scholarly journals Conformational states of vitronectin: preferential expression of an antigenic epitope when vitronectin is covalently and noncovalently complexed with thrombin-antithrombin III or treated with urea

Blood ◽  
1988 ◽  
Vol 72 (3) ◽  
pp. 903-912 ◽  
Author(s):  
BR Tomasini ◽  
DF Mosher
Keyword(s):  
Monoclonal Antibody ◽  
Antithrombin Iii ◽  
Dermatan Sulfate ◽  
Factor Xa ◽  
Keratan Sulfate ◽  
Urea Treatment ◽  
Preferential Expression ◽  
Covalently Linked ◽  
Thrombin Antithrombin Iii Complex ◽  
Protein Serum

Abstract A difference in recognition of the adhesive glycoprotein vitronectin (also called S-protein, serum spreading factor, and epibolin) by monoclonal antibody 8E6 (Hayman EG, et al, Proc Natl Acad Sci USA 80:4003, 1983) was investigated using a competitive enzyme- immunosorbent assay and immunoaffinity chromatography. Recognition of vitronectin in serum was approximately 50-fold greater than recognition of vitronectin in plasma. Recognition of vitronectin incubated with heparin, thrombin-antithrombin III complex, or heparin and thrombin- antithrombin III complex together was 2.5-, 7-, or 32-fold greater, respectively, than recognition of vitronectin alone. Thrombin or antithrombin III by itself did not induce the antigenic change. Factor Xa-antithrombin III was less effective than thrombin-antithrombin III in induction of the change. Dextran sulfate and fucoidan were more potent than heparin in induction of the antigenic change, whereas dermatan sulfate, hyaluronic acid, heparan sulfate, chondroitin sulfate, or keratan sulfate were less effective. Immunoblotting analysis of serum and of vitronectin incubated with thrombin and antithrombin III demonstrated the presence of complexes composed of vitronectin and thrombin-antithrombin III that could only be dissociated with reducing agent. N-ethylmaleimide completely blocked the formation of the presumably disulfide-bonded complexes and partially blocked the antigenic change. Both non-disulfide-bonded and disulfide-bonded vitronectin bound to antibody-Sepharose from a mixture of vitronectin and thrombin-antithrombin III. Treatment of vitronectin with 8 mol/L urea resulted in enhanced recognition by the monoclonal antibody. Thus, the 8E6 antibody reacts with an epitope that is preferentially expressed by noncovalently and covalently linked vitronectin/thrombin-antithrombin III complexes and by urea-treated vitronectin. The change in vitronectin induced by thrombin-antithrombin III, therefore, is a physiological correlate of urea treatment and of adsorption of vitronectin onto tissue culture plastic (as is done in cell adhesion assays). The change may be important for expression of vitronectin activity.

scholarly journals Conformational states of vitronectin: preferential expression of an antigenic epitope when vitronectin is covalently and noncovalently complexed with thrombin-antithrombin III or treated with urea

Blood ◽  
1988 ◽  
Vol 72 (3) ◽  
pp. 903-912 ◽  
Author(s):  
BR Tomasini ◽  
DF Mosher
Keyword(s):  
Monoclonal Antibody ◽  
Antithrombin Iii ◽  
Dermatan Sulfate ◽  
Factor Xa ◽  
Keratan Sulfate ◽  
Urea Treatment ◽  
Preferential Expression ◽  
Covalently Linked ◽  
Thrombin Antithrombin Iii Complex ◽  
Protein Serum

A difference in recognition of the adhesive glycoprotein vitronectin (also called S-protein, serum spreading factor, and epibolin) by monoclonal antibody 8E6 (Hayman EG, et al, Proc Natl Acad Sci USA 80:4003, 1983) was investigated using a competitive enzyme- immunosorbent assay and immunoaffinity chromatography. Recognition of vitronectin in serum was approximately 50-fold greater than recognition of vitronectin in plasma. Recognition of vitronectin incubated with heparin, thrombin-antithrombin III complex, or heparin and thrombin- antithrombin III complex together was 2.5-, 7-, or 32-fold greater, respectively, than recognition of vitronectin alone. Thrombin or antithrombin III by itself did not induce the antigenic change. Factor Xa-antithrombin III was less effective than thrombin-antithrombin III in induction of the change. Dextran sulfate and fucoidan were more potent than heparin in induction of the antigenic change, whereas dermatan sulfate, hyaluronic acid, heparan sulfate, chondroitin sulfate, or keratan sulfate were less effective. Immunoblotting analysis of serum and of vitronectin incubated with thrombin and antithrombin III demonstrated the presence of complexes composed of vitronectin and thrombin-antithrombin III that could only be dissociated with reducing agent. N-ethylmaleimide completely blocked the formation of the presumably disulfide-bonded complexes and partially blocked the antigenic change. Both non-disulfide-bonded and disulfide-bonded vitronectin bound to antibody-Sepharose from a mixture of vitronectin and thrombin-antithrombin III. Treatment of vitronectin with 8 mol/L urea resulted in enhanced recognition by the monoclonal antibody. Thus, the 8E6 antibody reacts with an epitope that is preferentially expressed by noncovalently and covalently linked vitronectin/thrombin-antithrombin III complexes and by urea-treated vitronectin. The change in vitronectin induced by thrombin-antithrombin III, therefore, is a physiological correlate of urea treatment and of adsorption of vitronectin onto tissue culture plastic (as is done in cell adhesion assays). The change may be important for expression of vitronectin activity.

scholarly journals Factor IXa Inhibition Contributes to the Heparin Effect

1991 ◽  
Vol 66 (03) ◽  
pp. 306-309 ◽  
Author(s):  
Suzette Béguin ◽  
Frédérique Dol ◽  
H Coenraad Hemker
Keyword(s):  
Antithrombin Iii ◽  
Dermatan Sulfate ◽  
Factor Xa ◽  
Inhibitory Effect ◽  
Heparin Cofactor Ii ◽  
Factor Ixa ◽  
Factor Viiia ◽  
At Iii ◽  
Inhibition Of Thrombin ◽  
Thrombin Formation

SummaryWe investigated whether the inactivation of factor IXa contributes to the partial inhibition of thrombin formation that is observed at therapeutic concentrations of heparin. The action of standard unfractionated heparin (0.05 U/ml) on thrombin formation in the intrinsic system was compared to that of a mixture of dermatan sulfate (DS) and a synthetic pentasaccharide (PS). DS enhances the action of heparin cofactor II which inhibits thrombin only. PS specifically enhances the anti-factor Xa activity of antithrombin III (AT III). The concentrations of DS and PS were chosen so as to obtain equal anti-thrombin and anti-factor Xa activities as in 0.05 U/ml heparin. An extra inhibitory effect of heparin over the mixture is observed in situations where free factor IXa, not bound to factor VIIIa and phospholipid, limits the rate of thrombin formation, notably in contact activated plasma. We conclude that the inactivation of free factor IXa by heparin contributes importantly to the inhibition of thrombin formation in the intrinsic system such as e.g. measured in the activated partial thromboplastin time.

scholarly journals Heparan sulfate and dermatan sulfate inhibit the generation of thrombin activity in plasma by complementary pathways

Blood ◽  
1984 ◽  
Vol 64 (3) ◽  
pp. 742-747 ◽  
Author(s):  
FA Ofosu ◽  
GJ Modi ◽  
LM Smith ◽  
AL Cerskus ◽  
J Hirsh ◽  
...  
Keyword(s):  
Heparan Sulfate ◽  
Thrombin Generation ◽  
Antithrombin Iii ◽  
Dermatan Sulfate ◽  
Factor Xa ◽  
Intrinsic Pathway ◽  
Heparin Cofactor Ii ◽  
Normal Plasma ◽  
Normal Vessel

Abstract Heparan with a low affinity for antithrombin III has previously been demonstrated to inhibit thrombin generation in both normal plasma and plasma depleted of antithrombin III. In addition, standard heparin and heparin with a low affinity for antithrombin III have been demonstrated to have equivalent inhibitory actions on thrombin generation in plasma depleted of antithrombin III. These observations prompted the investigation of the effects of four normal vessel wall glycosaminoglycans (heparan sulfate, dermatan sulfate, chondroitin-4- sulfate, and chondroitin-6-sulfate) on the intrinsic pathway generation of thrombin and factor Xa and on the inactivation of thrombin and factor Xa in plasma. Heparan sulfate inhibited thrombin generation and accelerated the inactivation of added thrombin and factor Xa in normal plasma but not in antithrombin III-depleted plasma. In contrast, dermatan sulfate inhibited thrombin generation in both normal and antithrombin III-depleted plasma. In addition, heparan sulfate was an effective inhibitor of factor Xa generation, while dermatan sulfate was not. Neither chondroitin-4-sulfate nor chondroitin-6-sulfate inhibited the generation of thrombin or factor Xa nor did they accelerate the inactivation of factor Xa or thrombin by plasma. These results suggest that heparan sulfate acts primarily by potentiating antithrombin III, while dermatan sulfate acts by potentiating heparin cofactor II. The inhibition of thrombin generation by heparan sulfate and dermatan sulfate thus appears to occur by complementary pathways, both of which may contribute to the anticoagulation of blood in vivo.

scholarly journals Heparan sulfate and dermatan sulfate inhibit the generation of thrombin activity in plasma by complementary pathways

Blood ◽  
1984 ◽  
Vol 64 (3) ◽  
pp. 742-747
Author(s):  
FA Ofosu ◽  
GJ Modi ◽  
LM Smith ◽  
AL Cerskus ◽  
J Hirsh ◽  
...  
Keyword(s):  
Heparan Sulfate ◽  
Thrombin Generation ◽  
Antithrombin Iii ◽  
Dermatan Sulfate ◽  
Factor Xa ◽  
Intrinsic Pathway ◽  
Heparin Cofactor Ii ◽  
Normal Plasma ◽  
Normal Vessel

Heparan with a low affinity for antithrombin III has previously been demonstrated to inhibit thrombin generation in both normal plasma and plasma depleted of antithrombin III. In addition, standard heparin and heparin with a low affinity for antithrombin III have been demonstrated to have equivalent inhibitory actions on thrombin generation in plasma depleted of antithrombin III. These observations prompted the investigation of the effects of four normal vessel wall glycosaminoglycans (heparan sulfate, dermatan sulfate, chondroitin-4- sulfate, and chondroitin-6-sulfate) on the intrinsic pathway generation of thrombin and factor Xa and on the inactivation of thrombin and factor Xa in plasma. Heparan sulfate inhibited thrombin generation and accelerated the inactivation of added thrombin and factor Xa in normal plasma but not in antithrombin III-depleted plasma. In contrast, dermatan sulfate inhibited thrombin generation in both normal and antithrombin III-depleted plasma. In addition, heparan sulfate was an effective inhibitor of factor Xa generation, while dermatan sulfate was not. Neither chondroitin-4-sulfate nor chondroitin-6-sulfate inhibited the generation of thrombin or factor Xa nor did they accelerate the inactivation of factor Xa or thrombin by plasma. These results suggest that heparan sulfate acts primarily by potentiating antithrombin III, while dermatan sulfate acts by potentiating heparin cofactor II. The inhibition of thrombin generation by heparan sulfate and dermatan sulfate thus appears to occur by complementary pathways, both of which may contribute to the anticoagulation of blood in vivo.

Standard Heparin Enhances the Antithrombotic Activity of Dermatan Sulfate in the Rabbit but CY 216 Does Not

1988 ◽  
Vol 59 (02) ◽  
pp. 295-298 ◽  
Author(s):  
Y Cadroy ◽  
F Dol ◽  
C Caranobe ◽  
M Petitou ◽  
J C Lormeau ◽  
...  
Keyword(s):  
Antithrombin Iii ◽  
Dermatan Sulfate ◽  
Factor Xa ◽  
Low Molecular Weight ◽  
Antithrombotic Agents ◽  
Antithrombotic Activity ◽  
Pharmacological Targets ◽  
At Iii ◽  
Binding Sequence ◽  
Higher Doses

SummaryStandard heparin (SH) and dermatan sulfate (DS) two gly- cosaminoglycans with different pharmacological targets are effective antithrombotic agents in the rabbit. We have investigated the antithrombotic activity of the association DS plus SH. It was found that doses as low as 25 µg/kg for DS and 10 µg/kg for SH were ineffective when injected separately but generated a high and significant antithrombotic activity when injected together. These results were confirmed when higher doses of each compound were delivered in association. Further experiments were performed to determine if the enhancement of the antithrombotic activity of DS by HS resulted from its anti-factor Ha or antifactor Xa activity or from its moiety without affinity to AT III. A low molecular weight heparin (CY 216) with an anti-factor Xa/ anti-factor Ha ratio of 5, the synthetic pentasaccharide bearing the minimum binding sequence to antithrombin III, and a low affinity fraction of SH to AT III did not increase the antithrombotic activity of DS; in contrast a high affinity fraction of SH to AT III had the same effect than SH. We conclude that the enhancement of the antithrombotic activity of DS by SH mainly results from its anti-factor IIa activity.

scholarly journals A Monoclonal Antibody That Triggers Deacylation of an Intermediate Thrombin-Antithrombin III Complex

1989 ◽  
Vol 264 (23) ◽  
pp. 13736-13739 ◽  
Author(s):  
S Asakura ◽  
M Matsuda ◽  
N Yoshida ◽  
S Terukina ◽  
H Kihara
Keyword(s):  
Monoclonal Antibody ◽  
Antithrombin Iii ◽  
Thrombin Antithrombin Iii Complex

The Anticoagulant Properties of a Modified Form of Protein S

1988 ◽  
Vol 60 (02) ◽  
pp. 298-304 ◽  
Author(s):  
C A Mitchell ◽  
S M Kelemen ◽  
H H Salem
Keyword(s):  
Monoclonal Antibody ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Factor Xa ◽  
Protein S ◽  
Human Neutrophil Elastase ◽  
Factor X ◽  
Sds Page

SummaryProtein S (PS) is a vitamin K-dependent anticoagulant that acts as a cofactor to activated protein C (APC). To date PS has not been shown to possess anticoagulant activity in the absence of APC.In this study, we have developed monoclonal antibody to protein S and used to purify the protein to homogeneity from plasma. Affinity purified protein S (PSM), although identical to the conventionally purified protein as judged by SDS-PAGE, had significant anticoagulant activity in the absence of APC when measured in a factor Xa recalcification time. Using SDS-PAGE we have demonstrated that prothrombin cleavage by factor X awas inhibited in the presence of PSM. Kinetic analysis of the reaction revealed that PSM competitively inhibited factor X amediated cleavage of prothrombin. PS preincubated with the monoclonal antibody, acquired similar anticoagulant properties. These results suggest that the interaction of the monoclonal antibody with PS results in an alteration in the protein exposing sites that mediate the observed anticoagulant effect. Support that the protein was altered was derived from the observation that PSM was eight fold more sensitive to cleavage by thrombin and human neutrophil elastase than conventionally purified protein S.These observations suggest that PS can be modified in vitro to a protein with APC-independent anticoagulant activity and raise the possibility that a similar alteration could occur in vivo through the binding protein S to a cellular or plasma protein.

The Effect of Antithrombin III on the Activity of the Coagulation Factors VII, IX and X

1976 ◽  
Vol 35 (02) ◽  
pp. 295-304 ◽  
Author(s):  
B Østerud ◽  
M Miller-Andersson ◽  
U Abildgaard ◽  
H Prydz
Keyword(s):  
Antithrombin Iii ◽  
Factor Xa ◽  
Coagulation Factors ◽  
Disc Electrophoresis ◽  
Polyacrylamide Gel ◽  
Factor Vii ◽  
Native Form ◽  
Factor Ixa ◽  
Plasma Factor

SummaryAntithrombin III, purified to homogeneity according to Polyacrylamide gel disc electrophoresis and immunoelectrophoresis, inhibited the activity of purified factor IXa and Xa, whereas factor VII was not inhibited either in the active or in the native form.Antithrombin III is the single most important inhibitor of factor Xa in plasma. Factor Xa does not, however, reduce the activity of antithrombin III against thrombin.

The Venous Antithrombotic Profile of Naroparcil in the Rabbit

1994 ◽  
Vol 72 (06) ◽  
pp. 874-879 ◽  
Author(s):  
Jean Millet ◽  
Jocelyne Theveniaux ◽  
Neil L Brown
Keyword(s):  
Oral Administration ◽  
Bleeding Time ◽  
Dermatan Sulfate ◽  
Factor Xa ◽  
Bleeding Risk ◽  
Thrombin Time ◽  
Heparin Cofactor Ii ◽  
Antithrombotic Activity ◽  
Thrombin Inhibition ◽  
Maximal Reduction

SummaryThe venous antithrombotic profile of naroparcil or (4-[4-cyanoben-zoyl]-phenyl)-1.5-dithio-β-D-xylopyranoside was investigated in the rabbit following single i. v. and oral administration. Naroparcil attenuated thrombus development in a Wessler stasis model of venous thrombosis (jugular vein) employing bovine factor Xa as a thrombogenic stimulus giving ED50 values of 21.9 mg/kg and 36.0 mg/kg after respectively i. v. and oral administration. Venous antithrombotic activity was maximal 2-3 h after i. v. administration and 4-8 h after oral administration. Four hours after the oral administration of maximal antithrombotic (Wessler model, factor Xa) doses (100 and 400 mg/kg), naroparcil had no significant effect on bleeding time. In platelet poor plasma obtained from animals treated 4 h previously with various doses (25 to 400 mg/kg) of naroparcil, there was no detectable anti-factor Xa nor antithrombin activity. Similarly, naroparcil had no effect on APTT nor on thrombin time. A sensitized thrombin time (to about 35 s) was modestly but significantly increased following oral administration of the compound at 400 mg/kg. However, thrombin generation by the intrinsic pathway was reduced in a dose-related manner, maximal reduction being 65% at 400 mg/kg. The same doses of naroparcil enhanced the formation of thrombin/heparin cofactor II complexes at the expense of thrombin/antithrombin III complexes in plasma incubated with (125I)-human a-thrombin and induced the appearance of dermatan sulfate-like material in the plasma of treated rabbits, as measured by a heparin cofactor II-mediated thrombin inhibition assay. The results suggest that naroparcil could have a safe venous antithrombotic profile following oral administration (antithrombotic effect compared to bleeding risk). It is probable that part of the mechanism of action of the β-D-xyloside, naroparcil, is due to the induction of chondroitin sulfate-like glycosaminoglycan biosynthesis, this material being detectable in the plasma.

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