Binding of thrombin to thrombomodulin accelerates inhibition of the enzyme by antithrombin III. Evidence for a heparin-independent mechanism

Abstract

The endothelial cell surface provides a receptor for thrombin-designated thrombomodulin (TM) which regulates thrombin formation and the activity of the enzyme at the vessel wall surface by serving as a potent cofactor for the activation of protein C by thrombin. Heparin-like structures of the vessel wall have been proposed as another regulatory mechanism catalyzing the inhibition of thrombin by antithrombin III. In the present study, the interaction of antithrombin III with the thrombin-TM complex and its interference with heparin and polycations were investigated by using human components and TM isolated from the microvasculature of rabbit lung. Purified TM bound thrombin and acted as a cofactor for protein C activation. The addition of heparin (0.5 unit/mL) to the reaction mixture interfered neither with the binding of thrombin to TM nor with the activation of protein C. However, the polycations protamine (1 unit/mL) as well as polybrene (0.1 mg/mL) affected the thrombin-TM interaction. This was documented by an increase in the Michaelis constant from 8.3 microM for thrombin alone to 19.5 microM for thrombin-TM with the chromogenic substrate compound S-2238 in the presence of 1 unit/mL protamine. When the inhibition of thrombin by antithrombin III was determined, the second-order rate constant k2 = 8.4 X 10(3) M-1 s-1 increased about 8-fold in the presence of TM, implying an accelerative function of TM in this reaction. Although purified TM did not bind to antithrombin III-Sepharose, suggesting the absence of heparin-like structures within the receptor molecule, protamine reversed the accelerative effect of TM in the inhibition reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Normal levels of anticoagulant heparan sulfate are not essential for normal hemostasis

“…Again, anticoagulant activity was not tightly linked to HS act production levels. This lack of correlation is consistent with observations that HS act is not the sole endothelial component capable of catalyzing AT activity (12,13).…”

“…The above examples, however, do not conclusively establish a critical role for HS act , because AT neutralization of T can also be enhanced by thrombomodulin bearing a chondroitin sulfate chain (12,13). Moreover, the probing of blood vessels with 125 I-AT reveals more than 95% of HS act are localized to the abluminal endothelial surface (14).…”

Detection of vitronectin mRNA in tissues and cells of the mouse.

“…For example, the Arg-Gly-Asp-containing sequence in Vn is recognized by receptors (integrins) on various cell types (4)(5)(6), and Vn is identical to the S protein of the complement cascade and thus inhibits complement-mediated cell lysis (7)(8)(9)(10). Vn also may regulate the blood coagulation pathway by inhibiting the rapid inactivation of thrombin by antithrombin in the presence of heparin (11,12). Vn was recently demonstrated to bind to and stabilize type 1 plasminogen activator inhibitor (PAI-1) (13)(14)(15)(16) and to alter the specificity (16) of this physiological inhibitor of both urinary-and tissue-type plasminogen activators (for review, see ref.…”

Eosinophil cationic granule proteins impair thrombomodulin function. A potential mechanism for thromboembolism in hypereosinophilic heart disease.

“…Moreover, at concentrations ofMBP > 10 -6 M the activities ofTMD-105 and TMD-75 are identical, suggesting that at these higher concentrations MBP obliterates any influence of the GAG moiety upon TM activity, and thereby renders it in effect functionally identical to TM-75. Second, in addition to impairing APC generation by TM, EPO and MBP also completely abrogate the capacity of fully glycosylated TM to prolong the thrombin clotting time (Table III), as do other cationic proteins (16)(17)(18)21 ). Others have previously noted that the ability of TM to prolong the thrombin clotting time is strongly influenced by presence ofthe GAG moiety, because its removal by chondroitinase ABC severely restricts ( 18,22) the capacity of TM to perform this function.…”

“…Of note, the complete TM molecule is quite anionic (pI 4 [131]), in part because ofextensive posttranslational glycation of the large extracellular domain of TM with an unusual hypersulfated, chondroitin sulfate E-like moiety (14)(15)(16)(17)(18)(19)(20). This bulky polyanionic domain strongly influences all three known anticoagulant functions of TM (14)(15)(16)(17)(18)(19)(20)(21)(22)(23).…”