the plasma membrane. The shedding of microvesicles correlated with the hydrolysis of actin-binding protein and the disruption of actin-membrane interactions. When the calpain-induced disruption of actin-membrane interactions was inhibited, the shedding of microvesicles was inhibited. These data are consistent with the hypothesis that association of the membrane skeleton with the plasma membrane maintains the integrity of the plasma membrane, preventing the shedding of procoagulant-rich microvesicles from the membrane of unstimulated platelets. They raise the possibility that the procoagulant-rich microvesicles that are released under a variety of physiological and pathological conditions may result from the dissociation of the platelet membrane skeleton from its membrane attachment sites.

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Genetic evidence for functional interactions between actin noncomplementing (Anc) gene products and actin cytoskeletal proteins in Saccharomyces cerevisiae.

Genetics ◽

10.1093/genetics/135.2.275 ◽

1993 ◽

Vol 135 (2) ◽

pp. 275-286

Author(s):

D B Vinh ◽

M D Welch ◽

A K Corsi ◽

K F Wertman ◽

D G Drubin

Keyword(s):

Synthetic Lethality ◽

Binding Protein ◽

Cytoskeletal Proteins ◽

Genetic Interactions ◽

Null Alleles ◽

Actin Binding ◽

Gene Products ◽

Actin Binding Protein ◽

Functional Interactions ◽

Allele Specific

Abstract We describe here genetic interactions between mutant alleles of Actin-NonComplementing (ANC) genes and actin (ACT1) or actin-binding protein (SAC6, ABP1, TPM1) genes. The anc mutations were found to exhibit allele-specific noncomplementing interactions with different act1 mutations. In addition, mutant alleles of four ANC genes (ANC1, ANC2, ANC3 and ANC4) were tested for interactions with null alleles of actin-binding protein genes. An anc1 mutant allele failed to complement null alleles of the SAC6 and TPM1 genes that encode yeast fimbrin and tropomyosin, respectively. Also, synthetic lethality between anc3 and sac6 mutations, and between anc4 and tpm1 mutations was observed. Taken together, the above results strongly suggest that the ANC gene products contribute to diverse aspects of actin function. Finally, we report the results of tests of two models previously proposed to explain extragenic noncomplementation.

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Presence of Clathrin at Adhesion Sites in Phagocytosing Macrophages

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100053309 ◽

1982 ◽

Vol 40 ◽

pp. 114-117

Author(s):

J. Aggeler ◽

J.E. Heuser ◽

Z. Werb

Keyword(s):

Plasma Membrane ◽

Actin Filaments ◽

Binding Proteins ◽

Binding Protein ◽

Cell Spreading ◽

Actin Binding ◽

Dense Network ◽

Actin Binding Protein ◽

Adhesion Sites ◽

Cytoplasmic Surface

Phagocytosis of particles by macrophages may be similar to cell spreading on a substratum, in that a dense network of actin filaments appears beneath the plasma membrane in both cases. When viewed in broken-open or detergent- extracted cells, cytoskeletal filaments are observed to form focal attachments to the plasma membrane and to the cytoplasmic surface of phagosomes. Hartwig et al. have presented a model of phagocytosis in which an actin-binding protein alters the organization of subplasmalemma1 actin filaments in such a way that the plasma membrane is forced up over the particle to form the phagosome. Their evidence indicates that similar actin-binding proteins may function during cell spreading.

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Cytoskeletal involvement in the sequential capping of rat thymocyte surface glycoproteins

Journal of Cell Science ◽

10.1242/jcs.89.3.309 ◽

1988 ◽

Vol 89 (3) ◽

pp. 309-319

Author(s):

C.E. Turner ◽

M.R. Newton ◽

D.M. Shotton

Keyword(s):

Monoclonal Antibodies ◽

Cell Surface ◽

Cytoskeletal Proteins ◽

The Other ◽

A Cell ◽

Major Surface ◽

Surface Glycoproteins

The independent capping of the three major rat thymocyte glycoproteins, the leucocyte-common (L-C) antigen, the leucocyte sialoglycoprotein (LSGP) and Thy-1, was investigated using specific monoclonal antibodies. The capping of each antigen did not require redistribution of the other major surface glycoproteins, and was accompanied by a partial co-capping of the cytoskeletal proteins fodrin and actin, but not of tubulin. A study of the ability of a cell that already possesses one glycoprotein cap to cap a second different glycoprotein showed that this was possible in all cases to varying degrees, the second cap always forming at the same position on the cell surface as the first. Colchicine failed to perturb this observed sequential capping polarity, indicating that microtubules did not direct this second capping event.

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