OKB7, a monoclonal antibody that reacts at or near the C3d binding site of human CR2

Heparin-induced thrombocytopenia/thrombosis (HIT/T) is a common complication of heparin therapy that is caused by antibodies to platelet factor 4 (PF4) complexed with heparin. The immune response is polyclonal and polyspecific, ie, more than one neoepitope on PF4 is recognized by HIT/T antibodies. One such epitope has been previously identified; it involves the domain between the third and fourth cysteine residues in PF4 (site 1). However, the binding sites for other HIT/T antibodies remain to be defined. To explore this issue, the binding site of KKO, an HIT/T-like murine monoclonal antibody, was defined. KKO shares a binding site with many HIT/T antibodies on PF4/heparin, but does not bind to site 1 or recognize mouse PF4/heparin. Therefore, the binding of KKO to a series of mouse/human PF4 chimeras complexed with heparin was examined. KKO recognizes a site that requires both the N terminus of PF4 and Pro34, which immediately precedes the third cysteine. Both regions lie on the surface of the PF4 tetramer in sufficient proximity (within 0.74 nm) to form a contiguous antigenic determinant. The 10 of 14 HIT/T sera that require the N terminus of PF4 for antigen recognition also require Pro34 to bind. This epitope, termed site 2, lies adjacent to site 1 in the crystal structure of the PF4 tetramer. Yet sites 1 and 2 can be recognized by distinct populations of antibodies. These studies further help to define a portion of the PF4 tetramer to which self-reactive antibodies develop in patients exposed to heparin.

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A monoclonal antibody to human insulin-like growth factor-I: characterization, use in radioimmunoassay and effect on the biological activities of the growth factor

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0020201 ◽

1989 ◽

Vol 2 (3) ◽

pp. 201-206 ◽

Cited By ~ 45

Author(s):

D. J. Morrell ◽

H. Dadi ◽

J. More ◽

A. M. Taylor ◽

A. Dabestani ◽

...

Keyword(s):

Monoclonal Antibody ◽

Growth Factor ◽

Binding Site ◽

Biological Activities ◽

Rabbit Antiserum ◽

Affinity Constant ◽

Insulin Like Growth Factor ◽

Factor I ◽

Igf I ◽

Growth Factor I

ABSTRACT A monoclonal antibody (BPL-M23) to insulin-like growth factor-I (IGF-I) was obtained following immunization of BALB/c mice with human IGF-I conjugated to ovalbumin. The affinity constant of BPL-M23 for IGF-I was 10·5 litres/nmol and the cross-reactivities of IGF-II, multiplication-stimulating activity III-2 and insulin were 08, 003 and less than 0·0001 % respectively. Porcine, bovine, ovine and rabbit sera, but not rat or mouse sera, showed substantial reactivity with the antibody. Comparison of radioimmunoassay analyses of 54 human serum samples from normal subjects and acromegalic and GH-deficient patients using BPL-M23 and a polyclonal rabbit antiserum (R557A) to human IGF-I showed a high correlation, indicating the usefulness of the monoclonal antibody in radioimmunoassay. Monoclonal antibody BPL-M23 was capable of abolishing the sulphation, mitogenic and insulin-like activities of IGF-I in in-vitro bioassays, suggesting that these activities may rely upon the same receptor-binding site which is near to the antibody-binding site.

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Plasminogen interactions with platelets in plasma

Blood ◽

10.1182/blood.v72.5.1530.1530 ◽

1988 ◽

Vol 72 (5) ◽

pp. 1530-1535 ◽

Cited By ~ 45

Author(s):

B Adelman ◽

A Rizk ◽

E Hanners

Keyword(s):

Monoclonal Antibody ◽

Binding Site ◽

Platelet Rich Plasma ◽

Aminocaproic Acid ◽

Binding Domains ◽

Fibrinogen Binding ◽

Platelet Binding ◽

Epsilon Aminocaproic Acid ◽

Von Willebrand ◽

Willebrand Factor

Abstract In this report we used a fluorescent flow cytometry-based assay to examine plasminogen binding to platelets in plasma. Our data indicate that platelets activated in platelet-rich plasma (PRP) by adenosine-5′- diphosphate (ADP) or thrombin bind plasminogen to their surface. Fab fragments of the monoclonal antibody LJ-CP8 that are directed against the fibrinogen binding site on the glycoprotein (GP) IIb-IIIa complex inhibit both plasminogen and fibrinogen binding to ADP-stimulated platelets as does 5 mmol/L EDTA. Platelet aggregation and plasminogen and fibrinogen binding are also concurrently inhibited by the Gly-Arg- Asp (RGD) analogue Gly-Arg-Gly-Asp-Ser (GRGDS) when it is added to PRP before ADP stimulation. The scrambled peptide analogue SDGRG has no effect. The monoclonal antibody 6D1, directed against the von Willebrand factor binding site on GPIb, has no effect on plasminogen- platelet binding, nor does antithrombospondin antibody. epsilon- Aminocaproic acid (EACA), however, inhibits plasminogen binding to ADP- activated platelets. These data indicate that plasminogen binds to platelets activated in plasma, that binding occurs on platelet GPIIb/IIIa, and that binding may be mediated via plasminogen association with fibrinogen via lysine binding domains. Finally, we found both plasminogen and fibrinogen on resting platelets in PRP and demonstrated that they are equally displaced by EDTA, LJ-CP8, and 10E5 (an additional anti-GPIIb/IIIa monoclonal antibody). Plasminogen is also equally displaced by EACA. These data suggest that plasminogen is also bound to GPIIb/IIIa on resting platelets, possibly also via interaction with fibrinogen.

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RELATION OP FIBRIN STIMULATION OF tPA MEDIATED PLASMINOGEN ACTIVATION AND FIBRIN BINDING TOWARDS FIBRONEKTIN AS REVEALED BY A MONOCLONAL ANTIBODY (MAB) AGAINST FCB-2 FIBRINOGEN FRAGMENTS

10.1055/s-0038-1644403 ◽

1987 ◽

Author(s):

S Meusburger ◽

R Beckmann ◽

J Wojta ◽

B R Binder

Keyword(s):

Monoclonal Antibody ◽

Binding Site ◽

Fluid Phase ◽

Plasminogen Activation ◽

Elisa System ◽

Matrix Bound ◽

Fibronectin Binding ◽

Fibrin Monomers ◽

Stimulation Of

Fibrin binds to the finger domain of fibronektin via the C-terminal end of the chain and it was reported previously by us that in a fluid phase assay fibronektin inhibits fibrin enhancement of plasminogen activation by tPA. However, other have shown that tPA binds to fibronectin thereby possibly mediating enhanced matrix bound plasmin formation. In the present study we tried to further characterize the interaction between fibronectin and fibrin in regard to fibrin dependent enhancement of plasminogen activation by tPA. For fibrin binding to fibronectin we have developed an ELISA system using fibronectin coated plates and antibodies against fibrin(ogen) to quantify bound fibrin. For determination of plasminogen activation we used a coupled spectrophotometric fluid phase assay with Glu-plasminogen as substrate and H-D-Val-Leu-Lys-pNA to quantify the formed plasmin. Fibrin binding to coated fibronectin was linear between 500ng and 1 mg/ml for fibrin monomers (reptilase), FCB-2 fragments and thrombin (3.3 U/ml) treated fibrinogen, respectively. A monoclonal antibody directed against the FCB-2 fibrinogen fragment which also could be shown to recognize fibirn but not fibrinogen did not recognize fibronectin bound fibrin and inhibited also the fibrin stimulatory effect on plasminogen activation indicating that the epitope against which the antibody is directed is closely related to both the fibronectin binding site and the site involved in t-PA stimulation.

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A new monoclonal antibody, mAb 204-11, that influences the binding of platelet GPVI to fibrous collagen

Thrombosis and Haemostasis ◽

10.1055/s-0037-1613401 ◽

2003 ◽

Vol 89 (06) ◽

pp. 996-1003 ◽

Cited By ~ 18

Author(s):

Jun Mizuguchi ◽

Sachiko Kawashima ◽

Michiko Nagamatsu ◽

Yoshiki Miura ◽

Tomohiro Nakagaki ◽

...

Keyword(s):

Monoclonal Antibody ◽

Platelet Aggregation ◽

Binding Site ◽

Tyrosine Phosphorylation ◽

Platelet Activation ◽

Collagen Binding ◽

Glycoprotein Vi ◽

Phosphorylation Of Proteins ◽

Dimeric Form ◽

Collagen Receptor

SummaryThe newly identified platelet collagen receptor glycoprotein VI binds to fibrous collagen, inducing platelet activation. Several antibodies against GPVI have been reported, including a patient’s auto-antibodies, that activates platelets through their ability to crosslink this glycoprotein. We have developed a monoclonal antibody (mAb) against GPVI using the recombinant extracellular domain of GPVI as an antigen. This antibody, mAb 204-11, induced platelet aggregation and tyrosine phosphorylation of proteins similar to those induced by GPVI-reactive proteins, collagen and convulxin. Its interaction with GPVI was analyzed by measuring the effect of the antibody on GPVI binding to collagen using a dimeric form of recombinant GPVI, GPVI-Fc2. MAb 204-11 inhibited the binding of GPVI-Fc2 to fibrous collagen particles, but enhanced the GPVI binding to immobilized collagen, suggesting that the antibody binds to a region near the collagen binding site of GPVI. MAb 204-11 also inhibited the GPVI binding to convulxin at a low concentration, but not completely. Since mAb 204-11 reacts specifically with GPVI and is applicable for immunoblotting and immunoprecipitation, this antibody would be useful for studies on GPVI.

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