Cyclic AMP-dependent phosphorylation of glycoprotein Ib inhibits collagen-induced polymerization of actin in platelets

Platelet function is inhibited by agents such as prostaglandin E1 (PGE1) that elevate the cytoplasmic concentration of cyclic AMP. Inhibition presumably results from the cyclic AMP-stimulated phosphorylation of intracellular proteins. Polypeptides that become phosphorylated are actin-binding protein, P51 (Mr = 51,000), P36 (Mr = 36,000), P24 (Mr = 24,000), and P22 (Mr = 22,000). Recently, we identified P24 as the beta-chain of glycoprotein (GP) Ib, a component of the plasma membrane GP Ib.IX complex. The existence of Bernard-Soulier syndrome, a hereditary disorder in which platelets selectively lack the GP Ib.IX complex, enabled us to examine whether the phosphorylation of GP Ib beta (P24) is responsible for any of the inhibitory effects of elevated cyclic AMP on platelet function. Exposure of control platelets to PGE1 increased phosphorylation of actin-binding protein, P51, P36, GP Ib beta, and P22. Prostaglandin E1 induced the same phosphorylation reactions in Bernard-Soulier platelets, except that of GP Ib beta, which is absent. In control platelets, PGE1 inhibited collagen-induced phosphorylation of myosin light chain, phosphorylation of P47 (an unidentified Mr 47,000 cytoplasmic protein that is phosphorylated by protein kinase C in stimulated platelets), aggregation, and the secretion of granule contents. Despite the absence of GP Ib beta, PGE1 also inhibited these collagen-induced responses in Bernard-Soulier platelets. However, while PGE1 inhibited collagen-induced polymerization of actin in control platelets, it did not inhibit actin polymerization in Bernard-Soulier platelets. These results suggest that cyclic AMP-induced phosphorylation of GP Ib inhibits collagen-induced actin polymerization in platelets. Because actin polymerization is required for at least some of the functional responses of platelets to an agonist, phosphorylation of Gp Ib beta may be one way in which cyclic AMP inhibits platelet function.     

Characterization of a novel membrane glycoprotein involved in platelet activation

When platelets bind certain specific ligands they are induced to secrete the contents of their cytoplasmic granules and to aggregate. Studies of the molecular events accompanying this vital physiological response have led to a greater understanding of cell activation in general since the pathways involved are common to a number of cell types. By contrast most of the information about the cell surface molecules that initiate signal transduction has emerged from work on T lymphocyte activation, a process essential to the initiation of the immune response. We have described an activation antigen on T lymphocytes that is involved in the differentiation of these cells. In the present report it is demonstrated that the antigen is expressed on the platelet membrane with about 1,200 copies/platelet. A monoclonal antibody detecting this antigen stimulates platelet secretion and aggregation with a half-maximal response at approximately 10(-8) M. Characterization of the antigen, termed PTA1, reveals a glycoprotein of Mr 67,000 showing extensive N-linked carbohydrate, much of which appears to be heavily sialated. The amino-terminal sequence of PTA1, EEVLWHTSVPFAEXMSLEXVYPSM, indicates that the protein has not previously been characterized. Preliminary investigation of the mechanism by which PTA1 mediates platelet activation suggests involvement of protein kinase C and the 47-kDa protein of platelets is rapidly phosphorylated upon antibody-mediated activation. During this process PTA1 is also phosphorylated, as it is following platelet activation by the other agonists, collagen, thrombin, and 12-O-tetradecanoylphorbol 13-acetate. These results provide the first example of a cell surface glycoprotein that is directly involved in both platelet and T lymphocyte activation.     

Pathological shear stress stimulates the tyrosine phosphorylation of alpha-actinin associated with the glycoprotein Ib-IX complex.

Shear-induced platelet responses are triggered by VWF binding to the platelet GpIb-IX complex, and there is evidence that this ligand-receptor coupling stimulates transmembranous signaling through the cytoplasmic tail of glycoprotein (Gp) Ib alpha. To investigate the mechanism by which signaling is effected, new molecular interactions involving GpIb-IX that develop in response to pathological shearing stress were examined in intact human platelets. Exposure to shear, but not alpha-thrombin, results in the co-immunoprecipitation of the actin cross-linking protein alpha-actinin with the GpIb-IX complex. Blockers of VWF binding to GpIb alpha or actin polymerization inhibit the association of alpha-actinin with the GpIb-IX complex, but the association of alpha-actinin with the GpIb-IX complex is not affected by inhibiting VWF binding to platelet integrin alpha IIb beta 3 (GpIIb-IIIa). alpha-Actinin becomes tyrosine phosphorylated in response to pathological shear stress, and phosphorylated alpha-actinin associates with GpIb-IX. In resting platelets, class IA heterodimeric phosphatidylinositol 3-kinase (PI 3-K) and protein kinase N (PKN) associate with nonphosphorylated alpha-actinin. Shear stress causes PI 3-K to disassociate from alpha-actinin, while it stimulates PKN binding to alpha-actinin. These results demonstrate that shear-induced VWF binding to GpIb alpha causes enhanced binding of cytoskeletal alpha-actinin to GpIb-IX and suggest that alpha-actinin, perhaps through tyrosine phosphorylation, serves as an adapter for a signaling complex that could regulate VWF-induced platelet aggregation.     

Role of actin binding protein phosphorylation in platelet cytoskeleton assembly

Actin binding protein from human blood platelets is shown to exist in the resting platelet as a phosphorylated protein and contains two residues of phosphate per 260,000 kd. Removal of one-half of these residues with E. coli alkaline phosphatase results in the loss of its ability to crosslink F-actin into a low speed sedimentable complex (its cytoskeleton) and to bind to an F-actin affinity column. Thus, phosphorylation-dephosphorylation of ABP may be an important regulatory mechanism by which the platelet regulates its shape via its cytoskeletal structure.     

Identification of shc as the primary protein binding to the tyrosine-phosphorylated beta 3 subunit of alpha IIbbeta 3 during outside-in integrin platelet signaling

Outside-in signaling mediated by the integrin alpha(IIb)beta(3) (GPIIbIIIa) is critical to platelet function and has been shown to involve the phosphorylation of tyrosine residues on the cytoplasmic tail of beta(3). To identify proteins that bind directly to phosphorylated beta(3), we utilized an affinity column consisting of a peptide modeled on the tyrosine-phosphorylated cytoplasmic domain of beta(3). Tandem mass spectrometric sequencing and immunoblotting demonstrated that Shc was the primary protein binding to phosphorylated beta(3). To determine the involvement of Shc in outside-in alpha(IIb)beta(3) signaling, the phosphorylation of Shc during platelet aggregation was examined; transient Shc phosphorylation was observed when thrombin-stimulated platelets were allowed to aggregate or when aggregation was induced by an LIBS (ligand-induced binding site) antibody, D3. Moreover, Shc was co-immunoprecipitated with tyrosine-phosphorylated beta(3) in detergent lysates of aggregated platelets. Using purified, recombinant protein, it was found that the binding of Shc to monophosphorylated (C-terminal tyrosine) and diphosphorylated beta(3) peptides was direct, demonstrating Shc recognition motifs on phospho-beta(3). Aggregation-induced Shc phosphorylation was also observed to be robust in platelets from wild-type mice, but not in those from mice expressing (Y747F,Y759F) beta(3), which are defective in outside-in alpha(IIb)beta(3) signaling. Thus, Shc is the primary downstream signaling partner of beta(3) in its tyrosine phosphorylation outside-in signaling pathway.     

Studies on Trypanosomatid Actin I. Immunochemical and Biochemical Identification

In this study, the presence of actin in cultured trypanosomatids was investigated using polyclonal antibodies to heterologous actin. Polyclonal antisera to rabbit muscle actin and a monospecific anti-actin antibody react with a 43-kDa polypeptide in extracts of Trypanosoma cruzi, Herpetomonas samuelpessoai and Leishmania mexicana amazonensis on protein immunoblots. The 43-kDa polypeptide co-migrates with skeletal muscle actin and is retained within trypanosomatid cytoskeletons. Attempts to isolate H. samuelpessoai actin through DNase I affinity chromatography showed that the 43-kDa polypeptide did not bind to the column. Instead, low yields of a 47-kDa polypeptide were obtained indicating that the trypanosomatid actin displays unusual DNase I binding behavior when compared to actins from higher eukaryotes. Immunofluorescence studies confirmed that cytoskeletons retain the actin-like protein. In H. samuelpessoai , actin is localized in the region close to the flagellum, whereas in T. cruzi it is more homogeneously distributed. The data presented here show that trypanosomatid actin displays biochemical characteristics similar to actins of other protozoa.     

Studies on trypanosomatid actin. I. Immunochemical and biochemical identification

In this study, the presence of actin in cultured trypanosomatids was investigated using polyclonal antibodies to heterologous actin. Polyclonal antisera to rabbit muscle actin and a monospecific anti-actin antibody react with a 43-kDa polypeptide in extracts of Trypanosoma cruzi, Herpetomonas samuelpessoai and Leishmania mexicana amazonensis on protein immunoblots. The 43-kDa polypeptide co-migrates with skeletal muscle actin and is retained within trypanosomatid cytoskeletons. Attempts to isolate H. samuelpessoai actin through DNase I affinity chromatography showed that the 43-kDa polypeptide did not bind to the column. Instead, low yields of a 47-kDa polypeptide were obtained indicating that the trypanosomatid actin displays unusual DNase I binding behavior when compared to actins from higher eukaryotes. Immunofluorescence studies confirmed that cytoskeletons retain the actin-like protein. In H. samuelpessoai, actin is localized in the region close to the flagellum, whereas in T. cruzi it is more homogeneously distributed. The data presented here show that trypanosomatid actin displays biochemical characteristics similar to actins of other protozoa.     

Purification of a cortical complex containing two unconventional actins from Acanthamoeba by affinity chromatography on profilin-agarose

We identified four polypeptides of 47, 44, 40, and 35 kD that bind to profilin-Sepharose and elute with high salt. When purified by conventional chromatography using an antibody to the 47-kD polypeptide, these four polypeptides copurified as a stoichiometric complex together with three additional polypeptides of 19, 18, and 13 kD that varied in their proportions to the other polypeptides. Partial protein sequences showed that the 47-kD polypeptide is a homologue of S. pombe act2 and the 44-kD polypeptide is a homologue of S. cerevisiae ACT2, both unconventional actins. The 40-kD polypeptide contains a sequence similar to the WD40 motif of the G beta subunit of a trimeric G-protein from Dictyostelium discoideum. From partial sequences, the 35-, 19-, and 18-kD polypeptides appear to be novel proteins. On gel filtration the complex of purified polypeptides cochromatograph with a Stokes' radius of 4.8 nm, a value consistent with a globular particle of 220 kD containing one copy of each polypeptide. Cell extracts also contain components of the complex that do not bind the profilin column. Affinity purified antibodies localize 47- and 18/19-kD polypeptides in the cortex and filopodia of Acanthamoeba. Antibodies to the 47-kD unconventional actin cross-react on immunoblots with polypeptides of similar size in Dictyostelium, rabbit muscle, and conventional preparations of rabbit muscle actin but do not react with actin.     

Bryostatins mimic the effects of phorbol esters in intact human platelets

Bryostatin-7 induces aggregation of human platelets and the phosphorylation of specific platelet proteins. Both the rate and extent of aggregation are similar to that induced by the tumor promoter phorbol ester 12-myristate 13-acetate (PMA); however, the rate of response is markedly reduced compared to that induced by thrombin. The addition of bryostatin-7 to 32P-labeled platelets results in a time-dependent incorporation of 32P into proteins of 20, 47 and 250 kDa; proteins of similar molecular mass are phosphorylated in response to the addition of thrombin or PMA. The time courses and dose responses of the phosphorylations induced by bryostatin-7 are similar to those found with PMA. In addition, bryostatin-7 increases the level of 32P incorporation into platelet polyphosphoinositides, which also occurs in response to PMA. These results suggest that, in intact human platelets, bryostatin-7 mimics the phorbol ester tumor promoter by directly activating protein kinase C.     

The phosphorylation of vinculin on tyrosine residues 100 and 1065, mediated by SRC kinases, affects cell spreading

Vinculin is a conserved actin binding protein localized in focal adhesions and cell-cell junctions. Here, we report that vinculin is tyrosine phosphorylated in platelets spread on fibrinogen and that the phosphorylation is Src kinases dependent. The phosphorylation of vinculin on tyrosine was reconstituted in vanadate treated COS-7 cells coexpressing c-Src. The tyrosine phosphorylation sites in vinculin were mapped to residues 100 and 1065. A phosphorylation-specific antibody directed against tyrosine residue 1065 reacted with phosphorylated platelet vinculin but failed to react with vinculin from unstimulated platelet lysates. Tyrosine residue 1065 located in the vinculin tail domain was phosphorylated by c-Src in vitro. When phosphorylated, the vinculin tail exhibited significantly less binding to the vinculin head domain than the unphosphorylated tail. In contrast, the phosphorylation did not affect the binding of vinculin to actin in vitro. A double vinculin mutant protein Y100F/Y1065F localized to focal adhesion plaques. Wild-type vinculin and single tyrosine phosphorylation mutant proteins Y100F and Y1065F were significantly more effective at rescuing the spreading defect of vinculin null cells than the double mutant Y100F/Y1065F. The phosphorylation of vinculin by Src kinases may be one mechanism by which these kinases regulate actin filament assembly and cell spreading.     

Phosphorylation-dependent regulation of Limulus myosin

Myosin from Limulus, the horseshoe crab, is shown to be regulated by a calcium-calmodulin-dependent phosphorylation of its regulatory light chains. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of a Limulus myosin preparation reveals three light chain bands. Two of these light chains have been termed regulatory light chains based on their ability to bind to light chain-denuded scallop myofibrils (Sellers, J. R., Chantler, P. D., and Szent-Gy?rgyi, A. G. (1980) J. Mol. Biol. 144, 223-245). Ths other light chain does not bind to these myofibrils and is thus termed the essential light chain. Both Limulus regulatory light chains can be phosphorylated with a highly purified turkey gizzard myosin light chain kinase or with a partially purified myosin light chain kinase which can be isolated from Limulus muscle by affinity chromatography on a calmodulin-Sepharose column. Phosphorylation with both of these enzymes requires calcium and calmodulin. Limulus myosin is isolated in an unphosphorylated form. The MgATPase of this unphosphorylated myosin is only slightly activated by rabbit skeletal muscle actin plus tropomyosin. The calcium-dependent phosphorylation of the myosin results in an increase in the actin-activated MgATPase rate. Once phosphorylated, the actin-activated MgATPase rate is only slightly modified by calcium. This suggests that calcium operates mainly at the level of the myosin kinase-calmodulin system.     

Interaction of purified actin-binding protein with the platelet membrane glycoprotein Ib-IX complex

The interaction of platelet membrane glycoprotein (GP) Ib-IX complex with the cytoplasmic membrane skeleton is potentially of major importance in regulating platelet function. Indirect evidence suggested that this interaction is mediated by actin-binding protein, but it is not known whether GP Ib-IX and actin-binding protein associate directly. To examine more closely the nature of this association, purified GP Ib-IX complex was specifically bound and oriented on the surface of impermeable polymer beads via a monoclonal antibody, AK 2, directed against the extracytoplasmic domain of GP Ib alpha (glycocalicin). Binding was specific since 1) it was abolished by excess unlabeled actin-binding protein; 2) there was no detectable specific binding of radiolabeled actin-binding protein to beads coated with glycocalicin, the major extracytoplasmic proteolytic fragment of GP Ib alpha; and 3) unlike actin-binding protein, there was no specific binding of bovine serum albumin or human platelet vinculin to the GP Ib-IX complex-coated beads. Binding of actin-binding protein to the GP Ib-IX complex-coated beads, but not to the glycocalicin-coated beads, was saturable and reversible (apparent Kd = 1 x 10(-7) M). These experiments provide direct evidence that actin-binding protein can bind to the cytoplasmic domain of a membrane glycoprotein. Because actin-binding protein is found submembranously in cells other than the platelet, it is possible that this protein may link actin filaments to the plasma membrane in those cells.     

Scapinin-induced inhibition of axon elongation is attenuated by phosphorylation and translocation to the cytoplasm

Scapinin is an actin- and PP1-binding protein that is exclusively expressed in the brain; however, its function in neurons has not been investigated. Here we show that expression of scapinin in primary rat cortical neurons inhibits axon elongation without affecting axon branching, dendritic outgrowth, or polarity. This inhibitory effect was dependent on its ability to bind actin because a mutant form that does not bind actin had no effect on axon elongation. Immunofluorescence analysis showed that scapinin is predominantly located in the distal axon shaft, cell body, and nucleus of neurons and displays a reciprocal staining pattern to phalloidin, consistent with previous reports that it binds actin monomers to inhibit polymerization. We show that scapinin is phosphorylated at a highly conserved site in the central region of the protein (Ser-277) by Cdk5 in vitro. Expression of a scapinin phospho-mimetic mutant (S277D) restored normal axon elongation without affecting actin binding. Instead, phosphorylated scapinin was sequestered in the cytoplasm of neurons and away from the axon. Because its expression is highest in relatively plastic regions of the adult brain (cortex, hippocampus), scapinin is a new regulator of neurite outgrowth and neuroplasticity in the brain.     

Phosphorylation of human platelet glycoprotein IIIa (GPIIIa). Dissociation from fibrinogen receptor activation and phosphorylation of GPIIIa in vitro.

Glycoprotein IIb-IIIa (GPIIb-IIIa) is the fibrinogen receptor on activated platelets. GPIIIa is phosphorylated in resting platelets and the incorporation of 32Pi increases with platelet activation. To address the functional significance of this modification, the stoichiometry of GPIIIa phosphorylation was determined in resting and activated platelets by estimating the specific activity of metabolic [gamma-32P]ATP from the specific activity of phosphatidic acid. Approximately 0.01 mol of P/mol of GPIIIa was phosphorylated in resting platelets and 0.03 mol of P/mol of GPIIIa was phosphorylated in thrombin-, phorbol ester-, or U46619-treated platelets. Myosin light chain (MLC) phosphorylation served as a positive control for this method (1.2 mol of P/mol of MLC). Phosphorylation of purified GPIIb-IIIa by human platelet protein kinase C (PKC) resulted in levels of GPIIIa phosphorylation similar to that in platelets (0.05 mol of P/mol of GPIIIa). However, while GPIIIa in platelets was phosphorylated primarily on threonine, purified GPIIIa treated with PKC was phosphorylated primarily on serine. These results suggest that PKC may not directly phosphorylate GPIIIa in intact platelets. Ca2+/calmodulin-dependent kinase II phosphorylated purified GPIIIa to higher levels (0.5 mol of P/mol of GPIIIa) with phosphorylation on both threonine and serine. The limited phosphorylation of GPIIIa in intact platelets suggests that this event is unlikely to affect functions involving large populations of GPIIb-IIIa, such as its conversion to a fibrinogen receptor. However, these results may suggest the existence of a more readily phosphorylated subpopulation of GPIIb-IIIa with potentially distinct structural or functional properties.     

Selective association of the tyrosine kinases Src, Fyn, and Lyn with integrin-rich actin cytoskeletons of activated, nonaggregated platelets.

Integrin-mediated interactions between cytoskeletal proteins and extracellular fibrinogen are required for platelet adhesion. We have previously demonstrated that the major platelet integrin, alpha(IIb)beta(3), becomes incorporated into the actin cytoskeleton of platelets in an activation-dependent, aggregation-independent manner. To determine if regulatory molecules are also associated with these integrin-rich cytoskeletal complexes, we examined actin cytoskeletons for the presence of kinases and phosphoproteins. Western immunoblot analysis revealed that the tyrosine kinases Src, Fyn, and Lyn are specifically associated with actin cytoskeletons of activated, nonaggregated platelets. However, as noted by others, the cytoskeletal association of focal adhesion kinase depends on platelet aggregation. Actin cytoskeletons isolated from (32)P-labeled platelets also contain a number of phosphorylated proteins. Interestingly, an approximately 18-kDa phosphoprotein was uniquely present in activated platelet cytoskeletons. Collectively, our results demonstrate that actin cytoskeletons of activated, nonaggregated platelets contain not only integrins, but also kinases and phosphoproteins that could regulate platelet adhesion and transmembrane communication.     

Interaction of the low-molecular-weight GTP-binding protein rap2 with the platelet cytoskeleton is mediated by direct binding to the actin filaments

The interaction of the low-molecular-weight GTP-binding protein rap2 with the cytoskeleton from thrombin-aggregated platelets was investigated by inducing depolymerization of the actin filaments, followed by in vitro-promoted repolymerization. We found that the association of rap2 with the cytoskeleton was spontaneously restored after one cycle of actin depolymerization and repolymerization. Exogenous rap2, but not unrelated proteins, added to depolymerized actin and solubilized actin-binding proteins, was also specifically incorporated into the in vitro reconstituted cytoskeleton. The incorporation of exogenous rap2 was also observed when the cytoskeleton from resting or thrombin-activated platelets was subjected to actin depolymerization-repolymerization. Moreover, such interaction occurred equally well when exogenous rap2 was loaded with either GDP or GTPgammaS. We also found that polyhistidine-tagged rap2 immobilized on Ni(2+)-Sepharose and loaded with either GDP or GTPgammaS, could specifically bind to cytoskeletal actin. Moreover, when purified monomeric actin was induced to polymerize in vitro in the presence of rap2, the small G-protein specifically associated with the actin filaments. Finally, rap2 loaded with either GDP or GTPgammaS was able to bind to purified F-actin immobilized on a plastic surface. These results demonstrate that rap2 interacts with the platelet cytoskeleton by direct binding to the actin filaments and that this interaction is not regulated by the activation state of the protein.     

Identification of glycoprotein Ib beta as one of the major proteins phosphorylated during exposure of intact platelets to agents that activate cyclic AMP-dependent protein kinase

Platelet function is inhibited by prostaglandin E1, prostaglandin I2, or forskolin, agents that increase the intracellular concentration of cyclic AMP. The inhibition appears to result from cyclic AMP-stimulated phosphorylation of specific intracellular proteins. One of the major increases in phosphorylation occurs in a polypeptide of Mr = 24,000 (P24). In this study, an effort was made to identify P24. Platelets prelabeled with [32P]phosphate were incubated with prostaglandin E1, prostaglandin I2, or forskolin. Proteins that became phosphorylated were detected by autoradiography of sodium dodecyl sulfate-polyacrylamide gels. Several lines of evidence indicated that P24 was the beta-subunit of the plasma membrane glycoprotein (GP) Ib, a glycoprotein that is essential for the adhesion of platelets to damaged subendothelium, for the rapid response of platelets to thrombin, and for the attachment of the membrane skeleton to the cytoplasmic face of the plasma membrane. P24 co-migrated with GP Ib beta on reduced gels (Mr = 24,000) and also on nonreduced gels (when GP Ib beta is disulfide-linked to GP Ib alpha and migrates with Mr = 170,000). Like GP Ib beta, P24 was associated with actin filaments in Triton X-100 lysates. Like GP Ib beta, it was selectively associated with filaments of the membrane skeleton and was 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 (which lack GP Ib) to prostaglandin E1 resulted in phosphorylation of other polypeptides, but not of P24. These studies show that P24, one of the major polypeptides phosphorylated when platelets are exposed to agents that inhibit platelet function by increasing the concentration of cyclic AMP, is the beta-subunit of GP Ib.     

Flow-cytometric detection of surface membrane alterations and concomitant changes in the cytoskeletal actin status of activated platelets.

Occlusive vascular diseases are promoted by a "prethrombotic state" with increased platelet activity. Polymerization of cytoskeletal proteins and exposure of subcellular structures or rebinding of secreted proteins have been characterized as early reactions after platelet activation preceding adhesion and aggregation. Here, we demonstrate the kinetic increase in specific binding of monoclonal antibodies to thrombospondin (P10) and to platelet membrane activation markers CD63 (GP53, a 53 kD lysosomal protein) and CD62 (GMP140, a 140 kD alpha granule protein) by using a flow-cytometric bio-assay and the related change in the actin status by using the DNase-I inhibition assay after stimulation of normal human platelets with 0.2 U/ml thrombin. F-actin was raised from 41% to 51% of total platelet actin content 30 s after stimulation and remained thereafter constant (50% at 60 s). Simultaneously, the percentage of P10, CD63, and CD62 positive platelets was elevated from 5.4%, 24.4%, and 9.1% to 67.4%, 80.2%, and 82.3% respectively. The mean number of P10, CD63, and CD62 antibody binding sites increased from 3,300, 1,715, and 2,146 to 6,400, 6,800, and 9,016 per platelet. Conclusively, changes in the organization of the cytoskeletal protein "actin" and exposure of subcellular structures indicating platelet secretion can be regarded as markers of early platelet activation. Thus, the parallel response in both analytical systems provides further support for the diagnostic concept of flow-cytometric detection of preactivated platelets in the peripheral blood by using fluochrome staining procedures detecting activation dependent structural alterations directly at the cellular level.     

Biochemical characterization of the Rho GTPase-regulated actin assembly by diaphanous-related formins, mDia1 and Daam1, in platelets

The diaphanous-related formins are actin nucleating and elongating factors. They are kept in an inactive state by an intramolecular interaction between the diaphanous inhibitory domain (DID) and the diaphanous-autoregulatory domain (DAD). It is considered that the dissociation of this autoinhibitory interaction upon binding of GTP-bound Rho to the GTPase binding domain next to DID induces exposure of the FH1-FH2 domains, which assemble actin filaments. Here, we isolated two diaphanous-related formins, mDia1 and Daam1, in platelet extracts by GTP-RhoA affinity column chromatography. We characterized them by a novel assay, where beads coated with the FH1-FH2-DAD domains of either mDia1 or Daam1 were incubated with platelet cytosol, and the assembled actin filaments were observed after staining with rhodamine-phalloidin. Both formins generated fluorescent filamentous structures on the beads. Quantification of the fluorescence intensity of the beads revealed that the initial velocity in the presence of mDia1 was more than 10 times faster than in the presence of Daam1. The actin assembly activities of both FH1-FH2-DADs were inhibited by adding cognate DID domains. GTP-RhoA, -RhoB, and -RhoC, but not GTP-Rac1 or -Cdc42, bound to both mDia1 and Daam1 and efficiently neutralized the inhibition by the DID domains. The association between RhoA and Daam1 was induced by thrombin stimulation in platelets, and RhoA-bound endogenous formins induced actin assembly, which was inhibited by the DID domains of Daam1 and mDia1. Thus, mDia1 and Daam1 are platelet actin assembly factors having distinct efficiencies, and they are directly regulated by Rho GTPases.     

Phosphorylation regulated ion-binding is a property shared by the acidic subclass dehydrins

Dehydrins are a family of proteins that accumulate in response to abiotic stresses. Little is known about the biochemical functions of these proteins. It is known that the Arabidopsis dehydrin, ERD14, is activated by phosphorylation to bind calcium and other ions. To begin to categorize the Arabidopsis dehydrins into functional families, we determined whether representative members of the dehydrin sub families share the properties of ERD14. When phosphorylated in vitro with casein kinase II; recombinant COR47, and ERD10 (and ERD14) become activated to bind calcium. ERD14 exhibited the highest calcium-binding activity followed by ERD10 and COR47. These dehydrins, when isolated from cold-treated Arabidopsis plants were also shown to have phosphorylation-dependent, calcium-binding activity. RAB18 showed very little calcium binding activity, even though it was phosphorylated by casein kinase II. XERO2 was not phosphorylated with CKII and did not bind calcium. Competition studies suggest that other divalent cations may bind to the dehydrins COR47, ERD10, and ERD14. Utilizing matrix-assisted laser desorption ionization – time of flight mass spectroscopy (MALDI-TOF), we determined that the poly serine region located in all three calcium-binding family members (COR47, ERD10, and ERD14) is the most likely phosphorylation site responsible for the activation of calcium binding. These results are consistent with a distinct biochemical function for the acidic subclass of dehydrins (COR47, ERD10, and ERD14) as ion (calcium)-interacting proteins.