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S J Shattil

Publications and source records attributed to S J Shattil.

At least 37 records · Page 2Linked to original sources
PubMed1999

Genetic and pharmacological analyses of Syk function in alphaIIbbeta3 signaling in platelets.

Agonists induce inside-out alphaIIbbeta3 signaling resulting in fibrinogen binding and platelet aggregation. These in turn trigger outside-in signaling resulting in further platelet stimulation. Because the Syk tyrosine kinase is activated during both phases of integrin signaling, we evaluated its role in alphaIIbbeta3 function in murine platelets rendered null for Syk by gene targeting and in human platelets incubated with piceatannol, a tyrosine kinase inhibitor reportedly selective for Syk. Both Syk null murine platelets and piceatannol-treated human platelets exhibited a partial, but statistically significant defect in activation of alphaIIbbeta3 by adenine diphosphate (ADP) +/- epinephrine as assessed by fibrinogen binding. Syk null platelets adhered normally to immobilized fibrinogen, and mice with these platelets exhibited normal tail bleeding times. In contrast, piceatannol treatment of human platelets completely inhibited platelet adhesion to immobilized fibrinogen. The discrepancy in extent of integrin dysfunction between murine and human platelet models may be due to lack of specificity of piceatannol, because this compound inhibited the activity of Src and FAK as well as Syk and also reduced tyrosine phosphorylation of multiple platelet proteins. These results provide genetic evidence that Syk plays a role in alphaIIbbeta3 signaling in platelets and pharmacological evidence that, although piceatannol also inhibits alphaIIbbeta3 signaling, it does so by inhibtion of multiple protein tyrosine kinases.

Animals↗
PubMed1998

Identification of a novel integrin signaling pathway involving the kinase Syk and the guanine nucleotide exchange factor Vav1.

BACKGROUND: . Integrins induce the formation of large complexes of cytoskeletal and signaling proteins, which regulate many intracellular processes. The activation and assembly of signaling complexes involving focal adhesion kinase (FAK) occurs late in integrin signaling, downstream from actin polymerization. Our previous studies indicated that integrin-mediated activation of the non-receptor tyrosine kinase Syk in hematopoietic cells is independent of FAK and actin polymerization, and suggested the existence of a distinct signaling pathway regulated by Syk. RESULTS: . Multiple proteins were found to be activated by Syk, downstream of engagement of the platelet/megakaryocyte-specific integrin alphaIIbbeta3. The guanine nucleotide exchange factor Vav1 was inducibly phosphorylated in a Syk-dependent manner in cells following their attachment to fibrinogen. Together, Syk and Vav1 triggered lamellipodia formation in fibrinogen-adherent cells and both Syk and Vav1 colocalized with alphaIIbbeta3 in lamellipodia but not in focal adhesions. Additionally, Syk and Vav1 cooperatively induced activation of Jun N-terminal kinase (JNK), extracellular-signal-regulated kinase 2 (ERK2) and the kinase Akt, and phosphorylation of the oncoprotein Cbl in fibrinogen-adherent cells. Activation of all of these proteins by Syk and Vav1 was not dependent on actin polymerization. CONCLUSIONS: . Syk and Vav1 regulate a unique integrin signaling pathway that differs from the FAK pathway in its proximity to the integrin itself, its localization to lamellipodia, and its activation, which is independent of actin polymerization. This pathway may regulate multiple downstream events in hematopoietic cells, including Rac-induced lamellipodia formation, tyrosine phosphorylation of Cbl, and activation of JNK, ERK2 and the phosphatidylinositol 3'-kinase-regulated kinase Akt.

Actins↗
PubMed1998

Complementary roles for receptor clustering and conformational change in the adhesive and signaling functions of integrin alphaIIb beta3.

Integrin alphaIIb beta3 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 alphaIIb beta3 function, alphaIIb was fused at its cytoplasmic tail to one or two FKBP12 repeats (FKBP). These modified alphaIIb subunits were expressed with beta3 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 alphaIIb beta3 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 alphaIIb beta3 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 alphaIIb beta3 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 alphaIIb beta3 in platelets.

Animals↗
PubMed1998

RhoA and the function of platelet integrin alphaIIbbeta3.

Integrins respond to "inside-out" signals, which enable them to bind adhesive ligands, and ligand binding initiates "outside-in" signals that mediate anchorage-dependent cellular responses. RhoA is a GTPase that regulates certain actin rearrangements and transcriptional events. It has also been implicated in integrin signaling, but the exact relationship is not understood. To examine this further, platelets were incubated with C3 exoenzyme to adenine diphosphate (ADP)-ribosylate and inactivate RhoA, and the function of integrin alphaIIbbeta3 was studied. Despite inactivation of >/= 90% of RhoA, platelets exhibited normal inside-out signaling, as monitored by agonist-induced binding of a fibrinogen-mimetic anti-alphaIIbbeta3 antibody and normal fibrinogen-dependent aggregation. On the other hand, RhoA inactivation decreased the adhesion of agonist-stimulated platelets to fibrinogen (P < .04) and the formation of vinculin-rich focal adhesions in platelets that did adhere (P < .001). These effects were selective because fibrin clot retraction, a response also dependent on alphaIIbbeta3 and actin contractility, was unaffected by C3, as was the content of F-actin in resting or agonist-stimulated platelets. Similar results were obtained in a Chinese hamster ovary (CHO) cell model system of alphaIIbbeta3: C3 exoenzyme (or overexpression of dominant-negative N19RhoA) failed to influence integrin activation state, but it blocked the formation of focal adhesions in cells spread on fibrinogen. These studies establish that RhoA plays a highly selective role in alphaIIbbeta3 signaling, and they identify a subset of responses to integrin ligation that may be uniquely dependent on the actin rearrangements regulated by this GTPase.

ADP Ribose Transferases↗
PubMed1997

Regulation of the pp72syk protein tyrosine kinase by platelet integrin alpha IIb beta 3.

pp72syk is essential for development and function of several hematopoietic cells, and it becomes activated through tandem SH2 interaction with ITAM motifs in immune response receptors. Since Syk is also activated through integrins, which do not contain ITAMs, a CHO cell model system was used to study Syk activation by the platelet integrin, alpha IIb beta 3. As in platelets, Syk underwent tyrosine phosphorylation and activation during CHO cell adhesion to alpha IIb beta 3 ligands, including fibrinogen. This involved Syk autophosphorylation and the tyrosine kinase activity of Src, and it exhibited two novel features. Firstly, unlike alpha IIb beta 3-mediated activation of pp125FAK, Syk activation could be triggered by the binding of soluble fibrinogen and abolished by truncation of the alpha IIb or beta 3 cytoplasmic tail, and it was resistant to inhibition by cytochalasin D. Secondly, it did not require phosphorylated ITAMs since it was unaffected by disruption of an ITAM-interaction motif in the SH2(C) domain of Syk or by simultaneous overexpression of the tandem SH2 domains. These studies demonstrate that Syk is a proximal component in alpha IIb beta 3 signaling and is regulated as a consequence of intimate functional relationships with the alpha IIb beta 3 cytoplasmic tails and with Src or a closely related kinase. Furthermore, there are fundamental differences in the activation of Syk by alpha IIb beta 3 and immune response receptors, suggesting a unique role for integrins in Syk function.

Amino Acid Sequence↗
PubMed1997

Truncation of the cytoplasmic domain of beta3 in a variant form of Glanzmann thrombasthenia abrogates signaling through the integrin alpha(IIb)beta3 complex.

Glanzmann thrombasthenia is an inherited bleeding disorder characterized by absence or dysfunction of the platelet integrin alpha(IIb)beta3. Patient RM is a thrombasthenic variant whose platelets fail to aggregate in response to physiological agonists, despite the fact that they express abundant levels of alpha(IIb)beta3 on their surface. Binding of soluble fibrinogen or fibrinogen mimetic antibodies to RM platelets did not occur, except in the presence of ligand-induced binding site (LIBS) antibodies that transformed the RM integrin complex into an active conformation from outside the cell. Sequence analysis of PCR-amplified genomic DNA and platelet mRNA revealed a C2268T nucleotide substitution in the gene encoding the integrin beta3 subunit that resulted in an Arg724Ter mutation, producing a truncated protein containing only the first eight of the 47 amino acids normally present in the cytoplasmic domain. Functional analysis of both RM platelets and CHO cells stably expressing this truncated integrin revealed that the alpha(IIb)beta3Arg724Ter complex is able to mediate binding to immobilized fibrinogen, though downstream events, including cytoskeletally-mediated cell spreading and tyrosine phosphorylation of focal adhesion kinase, pp125FAK, fail to occur. These studies establish the importance of the membrane-distal portion of the integrin beta3 cytoplasmic domain in bidirectional transmembrane signaling in human platelets, and the role of integrin signaling in maintaining normal hemostasis in vivo.

Amino Acid Sequence↗
PubMed1997

Effect of Ca2+ on GP IIb-IIIa interactions with integrilin: enhanced GP IIb-IIIa binding and inhibition of platelet aggregation by reductions in the concentration of ionized calcium in plasma anticoagulated with citrate.

BACKGROUND: Integrilin (eptifibatide), a potent inhibitor of the fibrinogen binding function of GP IIb-llla, has been shown to reduce the thrombotic complications of angioplasty and of acute coronary syndromes. The present study was designed to determine whether the reduced Ca2+ concentrations in plasma anticoagulated with citrate affect Integrilin binding to GP IIb-IIIa and the ex vivo pharmacodynamic measurements for this drug. METHODS AND RESULTS: Lower concentrations of Integrilin were found to inhibit platelet aggregation in plasma anticoagulated with citrate (for ADP, mean+/-SD IC(50)=140+/-40 nmol/L, n=6; Ca2+ =40 to 50 micromol/L) than with PPACK (IC(50)=570+/-70 nmol/L, P<.0001, n=6; Ca2+ approximately 1 mmol/L). Chelation of Ca2+ with EDTA or citrate caused a similar degree of enhancement in the inhibitory activity of Integrilin. Measurements of D3 LIBS epitope expression showed that the enhanced inhibitory activity was caused by enhanced GP IIb-IIIa occupancy by Integrilin. Citrate anticoagulation decreased the amounts of Integrilin required to inhibit the binding of PAC1, a monoclonal antibody that mimics the GP IIb-IIIa binding activity of fibrinogen. Reduced Ca2+ also increased Integrilin inhibition of the binding of biotinylated fibrinogen to purified, immobilized GP IIb-IIIa. CONCLUSIONS: These data suggest that citrate anticoagulation removes Ca2+ from GP IIb-IIIa and enhances the apparent inhibitory activity of Integrilin. This finding indicates that the inhibitory activity of Integrilin is overestimated in blood samples collected with citrate, suggesting that it may be possible to achieve greater antithrombotic efficacy beyond that observed in clinical trials to date with Integrilin.

Antibodies, Monoclonal↗
PubMed1997

Affinity modulation of platelet integrin alphaIIbbeta3 by beta3-endonexin, a selective binding partner of the beta3 integrin cytoplasmic tail.

Platelet agonists increase the affinity state of integrin alphaIIbbeta3, a prerequisite for fibrinogen binding and platelet aggregation. This process may be triggered by a regulatory molecule(s) that binds to the integrin cytoplasmic tails, causing a structural change in the receptor. beta3-Endonexin is a novel 111-amino acid protein that binds selectively to the beta3 tail. Since beta3-endonexin is present in platelets, we asked whether it can affect alphaIIbbeta3 function. When beta3-endonexin was fused to green fluorescent protein (GFP) and transfected into CHO cells, it was found in both the cytoplasm and the nucleus and could be detected on Western blots of cell lysates. PAC1, a fibrinogen-mimetic mAb, was used to monitor alphaIIbbeta3 affinity state in transfected cells by flow cytometry. Cells transfected with GFP and alphaIIbbeta3 bound little or no PAC1. However, those transfected with GFP/beta3-endonexin and alphaIIbbeta3 bound PAC1 specifically in an energy-dependent fashion, and they underwent fibrinogen-dependent aggregation. GFP/beta3-endonexin did not affect levels of surface expression of alphaIIbbeta3 nor did it modulate the affinity of an alphaIIbbeta3 mutant that is defective in binding to beta3-endonexin. Affinity modulation of alphaIIbbeta3 by GFP/beta3-endonexin was inhibited by coexpression of either a monomeric beta3 cytoplasmic tail chimera or an activated form of H-Ras. These results demonstrate that beta3-endonexin can modulate the affinity state of alphaIIbbeta3 in a manner that is structurally specific and subject to metabolic regulation. By analogy, the adhesive function of platelets may be regulated by such protein-protein interactions at the level of the cytoplasmic tails of alphaIIbbeta3.

Animals↗
PubMed1997

A conserved sequence motif in the integrin beta3 cytoplasmic domain is required for its specific interaction with beta3-endonexin.

Integrin signaling is mediated by interaction of integrin cytoplasmic domains with intracellular signaling molecules. Recently, we identified a novel 111-amino acid polypeptide, termed beta3-endonexin, which interacts selectively with the integrin beta3 cytoplasmic domain. In the present study we conducted a systematic mutational analysis of both the integrin beta3 cytoplasmic domain and beta3-endonexin to map sites required for interaction. The interaction of the full-length beta3 integrin subunit with beta3-endonexin in vitro required the beta3 cytoplasmic domain. In a yeast two-hybrid system, both membrane-proximal and membrane-distal residues of the beta3 cytoplasmic domain were necessary for interaction with beta3-endonexin. In particular, the membrane-distal NITY motif at beta3 756-759 was critical for the interaction. Exchange of beta3 residues 756-759 (NITY) for the corresponding residues in beta1 (NPKY) endowed the beta1 cytoplasmic domain with the ability to interact with beta3-endonexin. Conversely, exchange of the NPKY motif at beta1 772-775 for the NITY motif in beta3 abolished interaction of this chimeric cytoplasmic domain with beta3-endonexin. Because the NITY motif is present in the beta3 but not the beta1 cytoplasmic domain, these results explain the selective interaction of this cytoplasmic domain with beta3-endonexin. In addition, deletional analysis suggested that a core 91-residue sequence of beta3-endonexin is sufficient for specific binding to the beta3 cytoplasmic domain. These studies have identified a cytoplasmic domain sequence motif that specifies an integrin-specific protein-protein interaction.

Amino Acid Sequence↗
PubMed1997

A genetic analysis of integrin function: Glanzmann thrombasthenia in vitro.

Glanzmann thrombasthenia, an inherited bleeding disorder, can be caused by a defect or deficiency in platelet integrin alphaIIb beta3 (GPIIb-IIIa). Studies of thrombasthenia variants have facilitated identification of sites involved in the functions of alphaIIb beta3 and other integrins. Such sites include those that bind ligand and those that participate in the "activation" of alphaIIb beta3 required for high affinity binding of ligands such as fibrinogen or PAC1, a monoclonal antibody. Here we describe the isolation of such variants, created in vitro with Chinese hamster ovary cells that express an activated form of alphaIIb beta3. These cells were exposed to a mutagen, ethyl methane sulfonate, and variants that lost the capacity to bind PAC1 were isolated by fluorescence-activated cell sorting. These variants were grouped into three phenotypic classes. One comprised integrin mutations that disrupt ligand binding function; a second comprised mutations that interfere with the capacity of cells to activate the integrin. Most of these activation-defective mutations were in the integrin cytoplasmic domain, but surprisingly, several were caused by mutations affecting three closely spaced residues in the beta3 extracellular domain. A third class of mutants exhibited a defect in integrin activation not ascribable to changes in the integrin sequence. Thus, these may represent mutated signaling molecules required for integrin activation. This unbiased genetic approach provides new insights into the structural basis of integrin function and may assist in identifying the cellular events that regulate integrin function.

Amino Acid Sequence↗
PubMed1997

Not just another pretty face: regulation of platelet function at the cytoplasmic face of integrin alpha IIb beta 3.

Integrin alpha IIb beta 3 is necessary for platelet thrombus formation by virtue of its ability to bind fibrinogen and von Willebrand factor in a regulated fashion and to mediate platelet aggregation. Moreover, alpha IIb beta 3 transduces inward signals in response to ligand binding which promote cytoskeletal reorganization and other post-ligand binding events. Anchorage and signaling through alpha IIb beta 3 appear to be controlled by interactions of intracellular proteins with the cytoplasmic tails of alpha IIb and/or beta 3. The cytoplasmic tails and associated cytoskeletal components may function as a scaffold or nucleation site for the assembly of enzymes, substrates and adaptor molecules into a signaling complex. Identification of the components of this signaling complex and characterization of their dynamic interactions should lead to insights into the molecular basis of platelet activation defects and may provide a rationale for the development of new anti-thrombotic drugs.

Blood Platelets↗
PubMed1996

A molecular basis for affinity modulation of Fab ligand binding to integrin alphaIIb beta3.

The Arg-Gly-Asp (RGD) sequence within the third complementarity-determining region (CDR3) of the heavy chain (H3) is responsible for the binding of the recombinant murine Fab molecules, AP7 and PAC1.1, to the platelet integrin alphaIIbbeta3. AP7 binding is minimally influenced by the conformational state of this receptor, whereas PAC1.1 binds preferentially to the activated state of the receptor induced by platelet agonists. To study the molecular basis for this functional difference, we replaced the AP7 H3 loop (HPFYRGDGGN) with all or segments of the analogous sequence from PAC1.1 (RSPSYYRGDGAGP). AP7 Fd (VH domain + Cgamma1 domain) segments containing these H3 loop sequences were expressed as active Fab molecules by coinfection of Spodoptera frugiperda cell lines with recombinant baculoviruses containing Fd and AP7 kappa chain cDNA. Replacement of the entire AP7 H3 loop with that from PAC1.1 generated the mutant AP7.3 Fab molecule, which bound selectively to either activated, gel-filtered platelets or to purified alphaIIbbeta3 in a manner identical to that of PAC1.1. Identical results were obtained when solely the sequences flanking the amino side of RGD within the respective H3 loops were exchanged. AP7.3 and PAC1.1 exhibited saturable but submaximal binding to activated gel-filtered platelets. Relative to AP7, the number of AP7.3 or PAC1. 1 Fab molecules bound per platelet was 17% in the presence of 1 m Ca2+ + 1 mM Mg2+ or 40% in the presence of 10 microM Mn2+. The ratio of Fab molecules bound after versus before activation (mean =/- S.D.; n = 3) was: for AP7.3, 9.8 =/- 0.6; for PAC1.1, 8.8 +/- 0.3; and for AP7, 1.4 =/- 0.2. In addition, AP7 bound to the stably expressed integrin mutant alphaIIbbeta3(S123A), whereas AP7.3 and PAC1 did not. Because AP7.3 behaves in every respect like PAC1.1, we conclude that the ability of RGD-based ligands to distinguish activated from resting conformations of the integrin alphaIIbbeta3 can be regulated by limited amino acid sequences immediately adjacent to the RGD tripeptide. Furthermore, those Fab molecules that exhibit increased selectivity for the activated conformation of alphaIIbbeta3 bind to a subpopulation of this integrin on platelets that is modulated by divalent cations.

Amino Acid Sequence↗
PubMed1996

Glanzmann thrombasthenia due to a two amino acid deletion in the fourth calcium-binding domain of alpha IIb: demonstration of the importance of calcium-binding domains in the conformation of alpha IIb beta 3.

The integrin alpha IIb beta 3, a calcium-dependent heterodimer, plays a critical role in platelet aggregation. The alpha IIb subunit of the heterodimer contains four highly conserved putative calcium-binding domains in its extracellular portion. During studies of the molecular basis of Glanzmann thrombasthenia in a child of mixed Caucasian background whose platelets expressed little alpha IIb beta 3 on their surface, we found the patient heterozygous for a two amino acid deletion in the fourth alpha IIb calcium-binding domain. When this alpha IIb mutant was expressed in COS-1 cells, we found that the deletion did not interfere with the assembly of alpha IIb beta 3 heterodimers, but altered their conformation such that they were neither recognized by the heterodimer-specific antibody A2A9 nor able to undergo further intracellular processing or transport to the cell surface. These results suggest that the calcium-binding domains in alpha IIb play an important role maintaining the overall conformation of alpha IIb beta 3. To confirm this suggestion, we deleted each of the four 12 amino acid calcium-binding domains in alpha IIb by in vitro mutagenesis and expressed the mutants along with beta 3 in COS-1 cells. Each construct formed a heterodimer with beta 3, but none of the heterodimers interacted with A2A9 or underwent further intracellular processing. These data indicate that the calcium-binding domains in alpha IIb are not involved in alpha IIb beta 3 heterodimer formation, but their presence is required for the intracellular transport of alpha IIb beta 3 to the cell surface.

Adult↗
PubMed1996

Breaking the integrin hinge. A defined structural constraint regulates integrin signaling.

Integrins are heterodimeric (alpha, beta) cell adhesion receptors. We demonstrate that point mutations in the cytoplasmic domains of both the alpha and beta subunits promote constitutive signaling by the integrin alphaIIbbeta3. By generating charge reversal mutations, we show these "activating" mutations may act by disrupting a potential salt bridge between the membrane-proximal portions of the alpha and beta subunit cytoplasmic domains. Thus, the modulation of specific interactions between the alpha and beta subunit cytoplasmic domains may regulate transmembrane signaling through integrins. In addition, these activating mutations induce dominant alterations in cellular behavior, such as the assembly of the extracellular matrix. Consequently, somatic mutations in integrin cytoplasmic domains could have profound effects in vivo on integrin-dependent functions such as matrix assembly, cell migration, and anchorage-dependent cell growth and survival.

Amino Acid Sequence↗