Focal Adhesion Kinase pp125and the β1 Integrin Subunit Are Constitutively Complexed in HaCaT Cells

Binding of integrins to the extracellular matrix (ECM) activates various signal transduction pathways and regulates gene expression in many cell types. Integrin-dependent cytoplasmic protein/protein interactions are necessary for activation of those signal transduction cascades. In our studies we investigated a possible association of pp125^FAK, an adhesion involved tyrosine kinase, with the integrin β1 subunit. Further we wanted to know to which extent protein tyrosine phosphorylation affects cell adhesion to the ECM and the possible β1 integrin/pp125^FAKcomplex. We were able to show that in HaCaT cells (a human keratinocyte derived cell line) the integrin β1 subunit is associated with tyrosine kinase pp125^FAK. This association was observed in ECM-adherent cells and nonadherent cells and is independent of tyrosine phosphorylation. However, cell adhesion of HaCaT cells to specific substrates requires tyrosine phosphorylation since genistein treatment that blocks phosphorylation of many cellular proteins as pp125^FAKled to a reduced substrate adhesion.     

Integrin-Mediated Signal Transduction in Human Endothelial Cells: Analysis of Tyrosine Phosphorylation Events

Adhesion of human umbilical endothelial cells to fibronectin resulted in increased tyrosine phosphorylation of a group of proteins with molecular mass ranging from 100 to 130 kDa and of a 70 kDa protein. This pattern of tyrosine phosphorylation was also observed when endothelial cells adhered to vitronectin, collagen IV, collagen I and laminin or to culture dishes coated with antibodies directed to either βl, α3, α5, α6 or β3 integrin subunits. Increased phosphorylation of the 100–130 kDa proteins was detectable as early as 30 sec after adhesion, reached maximal level after 15 min, and remained high as long as the cells adhere to culture dishes. The 70 kDa protein was phosphorylated with a slower kinetics and its phosphorylation increased over a period of 3 h. Using specific monoclonal antibodies, the major component of the 100–130 kDa complex was identified as the focal adhesion tyrosine kinase p125^FAK. The phosphorylation of the pl25^FAK was also observed by inducing βl integrin clustering in rum adherent HEC, indicating that this is a primary signalling event induced by integrins. Using tyrosine kinase inhibitors, we show a direct correlation between integrin-stimulated tyrosine kinases and assembly of focal adhesions and actin fibres.     

Integrin-dependent phosphorylation and activation of the protein tyrosine kinase pp125FAK in platelets

We have investigated mechanisms involved in integrin-mediated signal transduction in platelets by examining integrin-dependent phosphorylation and activation of a newly identified protein tyrosine kinase, pp125FAK (FAK, focal adhesion kinase). This kinase was previously shown to be localized in focal adhesions in fibroblasts, and to be phosphorylated on tyrosine in normal and Src-transformed fibroblasts. We show that thrombin and collagen activation of platelets causes an induction of tyrosine phosphorylation of pp125FAK and that pp125FAK molecules isolated from activated platelets display enhanced levels of phosphorylation in immune-complex kinase assays. pp125FAK was not phosphorylated on tyrosine after thrombin or collagen treatment of Glanzmann's thrombasthenic platelets deficient in the fibrinogen receptor GPIIb-IIIa, or of platelets pretreated with an inhibitory monoclonal antibody to GP IIb-IIIa. Fibrinogen binding to GP IIb-IIIa was not sufficient to induce pp125FAK phosphorylation because pp125FAK was not phosphorylated on tyrosine in thrombin-treated platelets that were not allowed to aggregate. These results indicate that tyrosine phosphorylation of pp125FAK is dependent on platelet aggregation mediated by fibrinogen binding to the integrin receptor GP IIb-IIIa. The induction of tyrosine phosphorylation of pp125FAK was inhibited in thrombin- and collagen-treated platelets preincubated with cytochalasin D, which prevents actin polymerization following activation. Under all of these conditions, there was a strong correlation between the induction of tyrosine phosphorylation of pp125FAK in vivo and stimulation of the phosphorylation of pp125FAK in vitro in immune-complex kinase assays. This study provides the first genetic evidence that tyrosine phosphorylation of pp125FAK is dependent on integrin-mediated events, and demonstrates that there is a strong correlation between tyrosine phosphorylation of pp125FAK in platelets, and the activation of pp125FAK-associated phosphorylating activity in vitro.     

Mechanical Strain Increases Protein Tyrosine Phosphorylation in Airway Smooth Muscle Cells

Mechanical stress contributes to normal structure and function of the lung as well as pathology in such diseases as bronchopulmonary dysplasia and adult respiratory distress syndrome. Stress-related increases in airway smooth muscle (ASM) quantity are reflectedin vitrowhere cultured ASM cells respond to cyclic deformational strain with increased proliferation, cell reorientation, protein production, stress fibers, and focal adhesions. To understand the mechanisms of mechanical signaling in ASM cells, we investigated whether strain increased tyrosine phosphorylation of focal adhesion-related proteins. ASM cells were grown to confluence on collagen type I and subjected to 30 min of cyclic deformation strain (2 s of 25% deformation of the substratum, 2 s relaxation) and compared at various time points with identical cells not subjected to strain for phosphotyrosine content of three focal adhesion-concentrated proteins (pp125^FAK, paxillin, and talin) by Western blotting. Strain caused a rapid increase in tyrosine phosphorylation of pp125^FAKand paxillin. Tyrosine phosphorylation decreased by 4 h in pp125^FAKafter discontinuing strain but remained elevated in paxillin at 24 h. Increases in tyrosine phosphorylation of talin were not found. In separate studies, when cells were strained in the presence of tyrosine kinase inhibitors (genistein and herbimycin A), strain-induced reorientation and elongation were inhibited. Mechanochemical signal transduction appears to mediate cell morphologic changes through quantitative and possibly qualitative changes in tyrosine phosphorylation of adhesion-related proteins.     

Immobilised echistatin promotes platelet adhesion and protein tyrosine phosphorylation

Echistatin, a 5000-Da disintegrin, is a strong competitive inhibitor of platelet alpha(IIb)beta(3) binding to fibrinogen. In addition to its antiplatelet activity, echistatin also exhibits activating properties by inducing a switch of alpha(IIb)beta(3) conformation towards an active state. However, soluble echistatin, which is a monomeric ligand, provides only receptor affinity modulation, but it is unable to activate integrin-dependent intracellular signals. Since proteins may exhibit a multivalent functionality as a result of their absorption to a substrate, in this study we evaluated whether immobilised echistatin is able to stimulate platelet adhesion and signalling. The immobilisation process led to an increase of echistatin affinity for integrin(s) expressed on resting platelets. Unlike the soluble form, immobilised echistatin bound at comparable extent either unstimulated or ADP-activated platelets. Furthermore, echistatin presented in this manner was effective in stimulating integrin-dependent protein tyrosine phosphorylation. Platelets adhering to immobilised echistatin showed a pattern of total tyrosine phosphorylated proteins resembling that of fibrinogen-attached platelets. In particular, solid-phase echistatin induced a strong phosphorylation of tyrosine kinases pp72(syk) and pp125(FAK). Inhibitors of platelet signalling, such as apyrase, prostaglandin E(1), cytochalasin D and bisindolylmaleimide, while not affecting platelet adhesion to immobilised echistatin, abolished pp125(FAK) phosphorylation. This suggests that signals activating protein kinase C function, dense granule secretion and cytoskeleton assembly might be involved in echistatin-induced pp125(FAK) phosphorylation.     

Possible involvement of focal adhesion kinase,p125FAK,in osteoclastic bone resorption

Involvement of tyrosine phosphorylation in osteoclastic bone resorption was examined using osteoclast-like multinucleated cells prepared from co-cultures of mouse osteoblastic cells and bone marrow cells in the presence of 1α,25-dihydroxyvitamin D₃. When osteoclast-like cells were plated on culture dishes in the presence of 10% fetal bovine serum, they were sharply stained in their peripheral region by anti-phosphotyrosine antibody. Western blot analysis revealed that 115-to 130-kD proteins were tyrosine-phosphorylated in osteoclast-like cells. Using immunoprecipitation and immunoblotting, one of the proteins with 115–130 kD was identified as focal adhesion kinase (p125^FAK), a tyrosine kinase, which is localized in focal adhesions. Immunostaining with anti-p 125^FAK antibody revealed that p125^FAK was mainly localized at the periphery of osteoclast-like cells. Herbimycin A, a tyrosine kinase inhibitor, not only suppressed tyrosine phosphorylation of p125^FAK but also changed the intracellular localization of p125^FAK and disrupted a ringed structure of F-actin-containing podosomes in osteoclast-like cells. Antisense oligodeoxynucleotides to p125^FAK inhibited dentine resorption by osteoclast-like cells, whereas sense oligodeoxynucleotides did not. These results suggest that p125^FAK is involved in osteoclastic bone resorption and that tyrosine phosphorylation of p125^FAK is critical for regulating osteoclast function.     

Focal adhesion kinase pp125FAK is associated with both intercellular junctions and matrix adhesion sites in vivo

Previous studies have characterized pp125^FAK as a focal adhesion (FA)-associated non-receptor tyrosine kinase. However, there are few data available on the expression and localization of this kinase in tissues. In this study we show that in human tissues the highest expression of pp125^FAK is found in some developing epithelia, where pp125^FAK is associated with either intercellular junctions or with sites of adhesion to the basement membrane, whereas the same adult tissues show only a faint reactivity. Connective tissue cells do not show any reactivity for pp125^FAK in vivo, but developing arterial smooth muscle expresses pp125^FAK at high levels. The expression pattern in malignant tissues is variable, but most carcinomas do not express this kinase. In primary cultures of human amnion epithelial cells pp125^FAK first becomes associated with the polarized adhesion lamellae, but is subsequently translocated to the forming adherens junctions (AJs). Later upon culturing pp125^FAK becomes associated with prominent FAs, as in cultured cell lines. Taken together, our results suggest that the association of pp125^FAK with FAs in cultured cells is principally due to a process of adaptation, whereas in vivo pp125^FAK mainly functions as a regulatory component of intercellular AJs and cell-matrix adhesions of developing epithelia and also in developing arterial smooth muscle.     

H(2)O(2) activity on platelet adhesion to fibrinogen and protein tyrosine phosphorylation

Platelets represent a target of reactive oxygen species produced under oxidative stress conditions. Controversial data on the effect of these species on platelet functions have been reported so far. In this study we evaluated the effect of a wide range of H(2)O(2) concentrations on platelet adhesion to immobilized fibrinogen and on pp72(syk) and pp125(FAK) tyrosine phosphorylation. Our results demonstrate that: (1) H(2)O(2) does not affect the adhesion of unstimulated or apyrase-treated platelets to immobilized fibrinogen; (2) H(2)O(2) does not affect pp72(syk) phosphorylation induced by platelet adhesion to fibrinogen-coated dishes; (3) H(2)O(2) reduces, in a dose-dependent fashion, pp125(FAK) phosphorylation of fibrinogen-adherent platelets; (4) concentrations of H(2)O(2) near to physiological values (10-12 microM) are able to strengthen the subthreshold activation of pp125(FAK) induced by epinephrine in apyrase-treated platelets; (5) H(2)O(2) doses higher than 0.1 mM inhibit ADP-induced platelet aggregation and dense granule secretion. The ability of H(2)O(2) to modulate pp125(FAK) phosphorylation suggests a role of this molecule in physiological hemostasis as well as in thrombus generation.     

Adhesive ligand binding to integrin alpha IIb beta 3 stimulates tyrosine phosphorylation of novel protein substrates before phosphorylation of pp125FAK

Tyrosine phosphorylation of multiple platelet proteins is stimulated by thrombin and other agonists that cause platelet aggregation and secretion. The phosphorylation of a subset of these proteins, including a protein tyrosine kinase, pp125FAK, is dependent on the platelet aggregation that follows fibrinogen binding to integrin alpha IIb beta 3. In this report, we examined whether fibrinogen binding, per se, triggers a process of tyrosine phosphorylation in the absence of exogenous agonists. Binding of soluble fibrinogen was induced with Fab fragments of an anti-beta 3 antibody (anti-LIBS6) that directly exposes the fibrinogen binding site in alpha IIb beta3. Proteins of 50-68 KD and 140 kD became phosphorylated on tyrosine residues in a fibrinogen- dependent manner. This response did not require prostaglandin synthesis, an increase in cytosolic free calcium, platelet aggregation or granule secretion, nor was it associated with tyrosine phosphorylation of pp125FAK. Tyrosine phosphorylation of the 50-68-kD and 140-kD proteins was also observed when (a) fibrinogen binding was stimulated by agonists such as epinephrine, ADP, or thrombin instead of by anti-LIBS6; (b) fragment X, a dimeric plasmin-derived fragment of fibrinogen was used instead of fibrinogen; or (c) alpha IIb beta 3 complexes were cross-linked by antibodies, even in the absence of fibrinogen. In contrast, no tyrosine phosphorylation was observed when the ligand consisted of monomeric cell recognition peptides derived from fibrinogen (RGDS or gamma 400-411). Fibrinogen-dependent tyrosine phosphorylation was inhibited by cytochalasin D. These studies demonstrate that fibrinogen binding to alpha IIb beta 3 initiates a process of tyrosine phosphorylation that precedes platelet aggregation and the phosphorylation of pp125FAK. This reaction may depend on the oligomerization of integrin receptors and on the state of actin polymerization, organizational processes that may juxtapose tyrosine kinases with their substrates.     

PAF-Stimulated Protein Tyrosine Phosphorylation in Hippocampus: Involvement of NO Synthase

The effect of platelet-activating factor (PAF) on protein tyrosine phosphorylation was studied in rat hippocampal slices. PAF caused an increase in the tyrosine phosphorylation of two phosphoproteins, which we identified by immunoprecipitation assays as the focal adhesion kinase p125^FAK and crk-associated substrate p130^Cas. The PAF effect was time- and dose-dependent. In addition, the involvement of PAF receptor was demonstrated by using PCA-4248, a specific receptor antagonist. When NO synthase was inhibited by N^G-monomethyl-L-arginine (L-NMA), PAF-stimulated protein tyrosine phosphorylation was inhibited. In conclusion, our results indicate that PAF increased the tyrosine phosphorylation of both p125^FAK and p130^Cas proteins by the production of NO in hippocampus, suggesting that PAF may play a role in the functioning of this cerebral area.     

Store-operated Ca(2+) entry and tyrosine kinase pp60(src) hyperactivity are modulated by hyperglycemia in platelets from patients with non insulin-dependent diabetes mellitus

We have investigated the involvement of store-operated Ca(2+) entry (SOCE) in the abnormal platelet Ca(2+) homeostasis in patients with non insulin-dependent diabetes mellitus (NIDDM). In a medium containing 180 mg/dL glucose, platelets from NIDDM patients showed an increased SOCE compared to controls. We found that tyrosine phosphorylation was elevated in platelets from NIDDM patients. Consistent with this, the activity of the tyrosine kinase pp60(src) is enhanced in platelets from diabetic patients. When the experiments were performed in a medium containing 90 mg/dL both, SOCE and pp60(src) activity, were similar to those found in control platelets. Our results indicate that SOCE is altered in platelets from NIDDM patients probably due to the increased activity of the tyrosine kinase pp60(src). Both, SOCE and pp60(src) activity in platelets from NIDDM patients are more susceptible to the extracellular glucose concentration, which seems to be involved in the dysfunction of these mechanisms.     

Cyclic strain induces reorganization of integrin α5β1 and α2β1 in human umbilical vein endothelial cells

Cyclic strain has been shown to modulate endothelial cell (EC) morphology, proliferation, and function. We have recently reported that the focal adhesion proteins focal adhesion kinase (pp125^FAK) and paxillin, are tyrosine phosphorylated in EC exposed to strain and these events regulate the morphological change and migration induced by cyclic strain. Integrins are also localized on focal adhesion sites and have been reported to induce tyrosine phosphorylation of pp125^FAK under a variety of stimuli. To study the involvement of different integrins in signaling induced by cyclic strain, we first observed the redistribution of α and β integrins in EC subjected to 4 h cyclic strain. Human umbilical vein endothelial cells (HUVEC) seeded on either fibronectin or collagen surfaces were subjected to 10% average strain at a frequency 60 cycles/min. Confocal microscopy revealed that β₁ integrin reorganized in a linear pattern parallel with the long axis of the elongated cells creating a fusion of focal adhesion plaques in EC plated on either fibronectin (a ligand for α₅β₁) or collagen (a ligand for α₂β₁) coated plates after 4 h exposure to cyclic strain. β₃ integrin, which is a vitronectin receptor, did not redistribute in EC exposed to cyclic strain. Cyclic strain also led to a reorganization of α₅ and α₂ integrins in a linear pattern in HUVEC seeded on fibronectin or collagen, respectively. The expression of integrins α₅, α₂, and β₁ did not change even after 24 h exposure to strain when assessed by immunoprecipitation of these integrins. Cyclic strain-induced tyrosine phosphorylation of pp125^FAK occurred concomitant with the reorganization of β₁ integrin. We concluded that α₅β₁ and α₂β₁ integrins play an important role in transducing mechanical stimuli into intracellular signals. J. Cell. Biochem. 64:505–513. © 1997 Wiley-Liss, Inc.     

Complementary Roles for Receptor Clustering and Conformational Change in the Adhesive and Signaling Functions of Integrin αIIbβ3

Integrin α_IIbβ₃ mediates platelet aggregation and “outside-in” signaling. It is regulated by changes in receptor conformation and affinity and/or by lateral diffusion and receptor clustering. To document the relative contributions of conformation and clustering to α_IIbβ₃ function, α_IIb was fused at its cytoplasmic tail to one or two FKBP12 repeats (FKBP). These modified α_IIb subunits were expressed with β₃ in CHO cells, and the heterodimers could be clustered into morphologically detectable oligomers upon addition of AP1510, a membrane-permeable, bivalent FKBP ligand. Integrin clustering by AP1510 caused binding of fibrinogen and a multivalent (but not monovalent) fibrinogen-mimetic antibody. However, ligand binding due to clustering was only 25–50% of that observed when α_IIbβ₃ affinity was increased by an activating antibody or an activating mutation. The effects of integrin clustering and affinity modulation were additive, and clustering promoted irreversible ligand binding. Clustering of α_IIbβ₃ also promoted cell adhesion to fibrinogen or von Willebrand factor, but not as effectively as affinity modulation. However, clustering was sufficient to trigger fibrinogen-independent tyrosine phosphorylation of pp72^Syk and fibrinogen-dependent phosphorylation of pp125^FAK, even in non-adherent cells. Thus, receptor clustering and affinity modulation play complementary roles in α_IIbβ₃ function. Affinity modulation is the predominant regulator of ligand binding and cell adhesion, but clustering increases these responses further and triggers protein tyrosine phosphorylation, even in the absence of affinity modulation. Both affinity modulation and clustering may be needed for optimal function of α_IIbβ₃ in platelets.     

cGMP-independent inhibition of integrin alphaIIbbeta3-mediated platelet adhesion and outside-in signalling by nitric oxide

We examined the influence of S-nitrosoglutathione (GSNO) on alpha(IIb)beta(3) integrin-mediated platelet adhesion to immobilised fibrinogen. GSNO induced a time- and concentration-dependent inhibition of platelet adhesion. Inhibition was cGMP-independent and associated with both reduced platelet spreading and protein tyrosine phosphorylation. To investigate the cGMP-independent effects of NO we evaluated integrin beta(3) phosphorylation. Adhesion to fibrinogen induced rapid phosphorylation of beta(3) on tyrosines 773 and 785, which was reduced by GSNO in a cGMP independent manner. Similar results were observed in suspended platelets indicating that NO-induced effects were independent of spreading-induced signalling. This is the first demonstration that NO directly regulates integrin beta(3) phosphorylation.     

Orthovanadate decreases the leptin content in isolated mouse fat pads via proteasome activation

In a physiological milieu platelets continue to be exposed to agonists long after clot formation. We studied the regulation of postaggregation events consequent on protease-activated receptor (PAR) 1 ligation with either thrombin or the thrombin receptor-activating peptide (TRAP). Stimulation with TRAP (20 microM) but not with thrombin (1 U/ml) for 15 min evoked platelet disaggregation by about 30% and downregulation of high-affinity fibrinogen binding sites on integrin alpha(IIb)beta(3) to nearly prestimulation levels. Concurrently, only TRAP disorganized the actin-based cytoskeleton, with decrease in the cytoskeletal content of focal contact-associated proteins like integrin alpha(IIb)beta(3), Src, and focal adhesion kinase (FAK). While protein tyrosine kinases were activated during the initial period of platelet aggregation with either agonist, stimulation of protein tyrosine phosphatases determined the successive phase of reduced phosphotyrosine content. SHP-1, an abundant protein tyrosine phosphatase in the platelets, was tyrosine phosphorylated on challenge of PAR-1 and coprecipitated with two unidentified tyrosine phosphorylated proteins of 140 and 60 kDa; in addition, SHP-1 tyrosine phosphorylation (which is associated with enhanced phosphatase activity) was sustained until 15 min. Activity of calpain was upregulated following incubation with thrombin and not with TRAP. Collectively, these data suggest that signaling pathways elicited by PAR-1 agonists thrombin and TRAP are markedly different, which could have important implications on late platelet responses.     

An Examination of Focal Adhesion Formation and Tyrosine Phosphorylation in Fibroblasts Isolated from src¯, fyn¯, and yes¯ Mice

As cells adhere to extracellular matrix proteins, several focal adhesion proteins become tyrosine phosphorylated. One of the most prominent of these has been identified as the tyrosine kinase p125^FAK (focal adhesion kinase, FAK). An interaction between FAK and members of the Src family tyrosine kinases p59^fyn, pp60^v-src, and activated pp60^c-src (527F) has been demonstrated, raising the possibility that these kinases may regulate FAK activity. To explore the role of Src family kinases in focal adhesions and in the regulation of FAK activity, we isolated fibroblasts from transgenic mice that lack either pp60^c-src p59^fyn, or pp62^c-yes. These primary fibroblasts, and those of a control mouse, were passaged numerous times and resulted in spontaneously immortalized cell lines without the addition of transforming agents. After confirming the absence of the appropriate nonreceptor tyrosine kinases in the fyc¯, srn¯ and yes¯ fibroblasts, the ability of these fibroblasts to form focal adhesions and stress fibers was assessed by immunofluorescence microscopy and found to be comparable to that of normal fibroblasts. We investigated phosphotyrosine levels in response to adhesion to fibronectin and identified the pp60^src substrate p130 as the one major protein with reduced levels of tyrosine phosphorylation in the cells lacking p59^fyn and pp62^c-yes, and particularly in those lacking pp60^c-scr. We examined FAK phosphorylation and kinase activity and found that there were no significant differences between these cells.     

Regulation of adaptor protein GIT1 in platelets, leading to the interaction between GIT1 and integrin alpha(IIb)beta3

GIT1 is an adaptor protein, which links signaling proteins to focal adhesion, thereby regulating cytoskeletal reorganization. Platelets undergo dynamic cytoskeletal reorganization during platelet activation, for which a large number of adaptor proteins are required. However, there has been no report of GIT1 in platelets. We found that GIT1 was abundantly expressed in platelets and underwent tyrosine phosphorylation downstream of integrin α_IIbβ₃, which was inhibited by the Src kinase inhibitor PP2. Furthermore, GIT1 constitutively associated with βPIX, a guanine nucleotide exchange factor (GEF) for Rac. The GIT1/βPIX complex associated with α_IIbβ₃, concomitantly with GIT1 tyrosine phosphorylation. Moreover, both GIT1 and α_IIbβ₃ rapidly translocated to the cytoskeletal fraction during platelet aggregation, which was not observed in the absence of aggregation. These results suggest that tyrosine phosphorylation of GIT1 by Src kinases may regulate cytoskeletal reorganization downstream of α_IIbβ₃ by bringing the Rac GEF βPIX to the vicinity of the integrin.     

Differential effects of phosphotyrosine phosphatase expression on hormone-dependent and independent pp60 c-src activity

pp60 ^c-src kinase activity can be increased by phosphotyrosine dephosphorylation or growth factor-dependent phosphorylation reactions. Expression of the transmembrane phosphotyrosine phosphatase (PTPase) CD45 has been shown to inhibit growth factor receptor signal transduction (Mooney, RA, Freund, GG, Way, BA and Bordwell, KL (1992) J Biol Chem 267, 23443–23446). Here it is shown that PTPase expression decreased platelet-derived growth factor (PDGF)-dependant activation of pp60 ^c-src but failed to increase hormone independent (basal) pp60 ^c-src activity. PDGF-dependent tyrosine phosphorylation of its receptor was reduced by approximately 60% in cells expressing the PTPase. In contrast, a change in phosphotyrosine content of pp60 ^c-src was not detected in response to PDGF or in PTPase+cells. PDGF increased the intrinsic tyrosine kinase activity of pp60 ^c-src in both control and PTPase+cells but the effect was smaller in PTPase+cells. In anin vitro assay, hormone-stimulate pp60 ^c-src autophosphorylation from PTPase+ cells was decreased 64±22%, and substrate phosphorylation by pp60 ^c-src was reduced 54±16% compared to controls. Hormone-independent pp60 ^c-src kinase activity was unchanged by expression of the PTPase. pp60 ^c-src was, however, anin vitro substrate for CD45, being dephosphorylated at both the regulatory (Tyr⁵²⁷) and kinase domain (Tyr⁴¹⁶) residues. In addition,in vitro dephosphorylation by CD45 increased pp60 ^c-src activity. These findings suggest that the PDGF receptor was anin vivo substrate of CD45 but pp60 ^c-src was not. The lack of activation of pp60 ^c-src in the presence of expressed PTPase may demonstrate the importance of compartmentalization and/or accessory proteins to PTPase-substrate interactions.Abbreviations PTPase phosphotyrosine phosphatase - PDGF platelet-derived growth factor - PMSF phenylmethylsulfonyl fluoride - LCA, CD45 leukocyte common antigen - PBS phosphate buffered saline - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - DTT dithiothreitol - Na₃VO₄ sodium orthovanadate - PV pervanadate - -ME -mercaptoethanol     

Involvement of the antiplatelet activity of magnesium sulfate in suppression of protein kinase C and the Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchanger

Magnesium sulfate is widely used to prevent seizures in pregnant women with hypertension. The aim of this study was to examine the inhibitory mechanisms of magnesium sulfate in platelet aggregation in vitro. In this study, magnesium sulfate concentration-dependently (0.6–3.0 mM) inhibited platelet aggregation in human platelets stimulated by agonists. Magnesium sulfate (1.5 and 3.0 mM) also concentration-dependently inhibited phosphoinositide breakdown and intracellular Ca⁺² mobilization in human platelets stimulated by thrombin. Rapid phosphorylation of a platelet protein of M_r 47,000 (P47), a marker of protein kinase C activation, was triggered by phorbol-12-13-dibutyrate (PDBu, 50 nM). This phosphorylation was markedly inhibited by magnesium sulfate (3.0 mM). Magnesium sulfate (1.5 and 3.0 mM) further inhibited PDBu-stimulated platelet aggregation in human platelets. The thrombin-evoked increase in pHi was markedly inhibited in the presence of magnesium sulfate (3.0 mM). In conclusion, these results indicate that the antiplatelet activity of magnesium sulfate may be involved in the following two pathways: (1) Magnesium sulfate may inhibit the activation of protein kinase C, followed by inhibition of phosphoinositide breakdown and intracellular Ca⁺² mobilization, thereby leading to inhibition of the phosphorylation of P47. (2) On the other hand, magnesium sulfate inhibits the Na⁺/H⁺ exchanger, leading to reduced intracellular Ca⁺² mobilization, and ultimately to inhibition of platelet aggregation and the ATP-release reaction.