Experimental studies of the regulations of synthesis and release of tissue-type plasminogen activator from vascular endothelium

Abstract

The fibrinolytic system has several important functions and one crucial function is the prevention and removal of fibrin deposits in blood vessels. Tissue-type plasminogen activator (t-PA) plays a key role in this process, and in vitro data suggest that an acute release of t-PA is induced as a thromboprotective response whenever clotting mechanisms are initiated. However, the physiological regulation of its synthesis and secretion is incompletely understood. The aim of the present study was to develop human models appropriate for studies of the regulation of fibrinolytic proteins across an organ or in an intact blood vessel.The first two studies were performed in a human forearm model, which allows measurement of local release of t-PA in vivo. In essence, the model is based on simultaneous arterial and venous sampling from the antecubital region, and a simultaneous measurement of forearm blood flow. Net release per unit of time is calculated from the product of the arterio-venous concentration gradient of the respective protein and local plasma flow. Stimulated release of t-PA from the endothelium was triggered by mental stress and intraarterial infusions of methacholine (Mch), norepinephrine (NE), and the endothelium-independent vasodilator sodium nitroprusside (SNP). During basal conditions there was a net release of t-PA antigen of approximately 0.7 ng/min and dL of tissue. Mental stress and NE infusion induced a moderate increase, whereas infusion of Mch induced a 10-fold increase of the t-PA release rate. Increased flow by SNP had little effect.Biomechanical forces imposed on the vessel wall by flow and pressure have been suggested to regulate expression and release of antithrombotic factors. However, effects of biomechanical stimuli can only to a very limited extent be investigated in in vitro systems with cultured endothelial cells (ECs). To this end, a new computerised ex vivo perfusion system for intact conduit vessels was developed. Using quantitative real-time RT-PCR, we found that t-PA gene expression in human ECs was upregulated after 6, but not 1,5 or 3, hours exposure to high shear stress (25 dyn/cm2). Gene induction was associated with a marked increase in the intracellular storage pool of t-PA as assessed by immuno-histo-chemistry. Thus, shear stress appears to enhance the amount of t-PA available for acute stimulated release. Elevated intraluminal pressure was associated with a different pattern. In vessels exposed to high intraluminal pressure at physiological levels of shear stress, t-PA gene expression was suppressed and t-PA release from the endothelium was almost completely inhibited. This finding is corroborated by our recent finding that patients with essential hypertension have a markedly impaired capacity for t-PA release.In conclusion, these findings show that the healthy endo-thelium has a remarkable capacity for release of t-PA in vivo. Further, our findings show that high levels of shear stress, i.e. an inappropriately narrow lumen radius in relation to the prevailing flow, is a stimulus for induction of genes involved in the integrated thromboprotective program. By contrast, high intraluminal pressure causes repression of important thromboprotective factors.