Is protein C inhibitor antithrombotic and protective in pulmonary hypertension

In this issue, Nishii et al. [1] from Mie University in Japan provide interesting evidence that mice over-expressing protein C inhibitor (PCI) are protected from monocrotaline-induced pulmonary hypertension, due in part to the ability of PCI to inhibit thrombin and down-regulate coagulation. The transgenic mouse over-expressing PCI has enhanced lung secretion of PCI, and the clinical, biochemical and pathological parameters show a reduction in pulmonary hypertension in this experimental model. The pathophysiological role of the human plasma serine protease inhibitor (serpin), protein C inhibitor (PCI, also named plasminogen activator inhibitor-3, systematic name of SERPINA5) has remained elusive since its description in the 1980s [2,3]. From its name, PCI is an inhibitor of activated protein C (APC) [4–14]. However, PCI also inhibits thrombin (IIa) [8–10,12], factor Xa and factor XIa [8,11–14], kallikrein [8,10,12,15–17], urokinase-plasminogen activator and tissue-plasminogen activator (t-PA) [8,18–23], acrosin [24–26], prostate specific antigen [27,28], and remarkably, thrombin-thrombomodulin [29–31], which is responsible for generating APC (Fig. 1). The broad protease inhibitory profile of PCI has led many to postulate both specific and generic roles for this serpin. To further complicate matters, the tissue distribution of PCI in humans compared with mice is quite different. Humans show a broad tissue expression pattern for PCI, including the liver, kidney, pancreas, prostate, testes and ovaries [32–37]. Thus, this explains why human PCI (hPCI) is found not only in circulating blood, but also in urine, saliva, amniotic fluid, milk, tears and other body fluids [32]. In contrast, the mouse and rat express PCI only in the reproductive organs and it is not found in the circulating blood [34,38–42]. Through the creation of a PCI knockout mouse by homologous recombination, one non-hemostatic function of PCI was determined [26]. Male PCI−/− mice were infertile due to abnormal spermatogenesis caused by loss of the Sertoli cell barrier. Unopposed proteolytic activity in these mice brought about the degradation of the cell barrier [26]. Two transgenic mouse models expressing hPCI have been developed. The first was described by Wagenaar et al. [43], in which hPCI was expressed in the liver and found in the circulation. The second hPCI transgenic mouse was described by Hayashi et al. [44] and it expressed hPCI not only in the liver, but also in the kidney, heart, brain, lung and reproductive organs. Fig. 1 Role of protein C inhibitor and other serpins (antithrombin, heparin cofactor II, and plasminogen activator inhibitor-1) in the regulation of serine proteases thrombin, activated protein C (APC), APC generation by thrombin-thrombomodulin, and tissue plasminogen ... Concerning human health, the presence of PCI in various lung diseases has been described [45]. Fujimoto et al. [45] reported that bronchoalveolar lavage fluid contained increased amounts of both PCI and thrombin-activatable fibrinolysis inhibitor (TAFI) in patients with interstitial lung disease (ILD), particularly in patients with cryptogenic-organizing pneumonia, collagen vascular disease-associated ILD, and sarcoidosis. One explanation of their findings was that the levels of intra-alveolar PCI inhibited both APC activity and activation, which contributed to the pathogenesis of ILD. Therefore, a key question asked in the current study by Nishii et al. [1] was concerning the contribution of PCI to the pathogenesis of pulmonary hypertension. This study uses the hPCI over-expressing transgenic mouse described by Hayashi et al. [44] to begin to address this question regarding pulmonary hypertension, and also provides data on the physiological function of PCI (Fig. 1). Nishii et al. [1] treat mice with monocrotaline to induce pulmonary hypertension. This murine model is representative of pulmonary hypertension caused by a known etiology and not a secondary consequence of cardiovascular disease. Overall, hPCI reduces the disease state in the mouse lung compared with the wild-type mouse. The increase in pressure associated with pulmonary hypertension is not seen in the hPCI over-expressing transgenic mice. Pulmonary hypertension also results in endothelium dysfunction. The vessels in the lungs are hypercoagulant as a result of a decrease in prostaglandin and nitric oxide production. Platelets become activated and will adhere to the vessel wall. Hypercoagulability can be assessed by measuring the formation of thrombin:AT (TAT) complex. Although there is an increase of TAT complex in the hPCI over-expressing transgenic mice when treated with monocrotaline, this increase is significantly less than that in wild-type animals. These results suggest that either there is a decrease in thrombin production that would reduce TAT levels, or the increased presence of PCI is competing with AT to inhibit thrombin, which would also reduce the TAT levels. Either way, the hPCI over-expressing transgenic mouse does not exhibit an increase in activation of coagulation upon treatment with monocrotaline. Although PCI can be procoagulant through its inhibition of the protein C system of proteases, when hPCI is over-expressed in the mouse, its anticoagulant function is more prominent. Fibrinolysis is increased in mice upon treatment with monocrotaline, as indicated by an increase in t-PA activity. As PAI-1 levels are similar between the wild-type and hPCI over-expressing transgenic mice when treated with monocrotaline, the only explanation for a decrease in free t-PA and lowered fibrinolysis in the transgenic mice is the increase in hPCI. Pulmonary vascular endothelium dysfunction also results in the release of various cytokines, such as tumor necrosis factor-alpha (TNF-α) and monocyte chemoattractant protein-1 (MCP-1), and growth factors, such as platelet-derived growth factor (PDGF) and interleukin-13 (IL-13), that will promote inflammation and vascular wall thickening. The monocrotaline-treated wild-type mice show an increase in all of these protein levels. The hPCI over-expressing transgenic mice treated with monocrotaline exhibit little to no change in these same proteins. Therefore, hPCI reduces the endothelial dysfunction and inflammation associated with pulmonary hypertension. Furthermore, measurements of the pulmonary arteries in the hPCI over-expressing transgenic mouse show a smaller change in vessel wall and lumen area upon monocrotaline treatment. Overall, their results suggest that hPCI inhibits thrombin, a pro-inflammatory and pro-migratory factor, thus, reducing the effects of monocrotaline-induced pulmonary hypertension. The data presented in this paper raise more questions than answers about the in vivo protease specificity of PCI. Their results suggest that PCI exerts both anti-inflammatory and anticoagulant action by inhibiting thrombin, a known participant in coagulation, inflammation and tissue remodeling. This function of PCI is more prominent than its role as an inhibitor of APC. Inhibition of APC alone would result in an increase in coagulation and inflammation, and a reduction of tissue remodeling, which was not seen in the mouse model described. PCI is also antifibrinolytic through its inhibition of t-PA. Whether or not PCI can be used therapeutically for treating pulmonary hypertension remains to be studied. With the aid of new ELISAs reported for PCI-protease complexes [46–49], these tools can provide answers regarding the role of PCI in coagulation and inflammation.