A Monoclonal Antibody against a peptide sequence of Fibrinogen Gamma Chain Acts as an Inhibitor of factor XIII-Mediated Crosslinking of Human Fibrin

Taubenfeld, Stephen M.; Song, Yeqing; Sheng, Dequan; Ball, Evelyn L.; R, Matsueda

doi:10.1055/s-0038-1649848

Cited by 18 publications

(19 citation statements)

References 10 publications

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“…MAb P1D6 (34) against ␣ 5 was obtained from Life Technologies Inc. Two mAbs raised against RGD-containing synthetic peptides from Fg A␣-chain, anti-N directed to A␣ 87-100 (TTNIMEILRGDFSS), and anti-C di-rected to A␣ 566 -580 (SSTAYNRGDSTFESK) (3) were provided by Dr. Zaverio Ruggeri, The Scripps Research Institute, La Jolla, CA. MAb 4A5 (35), to the C terminus of the ␥-chain of Fg, was a gift from Dr. Gary Matsueda, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ. Anti-␤ 1 mAb 8A2 (36) was provided by Dr. Nicholas Kovach, Washington University, Seattle, WA.…”

Section: Methodsmentioning

confidence: 99%

Fibrinogen Is a Ligand for Integrin α5β1 on Endothelial Cells

Suehiro

Gailit

Plow

1997

Journal of Biological Chemistry

162

129

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Previous studies have shown that fibrinogen can associate with endothelial cells via an Arg-Gly-Asp (RGD) recognition specificity. In the present study, we have characterized the specificity of fibrinogen binding to endothelial cells under different cation conditions. Fibrinogen binding to suspended endothelial cells was selectively supported by Mn 2؉ and was suppressed by Ca 2؉ . The Mn 2؉ -supported interaction was completely inhibited by RGD peptides but not by ␣ v ␤ 3 blocking monoclonal antibodies. In contrast, the interaction was completely blocked by two ␣ 5 ␤ 1 monoclonal antibodies. This interaction was not mediated by fibronectin bound to the integrin; could be demonstrated with purified ␣ 5 ␤ 1 ; and also was observed with a second ␣ 5 ␤ 1 -bearing cell type, platelets. The binding of fibrinogen to ␣ 5 ␤ 1 on endothelial cells in the presence of Mn 2؉ was time-dependent, specific, saturable, and of high affinity (K d ‫؍‬ 65 nM). By employing anti-peptide monoclonal antibodies, the carboxyl-terminal RGD sequence at A␣ 572-574 was implicated in fibrinogen recognition by ␣ 5 ␤ 1 . Two circumstances were identified in which ␣ 5 ␤ 1 interacted with fibrinogen in the presence of Ca 2؉ : when the receptor was activated with monoclonal antibody (8A2) or when the fibrinogen was presented as an immobilized substratum. These results identify fibrinogen as a ligand for ␣ 5 ␤ 1 on endothelial and other cells, an interaction which may have broad biological implications.The luminal surface of endothelial cells (EC) 1 is continuously exposed to a high concentration of plasma fibrinogen (Fg). Disruption of the endothelium results in local thrombus formation, and Fg/fibrin accumulates at such sites of vascular injury. Based upon these proximal relationships, the molecular mechanisms and functional consequences of the interaction of Fg with EC have been topics of considerable interest and investigation (1-6). Indeed, it has been shown that Fg can induce EC attachment, spreading, and migration (1, 7) and can support an angiogenic response (8). Several distinct receptors have been implicated in mediating Fg binding to EC. These include ␣ v ␤ 3 (9, 10), a member of the integrin family of cell adhesion receptors, as well as several non-integrin binding sites (6, 11). Transglutaminase-mediated Fg cross-linking to EC also has been demonstrated (12).␣ v ␤ 3 interacts with Fg via an Arg-Gly-Asp (RGD) recognition specificity; i.e. RGD-containing peptides block Fg binding to this receptor (13). This tripeptide sequence is recognized not only by ␣ v ␤ 3 but also by several other integrins (14 -16), including ␣ 5 ␤ 1 , which serves as a fibronectin (Fn) receptor on EC and many other cell types (17)(18)(19)(20). Fg contains two RGD sequences within its A␣-chain: RGDF at A␣ 95-98 and RGDS at A␣ 572-575. Previous studies have shown that EC adhesion to immobilized Fg is blocked by a monoclonal antibody (mAb) to the carboxyl-terminal peptide containing RGD sequence at A␣ 572-574 (3). Moreover, this adhesion was blocked by mAbs against ␣...

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Section: Methodsmentioning

confidence: 99%

Fibrinogen Is a Ligand for Integrin α5β1 on Endothelial Cells

Suehiro

Gailit

Plow

1997

Journal of Biological Chemistry

162

129

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“…Antibodies. The monoclonal antibody (mAb) 4A5 (courtesy of Dr Gary R. Matsueda) used in enzyme‐linked immunosorbent assays (ELISA) binds to the carboxyl terminus of the fibrinogen γ‐chain (γ402–411) (Taubenfeld et al , 1995) and prevents platelet adhesion (Gartner et al , 1993). The mAb directed against β 3 of α IIb β 3 (C17), was a gift from Dr A. E. G. Kr.…”

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confidence: 99%

Reduced platelet adhesion in flowing blood to fibrinogen by alterations in segment γ316–322, part of the fibrin‐specific region*

et al. 2002

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Summary. The interaction of platelets with fibrinogen is a key event in the maintenance of a haemostatic response. It has been shown that the 12-carboxy-terminal residues of the c-chain of fibrinogen mediate platelet adhesion to immobilized fibrinogen. These studies, however, did not exclude the possibility that other domains of fibrinogen are involved in interactions with platelets. To obtain more insight into the involvement of other domains of fibrinogen in platelet adhesion, we studied platelet adhesion in flowing blood to patient dysfibrinogen Vlissingen/Frankfurt IV (V/FIV), to several variant recombinant fibrinogens with abnormalities in the c-chain segments c318-320 and c408-411. Perfusion studies at physiological shear rates showed that platelet adhesion was absent to cD408-411, slightly reduced to the heterozygous patient dysfibrinogen V/FIV and strongly reduced to the homozygous recombinant fibrinogens: cD319-320, c318Asp fi Ala and c320Asp fi Ala. Furthermore, antibodies raised against the sequences c308-322 and c316-333 inhibited platelet adhesion under shear conditions. These experiments indicated that the overlapping segment c316-322 contains amino acids that could be involved in platelet adhesion to immobilized fibrinogen under flow conditions. In soluble fibrinogen, this sequence is buried inside the fibrinogen molecule and becomes exposed after polymerization. In addition, we have shown that this fibrin-specific sequence also becomes exposed when fibrinogen is immobilized on a surface.

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“…Its primary structure and its three-dimensional structure including its active site are all known (14 -17). But still unknown are the determinants of substrate docking and specificity (18). Many of the domains are highly conserved and do not yield antibodies to probe their function.…”

mentioning

confidence: 99%

Coagulation Factor XIIIa Undergoes a Conformational Change Evoked by Glutamine Substrate

Mitkevich

Shainoff

DiBello

et al. 1998

Journal of Biological Chemistry

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Coagulation factor XIIIa, plasma transglutaminase (endo-␥-glutamine:⑀-lysine transferase EC 2.3.2.13) catalyzes isopeptide bond formation between glutamine and lysine residues and rapidly cross-links fibrin clots. A monoclonal antibody (5A2) directed to a fibrinogen A␣-chain segment 529 -539 was previously observed from analysis of end-stage plasma clots to block fibrin ␣-chain cross-linking. This prompted the study of its effect on nonfibrinogen substrates, with the prospect that 5A2 was inhibiting XIIIa directly. It inhibited XIIIa-catalyzed incorporation of the amine donor substrate dansylcadaverine into the glutamine acceptor dimethylcasein in an uncompetitive manner with respect to dimethylcasein utilization and competitively with respect to dansylcadaverine. Uncompetitive inhibition was also observed with the synthetic glutamine substrate, LGPGQSKVIG. Theoretically, uncompetitive inhibition arises from preferential interaction of the inhibitor with the enzyme-substrate complex but is also found to inhibit ␥-chain cross-linking. The conjunction of the uncompetitive and competitive modes of inhibition indicates in theory that this bireactant system involves an ordered reaction in which docking of the glutamine substrate precedes the amine exchange. The presence of substrate enhanced binding of 5A2 to XIIIa, an interaction deemed to occur through a C-terminal segment of the XIIIa A-chain (643-658, GSDMTVTVQFT-NPLKE), 55% of which comprises sequences occurring in the fibrinogen epitope A␣-(529 -540) (GSESGIFTNTKE). Removal of the C-terminal domain from XIIIa abolishes the inhibitory effect of 5A2 on activity. Crystallographic studies on recombinant XIIIa place the segment 643-658 in the region of the groove through which glutamine substrates access the active site and have predicted that for catalysis, a conformational change may accompany glutamine-substrate binding. The uncompetitive inhibition and the substrate-dependent binding of 5A2 provide evidence for the conformational change.

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A Monoclonal Antibody against a peptide sequence of Fibrinogen Gamma Chain Acts as an Inhibitor of factor XIII-Mediated Crosslinking of Human Fibrin

Cited by 18 publications

References 10 publications

Fibrinogen Is a Ligand for Integrin α5β1 on Endothelial Cells

Fibrinogen Is a Ligand for Integrin α5β1 on Endothelial Cells

Reduced platelet adhesion in flowing blood to fibrinogen by alterations in segment γ316–322, part of the fibrin‐specific region*

Coagulation Factor XIIIa Undergoes a Conformational Change Evoked by Glutamine Substrate

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