2. Molecular mechanisms of vWF secretion

#### 2.1. von Willebrand factor and Weibel-Palade bodies at a glance

WPBs are unique secretory granules in ECs, which were first discovered in the 1960s by transmission electron microscopy [30]. Later on, vWF was identified inside WPBs of cultured human ECs [4, 5, 31]. vWF synthesis and maturation trigger WPB biogenesis [32, 33]. vWF is the best example of a protein that drives its own organelle formation [5, 34, 35]. To date, the structure of vWF consists in domains arranged as follows: D1D2D'D3A1A2A3D4C1C2C3C4C5C6CK. Of these, D1D2 is the pro-peptide and, the rest, from D'-CK is the fully functional, mature vWF [36]. The high density of vWF in the secretory granules enables a bolus of fully functional vWF to be released at the site of vascular injury or inflammation, which will promote efficient platelet binding and hemostasis or lead to thrombosis [37]. The physiological importance of proper WPBs formation can be appreciated in the context of various disease states associated with the release of immature, defective or low levels of VWF due to altered Golgi multimerization or defective tubular packing in WPBs (i.e. type 2B von Willebrand disease (vWD)) [38, 39].

WPBs originate from clathrin and clathrin adaptor-protein1-coated vesicles, [35, 40] which, when loaded with vWF cargo, bud from the trans-Golgi network, continue to mature and vWF contained in them becomes densely pack and highly multimeric vWF [35]. Once the vesicles had formed, they lose the clathrin coat, and the interactions between WPBs and the actin cytoskeleton are thought to either tether WPBs to the perinuclear cytosolic compartment or to relocate WPBs in the proximity of the plasma membrane in preparation for fusion and exocytosis [41]. Upon secretion of its contents, WPBs dissociate from the membrane and are recycled and refilled [35].

pathway induced by agonists such as thrombin [8, 9]. Basal vWF secretion involves elements of regulated secretion, with vWF targeted to Weibel-Palade bodies (WPBs) after Golgi processing, but continuously secreted from the storage pool [7]. Release of WPB contents into the extracellular space upon thrombin stimulation of ECs, thought to be pathophysiologically relevant in inflammatory cardiovascular disease, occurs via several processes: (1) signaling via [Ca2+]/Gαq and Gα12/α-soluble NSF attachment protein (α-SNAP) [10] that (2) promotes engagement and activation of vesicle (v) and target (t) membrane-associated v-SNARE and t-SNARE proteins (vesicle- and target-soluble NSF attachment protein receptors) [11, 12] and (3) energydependent fusion of WPB with plasma membrane that promotes the formation of a secretion

Once secreted from WPBs, vWF circulates in a globular conformation under resting conditions [16] and is a carrier for circulating coagulation factor VIII, thereby protecting it from degradation [17–19]. The blood vWF level in health and inflammatory disease is predominantly EC derived, while the contribution of platelets is rather minimal [20]. In response to high shear stress and inflammatory mediators [21], normally quiescent ECs secrete long vWF multimers in large quantities from the storage pool into systemic circulation. vWF multimers are then extracellularly cleaved by a metalloprotease ADAMTS-13 (A Disintegrin and Metalloproteinase with a thrombospondin type 1 motif, member 13), unfold in the circulation and self-associate into particularly linked concatemers to form ultra-large vWF strings [22]. Platelets spontaneously bind to activated vWF via glycoprotein Ibα (GpIbα) interaction with the exposed A1 domain, initiating the thrombogenic process [23–26]. In addition to its classical role in platelet binding, the vWF A1 domain also plays a role in vWF-dependent inflammatory responses [27–29]. In this chapter, we will discuss the most recent findings related to vWF secretion from ECs, the finely tuned balance of vWF level and activity in hemostasis and thrombosis, and the mechanisms by which vWF/ADAMTS-13 axis links inflammation and thrombosis. The potential of vWF as a target in antithrombotic therapies is also discussed.

WPBs are unique secretory granules in ECs, which were first discovered in the 1960s by transmission electron microscopy [30]. Later on, vWF was identified inside WPBs of cultured human ECs [4, 5, 31]. vWF synthesis and maturation trigger WPB biogenesis [32, 33]. vWF is the best example of a protein that drives its own organelle formation [5, 34, 35]. To date, the structure of vWF consists in domains arranged as follows: D1D2D'D3A1A2A3D4C1C2C3C4C5C6CK. Of these, D1D2 is the pro-peptide and, the rest, from D'-CK is the fully functional, mature vWF [36]. The high density of vWF in the secretory granules enables a bolus of fully functional vWF to be released at the site of vascular injury or inflammation, which will promote efficient platelet binding and hemostasis or lead to thrombosis [37]. The physiological importance of proper WPBs formation can be appreciated in the context of various disease states associated with the release of immature, defective or low levels of VWF due to altered Golgi multimerization or

defective tubular packing in WPBs (i.e. type 2B von Willebrand disease (vWD)) [38, 39].

pore through which the exocytosis of WPB contents occurs [13–15].

148 Endothelial Dysfunction - Old Concepts and New Challenges

2. Molecular mechanisms of vWF secretion

2.1. von Willebrand factor and Weibel-Palade bodies at a glance

Interestingly, vWF secretion is influenced by plasma sodium level [42]. In mice subjected to mild water restriction, elevated endothelial vWF protein levels correlated with increased number of micro thrombi in capillaries [42]. Hypernatremia causes up-regulation of vWF gene expression via nuclear factor of activated T-cells 5 (NFAT5) binding to the vWF promoter [42]. Another regulator of vWF was found to be autophagy [43]. Endothelial cell-specific ATG7 (critical factor in autophagy) deficient mice have prolonged bleeding times as compared to control, and decreased vWF secretion of immature vWF [43].

At least two different secretion mechanisms of WPB cargo have been reported so far: selective, time-dependent 'kiss-and-run' release [13] and differential release of the distinct components of these storage granules [8]. Apart from vWF, WPBs also contain other inflammatory and vasoactive mediators: P-selectin, Rab-27, coagulation factor XIIIa, tissue plasminogen activator, interleukin-8 (IL-8), eotaxin, endothelin-1, endothelin-converting enzyme, calcitonin-generelated peptide, α1,3-fucosyltransferase VI and angiopoietin 2 [44]. vWF and the other components—stockpiled in and liberated as needed from WPBs—provide the ECs with the ability to react acutely to restore homeostasis; vWF plays a role in hemostasis and thrombosis, and also, in the regulation of inflammation [45], vascular permeability [27] and angiogenesis [46].

vWF is synthesized exclusively by ECs, megakaryocytes and platelets and stored in specialized secretory granules. EC WPBs and platelet α-granules share several characteristics, as both organelles contain vWF, P-selectin and CD63 [47, 48]. But, in platelets, vWF detection by immune-gold labeling is restricted to a subset of spherical α-granules [48]. In this vane, spherical secretory α-granules are quite distinct from WPBs. First, the vWF content of platelets is relatively low: 0.05–0.1 U per 3 <sup>10</sup><sup>8</sup> platelets [49]. For comparison, plasma vWF concentration is 1–1.8 U/mL (10 μg/mL blood), of which only 15% of plasma vWF comes from platelets, while the majority 85% of the total vWF comes from ECs [4, 20, 34, 50]. Second, ECs combine basal and regulated vWF release, while platelets do not have a mechanism of basal secretion and, thus, substantial platelet activation would be needed for platelet vWF secretion to significantly contribute to the elevated blood vWF observed in different disease states. Finally, excessive secretion of EC-derived vWF in inflammation is attributed to the large capacity of ECs to make and store vWF in EC-specific storage pool, that is, in WPBs.

Thus, EC secretory granules supply the vasculature with circulating vWF to control hemostasis and thrombosis, while platelets play only a secondary role in this process [20, 49]. A balanced level of EC-derived vWF is critical to overcome the thrombotic complications in pathological settings, therefore it is important to understand the underlying endothelial constitutive and evoked exocytosis mechanisms which provide the bloodstream vWF in health and disease.

#### 2.2. Basal versus regulated vWF secretion

Secretion of vWF occurs by one of the following three modes:

1 regulated secretion from WPBs, in response to a specific agonist (secretagogue) on-demand upon vascular injury [51, 52];

2.2.2. Regulated vWF secretion

ronment by finely tuning the rate of release.

2.3. G protein signaling in hemostasis and thrombosis

mechanisms of vWF secretion need to be elucidated.

The release of vWF from the regulated pathway occurs only following stimulation of ECs with an appropriate agonist, providing the endothelium with the means to react to its microenvi-

Endothelial Cell von Willebrand Factor Secretion in Health and Cardiovascular Disease

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The notion that blood collected under stress clots faster was known for a while. In the 1970s, it was found that desmopressin (a synthetic analog of vassopresin) can elevate vWF level twofolds in healthy individuals [58] The follow-up studies showed that desmopressin activates vasopressin receptor 2 (V2R) on ECs and triggers cyclic adenosine monophosphate (cAMP)-mediated release of vWF from EC WPBs [58]. A multitude of agonists have been reported to induce vWF release via regulated secretion. These include thrombin [51], calcium ionophores [34], hypoxia [59], vasopressin [58], histamine [14], complement [60], platelet-activating factor [61] and inter-

In order to understand how to control WPB secretion for therapeutic purposes, the signaling

The exocyst is a cytoplasmic protein complex which targets secretory granules from the trans-Golgi network to the plasma membrane; it facilitates docking and priming of the secretory granule to the plasma membrane, prior to SNARE-mediated fusion [64]. When WPBs are trafficked to the plasma membrane, the vesicles are aligned such that v-SNAREs from the vesicles and t-SNAREs from target membrane can assemble as α-helix zippers which pull the membranes together. This SNARE zipper model was studied in detail in synapses [65]. Interestingly, all major players of these complexes identified in neurons are also present in the endothelium, including SNAP23, syntaxin 2, 3 and 4, and vesicle-associated membrane protein

Release of WPBs contents into the extracellular space is thought to occur via GTPasedependent processes [10]. It has been postulated that G proteins mediate cell type and signaling microdomain-specific functions. According to the classic G protein signaling model, heterotrimeric G proteins are located in the proximity of the plasma membrane where they can be activated by seven transmembrane spanning receptors, the canonical G protein-coupled receptors (GPCRs), to provoke downstream signaling events. G proteins are GTPases that typically function through GTP hydrolysis and cycling between nucleotide free GDP-bound and GTP-bound forms. GTPases also control the timing and specificity of vesicle trafficking and the exocyst partners recognition events, without GTP hydrolysis [66, 67]; the distinction between cycling and non-cycling GTPases might be more obvious when examining the effect of GTP hydrolysis-deficient mutant proteins that would be expected to cause gain-of-function on non-cycling GTPases and loss-of-function on cycling GTPases [66]. Furthermore, several studies indicate heterotrimeric G proteins can rapidly shuttle between the plasma membrane and intracellular membranes to exercise their function upon cell-specific organelles, along the

leukins [62]. Importantly, high shear stress is modulating regulated vWF release [63].

3 (VAMP3) complex which are thought to regulate vWF exocytosis [11].


Over the years, there have been in the field opposite views about how much vWF is released through each of these three pathways and what determines the balance between basal (constitutive-like) and regulated vWF secretion is still under debate [34, 53, 54].

#### 2.2.1. Basal vWF secretion

In resting ECs, secretion of vWF was thought to take place predominantly via conventional constitutive vWF secretion which is susceptible to protein synthesis inhibitor cycloheximide [34]. However, the importance of this pathway was exaggerated initially, as it could be drawn from the more recently published studies listed below. A vWF multimeric analysis in ECs challenged this view, indicating that highly multimeric vWF might be released solely from WPB storage pool [53]. Most recently, the issue was revisited when a study using metabolic labeling of vWF in human umbilical vein ECs (HUVECs) showed that most of the vWF secreted from unstimulated cells is not mediated by conventional constitutive secretion, but rather by basal release from the WPBs [7, 55]. Giblin and colleagues [7] recently brought more evidence to dismiss the belief that multimeric vWF passes uninterrupted from the Golgi to plasma membrane. They showed that freshly synthetized vWF is held for at least 24 h in WPBs before unstimulated release occurred, escaping cycloheximide-induced inhibition of protein synthesis, and, thus, consistent with release from the storage pool [7]. Similarly, Romani de Wit and coworkers [56] studied the dynamics of GFP-vWF-positive WPBs in resting HUVECs over time, and they were able to capture multiple image frames of WPBs traveling long distances toward the cell periphery. Intriguingly, resting ECs displayed membrane-apposed accumulation of GFP-vWF-containing vesicles [33, 56, 57]. It is worth mentioning here that only whenever a component of the cytoplasmic machinery is ablated the constitutive vWF secretion becomes predominant [40]. Taken together, these studies of WPBs behavior suggest that only the multimeric form of vWF is stored and that ECs secrete multimeric vWF via a continuous secretory pathway, which is a source of vWF in the bloodstream. If the WPB storage compartment is the source of the continuously secreted form of vWF that travels in the bloodstream, then the ability to regulate this constitutive vWF secretory route has significant clinical implications. Disruption of continuous vWF secretion may be implicated in the pathophysiology of type 2 vWD, which is due to both reduced vWF-dependent platelet function and reduced highly multimeric vWF in plasma.
