Annexin V relocates to the platelet cytoskeleton upon activation and binds to a specific isoform of actin.
We have previously reported that stimulation of platelets causes a relocation of annexin V to the cytoplasmic side of the plasma membrane where it associates with actin. This study examined the association of annexin V with the platelet cytoskeleton and its binding to actin, following both physiological activation with thrombin and Ca2+ -ionophore activation. The time-dependence of annexin V incorporation into the detergent-extracted cytoskeleton following activation with thrombin was also measured. Although calcium from the intracellular stores was enough to relocate intracellular annexin V to the cytoskeleton, this relocation was further enhanced by influx of extracellular calcium. The association of annexin V with the cytoskeleton was found to be unaffected by the action of cytochalasin E, however, annexin V was solubilized when DNase I was used to depolymerize the membrane cytoskeleton, and spontaneously re-associated with the actin filaments when re-polymerization was induced in vitro. Using a bifunctional crosslinking reagent we have identified an 85-kDa complex in both membrane and cytoskeleton fractions containing annexin V and actin. Direct binding to actin filaments was only observed in high [Ca2+], however, inclusion of an extract from thrombin-stimulated platelets lowered the [Ca2+] requirement for the binding of annexin V to F-actin to physiological levels. We also show that GST-annexin V mimics the physiological binding of annexin V to membranes, and that this GST-annexin V binds directly to a specific isoform of actin. Immunoprecipitation using antibodies against annexin V copurify annexin V and gamma- but not beta-actin from activated platelets. This is the first report of a possible preferential binding of annexin V to a specific isoform of actin, namely gamma-actin. The results of this study suggest a model in which annexin V that relocates to the plasma membrane and binds to gamma-actin in an activation-dependent manner forms a strong association with the platelet cytoskeleton.
Tzima
E
E
School of Biochemistry and Molecular Biology, University of Leeds, UK.
Trotter
P J
PJ
Orchard
M A
MA
Walker
J H
JH
D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D017304:Annexin A5 / Q000378:metabolism*; D001792:Blood Platelets / Q000378:metabolism*; D015153:Blotting, Western; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003599:Cytoskeleton / Q000378:metabolism*; D003902:Detergents / Q000494:pharmacology; D004120:Dimethyl Suberimidate / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005982:Glutathione Transferase / Q000378:metabolism; D006490:Hemostatics / Q000494:pharmacology; D006801:Humans; D007476:Ionophores / Q000494:pharmacology; D008024:Ligands; D017830:Octoxynol / Q000494:pharmacology; D015539:Platelet Activation; D011233:Precipitin Tests; D020033:Protein Isoforms / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology; D013997:Time Factors
D000199:Actins; D017304:Annexin A5; D003432:Cross-Linking Reagents; D003902:Detergents; D006490:Hemostatics; D007476:Ionophores; D008024:Ligands; D020033:Protein Isoforms; D011994:Recombinant Proteins; D004120:Dimethyl Suberimidate; D000001:Calcimycin; D017830:Octoxynol; D005982:Glutathione Transferase; D013917:Thrombin; D002118:Calcium