Biomedical subjects

S J Shattil

Publications and source records attributed to S J Shattil.

At least 55 records · Page 3Linked to original sources

PubMed1996

The platelet cytoskeleton stabilizes the interaction between alphaIIbbeta3 and its ligand and induces selective movements of ligand-occupied integrin.

Previously, we showed that a subpopulation of the major platelet integrin, alphaIIbbeta3, co-sediments from detergent lysates with talin and other membrane skeleton proteins. Once alphaIIbbeta3 has bound adhesive ligand in a platelet aggregate, the detergent-insoluble alphaIIbbeta3 redistributes (along with the detergent-insoluble membrane skeleton proteins and a variety of signaling molecules) to a fraction that contains cytoplasmic actin filaments. Concomitantly, certain signaling molecules are activated. The present study shows that, in intact platelets, alphaIIbbeta3 forms clusters when occupied by ligand and is selectively moved into the open canalicular system; alphaIIbbeta3 that has not bound ligand remains diffusely distributed at the periphery of the cell. When cytoplasmic actin filaments are depolymerized by cytochalasins, the ability of alphaIIbbeta3 to bind ligand is decreased, and the movement of ligand-occupied alphaIIbbeta3 is prevented. Together with the previous findings, these results suggest that (i) membrane skeleton-associated alphaIIbbeta3 is selectively induced to bind ligand in activated platelets, (ii) ligand-induced transmembrane signaling causes an altered association of membrane skeleton-associated alphaIIbbeta3 with the cytoplasmic component of the cytoskeleton, (iii) ligand-induced cytoskeletal reorganizations stabilize the interaction between ligand and integrin, and (iv) ligand-occupancy triggers cytoskeletal reorganizations that result in selective movements of occupied ligand.

Actins↗

PubMed1996

Phosphoinositide 3-kinase gamma and p85/phosphoinositide 3-kinase in platelets. Relative activation by thrombin receptor or beta-phorbol myristate acetate and roles in promoting the ligand-binding function of alphaIIbbeta3 integrin.

Platelets exposed to thrombin or thrombin receptor agonist peptide (SFLLRN) activate phospholipase C and protein kinase C (PKC), and accumulate 3-phosphorylated phosphoinositides (3-PPI) as a function of the activation and relocalization of two cytoskeletally-associated phosphoinositide 3-kinases (PI 3-K): p85/PI 3-K and PI 3-Kgamma. We now report that exposure of platelets to PKC-activating beta-phorbol myristate acetate (betaPMA) does not stimulate PI 3-Kgamma, but rather stimulates p85/PI 3-K, which associates with the cytoskeleton. Wortmannin is an inhibitor of both PI 3-Ks, known to act with more potency on p85/PI 3-K. betaPMA-stimulated 3-PPI accumulation is more sensitive to wortmannin (IC50 = 1.3 nM) than is SFLLRN- or thrombin-stimulated 3-PPI accumulation (IC50 = 10 nM). The activity of p85/PI 3-K in immunoprecipitates or in cytoskeletal fractions is inhibited more potently by exposure of platelets to wortmannin than is the activity of PI 3-Kgamma. betaPMA or SFLLRN promotes the conversion of platelet integrin alphaIIb/beta3 into a fibrinogen-binding form required for platelet aggregation. Activation of alphaIIb/beta3 in response to betaPMA or SFLLRN is inhibited by wortmannin with an IC50 of 1 nM in each case. Wortmannin inhibits neither activation of alphaIIb/beta3 by ligand-induced binding site antibody (anti-LIBS6 Fab) nor anti-LIBS6 Fab-induced platelet aggregation in the presence of fibrinogen, indicating that this type of "outside-in" signaling by alphaIIb/beta3 is largely PI 3-K-independent. We conclude that p85/PI 3-K, in preference to PI 3-Kgamma, contributes to activation of alphaIIb/beta3 when the thrombin receptor or PKC is stimulated.

Amino Acid Sequence↗

PubMed1995

The conserved membrane-proximal region of an integrin cytoplasmic domain specifies ligand binding affinity.

Integrin affinities for ligands can change markedly via a process termed inside-out signaling. We expressed several truncations of the beta 3 cytoplasmic domain in conjunction with an "activating" alpha subunit chimera, alpha IIb alpha 6B. Deletion of the 4 C-terminal residues of the beta 2 tail blocked inside-out signaling as assessed by the binding of an activation-specific antibody, PAC1. Several additional truncations remained in the low affinity state, but complete truncation (beta 3 delta 717) caused PAC1 binding. Activation by this truncation mutant did not depend on the alpha subunit cytoplasmic domain and was resistant to inhibitors of cellular metabolism and the over-expression of an isolated beta 3 cytoplasmic domain. Since deletion of beta 3(Leu717-Asp723) results in a constitutively activated integrin, this membrane-proximal seven amino acids of the beta 3 cytoplasmic domain is required to maintain alpha IIb beta 3 in a default low affinity state. The amino acid sequence of this region is conserved among integrins. Moreover, the conserved membrane-proximal sequence in alpha subunit tails seems to serve a similar function. Consequently, the conserved membrane-proximal regions of both integrin cytoplasmic domains control the ligand binding affinity of the extracellular domain.

Amino Acid Sequence↗

PubMed1995

An enzyme-linked immunosorbent assay to identify inhibitors of activation of platelet integrin alpha IIb beta 3.

The affinity of integrin alpha IIb beta 3 for adhesive ligands is tightly regulated by the platelet such that fibrinogen binding is observed only after platelet activation. Ligand binding is necessary for platelet aggregation, which contributes to vascular occlusion in pathological states. Therefore, we have developed an ELISA assay to screen for compounds that inhibit alpha IIb beta 3 activation. Washed platelets were incubated in microtitre wells with potential inhibitory compounds and stimulated with an agonist to activate alpha IIb beta 3. After the addition of biotin-PAC1, a fibrinogen-mimetic monoclonal antibody, the activation state of alpha IIb beta 3 was measured by sedimenting the platelets and quantitating the residual biotin-PAC1 in the cell-free supernatant in a streptavidin-based ELISA. This assay detected (1) specific PAC1 binding to activated platelets in response to a variety of agonists, and (2) dose-dependent inhibition of PAC1 binding by function-blocking anti-alpha IIb beta 3 monoclonal antibodies, by the tetrapeptide, RGDS, and by an alpha IIb beta 3-selective RGD peptidomimetic. Furthermore, the assay detected inhibition of PAC1 binding by intracellular inhibitors of platelet activation, including bisindolylmaleimide, a selective protein kinase C antagonist, and wortmannin, an inhibitor of phosphatidylinositol 3-kinase. These studies demonstrate that this integrin activation ELISA can detect pharmacological blockade of platelet alpha IIb beta 3 by extracellular and intracellular inhibitors. Its use may facilitate the search for clinically useful anti-platelet drugs.

Blood Platelets↗

PubMed1995

Beta 3-endonexin, a novel polypeptide that interacts specifically with the cytoplasmic tail of the integrin beta 3 subunit.

The adhesive and signaling functions of integrins are regulated through their cytoplasmic domains. We identified a novel 111 residue polypeptide, designated beta 3-endonexin, that interacted with the cytoplasmic tail of the beta 3 integrin subunit in a yeast two-hybrid system. This interaction is structurally specific, since it was reduced by 64% by a point mutation in the beta 3 cytoplasmic tail (S752-->P) that disrupts integrin signaling. Moreover, this interaction is integrin subunit specific since it was not observed with the cytoplasmic tails of the alpha IIb, beta 1, or beta 2 subunits. beta 3-Endonexin fusion proteins bound selectively to detergent-solubilized beta 3 from platelets and human umbilical vein endothelial cells, and beta 3-endonexin mRNA and protein were detected in platelets and other tissues. A related mRNA encoded a larger polypeptide that failed to bind to beta integrin tails. The apparent specificity of beta 3-endonexin for the beta 3 integrin subunit suggests potential mechanisms for selective modulation of integrin functions.

Amino Acid Sequence↗

PubMed1995

Integrin signaling: roles for the cytoplasmic tails of alpha IIb beta 3 in the tyrosine phosphorylation of pp125FAK.

pp125FAK (focal adhesion kinase) a protein tyrosine kinase that may mediate cellular responses to adhesion, is activated and tyrosine-phosphorylated when platelets adhere to fibrinogen via the integrin, alpha IIb beta 3. To determine whether either of the cytoplasmic tails of alpha IIb beta 3 regulates FAK phosphorylation, CHO cells were stably transfected with alpha IIb beta 3 or various cytoplasmic tail truncation mutants. Cells expressing wild-type alpha IIb beta 3 or alpha IIb beta 3 that lacked the COOH-terminal 13 or 18 residues of the 20 residue alpha IIb tail adhered to and spread on fibrinogen or on an anti-alpha IIb antibody, and FAK became tyrosine-phosphorylated. FAK also became phosphorylated in adherent cells lacking the COOH-terminal 35 or 39 residues of the 47 residue beta 3 tail, although the extent of phosphorylation was reduced by about 50% in the latter mutant. Little or no FAK phosphorylation was observed if 46 residues were deleted from the beta 3 tail. None of these beta 3 truncation mutants spread on the anti-alpha IIb antibody. When cells with wild-type alpha IIb beta 3 or truncated beta 3 were detached from a surface, FAK became rapidly dephosphorylated. In contrast, FAK remained phosphorylated in the two alpha IIb truncation mutants for up to 90 minutes in suspension. This persistent phosphorylation was not due to occupancy of alpha IIb beta 3 by adhesive ligands because it was also observed with an alpha IIb tail truncation mutant that contained an additional mutation in the extracellular portion of the receptor that prevents ligand binding. These studies demonstrate that: (1) the beta 3 cytoplasmic tail, including the membrane-proximal portion, is involved in initiation of FAK phosphorylation; (2) FAK phosphorylation can be initiated by cell adhesion in the absence of cell spreading; and (3) the membrane-distal portion of the alpha IIb cytoplasmic tail may normally function to dampen FAK phosphorylation in non-anchored cells.

Amino Acid Sequence↗

PubMed1995

Function and regulation of the beta 3 integrins in hemostasis and vascular biology.

The beta 3 integrins, alpha IIb beta 3 and alpha V beta 3, are prominently expressed on the surfaces of platelets and endothelial cells, respectively. This vascular localization places them in a strategic position to perform adhesive functions necessary for hemostasis, wound healing and angiogenesis. In addition, these integrins are now known to serve a signaling function, whereby environmental cues on either side of the plasma membrane can be transmitted across the membrane to the other side. Indeed, preliminary studies indicate that these dual function receptors are wired to other signaling pathways within platelets and endothelial cells, thus helping to explain how integrin-mediated cell anchorage can affect cytoskeletal organization and cell motility as well as endothelial cell growth, differentiation and apoptosis.

Animals↗

PubMed1994

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

Agonist stimulation of platelets induces multiple waves of tyrosine phosphorylation, several of which are dependent on the integrin alpha IIb beta 3. At least two classes of protein tyrosine kinases are activated during various stages of platelet activation, 1) Src family tyrosine kinases are activated during an early phase of platelet activation by an integrin-independent mechanism and 2) pp125FAK is activated during a late stage of platelet activation, and it is dependent on platelet aggregation mediated by fibrinogen binding to alpha IIb beta 3. In this report, we examined the mechanism of agonist-induced phosphorylation and activation of the tyrosine kinase pp72syk, which is known to couple with immune response receptors in B cells and mast cells. pp72syk was found to be regulated by both agonist and integrin receptors in a pattern distinct from that of pp60src and pp125FAK. Specifically, thrombin induced the tyrosine phosphorylation and activation of pp72syk independent of platelet aggregation. However, full activation of pp72syk required integrin engagement since treatment with antibodies that block fibrinogen binding to alpha IIb beta 3 reduced pp72syk phosphorylation by 40%. Furthermore, fibrinogen binding to alpha IIb beta 3 stimulated directly with an anti-beta 3 antibody activated pp72syk 3-fold and stimulated its tyrosine phosphorylation. Thus, pp72syk is the only platelet tyrosine kinase identified to date that can be directly activated through integrin ligation. In addition, we found that the activation of pp72syk is dependent upon the state of actin polymerization and that pp72syk redistributes to actin-rich cytoskeletal complexes in an aggregation-dependent manner. These results suggest a role for pp72syk in both early, integrin-independent tyrosine phosphorylation events as well as those dependent upon subsequent integrin engagement.

Blood Platelets↗

PubMed1994

Determinants of specificity of a baculovirus-expressed antibody Fab fragment that binds selectively to the activated form of integrin alpha IIb beta 3.

PAC1 is an IgM kappa murine monoclonal antibody that, like the Arg-Gly-Asp-containing ligand fibrinogen, binds to integrin alpha IIb beta 3 only on activated platelets. The unique binding properties of PAC1 may be determined by its large size, its multivalency, and by variable region sequences, including an Arg-Tyr-Asp at residues 100A-C in H-CDR3. To study the molecular determinants of PAC1 function, baculoviruses containing cloned cDNA for the Fd heavy and kappa light chains of PAC1 were used to co-infect Sf9 insect cells. Infected cells secreted a soluble, monovalent, 50-kDa Fab fragment that bound saturably to agonist-stimulated platelets but not to resting cells. Fab binding was inhibited > 85% by 10 mM EDTA, 1 mM RGDS, 1 mM fibrinogen gamma 397-411, or 12 microM fibrinogen, but not by 1 mM RGES. Compared to PAC1 IgM, a 60-fold higher molar concentration of PAC1 Fab was required for half-maximal binding to platelets or for half-maximal inhibition of fibrinogen binding. PAC1 Fab bound to an activated form of alpha IIb beta 3 expressed in Chinese hamster ovary cells, but not to the resting form of the receptor in these cells or to alpha v beta 3 in human endothelial cells. Conversion of Asp100C to Glu by site-directed mutagenesis rendered the antibody inactive, indicating that the Arg-Tyr-Asp sequence in H-CDR3 is essential for PAC1 recognition of alpha IIb beta 3. Binding of fibrinogen or PAC1 IgM to platelets induced tyrosine phosphorylation of a 140-kDa platelet protein, but binding of PAC1 Fab did not. These studies demonstrate that the specificity of PAC1 for activated alpha IIb beta 3 is determined by an integrin recognition sequence within H-CDR3. However, the strength of this binding interaction and the ability of PAC1 to trigger signaling in platelets also depend on antibody valency.

Amino Acid Sequence↗

PubMed1994

Interaction of disintegrins with the alpha IIb beta 3 receptor on resting and activated human platelets.

Viper venom disintegrins contain the RGD/KGD motif. They inhibit platelet aggregation and cell adhesion, but show structural and functional heterogeneity. We investigated the interaction of four prototypic disintegrins with alpha IIb beta 3 expressed on the surface of resting and activated platelets. The binding affinity (Kd) of 125I-albolabrin, 125I-echistatin, 125I-bitistatin and 125I-eristostatin toward resting platelets was 294, 153, 48 and 18 nM respectively. The Kd value for albolabrin decreased 3-fold and 6-fold after ADP- or thrombin-induced activation. The Kd values for bitistatin and echistatin also decreased with ADP, but there was no further decrease with thrombin. In contrast, eristostatin bound with the same high affinity to resting and activated platelets. The pattern of fluorescein isothiocyanate (FITC)-eristostatin and FITC-albolabrin binding to resting and activated platelets was consistent with observations using radiolabelled material. Eristostatin showed faster and more irreversible binding to platelets, and greater potency compared with albolabrin in inducing conformational neo-epitopes in beta 3. The anti-alpha IIb beta 3 monoclonal antibody OP-G2 that is RGD-dependent inhibited disintegrin binding to activated platelets more strongly than binding to resting platelets and it inhibited the binding to platelets of albolabrin more strongly than eristostatin. The specificity of disintegrin interaction with alpha IIb beta 3 was confirmed by demonstrating cross-linking of these peptides to alpha IIb beta 3 on normal platelets, but not to thrombasthenic platelets deficient in alpha IIb beta 3.

Adenosine Diphosphate↗

PubMed1994

"Inside-out" signal transduction inhibited by isolated integrin cytoplasmic domains.

The affinities of integrin alpha beta heterodimers for extracellular ligands are important regulators of cell adhesion. Intracellular signals provoke changes in the integrin extracellular domain resulting in "activation," as manifested by an increase in affinity. Interactions of integrin cytoplasmic domains with intracellular elements may mediate this "inside-out signaling." Here we report that overexpression of chimeras of the cytoplasmic domain of integrin beta 3 or beta 1 subunits, joined to the extracellular and transmembrane domains of the Tac subunit of the interleukin-2 receptor, reduced integrin affinity. In contrast, chimeras containing the cytoplasmic domain of alpha 5 or alpha IIb or of beta 3 bearing a mutation that disrupts inside-out signaling lacked inhibitory activity. These data suggest that limiting quantities of intracellular factors bind to integrin beta 3 and beta 1 cytoplasmic domains to modulate ligand binding affinity. Structural mimics of these domains may provide a novel means to alter cell adhesion.

Amino Acid Sequence↗

PubMed1994

Tyrosine phosphorylation of pp125FAK in platelets requires coordinated signaling through integrin and agonist receptors.

FAK is a focal adhesion kinase that is phosphorylated on tyrosine in activated platelets. Induction of FAK phosphorylation requires both fibrinogen binding to integrin alpha IIb beta 3 and post-occupancy events during agonist-induced platelet aggregation or platelet spreading on a fibrinogen matrix. To identify the signaling pathways necessary for tyrosine phosphorylation of FAK, we have examined the conditions that stimulate or inhibit this phosphorylation in platelets in which fibrinogen binding to alpha IIb beta 3 and platelet aggregation were induced directly with an anti-beta 3 Fab fragment (anti-LIBS6). Apyrase was added to prevent effects of the endogenous platelet agonist, ADP. Under these conditions, neither fibrinogen binding nor primary platelet aggregation was sufficient to induce FAK phosphorylation, suggesting that a second "costimulatory" event was required. Indeed, when epinephrine was added with fibrinogen and anti-LIBS6, large platelet aggregates formed and FAK phosphorylation occurred. This response was prevented by blockade of cyclooxygenase with indomethacin or thromboxane A2 receptors with SQ 30,741. A stable thromboxane A2 analogue (U46619) could substitute for epinephrine as the costimulus. Epinephrine costimulation of FAK phosphorylation was also prevented by chelation of intracellular Ca2+ with BAPTA or selective inhibition of protein kinase C (PKC) with bisindolylmaleimide, indicating that Ca2+ and PKC are necessary for FAK phosphorylation under these conditions. Epinephrine also promoted FAK phosphorylation and adhesive spreading of apyrase-treated platelets on a fibrinogen matrix. Cytochalasin D, an inhibitor of actin polymerization, blocked FAK phosphorylation under all these conditions. Thus, tyrosine phosphorylation of FAK in platelets requires coordinated signaling through occupied integrin and agonist receptors. These separate pathways may converge to increase free Ca2+ and activate PKC and thus promote the cytoskeletal reorganization required for activation of FAK.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed1994

Adhesive signaling in platelets.

The anucleate platelet must perform its hemostatic functions in the absence of transcriptional regulation. Central among these functions is cell adhesion, which is mediated by multiple specialized plasma membrane receptors. The adhesive function of one of the key receptors, integrin alpha IIb beta 3, is regulated by intracellular signals triggered by platelet agonists and antagonists. Recent evidence indicates that adhesion receptors can transduce extracellular signals into the platelet to activate intracellular signaling pathways that affect hemostasis.

Amino Acid Sequence↗

PubMed1994

Regulation of protein tyrosine kinases in platelets.

Platelet activation is accompanied by a dramatic increase in tyrosine phosphorylation of many cellular proteins. Phosphorylation of these proteins occurs in successive waves during the activation process, suggesting that several distinct mechanisms, occurring in a temporal order, regulate protein tyrosine kinases and/or phosphatases in activated platelets. Several tyrosine kinases, including Src family kinases, Syk and FAK, have been implicated in these phosphorylation events. These kinases are regulated by distinct receptor-mediated events involving activation of their catalytic activity and alterations in their cellular localization.

Blood Platelets↗

PubMed1994

Integrin cytoplasmic domains mediate inside-out signal transduction.

We analyzed the binding of fibronectin to integrin alpha 5 beta 1 in various cells; in some cells fibronectin bound with low affinity (e.g., K562 cells) whereas in others (e.g., CHO), it bound with high affinity (Kd approximately 100 nM) in an energy-dependent manner. We constructed chimeras of the extracellular and transmembrane domains of alpha IIb beta 3 joined to the cytoplasmic domains of alpha 5 beta 1. The affinity state of these chimeras was assessed by binding of fibrinogen or the monoclonal antibody, PAC1. The cytoplasmic domains of alpha 5 beta 1 conferred an energy-dependent high affinity state on alpha IIb beta 3 in CHO but not K562 cells. Three additional alpha cytoplasmic domains (alpha 2, alpha 6A, alpha 6B) conferred PAC1 binding in CHO cells, while three others (alpha M, alpha L, alpha v) did not. In the high affinity alpha chimeras, cotransfection with a truncated (beta 3 delta 724) or mutated (beta 3(S752-->P)) beta 3 subunit abolished high affinity binding. Thus, both cytoplasmic domains are required for energy-dependent, cell type-specific affinity modulation. In addition, mutations that disrupted a highly conserved alpha subunit GFFKR motif, resulted in high affinity binding of ligands to alpha IIb beta 3. In contrast to the chimeras, the high affinity state of these mutants was independent of cellular metabolism, cell type, and the bulk of the beta subunit cytoplasmic domain. Thus, integrin cytoplasmic domains mediate inside-out signaling. Furthermore, the highly conserved GFFKR motif of the alpha subunit cytoplasmic domain maintains the default low affinity state.

Amino Acid Sequence↗

PubMed1994

Glanzmann thrombasthenia secondary to a Gly273-->Asp mutation adjacent to the first calcium-binding domain of platelet glycoprotein IIb.

We studied the defect responsible for Glanzmann thrombasthenia in a patient whose platelets expressed < 5% of the normal amount of GPIIb-IIIa. Genetic and biochemical evidence indicated that the patient's GPIIIa genes were normal. However, DNA analysis revealed the patient homozygous for a G818-->A substitution in her GPIIb genes, resulting in a Gly273-->Asp substitution adjacent to the first GPIIb calcium-binding domain. To determine how this mutation impaired GPIIb-IIIa expression, recombinant GPIIb containing the mutation was coexpressed with GPIIIa in COS-1 cells. The GPIIb mutant formed stable GPIIb-IIIa heterodimers that were not immunoprecipitated by either of two heterodimer-specific monoclonal antibodies, indicating that the mutation disrupted the epitopes for these antibodies. Moreover, the GPIIb in the heterodimers was not cleaved into heavy and light chains, indicating that the heterodimers were not transported from the endoplasmic reticulum to the Golgi complex where GPIIb cleavage occurs, nor were the mutant heterodimers expressed on the cell surface. These studies demonstrate that a Gly273-->Asp mutation in GPIIb does not prevent the assembly of GPIIb-IIIa heterodimers, but alters the conformation of these heterodimers sufficiently to impair their intracellular transport. The impaired GPIIb-IIIa transport is responsible for the thrombasthenia in this patient.

Adenosine Diphosphate↗

PubMed1994

Bioactive Arg-Gly-Asp conformations in anti-integrin GPIIb-IIIa antibodies.

Antibodies can mimic the biological function of physiological ligands, yet few examples indicate the structural similarity between antibodies and the ligands that they mimic. Originally, the competition of antibodies for ligand binding sites was conjectured to be through similar three-dimensional conformations, which represent the "internal image" of the given ligand. Here we show that residues in a complementary determining region (CDR) can adopt the same bioactive structures observed in ligands. Structure-function studies of three anti-GPIIb-IIIa murine monoclonal antibodies, PAC-1, LJ-CP3, and OP-G2, indicate that the RYD sequence in their H-CDR3 domain occupies the same conformational space as RGD in conformationally constrained, bioactive, GPIIb-IIIa cell-surface adhesion ligands. The relative location of the guanidinium and carboxylate groups in the RXD regions is identified as an important recognition feature, and the conformational space occupied by this region in the antibodies is only slightly larger than that in the most bioactive peptides. Additionally, we show that antibodies can unveil other potential bioactive sequences, which may impart specificity. Thus antibodies are an exquisite probe for identifying motifs of short adhesion stretches, thereby revealing amino acid sequences and restricted geometries that might be used as lead compounds in drug design.

Amino Acid Sequence↗

PubMed1993

Conformational changes in fibrinogen elicited by its interaction with platelet membrane glycoprotein GPIIb-IIIa.

The binding of fibrinogen to membrane glycoprotein GPIIb-IIIa on activated platelets leads to platelet aggregation. This interaction results in conformational changes in fibrinogen as evidenced by the expression of receptor-induced binding sites, RIBS, epitopes which are expressed by the bound but not the free ligand. In the present study, two RIBS epitopes have been localized. One sequence resides at gamma 112-119 and is recognized by mAb 9F9; the second is the RGDF sequence at A alpha 95-98 and is recognized by mAb 155B16. These epitopes are also exposed by adsorption of fibrinogen onto a plastic surface and digestion of the molecule by plasmin. Proteolytic exposure of the epitopes coincides with cleavage of the carboxyl-terminal aspects of the A alpha-chains to form fragment X2. The inaccessibility of the RGDF sequence at A alpha 95-98 in fibrinogen suggests that this sequence does not participate in the initial binding of the molecule to GPIIb-IIIa. The location of these RIBS epitopes suggests a model in which binding of fibrinogen to its receptor alters the conformation of the carboxyl-terminal aspects of the A alpha-chains, exposing the sequences which reside in the coiled-coil connector segments between the D and E domains of the molecule. These sequences may then serve as epitopes and may mediate unique functions of the receptor-bound molecule.

Amino Acid Sequence↗