The Residues AGDV of Recombinant γ Chains of Human Fibrinogen Must Be Carboxy-Terminal to Support Human Platelet Aggregation

Hettasch, Joann M.; Bolyard, Mark G.; Lord, Susan T.

doi:10.1055/s-0038-1646347

Cited by 41 publications

(35 citation statements)

References 29 publications

(46 reference statements)

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“…Studies with purified recombinant fibrinogen from transfected BHK cells showed that recombinant fibrinogen (a2p2y2) supported platelet aggregation, whereas the variant (cr2Dy'2) was defective in platelet aggregation [26]. In addition, studies using the recombinant fibrinogen y chain variants showed that the four C-terminal residues were required to support platelet aggregation [27]. In this study, the binding of recombinant ClfA and adherence of S. aurues cells occurs to a site present at the C-terminus of wild-type fibrinogen but not in fibrinogen variants defective in their interactions with platelets.…”

Section: Discussionmentioning

confidence: 99%

“…mitting multivalent binding interactions [39]. However, recent studies have demonstrated that the intact C-terminus of the fibrinogen y chain is critical for fibrinogen-dependent platelet aggregation [26,27,401. Since the C-terminus of the fibrinogen y chain contains the Clf33 binding site, this bacterial protein was assessed for its ability to interfere with fibrinogen-dependent platelet aggregation induced by ADP, and platelet adhesion to immobilized fibrinogen under shear conditions.…”

Section: Clf33 Binds To the C-terminus Of The Fibrinogen Y Chainmentioning

confidence: 99%

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Characterization of the Interaction Between the Staphylococcus Aureus Clumping Factor (ClfA) and Fibrinogen

McDevitt¹,

Nanavaty²,

House-Pompeo³

et al. 1997

European Journal of Biochemistry

200

168

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The ability of Staphylococcus aureus to adhere to adsorbed fibrinogen and fibrin is believed to be an important step in the initiation of bioinaterial and wound-associated infections. In this study, we show that the binding site in fibrinogen for the recently identified S. aureus fibrinogen-binding protein clumping factor (ClfA) is within the C-terminus of the fibrinogen y chain. S. aureus Newman cells expressing ClfA adhered to microtitre wells coated with recombinant fibrinogen purified from BHK cells, but did not adhere to wells coated with a purified recombinant fibrinogen variant where the 4 C-terminal residues of the y chain were replaced by 20 unrelated residues. In addition, a synthetic peptide corresponding to the 17 C-terminal amino acids of the fibrinogen y chain effectively inhibited adherence of ClfA-expressing cells to fibrinogen. In western ligand blots, a recombinant truncated ClfA protein called Clf33 (residues 221 -550) recognized intact recombinant fibrinogen y chains, but failed to recognize recombinant fibrinogen y chains where the 4 C-terminal amino acids were altered by deletion or substitution. Previous studies have shown that the C-terminal domain of fibrinogen y chains contains a binding site for the integrin . We now show that Clf33 inhibits ADPinduced, fibrinogen-dependent platelet aggregation in a concentration-dependent manner and inhibits adhesion of platelets to immobilized fibrinogen under fluid shear stress, indicating that the binding sites for the platelet integrin and the staphylococcal adhesin overlap. The interaction between Clf33 and fibrinogen was further characterized using the BIAcore biosensor. When soluble Clf33 was allowed to bind to immobilized fibrinogen, a Kd of 0.51 t-0.19 pM was experimentally determined using equilibrium binding data. It was also shown that the synthetic C-terminal y-chain peptide effectively inhibited this interaction.

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Section: Discussionmentioning

confidence: 99%

Section: Clf33 Binds To the C-terminus Of The Fibrinogen Y Chainmentioning

confidence: 99%

Characterization of the Interaction Between the Staphylococcus Aureus Clumping Factor (ClfA) and Fibrinogen

McDevitt¹,

Nanavaty²,

House-Pompeo³

et al. 1997

European Journal of Biochemistry

200

168

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“…[39][40][41][42] Subsequent studies showed that the ␥Ј chain does not promote platelet aggregation because it lacks the binding site required for platelet adhesion, ␥A residues 408 to 411. [43][44][45][46] Recently, Lancellotti et al reported that the ␥Ј chain reduces thrombin-induced platelet aggregation through a combined mechanism. 47 They showed that the ␥Ј chain, either in fragment D or the synthetic ␥Ј peptide, hinders both thrombin binding to GpIb␣ and thrombin cleavage of PAR1 on platelets.…”

Section: Influence Of the ␥ Chain On Platelet Aggregationmentioning

confidence: 99%

The pleiotropic role of the fibrinogen γ′ chain in hemostasis

Willige

Standeven

Philippou

et al. 2009

Blood

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“…The reaction was quenched by the addition of 100 l of 1 M Tris-HCl (pH 8.0), vortexed, and left at room temperature for 30 min. The fluoresceinated peptide was fractionated by reverse-phase chromatography on a DeltaPak C 18 Radial-Pak cartridge HPLC column connected to a Waters 486 multi-wavelength detector. The labeling procedure can yield three forms of fluoresceinated peptide with the probe attached to 1) the N-terminal amino group, 2) the amino group of the internal lysine residue, and 3) the amino groups in both positions.…”

Section: Analysis Of Fibrinogen-binding Activity Of Recombinant Proteinsmentioning

confidence: 99%

The Fibrinogen-binding MSCRAMM (Clumping Factor) of Staphylococcus aureus Has a Ca2+-dependent Inhibitory Site

O’Connell¹,

Nanavaty²,

McDevitt³

et al. 1998

Journal of Biological Chemistry

116

141

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The clumping factor (ClfA) is a cell surface-associated protein of Staphylococcus aureus that promotes binding of fibrinogen or fibrin to the bacterial cell. Previous studies have shown that ClfA and the platelet integrin ␣ IIb ␤ 3 recognize the same domain at the extreme C terminus of the fibrinogen ␥-chain. ␣ IIb ␤ 3 interaction with this domain is known to occur in close proximity to a Ca 2؉ -binding EF-hand structure in the ␣-subunit. Analysis of the primary structure of ClfA indicated the presence of a potential Ca Staphylococcus aureus causes a wide range of opportunistic infections that range from superficial skin infections to lifethreatening diseases including endocarditis, pneumonia, and septicemia. Adherence of bacteria to host matrix components that is mediated by bacterial surface adhesins is the initial critical event in the pathogenesis of most infections. The extracellular matrix (ECM) 1 contains numerous glycoproteins and proteoglycans assembled into insoluble matrices that serve as substrata for the adhesion and migration of tissue cells. These processes involve integrins, a family of heterodimeric (␣␤) cellsurface receptors that recognize specific ECM proteins. It has become increasingly evident that bacteria, including S. aureus, also utilize the ECM as substrata for their adhesion by way of a family of adhesins called MSCRAMM (microbial surface components recognizing adhesive matrix molecules) (1) that specifically recognize host matrix components. One important component of the ECM, also occurring in soluble form in blood plasma, is fibrinogen, a 340-kDa hexamer composed of 2␣-, 2␤-, and 2␥-chains linked by disulfide bonds. This protein is recognized by several integrins including the platelet integrin ␣ IIb ␤ 3 . Activation of platelets and integrin ␣ IIb ␤ 3 results in fibrinogen-dependent aggregation in vitro and the formation of platelet-fibrin thrombi in vivo.

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The Residues AGDV of Recombinant γ Chains of Human Fibrinogen Must Be Carboxy-Terminal to Support Human Platelet Aggregation

Cited by 41 publications

References 29 publications

Characterization of the Interaction Between the Staphylococcus Aureus Clumping Factor (ClfA) and Fibrinogen

Characterization of the Interaction Between the Staphylococcus Aureus Clumping Factor (ClfA) and Fibrinogen

The pleiotropic role of the fibrinogen γ′ chain in hemostasis

The Fibrinogen-binding MSCRAMM (Clumping Factor) of Staphylococcus aureus Has a Ca2+-dependent Inhibitory Site

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