Anticoagulant Activity of AT

AT, a glycoprotein, has a molecular weight of 58,200 Da with 432 amino acids and is mainly synthesized in the liver. AT is a physiological serine protease inhibitor that inhibits activated coagulation factors such as thrombin and factor Xa. The reactive site loop of AT includes a P1-P'1 (Arg393-

Ser394) bond (Figure 1). When thrombin cleaves this bond that resembles the substrate of thrombin, the protease is covalently linked to the P1 residue. Inhibition of these serine proteases by AT is accelerated approximately one-thousandfold by binding of heparin to arginine residues located at the heparin binding site of AT (Figure 1), with resultant conformational change leading to exposure of the P1-P'1 reactive center. Amino acid residues other than argi-nine shown in Figure 1 have also been found to be critical for interaction with heparin. AT is activated on the endothe-lial surface where thrombin generation is increased through binding to heparan sulfate molecules of ryudocan or synde-can. The physiological importance of AT is well illustrated by the presence of patients with congenital AT deficiency who developed recurrent thrombosis in their youth; 70 percent of the patients developed thrombosis before 35 years of age. Congenital AT deficiency usually presents as a heterozygous state associated with venous thrombosis, and the homozygous state is extremely rare, probably because of its presentation as lethal neonatal thrombosis. Similar observations have been reported for the pathologic sequelae of AT knockout mice [1]. The homozygous state of AT deficiency was only reported in patients who have variant AT molecules with heparin binding defects, and patients with such variant molecules have arterial thrombosis as well as venous thrombosis [2]. Possible mechanism(s) by which arterial

Arg13

Pro41 Arg46

Pro41 Arg46

Arg13

Heparin binding domain

Figure 1 Localization of heparin binding domains and the reactive site in the primary structure of antithrombin.

Heparin binding domain

Figure 1 Localization of heparin binding domains and the reactive site in the primary structure of antithrombin.

Enhancement of TNF-a production

Vicious cycle formation

Exudate

Figure 2 Pathologic mechanism(s) leading to the microcirculatory disturbance associated with sepsis or circulatory shock. I/R: ischemia-reperfusion.

thrombosis occurs in these patients will be discussed later in this chapter. These observations strongly suggested that AT plays a critical role in regulation of the coagulation system by inhibiting thrombin and other serine proteases, and the interaction of AT with the endothelial cell surface heparin-like substances is quite important for rapid and effective inhibition of such coagulation factors.

Anti-Inflammatory Activity of AT

Activation of the coagulation system leading to disseminated intravascular coagulation is frequently seen as a part of the inflammatory responses in pathologic conditions such as sepsis and circulatory shock. In these inflammatory responses, proinflammatory cytokines such as tumor necro sis factor-a (TNF-a) play an important role [3]. Although TNF-a is implicated in the activation of the coagulation system in sepsis, it also activates neutrophils, thereby promoting the release of a wide variety of inflammatory mediators such as neutrophil elastase and free oxygen radicals that are capable of damaging endothelial cells. The resultant endothelial cell damage increases microvascular permeability, leading to microcirculatory disturbance due to the hemo-concentration. Such microcirculatory disturbance precedes microthrombus formation [4]. Microthrombus formation further increases TNF-a production, thereby exacerbating the inflammatory responses to form a vicious cycle of progression of microcirculatory disturbances [5] (Figure 2).

We previously reported that AT reduced pulmonary endothelial injury by inhibiting neutrophil accumulation in the lung of rats given endotoxin. AT also reduced endotoxin-

induced hypotension in rats by inhibiting TNF-a production [6]. These therapeutic effects of AT could not be explained by the anticoagulant activity, but by its promotion of endothelial production of prostacyclin (PGI2), which potently inhibits leukocyte activation. Interaction of AT with glycosaminoglycans might be critical for promotion of endothelial production of PGI2. Ischemia-reperfusion is an important pathologic mechanism leading to the development of coagulation abnormalities and organ failure seen in patients with circulatory shock. TNF-a is also critically involved in this pathologic process. AT increased the hepatic tissue blood flow by inhibiting neutrophil activation in rats subjected to hepatic ischemia-reperfusion. AT also increased the renal tissue blood flow by inhibiting neu-trophil activation through inhibition of TNF-a production, thereby reducing renal injury [7]. These effects of AT were also independent of its anti-coagulant activity, but dependent on its capacity to promote endothelial production of PGI2. Although AT itself has been shown to inhibit TNF-a production by monocytes stimulated with endotoxin in vitro, inhibition by AT of TNF-a production in vivo was not observed when animals were pretreated with indomethacin, which inhibits prostaglandin biosynthesis. Iloprost, a stable derivative of PGI2, produced effects similar to those of AT in these animal models of sepsis and in those subjected to tissue ischemia-reperfusion. These observations strongly suggested that anti-inflammatory activity of AT might be mediated by PGI2 released from endothelial cells.

sensitive sensory neurons were activated during hepatic ischemia-reperfusion or water-immersion restraint stress in rats, leading to an increase in the endothelial production of PGI2 via activation of both endothelial nitric oxide synthase (NOS) and cyclooxygenase (COX)-1 [8] (Figure 3).

We previously reported that AT reduced ischemia-reperfusion-induced liver injury in rats by increasing endothelial production of PGI2. However, the mechanism(s) underlying this phenomenon remains to be fully elucidated. AT significantly enhanced the ischemia-reperfusion-induced increase in hepatic tissue levels of CGRP in rats. The increase in hepatic tissue levels of 6-keto-PGF1a, a stable metabolite of PGI2, increase in hepatic-tissue blood flow, and attenuation of both hepatic local inflammatory responses and liver injury in rats administered AT were completely reversed by administration of capsazepine, an inhibitor of the sensory neuron activation and CGRP(8-37), a CGRP antagonist. AT significantly increased CGRP release from cultured dorsal root ganglion neurons isolated from rats in the presence of capsaicin. These observations strongly suggested that AT might extravasate at the site of endothelial cell injury, thereby enhancing the activation of capsaicin-sensitive sensory neurons leading to increase in hepatic tissue levels of PGI2 [9] (Figure 3).

Essentials of Human Physiology

Essentials of Human Physiology

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