scholarly journals Farnesylcysteine analogues inhibit store-regulated Ca2+ entry in human platelets: evidence for involvement of small GTP-binding proteins and actin cytoskeleton

2000 ◽  
Vol 347 (1) ◽  
pp. 183-192 ◽  
Author(s):  
Juan A. ROSADO ◽  
Stewart O. SAGE
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Polymerization ◽  
Binding Proteins ◽  
Selective Inhibition ◽  
Human Platelets ◽  
Cytochalasin D ◽  
Dependent Manner ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Prenylated Proteins

We have investigated the mechanism of Ca2+ entry into fura-2-loaded human platelets by preventing the prenylation of proteins such as small GTP-binding proteins. The farnesylcysteine analogues farnesylthioacetic acid (FTA) and N-acetyl-S-geranylgeranyl-L-cysteine (AGGC), which are inhibitors of the methylation of prenylated and geranylgeranylated proteins respectively, significantly decreased thrombin-evoked increases in intracellular free Ca2+ concentration ([Ca2+]i) in the presence, but not in the absence, of external Ca2+, suggesting a relatively selective inhibition of Ca2+ entry over internal release. Both these compounds and N-acetyl-S-farnesyl-L-cysteine, which had similar effects to those of FTA, also decreased Ca2+ entry evoked by the depletion of intracellular Ca2+ stores with thapsigargin. The inactive control N-acetyl-S-geranyl-L-cysteine was without effect. Patulin, an inhibitor of prenylation that is inert with respect to methyltransferases, also decreased store-regulated Ca2+ entry. Cytochalasin D, an inhibitor of actin polymerization, significantly decreased store-regulated Ca2+ entry in a time-dependent manner. Both cytochalasin D and the farnesylcysteine analogues FTA and AGGC inhibited actin polymerization; however, when evoking the same extent of decrease in actin filament formation, FTA and AGGC showed greater inhibitory effects on Ca2+ entry, indicating a cytoskeleton-independent component in the regulation of Ca2+ entry by small GTP-binding-protein. These findings suggest that prenylated proteins such as small GTP-binding proteins are involved in store-regulated Ca2+ entry through actin cytoskeleton-dependent and cytoskeleton-independent mechanisms in human platelets.

scholarly journals Identification of the ral and rac1 Gene Products, Low Molecular Mass GTP-binding Proteins from Human Platelets

1989 ◽  
Vol 264 (28) ◽  
pp. 16383-16389
Author(s):  
P G Polakis ◽  
R F Weber ◽  
B Nevins ◽  
J R Didsbury ◽  
T Evans ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Binding Proteins ◽  
Human Platelets ◽  
Gene Products ◽  
Gtp Binding Proteins ◽  
Gtp Binding

Fibrinogen binding to the integrin αIIbβ3 modulates store-mediated calcium entry in human platelets

Blood ◽  
2001 ◽  
Vol 97 (9) ◽  
pp. 2648-2656 ◽  
Author(s):  
Juan A. Rosado ◽  
Else M. Y. Meijer ◽  
Karly Hamulyak ◽  
Irena Novakova ◽  
Johan W. M. Heemskerk ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Polymerization ◽  
Adenosine Diphosphate ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Integrin Αiibβ3 ◽  
Fibrinogen Binding

Abstract Effects of the occupation of integrin αIIbβ3 by fibrinogen on Ca++signaling in fura-2–loaded human platelets were investigated. Adding fibrinogen to washed platelet suspensions inhibited increases in cytosolic [Ca++] concentrations ([Ca++]i) evoked by adenosine diphosphate (ADP) and thrombin in a concentration-dependent manner in the presence of external Ca++ but not in the absence of external Ca++ or in the presence of the nonselective cation channel blocker SKF96365, indicating selective inhibition of Ca++entry. Fibrinogen also inhibited store-mediated Ca++ entry (SMCE) activated after Ca++ store depletion using thapsigargin. The inhibitory effect of fibrinogen was reversed if fibrinogen binding to αIIbβ3 was blocked using RDGS or abciximab and was absent in platelets from patients homozygous for Glanzmann thrombasthenia. Fibrinogen was without effect on SMCE once activated. Activation of SMCE in platelets occurs through conformational coupling between the intracellular stores and the plasma membrane and requires remodeling of the actin cytoskeleton. Fibrinogen inhibited actin polymerization evoked by ADP or thapsigargin in control cells and in cells loaded with the Ca++ chelator dimethyl BAPTA. It also inhibited the translocation of the tyrosine kinase p60src to the cytoskeleton. These results indicate that the binding of fibrinogen to integrin αIIbβ3 inhibits the activation of SMCE in platelets by a mechanism that may involve modulation of the reorganization of the actin cytoskeleton and the cytoskeletal association of p60src. This action may be important in intrinsic negative feedback to prevent the further activation of platelets subjected to low-level stimuli in vivo.

scholarly journals Actin filament organization in activated mast cells is regulated by heterotrimeric and small GTP-binding proteins.

1994 ◽  
Vol 126 (4) ◽  
pp. 1005-1015 ◽  
Author(s):  
J C Norman ◽  
L S Price ◽  
A J Ridley ◽  
A Hall ◽  
A Koffer
Keyword(s):  
Mast Cells ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Binding Proteins ◽  
Small Gtpases ◽  
Secretory Cells ◽  
Cortical Region ◽  
Permeabilized Cells ◽  
Gtp Binding Proteins ◽  
Gtp Binding

Rat peritoneal mast cells, both intact and permeabilized, have been used widely as model secretory cells. GTP-binding proteins and calcium play a major role in controlling their secretory response. Here we have examined changes in the organization of actin filaments in intact mast cells after activation by compound 48/80, and in permeabilized cells after direct activation of GTP-binding proteins by GTP-gamma-S. In both cases, a centripetal redistribution of cellular F-actin was observed: the content of F-actin was reduced in the cortical region and increased in the cell interior. The overall F-actin content was increased. Using permeabilized cells, we show that AIF4-, an activator of heterotrimeric G proteins, induces the disassembly of F-actin at the cortex, while the appearance of actin filaments in the interior of the cell is dependent on two small GTPases, rho and rac. Rho was found to be responsible for de novo actin polymerization, presumably from a membrane-bound monomeric pool, while rac was required for an entrapment of the released cortical filaments. Thus, a heterotrimeric G-protein and the small GTPases, rho and rac, participate in affecting the changes in the actin cytoskeleton observed after activation of mast cells.

GTP-binding Proteins in Human Platelets

Platelets ◽  
1990 ◽  
Vol 1 (2) ◽  
pp. 67-79 ◽  
Author(s):  
K. Nagata ◽  
Y. Nozawa
Keyword(s):  
Binding Proteins ◽  
Human Platelets ◽  
Gtp Binding Proteins ◽  
Gtp Binding

Shedding of Procoagulant Microparticles from Platelets through Integrin Outside-In Signaling Involving WASP and the Actin Cytoskeleton.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1529-1529
Author(s):  
Sandra Cauwenberghs ◽  
Marion A.H. Feijge ◽  
Alan G.H. Harper ◽  
Stewart O. Sage ◽  
Elisabeth van Pampus ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Cellular Response ◽  
Binding Proteins ◽  
Kinase Inhibitor ◽  
Rho Kinase ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Procoagulant Effect ◽  
Phosphoinositide 3 Kinase ◽  
Calpain Inhibition

Abstract Platelet activation by potent calcium-mobilizing agents, like collagen and thrombin or ionomycin, results in Ca2+- and calpain-dependent shedding of microparticles (MP) that are active in coagulation. Here, we describe and characterize a different mechanism of MP formation by platelets under storage in the absence of agonists. This shedding relies on integrin αIIbβ3 activity, since clinically used integrin blockers, like tirofiban or abciximab, reduce storage-induced MP shedding by 83±5.7% (p=0.006); inhibition of intracellular integrin signaling via Src kinase (PP1) or phosphoinositide 3-kinase (PI-3 kinase, wortmannin) inhibits this process by 52.3±14.3% (p=0.035) and 39±7% (p=0.005), respectively. Calpain inhibition suppresses MP formation by only 35±3.8% (p=0.003), while calpain activity is also not increased during storage. Levels of cytosolic Ca2+ concentration remain at 25–30 nM in the stored platelets. Treatment of platelets with recombinant WASP (Wiskott-Aldrich Syndrome Protein) peptide, activating the Arp2/3 complex and promoting actin nucleation, affects storage-induced MP shedding (p=0.001). Rho and Rac1, belonging to the family of Rho GTP-binding proteins, are involved in actin cytoskeleton regulation and platelet spreading. However, these GTP-binding proteins do not seem to be involved in MP shedding, as treatment of platelets with recombinant Rac1 peptide or with Rho kinase inhibitor is ineffective, as in the absence of these GTP-binding proteins. Furthermore, incubation of platelets with cytochalasin D, latrunculin B or jasplakinolide, which compounds affect actin filament polymerization, increases the amount of procoagulant MP (p<0.032). Interestingly, this effect is greatly reverted in the presence of integrin blockers or wortmannin. We determine that coagulation-active MP are abundantly present in stored platelet preparations that are used for transfusion purposes. The contribution of these MP to the coagulation process was studied after transfusion of these preparations to thrombocytopenic patients. Thrombin generation measurements indicate that the transfused MP initiate up to 25 nM concentrations of thrombin, which potentiate the procoagulant effect of platelets in these patients. Taken together, we conclude that the process of integrin-mediated MP shedding from apparently resting platelets is a uniquely regulated cellular response that operates independently of Ca2+ elevation and calpain activation, and contributes to coagulant activity in a clinically relevant way. It involves αIIbβ3 outside-in signaling, leading to WASP-mediated destabilization of the actin cytoskeleton, but independently of Rho and Rac1.

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