Thrombin Inhibiting Capacity Of Normal Aorta (original) (raw)
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The Journal of biological chemistry, 1984
A recirculating perfused rabbit heart preparation is used to study the reaction of antithrombin III (ATIII) with thrombin bound to the surface of the microvascular endothelium. Addition of ATIII to the system after thrombin is equilibrated with its binding sites results in inhibition of the enzyme as measured by disappearance of thrombin enzymatic activity from the circulation or by appearance of 125I-thrombin-ATIII complexes. The rate of inhibition of thrombin as reflected by either method is independent of the bound state of thrombin. Comparable results are obtained with ATIII modified at a single tryptophan residue. This modification does not alter the reaction rate of ATIII with thrombin but abolishes the capacity of heparin or heparan sulfate to enhance the reaction rate. From the kinetics and structural studies and the fit of the kinetics to a theoretical model relating binding equilibrium to thrombin inhibition, it is concluded that glycosaminoglycans are not involved in the ...
Mechanism of thrombin binding to endothelial cells
Blood, 1983
The interaction of human alpha-thrombin with mini-pig aortic endothelial cells was studied using 125I-labeled enzyme. Equilibrium between bound and free thrombin was attained within 1 min, and the Klotz-Hunston equations indicated two populations of binding sites. Approximately 30,000 sites/cell belonged to the high-affinity class with a Kd of about 3 x 10(-8) M. Modification of two lysine residues of thrombin with pyridoxal 5′-phosphate (PLP2-thrombin) destroyed the high- affinity binding and about three-fourths of the low-affinity bindings. When the lysine residue of thrombin involved in heparin binding was protected with heparin against chemical modification (PLP-thrombin), the modified enzyme remained similar to the native one with respect to cellular binding, with some loss of low-affinity binding only. Heparin, in a tenfold molar excess to enzyme, inhibited the binding of the native as well as the PLP-thrombin, whereas it did not influence the interaction between PLP2-thrombin...
An experimental model to study isolated effects of thrombin in vivo
Thrombosis Research, 2010
Background: In addition to a recognized role in the coagulation cascade and haemostasis, thrombin is known to have multiple functions. We hypothesized that protracted intravenous infusion of thrombin at steady state will allow to study isolated thrombin effects in vivo. Methods: Thrombin (0.05-0.9 U/kg/min) was continuously infused in Sprague Dawley rats over five hours (n = 38). The study consisted of three parts: dose escalation (n = 21), dose verification (n = 5) and a parallel group study to investigate whether thrombin effects can be antagonised by concomitant infusion of lepirudin (n = 12). Results: A thrombin dose of 0.9 U/kg/min decreased platelet counts by 70% compared to the control group (median 230 × 10^9/L vs. 752 × 10^9/L; p = 0.041). In accordance, infusion of 0.9 U/kg/min of thrombin decreased fibrinogen level by 75% compared to the control group (56 mg/dl vs. 220 mg/dl; p = 0.046). Cumulative thrombin doses of ≥ 0.1 U/kg/min caused bleedings but not thromboembolic events. Thrombin at doses ≥ 0.15 U/kg/min was lethal in four cases (30%). Platelet counts and fibrinogen levels after thrombin infusion correlated with bleeding events and mortality. Administration of thrombin at cumulative doses of 0.3-0.9 U/kg/min was associated with a 3 to 6.5-fold increase in IL-6 levels (139-306 pg/ml vs. 47 pg/ml, p b 0.05). In contrast, thrombin infusion did not alter other markers of inflammation (IL-10, MCP-1 or TNFalpha). In addition, lepirudin prevented thrombin-induced thrombocytopenia. Conclusion: Protracted intravenous infusion of thrombin offers a new experimental model, where consumption of fibrinogen and platelets correlates with bleedings and mortality. Infusion of thrombin increased only IL-6 levels but not other cytokines.
The Journal of clinical investigation, 1982
Two in vitro systems were used to identify an antithrombin III cofactor activity on vascular endothelium. Langendorff rat heart preparations or columns packed with endothelium cultured on microcarrier beads were perfused with mixtures of purified thrombin and antithrombin III. With each preparation, accelerated inhibition of thrombin by antithrombin III occurred during passage over endothelium. Platelet factor 4, protamine sulfate and diisopropylphosphoryl thrombin, all antagonists of the antithrombin III cofactor activity of heparin, significantly reduced the capacity of the preparation to inhibit thrombin. It is concluded that a substance with the functional properties of a stationary phase cofactor for antithrombin III is present on the microvascular endothelium and there catalyzes the inactivation of circulating free thrombin.
Endothelial Cells Exhibit Differential Chemokinetic and Mitogenic Responsiveness to α-Thrombin
Journal of Surgical Research, 1997
ate intimal regeneration through a dominant effect on Rapid regeneration of the endothelium is a critical limiting the HTR inhibitory effect on endothelial procomponent of vascular wall repair because limitations liferation. ᭧ 1997 Academic Press of this process enhance early thrombotic and vasospastic complications, as well as late sequelae of recurrent lesion formation. We have postulated that direct All forms of transcatheter or surgical-based intervenactivation of the thrombin receptor initiates both mitions for atherosclerotic occlusive disease are associtogenic and chemokinetic endothelial behavior which ated with the production of large areas of endothelial facilitates intimal repair. To characterize the role of loss. Consequently, rapid regeneration of the endothethe thrombin receptor in human endothelial cell (EC) lium is a critical component of vascular wall repair proliferation and migration, we investigated the efbecause limitations of this process enhance early fects of both a-thrombin (0.5-10 U/ml) and its receptorthrombotic and vasospastic complications. Moreover, activating peptide (TRAP; 1-100 mM). Responses of huchronic areas of endothelial denudation predispose to man aortic (HAEC) and umbilical vein (HUVEC) were the late sequelae of recurrent atherosclerosis and neocharacterized using [ 3 H]thymidine and 51 Cr microcarintimal hyperplasia. In this regard, cell migration and rier bead assays of proliferation and migration, reproliferation are important features of intimal repair spectively. Expression of motility-related genes was and clarification of the mechanisms which govern these evaluated using a ribonuclease protection assay. Thrombin exerts both of its chemokinetic and mito-responses essential for the development of rationale genic effects differentially in human endothelial cells. strategies directed at endothelial regeneration. Following 2 or 4 days in culture, HUVEC proliferation Thrombin is a multifunctional serine protease proincreased two-to threefold after exposure to thromduced during activation of the coagulation cascade at bin, primarily in the low concentration range (P õ sites of vascular wall injury. In addition to its central 0.05). However, HAEC proliferation was inhibited up role in hemostasis, thrombin has been shown to be an to 50% after a 4-day incubation period (P õ 0.005). agonist evoking diverse cellular responses which con-These mitogenic effects, including the inhibition of tribute to wound healing both at sites of vascular wall aortic endothelial cell proliferation, were reproduced, injury and elsewhere [1-3]. Many of these responses in part, by thrombin receptor activation with TRAP. most likely are mediated by a specific thrombin recep-In contrast, thrombin stimulates migratory responses tor which is a member of the seven-transmembrane in HAEC, but not HUVEC. However, this behavior was domain G-protein-coupled receptor family [4, 5]. Synnot reproduced by TRAP. It is noteworthy that urokithetic peptides mimicking the receptor's tethered linase-plasminogen activator (u-PA) expression was gand are sufficient for its activation [5]. much more strongly expressed in migrating HAEC It is well established that thrombin is a potent mithan in the HUVEC population. Moreover, when stimutogen for fibroblasts [6-8] and vascular smooth muslated with thrombin, u-PA gene expression was sigcle cells [6, 9, 10]. Its chemotactic effects on these nificantly augmented in HAEC. It has been speculated cells have also been reported [11, 12]. Endothelial that an effective human thrombin receptor (HTR) ancells, however, appear to be differentially responsive tagonist may reduce the proliferation of vascular to thrombin depending upon the organ type and spesmooth muscle cells and the development of a restecies of cell origin. For example, thrombin stimulates notic lesion following arterial wall injury. Our data growth of human umbilical vein endothelial cells suggest that such an inhibitor will likely also acceler-(HUVEC) and lung microvascular endothelial cells (MEC) while inhibiting proliferative responses of aor-1 Supported by the American College of Surgeons Faculty Fellowtic and iliac artery endothelial cells, as well as brain ship and the