Cellular uptake of coagulation factor VIII: Elusive role of the membrane-binding spikes in the C1 domain.

Low density lipoprotein receptor-related protein 1 (LRP1) is involved in the catabolism of many ligands, including factor VIII (FVIII) and alpha-2-macroglobulin (α2M). Transfer of FVIII to LRP1 is currently believed to be preceded by pre-concentration on the cell surface, by interacting with a so far unidentified component. In the present study, we used confocal microscopy and flow cytometry to compare endocytosis of FVIII and α2M using U87MG cells. The results show that α2M is rapidly internalized and does not compete for LRP1 mediated internalization of FVIII. FVIII endocytosis did not occur in the presence of receptor-associated-protein (RAP), but FVIII remained visible as a striated fluorescent pattern at the cell borders. In the presence of Von Willebrand Factor (VWF), no FVIII was observed on or within the cells, suggesting that VWF blocks interaction with both cell surface and LRP1. The same dual inhibition has previously been observed for FVIII C1 domain directed monoclonal antibody KM33. Elimination of the KM33 epitope by replacing FVIII C1 residues 2091-2095 and 2155-2160 for the homologues from factor V (FV), however, did not impair FVIII endocytosis. These membrane spikes alone were insufficient for cellular uptake, because FV was neither internalized by U87MG cells nor capable of effectively competing for FVIII endocytosis. These results show that FVIII endocytosis is driven by interaction with LRP1, but at the same time involves the spikes in the C1 domain that have been implicated in lipid binding.

Guanine exchange factor RalGDS mediates exocytosis of Weibel-Palade bodies from endothelial cells.

The small GTP-binding protein Ral has been implicated in regulated exocytosis via its interaction with the mammalian exocyst complex. We have previously demonstrated that Ral is involved in exocytosis of Weibel-Palade bodies (WPBs). Little is known about intracellular signaling pathways that promote activation of Ral in response to ligand binding of G protein-coupled receptors. Here we show that RNAi-mediated knockdown of RalGDS, an exchange factor for Ral, results in inhibition of thrombin- and epinephrine-induced exocytosis of WPBs, while overexpression of RalGDS promotes exocytosis of WPBs. A RalGDS variant lacking its exchange domain behaves in a dominant negative manner by blocking release of WPBs. We also provide evidence that RalGDS binds calmodulin (CaM) via an amino-terminal CaM-binding domain. RalGDS association to CaM is required for Ral activation because a cell-permeable peptide comprising this RalGDS CaM-binding domain inhibits Ral activation and WPB exocytosis. Together our findings suggest that RalGDS plays a vital role in the regulation of Ral-dependent WPB exocytosis after stimulation with Ca(2+)- or cAMP-raising agonists.

Dynamics and plasticity of Weibel-Palade bodies in endothelial cells.

Agonist-induced release of endothelial cell specific storage granules, designated Weibel-Palade bodies (WPBs), provides the endothelium with the ability to rapidly respond to changes in its micro-environment. Originally being defined as an intracellular storage pool for von Willebrand factor (VWF), it has recently been shown that an increasing number of other components, including P-selectin, interleukin (IL)-8, eotaxin-3, endothelin-1, and angiopoietin-2, is present within this subcellular organelle, implicating a role for WPB exocytosis in inflammation, hemostasis, regulation of vascular tone and angiogenesis. Recent studies emphasize that WPBs provide a dynamic storage compartment whose contents can be regulated depending on the presence of inflammatory mediators in the vascular micro-environment. Additionally, release of WPBs is tightly regulated and feedback mechanisms have been identified that prevent excessive release of bioactive components from this subcellular organelle. The ability to regulate both contents and exocytosis of WPBs endows these endothelial cell specific organelles with a remarkable plasticity. This is most likely needed to allow for controlled delivery of bioactive components into the circulation on vascular perturbation.

KLF2 provokes a gene expression pattern that establishes functional quiescent differentiation of the endothelium.

The flow-responsive transcription factor KLF2 is acquiring a leading role in the regulation of endothelial cell gene expression. A genome-wide microarray expression profiling is described employing lentivirus-mediated, 7-day overexpression of human KLF2 at levels observed under prolonged flow. KLF2 is not involved in lineage typing, as 42 endothelial-specific markers were unaffected. Rather, KLF2 generates a gene transcription profile (> 1000 genes) affecting key functional pathways such as cell migration, vasomotor function, inflammation, and hemostasis and induces a morphology change typical for shear exposure including stress fiber formation. Protein levels for thrombomodulin, endothelial nitric oxide synthase, and plasminogen activator inhibitor type-1 are altered to atheroprotective levels, even in the presence of the inflammatory cytokine TNF-alpha. KLF2 attenuates cell migration by affecting multiple genes including VEGFR2 and the potent antimigratory SEMA3F. The distribution of Weibel-Palade bodies in cultured cell populations is normalized at the single-cell level without interfering with their regulated, RalA-dependent release. In contrast, thrombin-induced release of Weibel-Palade bodies is significantly attenuated, consistent with the proposed role of VWF release at low-shear stress regions of the vasculature in atherosclerosis. These results establish that KLF2 acts as a central transcriptional switch point between the quiescent and activated states of the adult endothelial cell.

Dynein-dynactin complex mediates protein kinase A-dependent clustering of Weibel-Palade bodies in endothelial cells.

OBJECTIVE: Perinuclear clustering is observed for several different organelles and illustrates dynamic regulation of the secretory pathway and organelle distribution. Previously, we observed that a subset of Weibel-Palade bodies (WPBs), endothelial cell-specific storage organelles, undergo centralization when endothelial cells are stimulated with cAMP-raising agonists of von Willebrand factor (vWF) secretion. In this study, we investigated this phenomenon of WPB clustering in more detail. METHODS AND RESULTS: Our results demonstrate that the clustered WPBs are localized at the microtubule organizing center and that cluster formation depends on an intact microtubule network. Disruption of the microtubules by nocodazole completely abolished clustering, whereas treatment with the actin depolymerizing compound cytochalasin B had no effect on WPB clustering. Interfering with the dynein-dynactin interaction by overexpression of the p50 dynamitin subunit or the CC1 domain of the p150glued subunit of the dynactin complex completely inhibited perinuclear clustering of WPBs, suggesting that dynein activity mediates this process. Furthermore, we found that inhibition of dephosphorylation resulted in an increase in clustering, whereas inhibition of protein kinase A (PKA) markedly reduced WPB clustering. CONCLUSIONS: These results suggest that perinuclear clustering of WPBs involves PKA-dependent regulation of the dynein-dynactin complex. Endothelial cell stimulation with epinephrine results in retrograde movement of a subset of WPBs to the microtubule organizing center. This minus-end directed transport requires an intact microtubular network and is mediated by the motor protein dynein. Together, our results suggest that epinephrine-induced clustering of WPBs involves PKA-dependent regulation of the dynein-dynactin complex.

Small GTP-binding protein Ral is involved in cAMP-mediated release of von Willebrand factor from endothelial cells.

OBJECTIVE: von Willebrand factor (vWF) is synthesized by endothelial cells and stored in specialized vesicles called Weibel-Palade bodies (WPBs). Recently, we have shown that the small GTP-binding protein Ral is involved in thrombin-induced exocytosis of WPBs. In addition to Ca2+-elevating secretagogues such as histamine and thrombin, release of WPB is also observed after administration of cAMP-raising substances such as epinephrine and vasopressin. In the present study, we investigated whether Ral is also involved in cAMP-mediated vWF release. METHODS AND RESULTS: Activation of Ral was observed 15 to 20 minutes after stimulation of endothelial cells with epinephrine, forskolin, or dibutyryl-cAMP. A cell-permeable peptide comprising the carboxy-terminal part of the Ral protein reduced both thrombin-induced and epinephrine-induced vWF secretion supporting a crucial role for Ral in this process. Furthermore, inhibition of protein kinase A by H-89 resulted in a marked reduction of vWF release and greatly diminished levels of GTP-Ral on stimulation with epinephrine. Activation of Ral was independent of the activation of Epac, a cAMP-regulated exchange factor for the small GTPases Rap1 and Rap2. CONCLUSIONS: These results suggest that protein kinase A-dependent activation of Ral regulates cAMP-mediated exocytosis of WPB in endothelial cells.

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.