The inactivation of thrombin and plasmin by antithrombin III in the presence of Sepharose-heparin

Hatton, M. W. C.; Regoeczi, E.

doi:10.1016/0049-3848(77)90047-0

Cited by 84 publications

(32 citation statements)

References 17 publications

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“…Thrombin has been shown to bind to heparin with high affinity (KD = 1-100 nM), although the binding characteristics have been only partially delineated (19)(20)(21). It has been shown that thrombin must bind to heparin for the accelerated reaction to occur (19,20,(22)(23)(24)(25)(26); furthermore, as with the in vivo reaction, active siteblocked thrombin inhibits the heparin-accelerated reaction, but not the heparin-independent rate (27).…”

Section: Discussionmentioning

confidence: 99%

Clearance of Thrombin from Circulation in Rabbits by High-affinity Binding Sites on Endothelium

Lollar¹,

Owen²

1980

J. Clin. Invest.

197

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A B S T R A C T The clearance of 125I-thrombin and diisopropylphosphoryl-'251-thrombin (DIP-thrombin) from the circulation in rabbits was studied. When given either intraarterially or intravenously, DIP-thrombin, which is active-site blocked, was -90% cleared from the circulation by 1 min, the time of earliest sampling, indicating a large first-pass effect. DIP-thrombin given intravenously is found predominantly in the lungs, whereas DIP-thrombin injected into the aortic arch is distributed diffusely in approximate proportion to the blood supply. Renal artery, femoral artery, ear artery, left atrium, and portal vein infusions demonstrate that kidney, muscle, ear, heart, and liver, respectively, can remove DIP-thrombin from the circulation. These data imply that the clearance of DIP-thrombin is not a function of a specific organ but of the vascular bed per se. The clearance of DIP-thrombin was reversible since injection of 0.5 mg of unlabeled DIP-thrombin 10 min after the injection of a tracer dose of DIP-'251-thrombin resulted in the rapid reappearance ofthe DIP-'25I-thrombin into the circulation. In addition, the clearance of DIP-thrombin was saturable, i.e., clearance of DIP-1251-thrombin was inhibited by unlabeled DIP-thrombin in a dose-dependent fashion. In vivo Scatchard analysis of the saturation of the clearance process demonstrated that DIP-thrombin can be removed by binding to highaffinity binding sites, since dissociation constants (KD) of 10 and 13 nM were obtained for human and bovine DIP-thrombin, respectively.In contrast to DIP-thrombin, -75% of the radioacDr. Lollar is the recipient of National Institutes of Health grant P-32-HL-07344-2. Dr. Owen is the recipient of Career Development Award K04 HL 00348-03 from the National Heart, Lung, and Blood Institute.Received for publication 15 August 1979 and in revised form 11 August 1980. 1222 tivity associated with active thrombin remained in the circulation at 1 min. By 10 min 55% of 125I-thrombin had been removed from the circulation, and essentially all ofthe radioactivity can be accounted for in the liver. Sodium dodecyl sulfate-polyacrylamide gel radioelectrophoresis of plasma samples taken after injection of '25I-thrombin demonstrated that all ofthe active thrombin was converted to covalent thrombin-antithrombin III complex by the time of initial sampling (30 s). The in vitro conversion of 125I-thrombin to thrombin-antithrombin III complex was considerably slower (50+5% conversion at 30 s). The simultaneous injection of excess unlabeled DIP-thrombin inhibited the rate of formation of '25I-thrombin-antithrombin III complex formation in vivo (but not in vitro), which suggests that the binding of active thrombin to the high affinity binding sites is required for the rapid inactivation ofthrombin in vivo.We propose that (a) thrombin in the circulation binds to active site-independent high-affinity binding sites on the endothelial cell surface; (b) the inactivation of thrombin by antithrombin III is faster in vivo than in vitro because the high-affin...

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Section: Discussionmentioning

confidence: 99%

Clearance of Thrombin from Circulation in Rabbits by High-affinity Binding Sites on Endothelium

Lollar¹,

Owen²

1980

J. Clin. Invest.

197

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“…Approximately 10 6 dpm//xg protein was obtained by this method. Preparations of the radiolabeled isoforms were subjected to SDS-PAGE for comparison with unlabeled ATIII, and the gel, after it was stained and dried, was exposed to Kodak X-AR5 film For measurement of antithrombin activity, bovine a-thrombin was isolated and purified from Thrombostat by a procedure 15 derived from the method of Lundblad et al 16 After SDS-PAGE, purified thrombin appeared as a major band (36 kd) and a minor band (28 kd). A polyspecific antibody against bovine FPR-thrombin (i.e., thrombin inactivated by D-Phe-L-Pro-L-Arg-CH 2 C1) was raised in rabbits and was used to identify thrombin after blotting.…”

Section: Purity and Properties Of Antithrombin III Isoformsmentioning

confidence: 99%

Antithrombin III-beta associates more readily than antithrombin III-alpha with uninjured and de-endothelialized aortic wall in vitro and in vivo.

Witmer

Hatton

1991

Arterioscler Thromb

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The properties of two isoforms, or and fi, of rabbit antithrombin III (ATIII) were compared in the presence of undamaged or de-endothelialized rabbit aortic wall. Similar quantities of ATIII-tt and ATUI-fi bound to and rapidly saturated the endothelium in vitro, but the rate of transendothelial passage of ATIII-/3 exceeded that of ATIII-a by 22%. Furthermore, ATIII-0 was adsorbed approximately twice as rapidly as ATlII-a by the subendothelium of the de-endothelialized aorta. Binding of both isoforms was decreased (ATIII-/3 more than Al'lU-or) by pretreating the subendothelial surface with heparitinase. Also, subendothelium-bound ATIII-/J was desorbed more readily than bound ATlII-aby thrombin. In vivo, the rate of uptake of iodine-131-labeled ATI1I-/3 from the circulation by the aortic wall and the major organs was 30-50% faster than that of iodine-125-labeled ATIII-o: In contrast, the uptake of m I-ATIII-0 by the de-endothelialized aorta in vivo was three times faster than that of '"I-ATlII-a. By these criteria, ATIII-^ is the more active of the two isoforms. We surmise that plasma and, consequently, vessel wall levels of ATIII-/3 may be vital for controlling thrombogenic events caused by injury to the vascular wall. (Arteriosclerosis and Thrombosis 1991;ll:530-539) F rom recent reports, two forms of the glycoprotein antithrombin III (ATIII) have been isolated from rabbit 1 and human 2 plasma. Both the rabbit and human isoforms were separated by gradient elution from a heparin-Sepharose affinity column, the major human isoform, ATIII-a, eluting at 0.9-1.0 M NaCl, and the minor isoform, ATIII-ft at 1.4 M NaCl.2 Human ATIII-0 was shown to contain approximately 25-30% less carbohydrate than ATIII-a.2 More recently, Brennan et al 3 have reported that, unlike the human ATIII-a isoform, which has four biantennary /V-glycans (at asparagine [Asn] 96, 135, 155, and 172), the ATIII-0 isoform possesses only three 7V-glycans, lacking that at Asn 135. Can-ell et al 4 have suggested that the absence of the N-glycan at Asn 135 may extend the binding site for heparin and, consequently, increase the affinity of ATIII-/3 for heparin.

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“…In addition, the inherently slow formation of thrombin-antithrombin III complex is accelerated by heparin (1_1). Although the mechanism of this catalysis is s t i l l under investigation ( J^,21_,22^), it has been shown that the binding sites involved in the thrombin-antithrombin III interaction differ from those of heparin for the two proteins (23,24,25).…”

Section: Resultsmentioning

confidence: 99%

Interaction Mechanism of Thrombin with Functional Polystyrene Surfaces: A Study Using High-Performance Affinity Chromatography

Yu¹,

Müller²,

Fischer

et al. 1987

ACS Symposium Series

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Insoluble sulfonated polystyrenes (PSSO 3 ) and their L-arginyl methyl ester derivatives ( PAOM) have beeninvestigated by high-performance affinity chromatogra-phy.Highly active thrombin(Th) was found to be strongly adsorbed on both surfaces and was eluted by increasing ionic strength of the eluent. Compared to the PSSO 3 surface, desorption from the PAOM resin occurred at a lower salt concentration and with a more specific enzyme-resin interaction. Masking of the enzyme's active site by its natural inhibitor antithrombin III (AT III) caused a total disappearence of its affinity for both types of surface. It was demonstrated that the active seryl residue of thrombin was involved in enzyme-PAOM interactions. In contrast, this same site of the enzyme was always available after binding to PSSO 3 resin. This allowed a biospecific desorption procedure using AT III, heparin (Hep) and Hep-AT III complex. The present work provides further confirmation of the "AT III-like" and "heparin -like" properties, respectively, of PAOM and PSSO 3 materials.

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The inactivation of thrombin and plasmin by antithrombin III in the presence of Sepharose-heparin

Cited by 84 publications

References 17 publications

Clearance of Thrombin from Circulation in Rabbits by High-affinity Binding Sites on Endothelium

Clearance of Thrombin from Circulation in Rabbits by High-affinity Binding Sites on Endothelium

Antithrombin III-beta associates more readily than antithrombin III-alpha with uninjured and de-endothelialized aortic wall in vitro and in vivo.

Interaction Mechanism of Thrombin with Functional Polystyrene Surfaces: A Study Using High-Performance Affinity Chromatography

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