Role of the γ chain Ala–Gly–Asp–Val and Aα chain Arg–Gly–Asp–Ser sites of fibrinogen in coaggregation of platelets and fibrinogen-coated beads

Liu, Qingde; Rooney, Michael M.; Kasirer-Friede, Ana; Brown, Elizabeth A.; Lord, Susan T.; Frojmovic, Mony M.

doi:10.1016/s0167-4838(98)00039-9

Cited by 25 publications

(22 citation statements)

References 35 publications

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“…Previous studies have demonstrated the essential role of the C-terminal AGDV sequences in ␥C in platelet adhesion; deletion of this sequence in recombinant Fg or replacement with the 20-residue sequence occurring in the ␥Ј-chain of a human Fg variant impaired platelet adhesion under static (5) and flow (28) adhesion completely (5,29), and the binding of purified ␣ IIb ␤ 3 to Fg-agarose was not inhibited by ␥400 -411 (H12) or RGDS peptides (30), suggesting that other sites in Fg interact with platelets. The ability of the D 98 fragment and recombinant ␥C(⌬391-411) mutant, which lack AGDV, to support adhesion; the inhibition of platelet adhesion by P3; and the inhibition of platelet adhesion to P3 by function blocking anti-␣ IIb ␤ 3 are all consistent with a model in which P3 is a second site within ␥C that interacts with ␣ IIb ␤ 3 on platelets.…”

Section: Discussionmentioning

confidence: 99%

Identification of a Novel Binding Site for Platelet Integrins αIIbβ3 (GPIIbIIIa) and α5β1 in the γC-domain of Fibrinogen

Podolnikova

Yakubenko

Volkov

et al. 2003

Journal of Biological Chemistry

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The interactions of platelets with fibrinogen mediate a variety of responses including adhesion, platelet aggregation, and fibrin clot retraction. Whereas it was assumed that interactions of the platelet integrin ␣ IIb ␤ 3 with the AGDV sequence in the ␥C-domain of fibrinogen and/or RGD sites in the A␣ chains are involved in clot retraction and adhesion, recent data demonstrated that fibrinogen lacking these sites still supported clot retraction. These findings suggested that an unknown site in fibrinogen and/or other integrins participate in clot retraction. Here we have identified a sequence within ␥C that mediates binding of fibrinogen to platelets. Synthetic peptide duplicating the 365-383 sequence in ␥C, designated P3, efficiently inhibited clot retraction in a dose-dependent manner. Furthermore, P3 supported platelet adhesion and was an effective inhibitor of platelet adhesion to fibrinogen fragments. Analysis of overlapping peptides spanning P3 and mutant recombinant ␥C-domains demonstrated that the P3 activity is contained primarily within ␥370 -383. Integrins ␣ IIb ␤ 3 and ␣ 5 ␤ 1 were implicated in recognition of P3, since platelet adhesion to the peptide was blocked by function-blocking monoclonal antibodies against these receptors. Direct evidence that ␣ IIb ␤ 3 and ␣ 5 ␤ 1 bind P3 was obtained by selective capture of these integrins from platelet lysates using a P3 affinity matrix. Thus, these data suggest that the P3 sequence in the ␥C-domain of fibrinogen defines a previously unknown recognition specificity of ␣ IIb ␤ 3 and ␣ 5 ␤ 1 and may function as a binding site for these integrins.The process of thrombus formation upon vascular injury is a complex series of events that involves platelets and plasma proteins, including fibrinogen (Fg).1 Adhesive reactions of platelets with Fg are required for platelet aggregation, which triggers subsequent formation of a blood clot composed of insoluble fibrin and captured platelets. The interactions of platelets with fibrin within platelet-rich thrombi result in clot retraction, which is visually manifested in a dramatic reduction in fibrin gel volume. The mechanism and physiological significance of platelet-mediated fibrin clot retraction remain poorly understood, but it has been suggested that contraction of fibrin clots may be required for clearance of the thrombus and also may facilitate wound healing.The primary interactions of platelets with Fg and fibrin are mediated by the platelet-specific receptor ␣ IIb ␤ 3 (glycoprotein IIbIIIa), a member of the integrin family of receptors. ␣ IIb ␤ 3 is the most abundant integrin on the platelet surface and is expressed at ϳ80,000 copies/cell (1). Numerous studies using synthetic peptides and function-blocking antibodies have demonstrated that three sites in Fg can potentially interact with ␣ IIb ␤ 3 upon platelet adhesion and aggregation (1). Because Fg consists of two identical disulfide-bonded subunits, each of which is formed by three polypeptide chains (A␣, B␤, and ␥), two copies of ␣ IIb ␤ 3 -binding sites m...

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Section: Discussionmentioning

confidence: 99%

Identification of a Novel Binding Site for Platelet Integrins αIIbβ3 (GPIIbIIIa) and α5β1 in the γC-domain of Fibrinogen

Podolnikova

Yakubenko

Volkov

et al. 2003

Journal of Biological Chemistry

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“…Both ␤ 3 integrins bind multiple adhesive ligands, and many of the ligands bind to both receptors, including fibrinogen (8 -10). However, there is a fundamental difference in the specificity involved in fibrinogen recognition by the two integrins; ␣ llb ␤ 3 binds fibrinogen through the sequence at the carboxyl terminus of its ␥ chain 406 KQAGDV 411 (11,12), and ␣ v ␤ 3 binds to one of the two 572 RGD 574 sequences in its A␣ chain (13). In addition, sites within the ␥ chain, distinct from the KQAGDV, have been implicated in recognition of fibrinogen by ␣ v ␤ 3 (14).…”

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confidence: 99%

The Specificity and Function of the Metal-binding Sites in the Integrin β3 A-domain

Pesho

Błędzka

Michalec

et al. 2006

Journal of Biological Chemistry

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The A-domains within integrin ␤ subunits contain three metal sites termed the metal ion-dependent adhesion site (MIDAS), site adjacent to the metal ion-dependent adhesion site (ADMIDAS), and ligand-induced metal-binding site (LIMBS), and these sites are involved in ligand engagement. The ␤ 3 integrin subfamily consists of two members, ␣ v ␤ 3 and ␣ llb ␤ 3 . The two receptors share the same ␤ 3 subunit, and the amino acid sequences of their ␣ subunits are 36% identical (1). Both are typical integrin ␣/␤ heterodimers, in which each subunit is composed of a large extracellular region, a transmembrane region, and a short cytoplasmic tail. ␣ llb ␤ 3 expression is restricted primarily to platelets and their megakaryocyte precursors and plays an indispensable role in thrombus formation (2-4). ␣ v ␤ 3 is expressed by a variety of vascular cells, including endothelial cells, where it influences the adhesive and migratory properties of these cells (5-7). Both ␤ 3 integrins bind multiple adhesive ligands, and many of the ligands bind to both receptors, including fibrinogen (8 -10). However, there is a fundamental difference in the specificity involved in fibrinogen recognition by the two integrins; ␣ llb ␤ 3 binds fibrinogen through the sequence at the carboxyl terminus of its ␥ chain 406 KQAGDV 411 (11,12), and ␣ v ␤ 3 binds to one of the two 572 RGD 574 sequences in its A␣ chain (13). In addition, sites within the ␥ chain, distinct from the KQAGDV, have been implicated in recognition of fibrinogen by ␣ v ␤ 3 (14). According to recently published crystal structures of the extracellular regions of ␣ v ␤ 3 (15) and ␣ llb ␤ 3 (16) with bound ligand mimetics, the main area of ligand binding lies between the ␤-propeller of the ␣ subunit and the A-domain in the ␤ 3 subunit. This direct demonstration of ␤ 3 A-domain involvement in ligand engagement was preceded by numerous studies using mutational, immunological, and biochemical approaches (17-21), which indicated that this region played a critical role in ligand binding to the two ␤ 3 integrins.The binding of most ligands to integrins requires metal ions. This is also true for the ␤ 3 integrins and the cellular responses arising from ligand engagement (e.g. platelet aggregation (22-24)). The ␤-propeller domain of the ␣ subunit contains four divalent ion-binding sites within the loops of blades 4 -7 near the bottom of the propeller, but these are not directly involved in ligand engagement (25). The ␤ 3 A-domain contains three metal sites for binding ligands, which are more directly involved in ligand binding. These are referred to as the metal ion-dependent adhesion site (MIDAS), 4 a site adjacent to the MIDAS (ADMIDAS), and a ligand-induced metal-binding site (LIMBS). The ␣ v ␤ 3 and ␣ llb ␤ 3 crystal structures establish that cation bound in the MIDAS is involved directly in ligand engagement; the ADMIDAS cation is not directly involved in contacting ligand but may help to regulate ligand binding; and the LIMBS cation may aid in stabilizing the receptor-ligand complex (26). ...

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“…Such Abs may aid the further study of the ligand recognition specificity of the integrin. There are still many unknowns relating to the mechanisms leading to the activation of ␣ IIb ␤ 3 and to the structure of the ligand-binding pocket (65)(66)(67)(68). At least two ligand binding sites exist on ␣ IIb ␤ 3 , and the activation requirement for ligand binding appears to depend on the type of ligand being examined.…”

Section: Discussionmentioning

confidence: 99%

Human IgG Monoclonal Anti-αIIbβ3-Binding Fragments Derived from Immunized Donors Using Phage Display

Jacobin

Laroche‐Traineau

Little

et al. 2002

The Journal of Immunology

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Previous studies of the immune response in polytransfused Glanzmann thrombasthenia (GT) patients and in autoimmune thrombocytopenic purpura (AITP) have relied on serum analysis and have shown the frequent development of Abs directed against the αIIbβ3 integrin. However, little is known about the molecular diversity of the humoral immune response to αIIbβ3 due to the paucity of mAbs issuing from these pathologies. We have isolated human IgG anti-αIIbβ3 binding fragments using combinatorial libraries of single-chain IgG created from the B cells of a GT and an AITP patient, both with serum Abs. Ab screening was performed using activated platelets or activated αIIbβ3-expressing Chinese hamster ovary cells. Sequencing of selected phage Abs showed that a broad selection of genes from virtually all V gene families had been used, indicating the diversity of the immune response. About one-half of the VH and VL segments of our IgG anti-αIIbβ3 fragments displayed extensive hypermutations in the complementarity-determining region, supporting the idea that an Ag-driven immune response was occurring in both patients. The H chain complementarity-determining region 3 analysis of phage Abs revealed motifs other than the well-known RGD and KQAGDV integrin-binding sequences. To our knowledge, our study is the first to illustrate multiple human IgG anti-αIIbβ3 reactivities and structural variations linked to the anti-platelet human immune response. Human αIIbβ3 Abs preferentially directed against the activated form of the integrin were further characterized because platelet αIIbβ3 inhibitors are potential therapeutic reagents for treating acute coronary syndromes. Currently available αIIbβ3 antagonists do not specifically recognize the activated form of the integrin.

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Role of the γ chain Ala–Gly–Asp–Val and Aα chain Arg–Gly–Asp–Ser sites of fibrinogen in coaggregation of platelets and fibrinogen-coated beads

Cited by 25 publications

References 35 publications

Identification of a Novel Binding Site for Platelet Integrins αIIbβ3 (GPIIbIIIa) and α5β1 in the γC-domain of Fibrinogen

Identification of a Novel Binding Site for Platelet Integrins αIIbβ3 (GPIIbIIIa) and α5β1 in the γC-domain of Fibrinogen

The Specificity and Function of the Metal-binding Sites in the Integrin β3 A-domain

Human IgG Monoclonal Anti-αIIbβ3-Binding Fragments Derived from Immunized Donors Using Phage Display

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