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

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.

Aggregated, Conformationally Changed Fibrinogen Exposes the Stimulating Sites for t-PA-Catalysed Plasminogen Activation

10.1055/s-0038-1650269 ◽

1996 ◽

Vol 75 (02) ◽

pp. 326-331 ◽

Cited By ~ 7

Author(s):

Unni Haddeland ◽

Knut Sletten ◽

Anne Bennick ◽

Willem Nieuwenhuizen ◽

Frank Brosstad

Keyword(s):

Conformational Changes ◽

Gel Permeation Chromatography ◽

Tissue Type ◽

Plasminogen Activation ◽

Chromogenic Substrate ◽

Type Plasminogen Activator ◽

Gel Permeation ◽

Self Association ◽

Fibrin Monomers ◽

SummaryThe present paper shows that conformationally changed fibrinogen can expose the sites Aα-(148-160) and γ-(312-324) involved in stimulation of the tissue-type plasminogen activator (t-PA)-catalysed plasminogen activation. The exposure of the stimulating sites was determined by ELISA using mABs directed to these sites, and was shown to coincide with stimulation of t-PA-catalysed plasminogen activation as assessed in an assay using a chromogenic substrate for plasmin. Gel permeation chromatography of fibrinogen conformationally changed by heat (46.5° C for 25 min) demonstrated the presence of both aggregated and monomeric fibrinogen. The aggregated fibrinogen, but not the monomeric fibrinogen, had exposed the epitopes Aα-(148-160) and γ-(312-324) involved in t-PA-stimulation. Fibrinogen subjected to heat in the presence of 3 mM of the tetrapeptide GPRP neither aggregates nor exposes the rate-enhancing sites. Thus, aggregation and exposure of t-PA-stimulating sites in fibrinogen seem to be related phenomena, and it is tempting to believe that the exposure of stimulating sites is a consequence of the conformational changes that occur during aggregation, or self-association. Fibrin monomers kept in a monomeric state by a final GPRP concentration of 3 mM do not expose the epitopes Aα-(148-160) and γ-(312-324) involved in t-PA-stimulation, whereas dilution of GPRP to a concentration that is no longer anti-polymerizing, results in exposure of these sites. Consequently, the exposure of t-PA-stimulating sites in fibrin as well is due to the conformational changes that occur during selfassociation.

SITES IN TISSUE-TYPE PLASMINOGEN ACTIVATOR INVOLVED IN THE INTERACTION WITH FIBRIN, PLASMINOGEN AND LOW MOLECULAR WEIGHT LIGANDS

10.1055/s-0038-1644613 ◽

1987 ◽

Author(s):

J M Verheijen ◽

M P M Caspers ◽

G A W de Munk ◽

B E Enger-Valk ◽

G T G Chang ◽

...

Keyword(s):

Binding Site ◽

Plasminogen Activator ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Tissue Type ◽

Plasminogen Activation ◽

Tissue Type Plasminogen Activator ◽

Type Plasminogen Activator ◽

Arginine Residues ◽

Tissue-type plasminogen activator (t-PA) activates the proenzyme plasminogen to the active protease plasmin which degrades fibrin. The unique properties of t-PA, fibrin binding and stimulation of activity by fibrin make it an interesting molecule for specific thrombolysis. t-PA is thought to consist of five structural regions designated finger (F), growth factor (G), kringle 1 (Kl), kringle 2 (K2) and protease (P). Previous studies have shown that the interaction of t-PA with fibrin is mediated by the F and K2 regions.Mutated t-PA cDNA molecules were expressed in Chinese hamster ovary cells and t-PA analog proteins were purified from serum free culture media using affinity chromatography with immobilized monoclonal antibodies. Besides FGK1K2P (native t-PA) the following analogs were used GK1K2P, klK2P, K2P, P, FP and FGKlk2P (kl and k2 have partial deletions of the kringle). All the molecules comprising K2P could be stimulated in plasminogen activation activity by fibrinogen fragments comparable to normal t-PA. The activities of FP and FGKlk2P were only slightly influenced by these fragments. It was shown that the fibrin binding site in K2 was plasminogen dependent whereas that in F was not. K2 was found to contain a binding site for lysine, 6-amino-hexanoic acid but also 6-amino-hexane and thus to differ from the high affinity lysine binding sites in plasminogen.Chemical modification of lysine and arginine residues in t-PA with citraconic anhydride and cyclohexanedione respectively, revealed no involvement of these residues in interaction with lysine or analogs nor in stimulation of activity by fibrinogen fragments. Arginine modification led to inhibition of plasminogen activation activity, both in the presence and absence of fibrinogen fragments, but the amidolytic activity as measured with a tripeptide paranitroanilide was not changed. The involvement of one or more arginine residues in interaction of t-PA with plasminogen seems likely.

Complementary Approach for the Determination of Histidine in the Metal-binding Site of an Anti-DTPA-Indium Monoclonal Antibody

Annals of the New York Academy of Sciences ◽

10.1111/j.1749-6632.1995.tb19966.x ◽

1995 ◽

Vol 750 (1 Enzyme Engine) ◽

pp. 284-287

Author(s):

VALÉRIE BODEN ◽

CAROLE COLIN ◽

JACQUES BARBET ◽

JEAN MARC LE DOUSSAL ◽

MOOKAMBESWARAN VIJAYALAKSHMI

Keyword(s):

Monoclonal Antibody ◽

Binding Site ◽

Metal Binding ◽

Metal Binding Site ◽

Complementary Approach

Fibronectin binding to complement subcomponent C1q. Localization of their respective binding sites

Biochemical Journal ◽

10.1042/bj2260207 ◽

1985 ◽

Vol 226 (1) ◽

pp. 207-215 ◽

Cited By ~ 26

Author(s):

J Sorvillo ◽

I Gigli ◽

E Pearlstein

Keyword(s):

Amino Acids ◽

Binding Site ◽

Binding Sites ◽

Solid Phase ◽

Fluid Phase ◽

Globular Domain ◽

Plasma Fibronectin ◽

Fibronectin Binding ◽

Inhibition Studies ◽

Radiobinding Assay

The interaction of purified human plasma fibronectin with the C1q subcomponent of complement was investigated by using a solid-phase radiobinding assay. 125I-fibronectin binding to native C1q, purified collagen domain (C1q-c) or globular domain (C1q-g) was compared. When the purified domains were insolubilized by binding to plastic, the C1q-c exhibited 59% of the binding demonstrated with intact C1q, whereas the C1q-g exhibited 35% of the binding. N-Terminal sequencing of the globular domain showed that a sequence of seven collagen-like amino acids was retained on each chain of the C1q-g fragment. 125I-fibronectin binding to C1q could be inhibited equally well by fluid-phase C1q and C1q-c, but not by fluid-phase C1q-g, implying that the collagen-like region retained on the C1q-g is masked in the fluid phase. In addition, studies were performed to determine which subunit(s) of C1q bind(s) fibronectin. The percentages of fibronectin bound by the A, B, and C chain of C1q were found to be 38, 21 and 41% respectively. Inhibition studies with purified 200-180 kDa, 50 kDa or 29 kDa fragments of fibronectin show that the binding site on fibronectin for C1q is the 50 kDa gelatin-binding domain.

The Interaction of TSH Receptor Autoantibodies with 125I-Labelled TSH Receptor

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jcem.84.10.6071 ◽

1999 ◽

Vol 84 (10) ◽

pp. 3797-3802 ◽

Cited By ~ 21

Author(s):

Jane Sanders ◽

Yasuo Oda ◽

Sara Roberts ◽

Angela Kiddie ◽

Tonya Richards ◽

...

Keyword(s):

Monoclonal Antibody ◽

Cyclic Amp ◽

Binding Site ◽

Tsh Receptor ◽

Thyroid Cells ◽

Terminal Domain ◽

Close Relationship ◽

Immunoprecipitation Reaction ◽

Abstract Detergent-solubilized porcine TSH receptor (TSHR) has been labeled with 125I using a monoclonal antibody to the C-terminal domain of the receptor. The ability of sera containing TSHR autoantibody to immunoprecipitate the labeled receptor was then investigated. Sera negative for TSHR autoantibody (as judged by assays based on inhibition of labeled TSH binding to detergent-solubilized porcine TSHR) immunoprecipitated about 4% of the labeled receptor, whereas sera with high levels of receptor autoantibody immunoprecipitated more than 25% of the labeled receptor. The ability to immunoprecipitate labeled TSHR correlated well with ability of the sera to inhibit labeled TSH binding to the receptor (r = 0.92; n = 63), and this is consistent with TSHR autoantibodies in these samples being directed principally to a region of the receptor closely related to the TSH binding site. Preincubation of labeled TSHR with unlabeled TSH before reaction with test sera inhibited the immunoprecipitation reaction, providing further evidence for a close relationship between the TSHR autoantibody binding site(s) and the TSH binding site. This was the case whether the sera had TSH agonist (i.e., thyroid stimulating) or TSH antagonist (i.e., blocking) activities, thus, providing no clear evidence for different regions of the TSHR being involved in forming the binding site(s) for TSHR autoantibodies with stimulating and with blocking activities. The ability of TSHR autoantibodies to stimulate cyclic AMP production in isolated porcine thyroid cells was compared with their ability to immunoprecipitate labeled porcine TSHR. A significant correlation was observed (r = 0.58; n = 50; P < 0.001) and the correlation was improved when stimulation of cyclic AMP production was compared with inhibition of labeled TSH binding to porcine TSHR (r = 0.76). Overall, our results indicate that TSHR autoantibodies bind principally to a region on the TSHR closely related to the TSH binding site, and this seems to be the case whether the autoantibodies act as TSH agonists or antagonists.

Effects of an Antifibrin Monoclonal Antibody and Fragments thereof on Some Properties of Fibrin

10.1055/s-0038-1645683 ◽

1990 ◽

Vol 63 (01) ◽

pp. 039-043 ◽

Cited By ~ 5

Author(s):

M N J M Wasser ◽

M Welling ◽

G Lamers ◽

E K J Pauwels ◽

W Nieuwenhuizen

Keyword(s):

Monoclonal Antibody ◽

Steric Hindrance ◽

Anticoagulant Effect ◽

Thrombin Time ◽

Plasminogen Activation ◽

Fibrin Formation ◽

Fab Fragments ◽

Parabolic Rate ◽

Igg1 Subclass ◽

Fibrin Monomers

SummaryAntifibrin monoclonal antibody Y22, of IgG1-subclass, has its epitope in the D-domain of fibrin. In a thrombin time assay, Y22 and its F(ab)2 fragments interfere with clotting of citrated plasma. Transmission and scanning electronmicroscopic studies show that clotting of citrated blood or plasma in the presence of Y22 results in formation of thin, short fibrin fibres. The (smaller) Fab fragments of Y22 did not have an anti-clotting effect. This suggests that the anticoagulant effect of Y22 is due to steric hindrance of the association of fibrin monomers. A control antibody and its F(ab)2 and Fab fragments have no effect on fibrin formation.In a parabolic rate assay, Y22 Fab fragments interfered strongly with the fibrin-induced enhancement of the t-PA-catalyzed plasminogen activation, whereas intact Y22 and a control antibody did not. In contrast with their effects on the fibrin assembly, the effects of Y22, Y22-F(ab)2 and Y22-Fab on the capacity of fibrin to act as a rate-enhancer in the plasminogen activation by t-PA appears to decrease with the size of the immunoreactive entity. As is discussed, this may be due to the differential accessibility of sites involved in stimulation and polymerization which are located in the fibrin D-domain.

Determination of binding site of anti-tumour necrosis factor-α monoclonal antibody using hybrid and mutant proteins

FEBS Letters ◽

10.1016/0014-5793(96)00396-1 ◽

1996 ◽

Vol 386 (1) ◽

pp. 72-74 ◽

Cited By ~ 3

Author(s):

Ludmila N. Shingarova ◽

Lubov N. Sagaidak ◽

Nadia Berkova ◽

Vyacheslav G. Korobko

Keyword(s):

Monoclonal Antibody ◽

Binding Site ◽

Tumour Necrosis Factor ◽

Tumour Necrosis ◽

Tumour Necrosis Factor Α ◽

Mutant Proteins ◽

Factor Α ◽

Necrosis Factor

Functional Characterization of PM6/13, a β3-specific (GPIIIa/CD61) Monoclonal Antibody that Shows Preferential Inhibition of Fibrinogen Binding over Fibronectin Binding to Activated Human Platelets

10.1055/s-0037-1614247 ◽

1998 ◽

Vol 79 (01) ◽

pp. 177-185 ◽

Cited By ~ 10

Author(s):

Ashia Siddiqua ◽

Michael Wilkinson ◽

Vijay Kakkar ◽

Yatin Patel ◽

Salman Rahman ◽

...

Keyword(s):

Monoclonal Antibody ◽

Platelet Aggregation ◽

Platelet Rich Plasma ◽

Functional Characterization ◽

Human Platelets ◽

Western Blots ◽

Activated Platelets ◽

Reducing Conditions ◽

Fibronectin Binding

SummaryWe report the characterization of a monoclonal antibody (MAb) PM6/13 which recognises glycoprotein IIIa (GPIIIa) on platelet membranes and in functional studies inhibits platelet aggregation induced by all agonists examined. In platelet-rich plasma, inhibition of aggregation induced by ADP or low concentrations of collagen was accompanied by inhibition of 5-hydroxytryptamine secretion. EC50 values were 10 and 9 [H9262]g/ml antibody against ADP and collagen induced responses respectively. In washed platelets treated with the cyclooxygenase inhibitor, indomethacin, PM6/13 inhibited platelet aggregation induced by thrombin (0.2 U/ml), collagen (10 [H9262]g/ml) and U46619 (3 [H9262]M) with EC50 = 4, 8 and 4 [H9262]g/ml respectively, without affecting [14C]5-hydroxytryptamine secretion or [3H]arachidonate release in appropriately labelled cells. Studies in Fura 2-labelled platelets revealed that elevation of intracellular calcium by ADP, thrombin or U46619 was unaffected by PM6/13 suggesting that the epitope recognised by the antibody did not influence Ca2+ regulation. In agreement with the results from the platelet aggregation studies, PM6/13 was found to potently inhibit binding of 125I-fibrinogen to ADP activated platelets. Binding of this ligand was also inhibited by two other MAbs tested, namely SZ-21 (also to GPIIIa) and PM6/248 (to the GPIIb-IIIa complex). However when tested against binding of 125I-fibronectin to thrombin stimulated platelets, PM6/13 was ineffective in contrast with SZ-21 and PM6/248, that were both potent inhibitors. This suggested that the epitopes recognised by PM6/13 and SZ-21 on GPIIIa were distinct. Studies employing proteolytic dissection of 125I-labelled GPIIIa by trypsin followed by immunoprecipitation with PM6/13 and analysis by SDS-PAGE, revealed the presence of four fragments at 70, 55, 30 and 28 kDa. PM6/13 did not recognize any protein bands on Western blots performed under reducing conditions. However Western blotting analysis with PM6/13 under non-reducing conditions revealed strong detection of the parent GP IIIa molecule, of trypsin treated samples revealed recognition of an 80 kDa fragment at 1 min, faint recognition of a 60 kDa fragment at 60 min and no recognition of any product at 18 h treatment. Under similar conditions, SZ-21 recognized fragments at 80, 75 and 55 kDa with the 55kDa species persisting even after 18 h trypsin treatment. These studies confirm the epitopes recognised by PM6/13 and SZ-21 to be distinct and that PM6/13 represents a useful tool to differentiate the characteristics of fibrinogen and fibronectin binding to the GPIIb-IIIa complex on activated platelets.

Antithrombin-mediated Inhibition of Factor Vlla-Tissue Factor Complex by the Synthetic Pentasaccharide Representing the Heparin Binding Site to Antithrombin

10.1055/s-0038-1650512 ◽

1996 ◽

Vol 76 (01) ◽

pp. 005-008 ◽

Cited By ~ 22

Author(s):

Jean Claude Lormeau ◽

Jean Pascal Herault ◽

Jean Marc Herbert

Keyword(s):

Binding Site ◽

Tissue Factor ◽

Residual Activity ◽

Coagulation Factors ◽

Heparin Binding ◽

Experimental Conditions ◽

First Order ◽

Heparin Binding Site ◽

Coagulant Activity

SummaryWe examined the effect of the synthetic pentasaccharide representing the minimal binding site of heparin to antithrombin on the antithrombin-mediated inactivation of factor Vila bound to tissue factor. This effect was compared to the effect of unfractionated heparin. Using purified recombinant human coagulation factors and either a clotting or an amidolytic assay for the determination of the residual activity of factor Vila, we showed that the pentasaccharide was an efficient antithrombin-dependent inhibitor of the coagulant activity of tissue factor-factor Vila complex. In our experimental conditions, assuming a mean MW of 14,000 for heparin, the molar pseudo-first order rate constants for ATIII-mediated FVIIa inhibition by ATIII-binding heparin and by the synthetic pentasaccharide were found to be similar with respective values of 104,000 ± 10,500 min-1 and 112,000 ± 12,000 min-1 (mean ± s.e.m., n = 3)

On The Mechanism Of Heparin-Induced Potentiation Of Platelet Aggregation

10.1055/s-0038-1652598 ◽

1981 ◽

Author(s):

M Yamamoto ◽

K Watanabe ◽

Y Ando ◽

H Iri ◽

N Fujiyama ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Coagulation Factors ◽

Marked Enhancement ◽

Matrix Bound ◽

Induced Aggregation ◽

Aggregation Of Platelets ◽

Plasma Heparin

It has been suggested that heparin caused potentiation of aggregation induced by ADP or epinephrine. The exact mechanism of heparin-induced platelet activation, however, remained unknown. In this paper, we have investigated the role of anti-thrombin III ( AT ) in heparin-induced platelet activation using purified AT and AT depleted plasma. When ADP or epinephrine was added to citrated PRP one minute after addition of heparin ( 1 u/ml, porcine intestinal mucosal heparin, Sigma Co. USA ), marked enhancement of platelet aggregation was observed, compared with the degree of aggregation in the absence of heparin. However, in platelet suspensions prepared in modified Tyrode’s solution, heparin exhibited no potentiating effect on platelet aggregation induced by epinephrine or ADP. Potentiation of epinephrine- or ADP-induced platelet aggregation by heparin was demonstrated when purified AT was added to platelet suspensions at a concentration of 20 μg/ml. AT depleted plasma, which was prepared by immunosorption using matrix-bound antibodies to AT, retained no AT, while determination of α1-antitrypsinα2- macroglobulin and fibrinogen in AT depleted plasma produced values which corresponded to those of the original plasma when dilution factor was taken into account. The activities of coagulation factors were also comparable to those of the original plasma. Heparin exhibited potentiating effect on ADP- or epinephrine-induced aggregation of platelets in original plasma, but no effect in AT depleted plasma. When purified AT was added back to AT depleted plasma at a concentration of 20 μg/ml, potentiation of platelet aggregation by heparin was clearly demonstrated.Our results suggest that effect of heparin on platelet aggregation is also mediated by anti-thrombin III.