Phosphorylation Of Platelet Actin-Binding Protein During Platelet Activation

Actin Binding ◽

Actin Binding Protein ◽

Platelet Protein ◽

External Calcium

We have followed the 32P-labelling of actin-binding protein as a function of platelet activation. Utilizing polyacrylamide sodium dodecyl sulfate gel electrophoresis to resolve total platelet protein samples we found 2 to 3 fold labelling increases in actin-binding protein 30 to 60 seconds after thrombin stimulation. Somewhat larger increases were observed for 40,000 and 20,000 apparent molecular weight peptides. The actin-binding protein was identified on the gels by coelectrophoresis of purified actin-binding protein as well as cytoskeletal cores prepared by detergent extraction of activated 32p-iabelled platelets. In addition, these cytoskeletal cores indicated that the 32P-labelled actin-binding protein was closely associated with the activated platelet's cytoskeleton. Following the 32P-labelling of actin-binding protein over an 8 minute time course revealed that in aggregating platelet samples rapid desphosphorylation to almost initial levels occurred between 3 and 5 minutes. A similar curve was obtained for the 20,000 apparent molecular weight peptide. This rapid dephosphorylation was shown to be dependent on platelet aggregation in the absence of external calcium or in thrombastenic platelets lacking the aggregation response to activation. These results suggest that phosphorylation of actin-binding protein initiates its association with the platelet cytoskeleton during activation.

Phosphorylation of platelet actin-binding protein during platelet activation

Blood ◽

10.1182/blood.v59.3.466.466 ◽

1982 ◽

Vol 59 (3) ◽

pp. 466-471 ◽

Cited By ~ 45

Author(s):

RC Carroll ◽

JM Gerrard

Keyword(s):

Molecular Weight ◽

Platelet Activation ◽

Time Course ◽

Binding Protein ◽

Sodium Dodecyl ◽

Actin Binding ◽

Cell Contact ◽

Actin Binding Protein ◽

Abstract In this study we have followed the 32P-labeling of actin-binding protein as a function of platelet activation. Utilizing polyacrylamide- sodium dodecyl sulfate gel electrophoresis to resolve total platelet protein samples, we found 2--3-fold labeling increases in actin-binding protein 30--60 sec after thrombin stimulation. Somewhat larger increases were observed for 40,000 and 20,000 apparent molecular weight peptides. The actin-binding protein was identified on the gels by coelectrophoresis with purified actin-binding protein, its presence in cytoskeletal cores prepared by detergent extraction of activated 32P- labeled platelets, and by direct immunoprecipitation with antibodies against guinea pig vas deferens filamin (actin-binding protein). In addition, these cytoskeletal cores indicated that the 32P-labeled actin- binding protein was closely associated with the activated platelet's cytoskeleton. Following the 32P-labeling of actin-binding protein over an 8-min time course revealed that in aggregating platelet samples rapid dephosphorylation to almost initial levels occurred between 3 and 5 min. A similar curve was obtained for the 20,000 apparent molecular weight peptide. However, rapid dephosphorylation was not observed if platelet aggregation was prevented by chelating external calcium or by using thrombasthenic platelets lacking the aggregation response. Thus, cell-cell contact would seem to be crucial in initiating the rapid dephosphorylation response.

Phosphorylation of platelet actin-binding protein during platelet activation

Blood ◽

10.1182/blood.v59.3.466.bloodjournal593466 ◽

1982 ◽

Vol 59 (3) ◽

pp. 466-471 ◽

Cited By ~ 3

Author(s):

RC Carroll ◽

JM Gerrard

Keyword(s):

Molecular Weight ◽

Platelet Activation ◽

Time Course ◽

Binding Protein ◽

Sodium Dodecyl ◽

Actin Binding ◽

Cell Contact ◽

Actin Binding Protein ◽

In this study we have followed the 32P-labeling of actin-binding protein as a function of platelet activation. Utilizing polyacrylamide- sodium dodecyl sulfate gel electrophoresis to resolve total platelet protein samples, we found 2--3-fold labeling increases in actin-binding protein 30--60 sec after thrombin stimulation. Somewhat larger increases were observed for 40,000 and 20,000 apparent molecular weight peptides. The actin-binding protein was identified on the gels by coelectrophoresis with purified actin-binding protein, its presence in cytoskeletal cores prepared by detergent extraction of activated 32P- labeled platelets, and by direct immunoprecipitation with antibodies against guinea pig vas deferens filamin (actin-binding protein). In addition, these cytoskeletal cores indicated that the 32P-labeled actin- binding protein was closely associated with the activated platelet's cytoskeleton. Following the 32P-labeling of actin-binding protein over an 8-min time course revealed that in aggregating platelet samples rapid dephosphorylation to almost initial levels occurred between 3 and 5 min. A similar curve was obtained for the 20,000 apparent molecular weight peptide. However, rapid dephosphorylation was not observed if platelet aggregation was prevented by chelating external calcium or by using thrombasthenic platelets lacking the aggregation response. Thus, cell-cell contact would seem to be crucial in initiating the rapid dephosphorylation response.

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

10.1055/s-0038-1652410 ◽

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.

Identification of a high molecular weight actin-binding protein in skeletal muscle.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)37713-x ◽

1979 ◽

Vol 254 (6) ◽

pp. 1755-1758

Author(s):

P.J. Bechtel

Keyword(s):

Skeletal Muscle ◽

Molecular Weight ◽

High Molecular Weight ◽

Binding Protein ◽

Actin Binding ◽

Actin Binding Protein

Platelets contain proteins immunologically related to red cell spectrin and protein 4.1

Blood ◽

10.1182/blood.v65.1.52.bloodjournal65152 ◽

1985 ◽

Vol 65 (1) ◽

pp. 52-59

Author(s):

GE Davies ◽

CM Cohen

Keyword(s):

Molecular Weight ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Human Platelets ◽

Cell Protein ◽

Immunoperoxidase Staining ◽

Red Cell ◽

Protein 4.1 ◽

Human platelets were tested for the presence of proteins immunologically cross-reactive with red cell spectrin and protein 4.1. As assessed by indirect immunofluorescence microscopy, platelets were specifically reactive with affinity-purified rabbit antisera against red cell spectrin and protein 4.1. The immunoreactive platelet constituents were further analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis, followed by electrophoretic transfer to nitrocellulose paper and immunoperoxidase staining. We found that whole platelets, membranes, and cytoskeletal preparations isolated by Triton X-100 extraction contain small amounts of proteins reacting with anti-spectrin or anti-protein 4.1 antiserum. The immunoreactive spectrin-like platelet protein has an apparent molecular weight of 240,000 and comigrates with the alpha-subunit of red cell spectrin. The major immunoreactive protein 4.1-like constituent has an apparent molecular weight of 78,000, which is slightly less than that of red cell protein 4.1. We conclude that platelets contain a spectrin- like protein which, by analogy with red cell spectrin, may have a role in membrane-cytoskeletal attachment. The properties and function of the platelet protein 4.1-like constituent are not yet known.

Isolation of a high molecular weight actin-binding protein from baby hamster kidney (BHK-21) cells.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.76.9.4484 ◽

1979 ◽

Vol 76 (9) ◽

pp. 4484-4488 ◽

Cited By ~ 23

Author(s):

J. A. Schloss ◽

R. D. Goldman

Keyword(s):

Molecular Weight ◽

High Molecular Weight ◽

Binding Protein ◽

Actin Binding ◽

Baby Hamster Kidney ◽

Actin Binding Protein

Identification by monoclonal antibodies and characterization of human platelet caldesmon.

The Journal of Cell Biology ◽

10.1083/jcb.102.5.1748 ◽

1986 ◽

Vol 102 (5) ◽

pp. 1748-1757 ◽

Cited By ~ 49

Author(s):

J Dingus ◽

S Hwo ◽

J Bryan

Keyword(s):

Molecular Weight ◽

Smooth Muscle ◽

Monoclonal Antibodies ◽

Cultured Cells ◽

Human Platelets ◽

Binding Activity ◽

Actin Binding ◽

Dependent Manner ◽

Actin-based gels were prepared from clarified high-salt extracts of human platelets by dialysis against physiological salt buffers. The gel was partially solubilized with 0.3 M KCl. Mice were immunized with the 0.3 M KCl extract of the actin gel, and hybridomas were produced by fusion of spleen cells with myeloma cells. Three hybridomas were generated that secrete antibodies against an 80-kD protein. These monoclonal antibodies stained stress fibers in cultured cells and cross-reacted with proteins in several tissue types, including smooth muscle. The cross-reacting protein in chicken gizzard smooth muscle had an apparent molecular weight of 140,000 and was demonstrated to be caldesmon, a calmodulin and actin-binding protein (Sobue, K., Y. Muramoto, M. Fujita, and S. Kakiuchi, Proc. Natl. Acad. Sci. USA, 78:5652-5655). No proteins of molecular weight greater than 80 kD were detectable in platelets by immunoblotting using the monoclonal antibodies. The 80-kD protein is heat stable and was purified using modifications of the procedure reported by Bretscher for the rapid purification of smooth muscle caldesmon (Bretscher, A., 1985, J. Biol. Chem., 259:12873-12880). The 80-kD protein bound to calmodulin-Sepharose in a Ca++-dependent manner and sedimented with actin filaments, but did not greatly increase the viscosity of F-actin solutions. The actin-binding activity was inhibited by calmodulin in the presence of calcium. Except for the molecular weight difference, the 80-kD platelet protein appears functionally similar to 140-kD smooth muscle caldesmon. We propose that the 80-kD protein is platelet caldesmon.