IDENTIFICATION OF GLYCOPROTEIN Ib8 AS THE Mr = 24,000 PLATELET POLYPEPTIDE PHOSPHORYLATED BY AGENTS THAT ELEVATE CYCLIC AMP

1987 ◽  
Author(s):  
J E B Fox ◽  
C C Reynolds ◽  
J K Boyles ◽  
R A Abel ◽  
M M Johnson
Keyword(s):  
Cyclic Amp ◽  
Platelet Function ◽  
Actin Polymerization ◽  
Binding Protein ◽  
Membrane Skeleton ◽  
Actin Binding ◽  
Inhibitory Effects ◽  
Actin Binding Protein ◽  
Triton X ◽  
Β Chain

Platelet function is inhibited by agents that elevate intracellular cyclic AMP concentrations, presumably as a result of the cyclic AMP-stimulated phosphorylation of intracellular proteins. Polypeptides that become phosphorylated are of Mr = 250,000, Mr = 51.000 (P51), Mr = 36,000 (P36), Mr = 24,000 (P24), and Mr = 22.000 (P22). The Mr = 250,000 polypeptide is actin-binding protein, but the identity of the other polypeptides 1s unknown. In the present study, we identified the P24 polypeptide. Platelets were radiolabeled with [32P]P1 and then Incubated for 2-5 min in the presence or absence of 5 μM prostaglandin E1 (PGE1). The PGE1-induced phosphorylation of P24 was detected on autoradiograms of SDS-gels. Since P24 has been shown to be membrane-associated, its molecular weight was compared with those of known membrane proteins. P24 comigrated with the β-chain of purified GP Ib on reduced gels (Mr = 24,000) and also on nonreduced gels (when GP Ibβ is disulfide-linked to GP Ibα and migrates with Mr = 170,000). Like GP Ibβ, P24 was associated with actin filaments in Triton X-100 lysates. Both GP Ibβ and P24 were selectively associated with filaments of the membrane skeleton and were released from filaments when the Ca2+-dependent protease was active. Antibodies against GP Ib immunoprecipitated P24 from platelet lysates. Finally, exposure of Bernard-Soulier platelets (that lacked GP Ib) to PGE1 resulted in phosphorylation of actin-binding protein, P51, P36, and P22, but not P24. We conclude that P24 is GP Ibβ. To determine whether phosphorylation of GP Ibβ is responsible for the inhibitory effects of PGE1 on platelets, we compared the action of PGE1 on control platelets with that on Bernard-Soulier platelets. One of the ways in which PGE1 inhibits platelet activation is by inhibiting the polymerization of actin. While PGE1 inhibited actin polymerization in control platelets, it did not in Bernard-Soulier platelets. We conclude that GP Ibβ is phosphorylated by agents that elevate cyclic AMP and that phosphorylation of this glycoprotein results in inhibition of platelet function.

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

1992 ◽  
Vol 67 (02) ◽  
pp. 252-257 ◽  
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.

scholarly journals Actin-binding protein promotes the bipolar and perpendicular branching of actin filaments.

1980 ◽  
Vol 87 (3) ◽  
pp. 841-848 ◽  
Author(s):  
J H Hartwig ◽  
J Tyler ◽  
T P Stossel
Keyword(s):  
Actin Filaments ◽  
Actin Polymerization ◽  
Average Length ◽  
Binding Protein ◽  
Actin Binding ◽  
Branch Points ◽  
Heavy Meromyosin ◽  
Actin Binding Protein ◽  
Protein Dimers ◽  
Protein Molecules

Branching filaments with striking perpendicularity form when actin polymerizes in the presence of macrophage actin-binding protein. Actin-binding protein molecules are visible at the branch points. Compared with actin polymerized in the absence of actin-binding proteins, not only do the filaments branch but the average length of the actin filaments decreases from 3.2 to 0.63 micrometer. Arrowhead complexes formed by addition of heavy meromyosin molecules to the branching actin filaments point toward the branch points. Actin-binding protein also accelerates the onset of actin polymerization. All of these findings show that actin filaments assemble from nucleating sites on actin-binding protein dimers. A branching polymerization of actin filaments from a preexisting lattice of actin filaments joined by actin-binding protein molecules could generate expansion of cortical cytoplasm in amoeboid cells.

scholarly journals Actin-binding Protein Drebrin Regulates HIV-1-triggered Actin Polymerization and Viral Infection

2013 ◽  
Vol 288 (39) ◽  
pp. 28382-28397 ◽  
Author(s):  
Mónica Gordón-Alonso ◽  
Vera Rocha-Perugini ◽  
Susana Álvarez ◽  
Ángeles Ursa ◽  
Nuria Izquierdo-Useros ◽  
...  
Keyword(s):  
Viral Infection ◽  
Actin Polymerization ◽  
Binding Protein ◽  
Actin Binding ◽  
Actin Binding Protein ◽  
Hiv 1

scholarly journals Recycling of platelet phosphorylation and cytoskeletal assembly.

1984 ◽  
Vol 98 (1) ◽  
pp. 8-15 ◽  
Author(s):  
A C Cox ◽  
R C Carroll ◽  
J G White ◽  
G H Rao
Keyword(s):  
Protein Phosphorylation ◽  
Myosin Light Chain ◽  
Shape Change ◽  
Binding Protein ◽  
Actin Binding ◽  
Dibutyryl Camp ◽  
Actin Binding Protein ◽  
Fibrinogen Binding ◽  
Triton X ◽  
Subsequent Addition

The shape change and aggregation of washed platelets induced by 10 microM arachidonic acid (AA) can be reversed by 20 ng/ml prostacyclin (PGI2), but these platelets can be reactivated by treatment with 30 microM epinephrine and subsequent addition of 10 microM AA mixture. These events may be modulated by cAMP since 2 mM dibutyryl cAMP also reversed activation without reactivation by epinephrine and AA. We examined protein phosphorylation and formation of cytoskeletal cores resistant to 1% Triton X-100 extraction of these platelets and correlated these processes with aggregation, fibrinogen binding, and changes in ultrastructure. Unactivated platelet cores contained less than 15% of the total actin and no detectable myosin or actin-binding protein. AA-induced cytoskeletal cores, which contained 60-80% of the total actin, myosin, and actin-binding protein as the major components, were disassembled back to unactivated levels by PGI2 and then fully reassembled by epinephrine and AA. Phosphorylation of myosin light chain and a 40,000-dalton protein triggered by AA (two- to fivefold) was reversed to basal levels by PGI2 but was completely restored to peak levels upon addition of the epinephrine and AA mixture. The reversibility of actin-binding protein phosphorylation could not be established clearly because both PGI2 and dibutyryl cAMP caused its phosphorylation independent of activation. With this possible exception, cytoskeletal assembly with associated protein phosphorylation, aggregation, fibrinogen binding, and changes in ultrastructure triggered by activation are readily and concertedly recyclable.

scholarly journals αII-Spectrin interacts with Tes and EVL, two actin-binding proteins located at cell contacts

2005 ◽  
Vol 388 (2) ◽  
pp. 631-638 ◽  
Author(s):  
Björn ROTTER ◽  
Odile BOURNIER ◽  
Gael NICOLAS ◽  
Didier DHERMY ◽  
Marie-Christine LECOMTE
Keyword(s):  
Binding Protein ◽  
Focal Adhesions ◽  
Membrane Skeleton ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Actin Binding Protein ◽  
Src Homology 3 ◽  
Src Homology 3 Domain ◽  
Src Homology

The spectrin-based membrane skeleton, a multi-protein scaffold attached to diverse cellular membranes, is presumed to be involved in the stabilization of membranes, the establishment of membrane domains as well as in vesicle trafficking and nuclear functions. Spectrin tetramers made of α- and β-subunits are linked to actin microfilaments, forming a network that binds a multitude of proteins. The most prevalent α-spectrin subunit in non-erythroid cells, αII-spectrin, contains two particular spectrin repeats in its central region, α9 and α10, which host an Src homology 3 domain, a tissue-specific spliced sequence of 20 residues, a calmodulin-binding site and major cleavage sites for caspases and calpains. Using yeast two-hybrid screening of kidney libraries, we identified two partners of the α9-α10 repeats: the potential tumour suppressor Tes, an actin-binding protein mainly located at focal adhesions; and EVL (Ena/vasodilator-stimulated phosphoprotein-like protein), another actin-binding protein, equally recruited at focal adhesions. Interactions between spectrin and overexpressed Tes and EVL were confirmed by co-immunoprecipitation. In vitro studies showed that the interaction between Tes and spectrin is mediated by a LIM (Lin-11, Isl-1 and Mec3) domain of Tes and by the α10 repeat of αII-spectrin whereas EVL interacts with the Src homology 3 domain located within the α9 repeat. Moreover, we describe an in vitro interaction between Tes and EVL, and a co-localization of these two proteins at focal adhesions. These interactions between αII-spectrin, Tes and EVL indicate new functions for spectrin in actin dynamics and focal adhesions.

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

1990 ◽  
Vol 111 (2) ◽  
pp. 483-493 ◽  
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.

scholarly journals Identification of a membrane skeleton in platelets.

1988 ◽  
Vol 106 (5) ◽  
pp. 1525-1538 ◽  
Author(s):  
J E Fox ◽  
J K Boyles ◽  
M C Berndt ◽  
P K Steffen ◽  
L K Anderson
Keyword(s):  
Plasma Membrane ◽  
Actin Filaments ◽  
Binding Protein ◽  
Amorphous Material ◽  
Three Dimensional ◽  
Amorphous Layer ◽  
Membrane Skeleton ◽  
Actin Binding ◽  
Filamentous Form ◽  
Actin Binding Protein

Platelets have previously been shown to contain actin filaments that are linked, through actin-binding protein, to the glycoprotein (GP) Ib-IX complex, GP Ia, GP IIa, and an unidentified GP of Mr 250,000 on the plasma membrane. The objective of the present study was to use a morphological approach to examine the distribution of these membrane-bound filaments within platelets. Preliminary experiments showed that the Triton X-100 lysis buffers used previously to solubilize platelets completely disrupt the three-dimensional organization of the cytoskeletons. Conditions were established that minimized these postlysis changes. The cytoskeletons remained as platelet-shaped structures. These structures consisted of a network of long actin filaments and a more amorphous layer that outlined the periphery. When Ca2+ was present, the long actin filaments were lost but the amorphous layer at the periphery remained; conditions were established in which this amorphous layer retained the outline of the platelet from which it originated. Immunocytochemical experiments showed that the GP Ib-IX complex and actin-binding protein were associated with the amorphous layer. Analysis of the amorphous material on SDS-polyacrylamide gels showed that it contained actin, actin-binding protein, and all actin-bound GP Ib-IX. Although actin filaments could not be visualized in thin section, the actin presumably was in a filamentous form because it was solubilized by DNase I and bound phalloidin. These studies show that platelets contain a membrane skeleton and suggest that it is distinct from the network of cytoplasmic actin filaments. This membrane skeleton exists as a submembranous lining that, by analogy to the erythrocyte membrane skeleton, may stabilize the plasma membrane and contribute to determining its shape.

Examination of irreversible platelet-fibrinogen interactions

1985 ◽  
Vol 248 (5) ◽  
pp. C466-C472 ◽  
Author(s):  
E. I. Peerschke ◽  
J. A. Wainer
Keyword(s):  
Lactate Dehydrogenase ◽  
Cytochalasin B ◽  
Ethylenediaminetetraacetic Acid ◽  
Binding Protein ◽  
Actin Binding ◽  
Actin Binding Protein ◽  
Fibrinogen Binding ◽  
Human Thrombin ◽  
Significant Release ◽  
Triton X

The progressive stabilization of fibrinogen binding to ADP-treated platelets has been well described, but the nature of this interaction remains obscure. In the present study, irreversibly bound fibrinogen was defined as that fraction of bound iodinated fibrinogen that failed to dissociate from stimulated human gel-filtered platelets within 10 min of adding 10 mM ethylenediaminetetraacetic acid. It represented 16 +/- 11% (mean +/- SD, n = 10) of fibrinogen bound to ADP-treated platelets after 1 min and 52 +/- 11% of fibrinogen bound to these platelets after 60 min. Similar results were obtained if platelets were stimulated with purified human thrombin (0.1 U/ml) or epinephrine (10 microM). Irreversible fibrinogen binding was significantly reduced at 4 degrees C (27 +/- 9%, mean +/- SD, n = 6) if platelets were preincubated (30 min, 25 degrees C) with 30 micrograms/ml cytochalasin B or D (18 +/- 8%) or stimulated with chymotrypsin (0.5 mg/2-3 X 10(8) platelets) (31 +/- 8%). Formation of irreversible platelet-fibrinogen interactions correlated with the incorporation of actin and actin-binding protein into the Triton X-100-insoluble platelet cytoskeleton and the ability of platelets to retract fibrin clots. Irreversibly bound fibrinogen was available on platelets for digestion by 0.2 U/ml plasmin. The enzyme removed 96 +/- 6% (mean +/- SD, n = 6) of all bound fibrinogen from platelets after 30 min at 25 degrees C. This was not accompanied by significant release of [14C]serotonin or lactate dehydrogenase. Furthermore, platelets incubated with plasmin could bind fibrinogen normally after the enzyme had been neutralized with aprotinin.(ABSTRACT TRUNCATED AT 250 WORDS)

Sign in / Sign up

Export Citation Format

Share Document