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.

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)

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.

Cdc42/Rac1-dependent activation of the p21-activated kinase (PAK) regulates human platelet lamellipodia spreading: implication of the cortical-actin binding protein cortactin

Blood ◽  
2002 ◽  
Vol 100 (13) ◽  
pp. 4462-4469 ◽  
Author(s):  
Catherine Vidal ◽  
Blandine Geny ◽  
Josiane Melle ◽  
Martine Jandrot-Perrus ◽  
Michaëla Fontenay-Roupie
Keyword(s):  
Shape Change ◽  
Binding Protein ◽  
Actin Binding ◽  
Cortical Actin ◽  
Toxin B ◽  
Actin Reorganization ◽  
Actin Binding Protein ◽  
Direct Binding ◽  
P21 Activated Kinase ◽  
The Relationship

Platelet activation by thrombin or thrombin receptor-activating peptide (TRAP) results in extensive actin reorganization that leads to filopodia emission and lamellae spreading concomitantly with activation of the Rho family small G proteins, Cdc42 and Rac1. Evidence has been provided that direct binding of Cdc42-guanosine triphosphate (GTP) and Rac1-GTP to the N-terminal regulatory domain of the p21-activated kinase (PAK) stimulates PAK activation and actin reorganization. In the present study, we have investigated the relationship between shape change and PAK activation. We show that thrombin, TRAP, or monoclonal antibody (MoAb) anti-FcγRIIA IV.3 induces an activation of Cdc42 and Rac1. The GpVI ligand, convulxin (CVX), that forces platelets to lamellae spreading efficiently activates Rac1. Thrombin, TRAP, MoAb IV.3, and CVX stimulate autophosphorylation and kinase activity of PAK. Inhibition of Cdc42 and Rac1 with clostridial toxin B inhibits PAK activation and lamellae spreading. The cortical-actin binding protein, p80/85 cortactin, is constitutively associated with PAK in resting platelets and dissociates from PAK after thrombin stimulation. Inhibition of PAK autophosphorylation by toxin B prevents the dissociation of cortactin. These results suggest that Cdc42/Rac1-dependent activation of PAK may trigger early platelet shape change, at least in part through the regulation of cortactin binding to PAK.

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.

scholarly journals A novel 36,000-dalton actin-binding protein purified from microfilaments in Physarum plasmodia which aggregates actin filaments and blocks actin-myosin interaction.

1982 ◽  
Vol 93 (3) ◽  
pp. 604-614 ◽  
Author(s):  
S Ogihara ◽  
Y Tonomura
Keyword(s):  
Actin Filaments ◽  
Binding Protein ◽  
Actin Binding ◽  
Cyclic Contraction ◽  
Actin Binding Protein ◽  
Gel Layer ◽  
Skeletal Myosin ◽  
Myosin Interaction ◽  
Physarum Plasmodia ◽  
Triton X

In the plasmodia of Physarum polycephalum, which show a cyclic contraction-relaxation rhythm of the gel layer, huge aggregates of entangled actin microfilaments are formed at about the onset of the relaxation (R. Nagai, Y. Yoshimoto, and N. Kamiya. 1978. J. Cell Sci. 33:205-225). By treating the plasmodia with Triton X-100, we prepared a demembranated cytoskeleton consisting of entangled actin filaments and found that the actin filaments hardly interact with rabbit skeletal myosin. From the cytoskeleton we purified a novel actin-binding protein which binds stoichiometrically to actin and makes actin filaments curled and aggregated. It also inhibits the ATPase activity as well as the superprecipitation of reconstituted rabbit skeletal muscle actomyosin. This protein has a polypeptide molecular weight of 36,000 and binds 7 mol of actin/mol 36,000 polypeptide.

Phosphorylation Of Platelet Proteins In Response To Activation By Phorbol 12-Myristate 13-Acetate

1981 ◽  
Author(s):  
Roger C Carroll ◽  
Jonathan M Gerrard
Keyword(s):  
Molecular Weight ◽  
Light Chain ◽  
Time Course ◽  
Myosin Light Chain ◽  
Binding Protein ◽  
Sodium Dodecyl ◽  
Actin Binding ◽  
Actin Binding Protein ◽  
Activated Platelets ◽  
Platelet Proteins

We have investigated the 32P-1abelling of platelet proteins in response to 5uM to l0uM phorbol 12-myristate 13- acetate (PMA) which triggers pseudopod formation and aggregation but an atypical release without granule centralization by a contractile gel. Total platelet protein samples resolved on polyacrylamide-sodium dodecyl sulfate gels showed greater than 3 fold increases sustained over a 15 minute time course in the 32p-abelling of 260,000; 40,000; and 20,000 apparent molecular weight peptides. While similar increases in 32p-labelling are observed with other activators, such as thrombin, arachidonate, and A23187, peak phosphorylation routinely occurs between 30 to 60 seconds followed by an aggregation dependent dephosphorylation to less than 50% of peak levels between 3 to 5 minutes. The cytoskeletal cores which remain after 1% Triton X-100 extraction of platelets activated by typical stimuli contain mostly actin, myosin, and actin-binding protein. The presence in this cytoskeletal core most of the 32p-label associated with the 260,000 and 20,000 molecular weight peptides suggests that these phosphopeptide are the 260,000 molecular weight actin binding protein, and the 20,000 molecular weight myosin light chain subunits. Cytoskeletal cores prepared from PMA activated platelets still contain greater than 90% of the 32p-labelled 260,000 molecular weight peptide but less than 20% of the 32p-labelled 20,000 molecular weight peptide, most of which is found in the solubilized fraction. These results suggest that the lack of granule centralization by a contractile gel is due to a disruption of actin-myosin interaction even though the myosin light chain is phosphorylated. This effect seems to be specific in that actin-binding protein - actin interaction believed to be responsible for pseudopod formation is still present in PMA activated platelets.

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