Storage of Tissue-Type Plasminogen Activator in Weibel-Palade Bodies of Human Endothelial Cells

Abstract

Abstract-Tissue-type plasminogen activator (t-PA) is acutely released by endothelial cells. Although its endothelial storage compartment is still not well defined, t-PA release is often accompanied by release of von Willebrand factor (vWf), a protein stored in Weibel-Palade bodies. We investigated, therefore, whether t-PA is stored in these secretory organelles. Under basal culture conditions, a minority of human umbilical vein endothelial cells (HUVEC) exhibited immunofluorescent staining for t-PA, which was observed only in Weibel-Palade bodies. To increase t-PA expression, HUVEC were infected with a t-PA recombinant adenovirus (AdCMVt-PA issue-type plasminogen activator (t-PA) is a key enzyme for the removal of incipient thrombi in the vascular system, and recombinant t-PA is now widely used for the thrombolytic therapy of myocardial infarction. Endothelial cells (EC) are the main source for plasma t-PA. 1-4 Although a variety of stimuli such as venous occlusion, exercise, or injection of vasoactive substances are known to acutely increase plasma levels of t-PA, the precise mechanisms responsible for this increase are poorly defined. They may involve an acute release of t-PA from EC, variations in the rate of production of t-PA by EC, or changes in the clearance rate. 5,6 Several in vivo and ex vivo studies give clear evidence for the occurrence of acute release of t-PA. Experimental induction of disseminated intravascular coagulation in chimpanzees or baboons results in a 50-fold increase in t-PA plasma levels within a few minutes. 7,8 The rapidity and the magnitude of the increase in the t-PA concentration, as well as the rapid return to normal levels, are consistent only with a massive release of t-PA from storage pools. The findings that injection of vasoactive agents such as thrombin or calcium ionophore leads to an acute increase of t-PA in isolated vascular systems are also consistent with an endothelial t-PA storage pool. 9 -11

Differential regulation of endothelial exocytosis of P-selectin and von Willebrand factor by protease-activated receptors and cAMP

“…32,52 Weibel-Palade bodies also contain premature von Willebrand factor, interleukin-8, endothelin-1, ␣1,3 fucosyltransferase, CD63, 53,54 and possibly tissue plasminogen activator (t-PA). 40,55 Our findings raise the question of whether the other contents of WPBs also display differential signaling. Endothelial t-PA is released in response to cAMP-raising agents, suggesting that t-PA secretion more closely resembles VWF than P-selectin.…”

“…38 However, this observation is controversial because other studies have shown that t-PA is stored in WPBs in HUVECs. [39][40][41] Indeed, some of the observed differences in localization of t-PA have been ascribed to subtle differences in tissue culture methods. 39 Accordingly, we examined the localization of VWF and P-selectin in our cell-culture system by performing immunostaining and confocal microscopy.…”

Inhibition of carboxypeptidase U (TAFIa) activity improves rt-PA induced thrombolysis in a dog model of coronary artery thrombosis

“…By constitutive secretion, the newly synthesised protein continuously leaves the Golgi compartment in transport vesicles that fuse with the cell membrane. By regulated secretion, on the other hand, t-PA is released from small dense vesicles and/or the Weibel-Palade bodies [38][39][40]. The intracellular storage pool of t-PA is quite large, and it follows that the regulated pathway allows local intravascular t-PA concentrations to increase rapidly and substantially [41,42].…”

The influence of unfractionated and low-molecular weight heparins on the properties of human umbilical vein endothelial cells (HUVEC).

“…The studies were also carried out to discover the mechanism responsible for the release of t-PA. They confirmed its release from endothelial cell and additionally indicated Weibel-Pallade bodies as the site of t-PA reservoirs (similarly to vWF) [14,15].…”