Endothelial cell activation in thrombotic thrombocytopenic purpura (TTP): a prospective analysis.

It is generally assumed that endothelial cell injury is the primary event in the pathogenesis of thrombotic thrombocytopenic purpura (TTP). In this study, we have determined the extent of vascular perturbation during acute episodes of the disease. We performed a prospective, serial study of nine patients with relapsing TTP during hospitalization and treatment, and assessed the degree of endothelial cell involvement at admission, exacerbation and remission by measurement of von Willebrand factor (VWF) and VWF-propeptide levels. Measurement of both VWF and its propeptide enabled discrimination between acute and chronic perturbation of the endothelium. Elevated levels of both VWF and propeptide were found at admission. These levels decreased immediately upon plasma exchange therapy. However, plasma VWF and propeptide concentrations did not change, even at the time of acute exacerbation. These observations suggest that endothelial cell activation is not the primary event leading to TTP. Vascular perturbation seems to be a consequence, rather than a cause, of the disease.

Von Willebrand factor targets IL-8 to Weibel-Palade bodies in an endothelial cell line.

Vascular endothelial cells are able to store the chemotactic cytokine interleukin-8 (IL-8) in specialized storage vesicles, Weibel-Palade bodies, together with von Willebrand factor (VWF) and P-selectin. We investigated whether VWF plays a role in the sorting of IL-8 into these organelles. We examined the effect of VWF expression on IL-8 targeting in an endothelial cell line (EC-RF24). This cell line has retained the typical phenotypic characteristics of primary endothelial cells but has lost the capacity to produce VWF in appreciable amounts. EC-RF24 cells were retrovirally transduced with a vector encoding a VWF-green fluorescent protein chimera (VWF-GFP). This approach enables direct visualization of the cellular distribution and secretory behavior of the VWF-GFP hybrid. Expression of VWF-GFP resulted in the generation of Weibel-Palade body-like organelles as shown by the colocalization of VWF-GFP and P-selectin. VWF-GFP expressing EC-RF24 cells also showed significant colocalization of VWF-GFP with IL-8 in these storage vesicles. Live cell imaging revealed that the number of VWF-GFP-containing granules decreased upon cell stimulation. These observations indicate that VWF plays an active role in sequestering IL-8 into Weibel-Palade bodies.

Real-time imaging of the dynamics and secretory behavior of Weibel-Palade bodies.

OBJECTIVE: Weibel-Palade bodies (WPBs) are specialized secretory granules found in endothelial cells. These vesicles store hormones, enzymes, and receptors and exhibit regulated exocytosis on cellular stimulation. Here we have directly visualized intracellular trafficking and the secretory behavior of WPBs in living cells by using a hybrid protein consisting of von Willebrand factor (vWF), a prominent WPB constituent, and green fluorescent protein (GFP). METHODS AND RESULTS: Immunofluorescence microscopy demonstrated that this chimera was targeted into WPBs. In resting cells, some WPBs seemed motionless, whereas others moved at low speed in a stochastic manner. On stimulation of cells with [Ca2+]i- or cAMP-raising secretagogues, membrane-apposed patches were formed, suggesting fusion of WPBs with the plasma membrane. Patches remained visible for >20 minutes. This sustained, membrane-associated retention of vWF might play a role in focal adhesion of blood constituents to the endothelium after vascular injury. In addition, stimulation with cAMP-raising agonists resulted in clustering of a subset of WPBs in the perinuclear region of the cell. Apparently, these WPBs escaped secretion. This feature might provide a mechanism to control regulated exocytosis. CONCLUSIONS: In conclusion, the fusion protein vWF-GFP provides a powerful tool to study, in real time, signal-mediated trafficking of WPBs.

Biogenesis and exocytosis of Weibel-Palade bodies.

Vascular endothelial cells contain typical, elongated vesicles, the so-called Weibel-Palade bodies, which serve as a storage compartment for von Willebrand factor (VWF), a plasma protein that plays an essential role in controlling the adhesion and aggregation of platelets at sites of vascular injury. Upon activation of endothelial cells by agonists such as thrombin, epinephrine or histamine, the Weibel-Palade bodies fuse with the plasma membrane and release their contents into the blood circulation. This process provides an adequate means by which endothelial cells can actively participate in controlling the arrest of bleeding upon vascular damage. Besides VWF, Weibel-Palade bodies contain a subset of other proteins, including interleukin-8 (IL-8), P-selectin and endothelin. Similar to VWF, these proteins are transported to the outside of the cell upon stimulation and may control local or systemic biological effects, including inflammatory and vasoactive responses. Apparently, endothelial cells are able to create a storage pool for a variety of bioactive molecules which can be mobilised upon demand. Endothelial cells that are deficient of VWF synthesis are not only unable to form Weibel-Palade bodies, but also lack the ability to store IL-8 or P-selectin or release these proteins in a regulated manner. It thus appears that VWF not only plays a prominent role in controlling primary haemostasis, but also may modulate inflammatory processes through its ability to target inflammatory mediators to the regulated secretion pathway of the endothelium.