Abstract-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.

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-␣.

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Weibel-Palade bodies (WPBs) are elongated secretory organelles specific to endothelial cells that contain von Willebrand factor (VWF) and a variety of other proteins that contribute to inflammation, angiogenesis, and tissue repair. The remarkable architecture of WPBs is because of the unique properties of their major constituent VWF. VWF is stored inside WPBs as tubules, but on its release, forms strikingly long strings that arrest bleeding by recruiting blood platelets to sites of vascular injury. In recent years considerable progress has been made regarding the molecular events that underlie the packaging of VWF multimers into tubules and the processes leading to the formation of elongated WPBs. Mechanisms directing the conversion of tightly packaged VWF tubules into VWF strings on the surface of endothelial cells are starting to be unraveled. Several modes of exocytosis have now been described for WPBs, emphasizing the plasticity of IntroductionIn 1964 Ewald Weibel and George Palade used transmission electron microscopy (EM) to discover that "a hitherto unknown rod-shaped cytoplasmic component which consists of a bundle of fine tubules, enveloped by a tightly fitted membrane, was regularly found in endothelial cells of small arteries in various organs in rat and man." 1 Subsequent studies confirmed the presence of those organelles in a variety of vertebrates, including hagfish, 2 which suggests they were present at least 500 million years ago. These organelles have a diameter of 0.1-0.3 m, length of 1-5 m, and characteristic longitudinal striations (Figure 1). 3 In cross section they consist of electron dense tubules with an inside diameter of 12 nm, surrounded by a less dense matrix, and packed in parallel bundles that are surrounded by a lipid bilayer. We now refer to these organelles as Weibel-Palade bodies (WPBs).At the time of their discovery, the biologic function of WPBs was unknown, although their conserved ultrastructure and wide distribution suggested that they must play a significant role in vertebrate endothelium. Almost 20 years later WPBs were shown to contain von Willebrand factor (VWF), 4 a multimeric hemostatic protein that is secreted into the blood in response to a variety of agonists and mediates platelet adhesion at sites of vascular injury. Inherited defects in VWF cause von Willebrand disease (VWD), the most common inherited bleeding disorder. 5 Interestingly, VWF is also produced by megakaryocytes and platelets, 6 the latter containing tubular structures similar to those found in WPBs but eccentrically localized in the ␣-granule, and shown to be positive for VWF by immunogold EM techniques. 7 In fact, VWF is the only protein readily detected in WPBs after biosynthetic labeling of endothelial cells. 8 Several other proteins have been identified as constituents of WPBs including tissue-type plasminogen activator (tPA), P-selectin, interleukin-8 (IL-8), eotaxin-3, angiopoietin-2, osteoprotegerin, endothelin-1, endothelin-converting enzyme, and calcitonin generelated peptide. 9,10 In addi...

The results of studies with cultured endothelial cells have shown that most von Willebrand factor (vWF) synthesized is directly secreted (constitutive pathway) and consists of both mature vWF, its precursor molecule pro-vWF, and the cleaved vWF prosequence. Only fully processed, functionally mature vWF is stored within the cell, together with the propeptide, and leaves the cell only on stimulation (regulated secretion). Both in resting and stimulated cultured endothelial cells, the stoichiometry of the released propeptide to the released mature vWF is essentially equimolar. In the present study, we have measured the molar ratio of propeptide to mature vWF in vivo, both under resting conditions and conditions that reflect activation of the endothelium. To this end, we devised a method that allows the measurement of the propeptide (vW antigen II) on a quantitative, is, molar basis, using purified recombinant propeptide as a standard. Our results show that the molar concentration of the propeptide in normal plasma is about one tenth of the concentration of mature vWF (expressed as half-dimer concentration). This ratio is approximately 1:1 in the medium of cultured endothelial cells. On administration in healthy subjects of either 1-deamino-8-D-arginine vasopressin or endotoxin, both agents being known to elicit an intravascular increase of vWF, the molar ratio of propeptide to mature vWF increased fourfold to fivefold. The propeptide concentration returned to baseline values after about 6 to 7 hours of injection of each stimulus, whereas the increase of mature vWF was much more sustained. Because the respective half-lives of mature vWF and its propeptide clearly differ, measurement of the concentration of these proteins could provide a means to assess the extent of activation of the endothelium under physiological and pathophysiological conditions.

Immune thrombocytopenia (ITP) is an autoimmune disease defined by low platelet counts which presents with an increased bleeding risk. Several genetic risk factors (e.g., polymorphisms in immunity-related genes) predispose to ITP. Autoantibodies and cytotoxic CD8+ T cells (Tc) mediate the anti-platelet response leading to thrombocytopenia. Both effector arms enhance platelet clearance through phagocytosis by splenic macrophages or dendritic cells and by induction of apoptosis. Meanwhile, platelet production is inhibited by CD8+ Tc targeting megakaryocytes in the bone marrow. CD4+ T helper cells are important for B cell differentiation into autoantibody secreting plasma cells. Regulatory Tc are essential to secure immune tolerance, and reduced levels have been implicated in the development of ITP. Both Fcγ-receptor-dependent and -independent pathways are involved in the etiology of ITP. In this review, we present a simplified model for the pathogenesis of ITP, in which exposure of platelet surface antigens and a loss of tolerance are required for development of chronic anti-platelet responses. We also suggest that infections may comprise an important trigger for the development of auto-immunity against platelets in ITP. Post-translational modification of autoantigens has been firmly implicated in the development of autoimmune disorders like rheumatoid arthritis and type 1 diabetes. Based on these findings, we propose that post-translational modifications of platelet antigens may also contribute to the pathogenesis of ITP.

In the majority of patients with acquired thrombotic thrombocytopenic purpura (TTP), antibodies are directed toward the spacer domain of ADAMTS13. We have previously shown that region Y658-Y665 is involved. We now show that replacement of R660, Y661, or Y665 with alanine in ADAMTS13 reduced/abolished the binding of 2 previously isolated human monoclonal antibodies and polyclonal antibodies derived from plasma of 6 patients with acquired TTP. We investigated whether these residues also influenced cleavage IntroductionVon Willebrand factor (VWF) is a key hemostatic glycoprotein involved in the adhesion of platelets to sites of vascular perturbation. 1 During its biosynthesis in endothelial cells, VWF undergoes a number of posttranslational modifications that include the formation of intermolecular disulfide bonds between the carboxylterminal cysteine knot domains and the amino-terminal D3 domains. 2,3 Current findings suggest that the resulting VWF polymers condense into tubular structures in the trans Golgi network and are subsequently packaged into Weibel-Palade bodies, rod-shaped subcellular organelles. 4,5 Upon release of Weibel-Palade body contents, the VWF tubules rapidly unfold, and the fluid shear stress in the flowing blood induces the formation of ultra-large VWF (UL-VWF) strings on the surface of endothelial cells. 6,7 Andre et al 6 have shown that the appearance of platelet-decorated strings is a transient process in vivo. This transient nature is attributed to the rapid proteolysis of UL-VWF multimers by the metalloprotease ADAMTS13. 8,9 In the absence of ADAMTS13, the rate at which platelet strings disappear from the endothelium is markedly reduced.Thrombotic thrombocytopenic purpura (TTP) is a thrombotic microangiopathy characterized by hemolytic anemia, severe thrombocytopenia, and the presence of schistocytes in blood smears and is accompanied by a deficiency in ADAMTS13. ADAMTS13 is a large multidomain protein that consists of a propeptide, a catalytic metalloprotease domain, a disintegrin-like domain, a thrombospondin type I repeat (TSP), a cysteine-rich domain, a spacer domain, 7 additional TSP repeats, and 2 carboxyl-terminal CUB domains. [10][11][12] In the majority of patients with TTP, inhibitory antibodies targeting ADAMTS13 have been found. [13][14][15][16] In addition to inhibition of ADAMTS13 activity, anti-ADAMTS13 antibodies may also accelerate ADAMTS13 clearance. 17 Epitope mapping studies have shown that the cysteine-rich/spacer domains contain the major binding site for human anti-ADAMTS13 antibodies. [18][19][20][21] Additional epitopes for human anti-ADAMTS13 antibodies located outside the spacer domain have also been identified. 18,22,23 In a previous study, we have shown that amino acid residues Y658-Y665 within the spacer domain comprise part of a core binding site for anti-ADAMTS13 antibodies. 24 The ADAMTS13 metalloprotease domain cleaves the VWF A2 domain at the Y1605-M1606 scissile bond. The metalloprotease domain by itself cannot efficiently proteolyse VWF; the proximal ...

The human fur gene encodes a protein, designated furin, the C-terminal half of which contains a transmembrane and a cysteine-rich receptor-like domain. The N-terminal half of furin exhibits striking primary amino acid sequence similarity to the catalytic domains of members of the subtilisin family of serine proteases. We here report characteristics of the furin protein and propose a three-dimensional model for its presumptive catalytic domain with characteristics, that predict furin to exhibit an endoproteolytic cleavage selectivity at paired basic residues. This prediction is substantiated by transfection and cotransfection experiments, using COS-1 cells. Full length fur cDNA evokes the specific synthesis of two polypeptides of about 100 kDa and 90 kDa as appeared from Western blot analysis of transfected COS-1 cells using a polyclonal anti-furin antiserum. Functional analysis of furin was performed by cotransfection of fur cDNA with cDNA encoding the 'wild type' precursor of von Willebrand factor (pro-vWF) and revealed an increased proteolytic processing of provWF. In contrast, cotransfection of fur cDNA with a recombinant derivative (provWFgly763), having the arginine residue adjacent to the proteolytic cleavage site (arg-ser-lys-arg) replaced by glycine, revealed that provWFgly763 is not processed by the fur gene product. We conclude that in higher eukaryotes, furin is the prototype of a subtilisin-like class of proprotein processing enzymes with substrate specificity for paired basic residues.