Regulation of Thrombin Generation and Functions

Thrombin (EC 3.4.21.5) is the activation product of prothrombin. Unlike related serine proteinases of the blood coagulation pathways, procoagulant αthrombin has several seemingly diverse, yet central functions in hemostasis. It also participates in wound healing and is implicated in various disease processes. Human disability and death attributable to throm­ botic or hemostatic disorders (such as coronary throm­ bosis, pulmonary embolism, stroke, atherosclerosis) are of considerable social and economic concern. Yet, despite involvement in such maladies, its underlying functions appear to be those of controlling normal physiologic processes for the benefit and survival of higher animals. Hemostasis is a necessary adaptation for the con­ tainment of circulating blood in order to supply nu­ trients and remove wastes from dependent tissues. Thrombin functions at all three levels of hemostasis and does so by enzymic (proteolytic), nonenzymic (hormonal), or coupled mechanisms. In the case of coupled mechanisms, both the proteolytic active site and a noncontiguous site (exosite) are involved, such as a-thrombin cleavage of fibrinopeptide A from fi­ brinogen and stimulation of platelets, as well as other cells. Such mechanisms, including use of the anion-binding exosite, are among the distinguishing properties of α-thrombin from those of the pancreatic serine proteinases with primarily digestive functions (such as α-chymotrypsin, β-trypsin). Whereas α-thrombin is a multifunctional en­ zyme, its physiologic control is to a large part directed at regulation of thrombin generation. First of all, pro­ thrombin is a vitamin K-dependent protein synthe­ sized by the parenchymal cells in the liver, where the vitamin is required for postribosomal formation of ten 7-carboxylglutamic acids. The vitamin K antagonist warfarin has been a widely used rodenticide, as well as an orally administered pharmaceutical agent for treatment of patients with thrombotic ten­ dencies. Prothrombin circulates in plasma with a half-life of about 3 days. The desialidation of its three carbohydrate side chains is presumptively im­ portant in determining its resorption by the liver. On the other hand, the single carbohydrate side chain retained by thrombin (Fig. 1) has no known function and is removed from active-site regions. Plasma contains sufficient prothrombin to gener­ ate 150,000 US NIH units of α-thrombin per liter, corresponding to 1.37 μM. During the activation in whole blood or recalcified plasma only approximately 10% of the potential α-thrombin concentration is ever achieved, despite more than 80% consumption of the proenzyme. ' This apparent consumption of αthrombin is believed to be largely due to its active incorporation into fibrin clots, where it is sequestered and can be released on fibrinolysis. Plasma con­ tains various proteinase inhibitors (such as antithrombin III, heparin cofactor II, α2-macroglobulin) that inactivate various thrombin forms. Cultured fibro­ blasts release into media various proteinase inhibitors, or "nexins," which also inhibit thrombin. Further­ more, the activities of the nexin PN1 are enhanced by heparin, analogous to antithrombin III. Heparin is a sulfonated, negatively charged, heterogeneous car­ bohydrate found on the surface of various cells (such as endothelial cells). It binds both α-thrombin and antithrombin III and does so in a catalytic manner, where a minimum of approximately 18 saccharide units are needed. In plasma, heparin greatly en­ hances the activity of antithrombin III in thrombin inactivation. Perhaps its primary function, when ad­ ministered therapeutically, is to lower α-thrombin concentrations to those where it no longer activates From the Wadsworth Center for Laboratories and Research, School of Public Health Sciences, New York State Department of Health, and the Department of Physiology, The Albany Medical College of Union University, Albany, New York. Reprint requests: Dr. Fenton, Wadsworth Center for Labo­ ratories and Research, New York State Department of Health, Empire State Plaza, Albany, NY 12201.