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

Abstract

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...

Identification of an endothelial cell cofactor for thrombin-catalyzed activation of protein C.

“…The data (Fig. 4A) suggest that the system is not saturated with protein C at physiological (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) ,tg/ml) concentrations of the zymogen, although species specificity may be an important qualification. The effect of thrombin concentration on the rate of protein C activation was also evaluated.…”

“…Thrombin is known to bind reversibly in the microcirculation in vivo (11). If reversibly bound thrombin is the protein C activator, then it should be eluted slowly from the heart.…”

“…Finally, the role of antithrombin III in the process must be addressed. Although the microcirculation can promote very rapid inhibition ofcirculating thrombin, this capacity can be saturated (11). Perhaps protein C represents a second-tier control system that becomes active in proportion to the saturation of the primary inactivation catalyst.…”

“…However, the rate of activation ofpurified protein C by thrombin (EC 3.4.21.5), the only known blood-derived activator, is inappropriately slow for a physiologically important reaction (4,6,9); this raises the possibility that an unknown activator or cofactor is involved in the activation ofprotein C in vivo. Recently, an interaction ofthrombin with an endothelial cell surface antithrombin III cofactor was demonstrated in vivo (11). In the large vascular.…”

Transient activation of hepatic glycogenolysis by thrombin in perfused rat livers

“…Furthermore, coincubation of thrombin with diisopropyl fluorophosphate prior to infusion into liver, a procedure which blocks the active sites of thrombin as a serine protease without altering protein structure [27], prevented the increase in carbohydrate output almost completely (Table 1). In addition, nisoldipine (10 pM), a dihydropyridine-type calcium channel blocker, decreased thrombin-induced glycogenolysis by about 50% (Table l).…”