Hepatocyte-Derived Tissue Plasminogen Activator Regulates Systemic Fibrinolysis

Abstract

Tissue-type plasminogen activator (tPA) is a secreted serine protease that initiates the dissolution of a fibrin clot in a process called fibrinolysis. When a fibrin clot forms on the wall of an injured vessel, tPA binds to the fibrin and converts plasminogen to plasmin, which proteolytically degrades the fibrin clot. tPA is released locally by vascular endothelial cells in response to injury, preventing excessive fibrin deposition and thrombosis. However, freshly isolated blood from healthy subjects undergoes spontaneous fibrinolysis, suggesting the presence of basal tPA, i.e., tPA activity present before injury occurs. A major gap in fibrinolysis research is the roles, regulation, and sources of basal plasma tPA. A key question that raises the possibility that a non-endothelial source of tPA may be important, is how medium-sized and large vessels respond to injury with respect to fibrinolysis. Endothelial cells of these vessels express less tPA than small vessels, and the tPA that is secreted by these cells gets rapidly diluted owing to the rapid flow and a much lower surface-to-volume ratio of large vessels. Thus, a non-endothelial source of tPA may be necessary for limiting clot extension and thrombosis after an injury to medium-sized and large arteries.The fibrinolytic activity of plasma is determined mostly by the relative concentrations of plasma tPA and its inhibitor, plasminogen activator inhibitor-1. Longitudinal studies have revealed an association between lower plasminogen activator and fibrinolytic activity in plasma with future recurrent myocardial infarction. Moreover, basal plasma fibrinolytic activity was shown to have a diurnal variation with a nadir in the morning, which is the time of day of highest risk for coronary artery disease. More recently, studies have shown that low plasma tPA activity per se predicts cardiovascular disease in humans. Collectively, these studies suggest that basal plasma fibrinolytic activity, determined in part of tPA concentration, is a functionally important mechanism to prevent pathological fibrin clot formation and thrombosis.Although hepatocytes have been shown to express tPA protein and mRNA, the fibrinolytic function of hepatocyte tPA, either in the liver or systemically, and its mode of regulation remain unknown. Therefore, we conducted a series of tests to explore the relative level of tPA expression by hepatocytes and its role in wildtype mice. Using flow cytometry, we found that hepatocytes express high levels of tPA relative to non-parenchymal cells of liver. Comparing with well-known sources of tPA, the levels of tPA mRNA, immunoreactive tPA protein, and tPA activity in liver were comparable to those in arterial tissue. By silencing tPA specifically in hepatocytes, we were able to lower plasma tPA protein by ~50% and lower plasma tPA activity by ~30%. Despite this only partially silencing efficiency, there was a 43% decrease in bleeding time (P=0.0003) and a 25% decrease in the time to occlusive carotid arterial thrombosis induced by photochemical injury (P=0.001).tPA is released rapidly from endothelial storage granules in response to endothelial injury-induced thrombosis, which we predicted would not be affected by silencing hepatocyte tPA. Indeed, the data show that although tPA release after FeCl3-induced carotid thrombosis varied among individual mice in both cohorts, the average release values were similar between control and hepatocyte-tPA-silenced mice. These data suggest that the release of tPA from the endothelium after injury is independent of hepatocyte tPA and that hepatocyte tPA works in concert with tPA released from the endothelium to affect post-injury fibrinolysis. As a final test of the role of hepatocyte tPA, we restored tPA only in the hepatocyte of holo-tPA-KO mice, resulting a substantial increase in plasma tPA protein and a significant decrease in clot lysis time.In summary, we show that hepatocytes are a significant source of plasma tPA protein and tPA-mediated fibrinolytic activity under basal conditions and that hepatocyte-derived tPA complements locally released vessel wall-derived tPA to limit clotting after arterial injury. We imagine that these insights and ones that will follow from future studies in this area will suggest new therapeutic strategies for diseases of hemostatic imbalance. DisclosuresNo relevant conflicts of interest to declare.