2.3. G protein signaling in hemostasis and thrombosis

In order to understand how to control WPB secretion for therapeutic purposes, the signaling mechanisms of vWF secretion need to be elucidated.

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

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 secretory routes. Activation of GPCRs and G protein α and βγ subunits of Gs, Gi, Gq/11 and G12/13 can stimulate secretory granule release [10, 68]. It has been shown, for example, by fluorescence polarization, that G protein i/o βγ subunit competes with synaptotagmin for specific interaction sites on t-SNAREs, namely syntaxin 1 and SNAP25B (Synaptosomal-associated protein 25) [69]. G protein i/o binds to the SNARE at the plasma membrane, but in the presence of synaptotagmin and calcium, inhibits vesicle fusion with the plasma membrane, suggesting the G βγ-SNARE axis has an inhibitory role during synaptic exocytosis [69]. In the exocrine pancreas, G proteins have been shown to play a role in early transport events [68].

SNARE complex [81]. Therefore, the rate of exocytosis depends on α-SNAP and NSF activity [78]. Using PC12 cells as an α-SNAP-regulated exocytosis model, it was shown that α-SNAP, in absence of NSF activity, can actually block exocytosis and that this α-SNAP-dependent event occurs by direct binding of α-SNAP to free syntaxin, thus preventing SNARE complex assembly [82]. It is documented that α-SNAP is activated in the exocyst complex by phosphorylation [83], that α-SNAP binds and stimulates NSF ATPase activity, [84] that Gα12 interacts with α-SNAP, [66] and that S-nitrosylation of NSF inhibits WPB exocytosis [85]. α-SNAP regulates exocytosis of granules from different types of cells [86]. α-SNAP binding to the SNARE complex in the fused membrane mediates recruitment and activation of NSF resulting of exocytosis from ECs [10, 87]. The Gα12 binding site for α-SNAP was recently identified by using a library of substitution mutants within myc-tagged Gα12QL in which regions of the cDNA encoding consecutive six aminoacids were replaced with a sequence encoding the following six aminoacids: asparagine-alanine-alanine-isoleucine-arginine-serine (NAAIRS) by oligonucleotide-directed mutagenesis and expressed in a cell line as described previously [88], followed by evaluation of direct binding between Gα12 NAAIRS mutants and α-SNAP by glutathione S-transferase (GST) pulldown [10]. Based on the evidence generated by the GST pulldown assay, we constructed an α-SNAP Binding Domain peptide to which we added a myristoyl group and micellar nanoformulation for cellular entry, to further assess the role of

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Secretion of vWF from ECs is mediated by small GTPases via both second messengers Ca2+ and cAMP-dependent signaling pathways [91–93]. Small GTPases are the effectors of the exocyst complex, involved in temporal coordination, spatial segregation and proof-reading of membrane trafficking events [94]. Rab and Ral GTPases are thought to be involved in vesicle

RalA was the first GTPase found to co-sediment with WPBs in density gradients [96]. Ral A is activated by its exchange factor, Ral guanine-nucleotide dissociation stimulator (RalGDS) [97], which is kept inactive by β-arrestin under static conditions [98]. Upon GPCR activation, Ral GDS uncouples from β-arrestin and functions in its GTP-bound state. RalGDS downregulation with siRNA in thrombin-stimulated HUVECs leads to accumulation of WPBs in the proximity of the plasma membrane, but exocytosis is incomplete, suggesting RalGDS/ β-arrestin complex is necessary to link thrombin receptors to WPBs exocytosis [98]. Ral A facilitates WPBs trafficking and delivery to the plasma membrane [91]. Ral A has been implicated in actin cytoskeleton dynamic rearrangements through its direct interactions with effectors filamin A and RalBP1/RLIP76 (a RAC/CDC42 guanine-nucleotide activating protein (GAP) [99]. RalBP1 links RhoGTPases and RalGTPases, and, of note, RalBP1 GAP has an ATP binding domain, although it is not clear whether this domain is a motor required for assembly of the exocyst [99]. Studies conducted in our laboratory suggest that human pulmonary artery ECs treated with filamin A siRNA attenuates constitutive and thrombin-induced vWF secretion [100]. It has been proposed that Ral A/Ca2+-dependent signaling might be a prerequisite for the exocy-

tethering, whereas RhoGTPases are spatial regulators of the exocyst complex [95].

Gα12 interaction with α-SNAP in vWF secretion [89, 90].

totic machinery [101].

2.5. Spatial and temporal regulators of the exocyst complex

A role for G proteins Gαq/11 and Gα12/13 in hemostasis has been previously reported [70, 71]. Platelets from Gαq-deficient mice fail to respond to low doses of platelet-activating agonists and these mice have prolonged bleeding times and reduced thrombus formation after intravenous administration of adrenaline/collagen [71, 72]. Studies in megakaryocyterestricted Gα12/13 double knockout mice reveal that these mice have prolonged bleeding time and reduced thrombus formation, suggesting that Gα13-dependent signaling in platelets is also relevant for hemostasis and thrombosis [70]. Interestingly, we found that, in addition to Gαs stimulatory-G protein [58, 73], Gα12 and Gαq/11 facilitate exocytosis of vWF from ECs [10]. Importantly, in Gα12 overexpression studies, Gα12 is able to localize to the plasma membrane because of its palmitoylation at cysteine 11 [74]. Palmitoylation targets Gα12 to lipid rafts fractions [75]. Both wild-type Gα12wt and Gα12QL (constitutive active) were observed in lipid rafts fractions, and therefore, the localization of Gα12 to discreet endothelial microdomains may be independent of Gα12 activation [76]. Other studies reported direct G protein-dependent regulation of the SNARE protein fusion machinery is required for secretory granule exocytosis [77], and Gα12 was shown by the yeast twohybrid method to interact with a member of the exocytotic assembly, α-SNAP [66]. Using cultured human ECs and knockout mouse models, we showed that depletion of Gα12 or α-SNAP inhibited both basal and thrombin-induced vWF secretion and that Gα12/ mice exhibit mildly reduced blood levels of vWF, but intact vWF multimeric pattern, and impaired thrombus formation [10]. Our studies suggest that Gα12 may interact directly with α-SNAP to promote the docking and fusion of WPBs [10]. Furthermore, Gα12 and Gαq subunits, which are known to regulate actin cytoskeleton rearrangements in ECs via activation of RhoA GTPase, promote WPB docking on the plasma membrane, providing both direct and indirect mechanisms linking GPCR activation and SNARE complex fusion [10].

#### 2.4. Kinetics of WPB secretion

Secretion of vWF from ECs is mediated by fusion of WPBs with the plasma membrane in a manner dependent on the ATPase N-ethylmaleimide-sensitive-factor (NSF), soluble-NSFattachment protein alpha (α-SNAP) and SNAREs [78]. NSF binds to SNARE complexes to facilitate the disassembly of the zippered bundles [79]. Because NSF lacks a direct binding domain for members of the SNARE family, it connects via an adaptor, α-SNAP [80]. Six NSF proteins assemble together at the plasma membrane, and each NSF hexamer requires three α-SNAPs to mediate binding to the SNAREs [78]. Once the NSF/α-SNAP/SNARE complex is formed, NSF hydrolyses ATP, providing the energy necessary for the disassembly of the SNARE complex [81]. Therefore, the rate of exocytosis depends on α-SNAP and NSF activity [78]. Using PC12 cells as an α-SNAP-regulated exocytosis model, it was shown that α-SNAP, in absence of NSF activity, can actually block exocytosis and that this α-SNAP-dependent event occurs by direct binding of α-SNAP to free syntaxin, thus preventing SNARE complex assembly [82]. It is documented that α-SNAP is activated in the exocyst complex by phosphorylation [83], that α-SNAP binds and stimulates NSF ATPase activity, [84] that Gα12 interacts with α-SNAP, [66] and that S-nitrosylation of NSF inhibits WPB exocytosis [85]. α-SNAP regulates exocytosis of granules from different types of cells [86]. α-SNAP binding to the SNARE complex in the fused membrane mediates recruitment and activation of NSF resulting of exocytosis from ECs [10, 87]. The Gα12 binding site for α-SNAP was recently identified by using a library of substitution mutants within myc-tagged Gα12QL in which regions of the cDNA encoding consecutive six aminoacids were replaced with a sequence encoding the following six aminoacids: asparagine-alanine-alanine-isoleucine-arginine-serine (NAAIRS) by oligonucleotide-directed mutagenesis and expressed in a cell line as described previously [88], followed by evaluation of direct binding between Gα12 NAAIRS mutants and α-SNAP by glutathione S-transferase (GST) pulldown [10]. Based on the evidence generated by the GST pulldown assay, we constructed an α-SNAP Binding Domain peptide to which we added a myristoyl group and micellar nanoformulation for cellular entry, to further assess the role of Gα12 interaction with α-SNAP in vWF secretion [89, 90].

#### 2.5. Spatial and temporal regulators of the exocyst complex

secretory routes. Activation of GPCRs and G protein α and βγ subunits of Gs, Gi, Gq/11 and G12/13 can stimulate secretory granule release [10, 68]. It has been shown, for example, by fluorescence polarization, that G protein i/o βγ subunit competes with synaptotagmin for specific interaction sites on t-SNAREs, namely syntaxin 1 and SNAP25B (Synaptosomal-associated protein 25) [69]. G protein i/o binds to the SNARE at the plasma membrane, but in the presence of synaptotagmin and calcium, inhibits vesicle fusion with the plasma membrane, suggesting the G βγ-SNARE axis has an inhibitory role during synaptic exocytosis [69]. In the exocrine pancreas, G proteins have been shown to play a role in early transport events [68].

152 Endothelial Dysfunction - Old Concepts and New Challenges

A role for G proteins Gαq/11 and Gα12/13 in hemostasis has been previously reported [70, 71]. Platelets from Gαq-deficient mice fail to respond to low doses of platelet-activating agonists and these mice have prolonged bleeding times and reduced thrombus formation after intravenous administration of adrenaline/collagen [71, 72]. Studies in megakaryocyterestricted Gα12/13 double knockout mice reveal that these mice have prolonged bleeding time and reduced thrombus formation, suggesting that Gα13-dependent signaling in platelets is also relevant for hemostasis and thrombosis [70]. Interestingly, we found that, in addition to Gαs stimulatory-G protein [58, 73], Gα12 and Gαq/11 facilitate exocytosis of vWF from ECs [10]. Importantly, in Gα12 overexpression studies, Gα12 is able to localize to the plasma membrane because of its palmitoylation at cysteine 11 [74]. Palmitoylation targets Gα12 to lipid rafts fractions [75]. Both wild-type Gα12wt and Gα12QL (constitutive active) were observed in lipid rafts fractions, and therefore, the localization of Gα12 to discreet endothelial microdomains may be independent of Gα12 activation [76]. Other studies reported direct G protein-dependent regulation of the SNARE protein fusion machinery is required for secretory granule exocytosis [77], and Gα12 was shown by the yeast twohybrid method to interact with a member of the exocytotic assembly, α-SNAP [66]. Using cultured human ECs and knockout mouse models, we showed that depletion of Gα12 or α-SNAP inhibited both basal and thrombin-induced vWF secretion and that Gα12/ mice exhibit mildly reduced blood levels of vWF, but intact vWF multimeric pattern, and impaired thrombus formation [10]. Our studies suggest that Gα12 may interact directly with α-SNAP to promote the docking and fusion of WPBs [10]. Furthermore, Gα12 and Gαq subunits, which are known to regulate actin cytoskeleton rearrangements in ECs via activation of RhoA GTPase, promote WPB docking on the plasma membrane, providing both direct and indirect mechanisms linking GPCR activation and SNARE complex fusion [10].

Secretion of vWF from ECs is mediated by fusion of WPBs with the plasma membrane in a manner dependent on the ATPase N-ethylmaleimide-sensitive-factor (NSF), soluble-NSFattachment protein alpha (α-SNAP) and SNAREs [78]. NSF binds to SNARE complexes to facilitate the disassembly of the zippered bundles [79]. Because NSF lacks a direct binding domain for members of the SNARE family, it connects via an adaptor, α-SNAP [80]. Six NSF proteins assemble together at the plasma membrane, and each NSF hexamer requires three α-SNAPs to mediate binding to the SNAREs [78]. Once the NSF/α-SNAP/SNARE complex is formed, NSF hydrolyses ATP, providing the energy necessary for the disassembly of the

2.4. Kinetics of WPB secretion

Secretion of vWF from ECs is mediated by small GTPases via both second messengers Ca2+ and cAMP-dependent signaling pathways [91–93]. Small GTPases are the effectors of the exocyst complex, involved in temporal coordination, spatial segregation and proof-reading of membrane trafficking events [94]. Rab and Ral GTPases are thought to be involved in vesicle tethering, whereas RhoGTPases are spatial regulators of the exocyst complex [95].

RalA was the first GTPase found to co-sediment with WPBs in density gradients [96]. Ral A is activated by its exchange factor, Ral guanine-nucleotide dissociation stimulator (RalGDS) [97], which is kept inactive by β-arrestin under static conditions [98]. Upon GPCR activation, Ral GDS uncouples from β-arrestin and functions in its GTP-bound state. RalGDS downregulation with siRNA in thrombin-stimulated HUVECs leads to accumulation of WPBs in the proximity of the plasma membrane, but exocytosis is incomplete, suggesting RalGDS/ β-arrestin complex is necessary to link thrombin receptors to WPBs exocytosis [98]. Ral A facilitates WPBs trafficking and delivery to the plasma membrane [91]. Ral A has been implicated in actin cytoskeleton dynamic rearrangements through its direct interactions with effectors filamin A and RalBP1/RLIP76 (a RAC/CDC42 guanine-nucleotide activating protein (GAP) [99]. RalBP1 links RhoGTPases and RalGTPases, and, of note, RalBP1 GAP has an ATP binding domain, although it is not clear whether this domain is a motor required for assembly of the exocyst [99]. Studies conducted in our laboratory suggest that human pulmonary artery ECs treated with filamin A siRNA attenuates constitutive and thrombin-induced vWF secretion [100]. It has been proposed that Ral A/Ca2+-dependent signaling might be a prerequisite for the exocytotic machinery [101].

The second point of intervention for the Rab and Ral GTPases is anchoring and fusion of WPBs with the plasma membrane [8]. Ral A also promotes exocytosis by increasing phospholipase D1 (PLD1) activity and subsequent phosphatidic acid (PA) production, thereby facilitating plasma membrane fusion as described in more detail in Section 2.6 of this chapter. The Rabs are a family of over 60 members of small GTPases that control membrane identity and the actions of the intracellular vesicles; each type of secretory vesicle in the cells has a unique set of Rab family members, as reviewed in [102]; of these, six Rab proteins were found in association with WPBs:Rab3 isoform b [103], Rab3 isoform d [104], Rab27a [105], Rab3 isoform a, Rab15, Rab33a, Rab37 [106] and Rab35 [107]. Rab 27a is located on the cytosolic face of WPBs, is used as a marker of organelle identity, and has multifunctional capacities: it could either mildly inhibit the secretion of WPBs by associating with its effector MyRIP (myosin VIIa and Rabinteracting protein), thus anchoring WPBs to actin filaments and keeping them from attaching to the plasma membrane until the right timing, [57] or it could strongly activate the secretion of WPBs by associating with an alternate effector, synaptotagmin-like protein 4 (or granuphilin) (Slp4-a), [108] which links the secretory granules to the plasma membrane via syntaxinbinding protein 1 and syntaxin 2 and 3 [108, 109]. The ratio of Rab27a occupancy by Slp4-a or MyRIP dictates whether Rab27a is stimulatory or inhibitory with regards to WPB exocytosis [103]. Furthermore, Rab27 was shown to work synergistically with Rab15 to control exocytosis; Zografou et al. recently showed that simultaneous knockdown of the two Rabs using siRNA in HUVEC leads to a greater reduction in vWF secretion compared with knockdown of either Rab alone [106]. Their experiments further showed that Munc13-4, a known effector of Rab27a, co-localized with Rab15 on WPBs. The three proteins, Rab27a, Rab15 and Munc13- 4, thus form a complex and work in tandem to help regulate exocytosis of vWF [106]. Rab 33b has an inhibitory effect on vWF secretion [106].

PA production in HUVECs upon stimulation with histamine, which is a known agonist to induce exocytosis of WPB contents [111]. The increase in PA production is mediated by recruitment and activation of PLD1, an enzyme that hydrolyses phosphatidylcholine to produce PA [111]. PLD1 downregulation using shRNA resulted in a reduced secretion of vWF upon histamine stimulation [111]. PLD1 is commonly thought of as a general promoter of membrane fusion because of its role in producing fusogenic conical lipids such as PA [112]. PLD1 requires activation by one or more factors specific to the cell type and activation pathway, including small GTPases such as those of the ADP-ribosylation factor/Rho families as well as RalA, RalGDS or protein kinase C [113]. Thus, RalA and RalGDS not only play a role in the exocytosis process itself, as discussed in Section 2.5, but are also directly associated with the cytosolic face of WPBs. Therefore, it has been suggested that RalA could serve as an upstream activator of PLD1, promoting PLD1 movement to the membrane and subsequent generation of PA-enriched membrane microdomains important for membrane fusion [97].

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2.7. Zyxin and other proteins that regulate vWF release from endothelial cells

Data published by Han et al. show that zyxin, a focal adhesion LIM domain-containing protein, is involved in thrombin-mediated remodeling of the actin cytoskeleton [114]. The molecular structure of zyxin predicts its function in cytoskeletal dynamics [115]. Zyxin has proline-rich repeats at the N terminus followed by a leucine-rich nuclear export signal (NES) and three copies of a cysteine- and histidine-rich motif called the LIM domain at the C terminus [115]. Regulators of cytoskeletal dynamics, such as Enap/vasodilator-stimulated phosphoprotein (VASP) family members and α-actinin interact with the proline-rich region of zyxin. Zyxin can generate new actin structures in a VASP-dependent manner, independently of the Arp2/3 complex that cooperates with members of the Wiskott-Aldrich syndrome family of proteins (WASP) to nucleate actin filaments [114, 116]. Zyxin is a VASP-dependent actin polymerization machine in cells [117], and Han et al. showed zyxin binds to the C-terminal domain of protease-activated receptor 1 (PAR-1) [114]. Upon disruption of PAR-1-zyxin interaction, thrombin-induced formation of actin stress fibers was inhibited further supporting the hypothesis that zyxin functions as a signal transducer in PAR-1 signaling. In contrast, downregulation of zyxin did not affect thrombin-induced activation of RhoA or Gi, Gq and G12/13 heterotrimeric G proteins, implicating a novel signaling pathway regulated by PAR-1 that is not mediated by G proteins. Depletion of zyxin using siRNA inhibited thrombininduced actin stress fiber formation and serum response element (SRE)-dependent gene transcription. In addition, depletion of zyxin resulted in delay of endothelial barrier restoration after thrombin treatment. In 2017, Han et al. reported that downregulation of zyxin in HUVECs with shRNA inhibits cAMP-dependent secretion of vWF [116]. In zyxin shRNAexpressing cells, formation of the actin framework around exocytic WPBs was scarce. Moreover, phosphorylation of zyxin at serines 142 and 143 (S142/S143) is critical for vWF secretion since the zyxin mutant could not rescue the defect in zyxin shRNA-treated cells [116]. They showed that a protein kinase A (PKA)-specific inhibitor blocked zyxin phosphorylation at S142/S143 and concluded that zyxin acts downstream of PKA [116]. Han et al. thus proposed a novel model for cytoskeleton reorganization around WPBs undergoing exocytosis. Upon epinephrine stimulation, pre-existing filaments are reorganized to form actin frameworks

A recent genome-wide screen identified a completely new signaling pathway associated with WPB exocytosis. Rab 35, which is controlled by Rab GAP TBC1D10A, promotes ACAP2 (ArfGAP with coiled-coil, Ank repeat and pleckstrin homology domain-containing protein) activation, which inhibits histamine-induced Ca2+-dependent vWF and P-selectin expression in human ECs. This study used constitutively active mutants of Rab35, downregulation with siRNA and a fluorescence activated cell sorting (FACS)-based vWF secretory assay to prove that Rab35 promotes histamine-induced vWF secretion in a TBC1D10A- and ACAP2-dependent manner [107]. Of note, ACAP2 GAP targets Arf 6 which is a positive regulator of vWF secretion from human ECs, as shown by total internal reflection fluorescence (TIRF) microscopy and FACS-based vWF secretory essay [107]. Arf 6 GTPase activity at the plasma membrane elevates phosphatidylinositol 4,5-bisphosphate (PI (4,5) P2) levels via PI (4)P5-kinase activation, acting antagonistically to Rab35 through TBC1D10A Rab GAP [107]. Finally, among the Rabs found to be in association with WPBs, only Rab27 is known to be involved in basal secretion [57, 108].

#### 2.6. Endothelial cell secretory microdomains

GTPase-mediated exocyst activity of the SNARE assembly occurs in specific regions of the plasma membrane with distinct lipid profiles [110]. Namely, we know there is an increase in PA production in HUVECs upon stimulation with histamine, which is a known agonist to induce exocytosis of WPB contents [111]. The increase in PA production is mediated by recruitment and activation of PLD1, an enzyme that hydrolyses phosphatidylcholine to produce PA [111]. PLD1 downregulation using shRNA resulted in a reduced secretion of vWF upon histamine stimulation [111]. PLD1 is commonly thought of as a general promoter of membrane fusion because of its role in producing fusogenic conical lipids such as PA [112]. PLD1 requires activation by one or more factors specific to the cell type and activation pathway, including small GTPases such as those of the ADP-ribosylation factor/Rho families as well as RalA, RalGDS or protein kinase C [113]. Thus, RalA and RalGDS not only play a role in the exocytosis process itself, as discussed in Section 2.5, but are also directly associated with the cytosolic face of WPBs. Therefore, it has been suggested that RalA could serve as an upstream activator of PLD1, promoting PLD1 movement to the membrane and subsequent generation of PA-enriched membrane microdomains important for membrane fusion [97].

#### 2.7. Zyxin and other proteins that regulate vWF release from endothelial cells

The second point of intervention for the Rab and Ral GTPases is anchoring and fusion of WPBs with the plasma membrane [8]. Ral A also promotes exocytosis by increasing phospholipase D1 (PLD1) activity and subsequent phosphatidic acid (PA) production, thereby facilitating plasma membrane fusion as described in more detail in Section 2.6 of this chapter. The Rabs are a family of over 60 members of small GTPases that control membrane identity and the actions of the intracellular vesicles; each type of secretory vesicle in the cells has a unique set of Rab family members, as reviewed in [102]; of these, six Rab proteins were found in association with WPBs:Rab3 isoform b [103], Rab3 isoform d [104], Rab27a [105], Rab3 isoform a, Rab15, Rab33a, Rab37 [106] and Rab35 [107]. Rab 27a is located on the cytosolic face of WPBs, is used as a marker of organelle identity, and has multifunctional capacities: it could either mildly inhibit the secretion of WPBs by associating with its effector MyRIP (myosin VIIa and Rabinteracting protein), thus anchoring WPBs to actin filaments and keeping them from attaching to the plasma membrane until the right timing, [57] or it could strongly activate the secretion of WPBs by associating with an alternate effector, synaptotagmin-like protein 4 (or granuphilin) (Slp4-a), [108] which links the secretory granules to the plasma membrane via syntaxinbinding protein 1 and syntaxin 2 and 3 [108, 109]. The ratio of Rab27a occupancy by Slp4-a or MyRIP dictates whether Rab27a is stimulatory or inhibitory with regards to WPB exocytosis [103]. Furthermore, Rab27 was shown to work synergistically with Rab15 to control exocytosis; Zografou et al. recently showed that simultaneous knockdown of the two Rabs using siRNA in HUVEC leads to a greater reduction in vWF secretion compared with knockdown of either Rab alone [106]. Their experiments further showed that Munc13-4, a known effector of Rab27a, co-localized with Rab15 on WPBs. The three proteins, Rab27a, Rab15 and Munc13- 4, thus form a complex and work in tandem to help regulate exocytosis of vWF [106]. Rab 33b

A recent genome-wide screen identified a completely new signaling pathway associated with WPB exocytosis. Rab 35, which is controlled by Rab GAP TBC1D10A, promotes ACAP2 (ArfGAP with coiled-coil, Ank repeat and pleckstrin homology domain-containing protein) activation, which inhibits histamine-induced Ca2+-dependent vWF and P-selectin expression in human ECs. This study used constitutively active mutants of Rab35, downregulation with siRNA and a fluorescence activated cell sorting (FACS)-based vWF secretory assay to prove that Rab35 promotes histamine-induced vWF secretion in a TBC1D10A- and ACAP2-dependent manner [107]. Of note, ACAP2 GAP targets Arf 6 which is a positive regulator of vWF secretion from human ECs, as shown by total internal reflection fluorescence (TIRF) microscopy and FACS-based vWF secretory essay [107]. Arf 6 GTPase activity at the plasma membrane elevates phosphatidylinositol 4,5-bisphosphate (PI (4,5) P2) levels via PI (4)P5-kinase activation, acting antagonistically to Rab35 through TBC1D10A Rab GAP [107]. Finally, among the Rabs found to be in association with WPBs, only Rab27 is known to be involved in basal

GTPase-mediated exocyst activity of the SNARE assembly occurs in specific regions of the plasma membrane with distinct lipid profiles [110]. Namely, we know there is an increase in

has an inhibitory effect on vWF secretion [106].

154 Endothelial Dysfunction - Old Concepts and New Challenges

2.6. Endothelial cell secretory microdomains

secretion [57, 108].

Data published by Han et al. show that zyxin, a focal adhesion LIM domain-containing protein, is involved in thrombin-mediated remodeling of the actin cytoskeleton [114]. The molecular structure of zyxin predicts its function in cytoskeletal dynamics [115]. Zyxin has proline-rich repeats at the N terminus followed by a leucine-rich nuclear export signal (NES) and three copies of a cysteine- and histidine-rich motif called the LIM domain at the C terminus [115]. Regulators of cytoskeletal dynamics, such as Enap/vasodilator-stimulated phosphoprotein (VASP) family members and α-actinin interact with the proline-rich region of zyxin. Zyxin can generate new actin structures in a VASP-dependent manner, independently of the Arp2/3 complex that cooperates with members of the Wiskott-Aldrich syndrome family of proteins (WASP) to nucleate actin filaments [114, 116]. Zyxin is a VASP-dependent actin polymerization machine in cells [117], and Han et al. showed zyxin binds to the C-terminal domain of protease-activated receptor 1 (PAR-1) [114]. Upon disruption of PAR-1-zyxin interaction, thrombin-induced formation of actin stress fibers was inhibited further supporting the hypothesis that zyxin functions as a signal transducer in PAR-1 signaling. In contrast, downregulation of zyxin did not affect thrombin-induced activation of RhoA or Gi, Gq and G12/13 heterotrimeric G proteins, implicating a novel signaling pathway regulated by PAR-1 that is not mediated by G proteins. Depletion of zyxin using siRNA inhibited thrombininduced actin stress fiber formation and serum response element (SRE)-dependent gene transcription. In addition, depletion of zyxin resulted in delay of endothelial barrier restoration after thrombin treatment. In 2017, Han et al. reported that downregulation of zyxin in HUVECs with shRNA inhibits cAMP-dependent secretion of vWF [116]. In zyxin shRNAexpressing cells, formation of the actin framework around exocytic WPBs was scarce. Moreover, phosphorylation of zyxin at serines 142 and 143 (S142/S143) is critical for vWF secretion since the zyxin mutant could not rescue the defect in zyxin shRNA-treated cells [116]. They showed that a protein kinase A (PKA)-specific inhibitor blocked zyxin phosphorylation at S142/S143 and concluded that zyxin acts downstream of PKA [116]. Han et al. thus proposed a novel model for cytoskeleton reorganization around WPBs undergoing exocytosis. Upon epinephrine stimulation, pre-existing filaments are reorganized to form actin frameworks around exocytotic granules, limiting granule movement and promoting their localization in close proximity to the plasma membrane [116]. Then, actin monomers are recruited from the cytosol to form coat structures around granules within actin frameworks that promote fusion [116]. It was postulated that ECs use this synergistic strategy for effective and precise exocytosis. Under their experimental conditions, zyxin downregulation with shRNA had no effect on vWF release upon thrombin or histamine stimulation, whereas these mice exhibited impaired epinephrine-stimulated vWF release, prolonged bleeding time and thrombosis. Live cell super-resolution microscopy allowed visualization of zyxin-dependent reorganization of pre-existing actin filaments around WPBs before fusion. Using the total internal reflection fluorescence structured illumination microscopy (TIRF-SIM) technique, it was possible to achieve simultaneous visualization of the dynamics of fine cortical actin filaments and the behavior of the exocytotic granule in close proximity of the plasma membrane. Zyxin promotes the recruitment of the actin regulatory protein α-actinin; α-actinin is an actin crosslinking protein [114]. To prove the co-localization of zyxin with its interacting partners, they co-expressed zyxin construct tagged with mCherry for fluorescence microscopy detection (zyxin-mCherry) and Lifeact tagged with green fluorescence protein (GFP-Lifeact) (Life act is an actin binding peptide used in microscopy to monitor the behavior of actin filaments). Interestingly, the assembly of the pre-existing filaments started when WPBs were still tubular, so the formation of the actin framework appeared as a pre-fusion event by TIRF-SIM. Alexa Fluor 647-G actin incorporation assay indicated that pre-existing actin filaments reorganize to form the actin framework around the tubular WPBs, and G-actin was also recruited to form the actin coat structure in proximity to WPBs fused to the membrane and connected with the actin frameworks. Once WPBs became spherical and fluorescentlylabeled, vWF was expulsed and fluorescence intensity declined in the expulsed area. The authors explain that the exocytotic events shown by variable-angle TIRF are mediated by the contraction of the actin coat which squeezes out WPB contents, followed by retraction of the depleted WPBs in the cytoplasm [116].

Detection of active vWF is now possible using an assay based on a nanobody AU/vWF a11 which allows investigators to distinguish between the active and latent conformations of the vWF A1 domain [123]. Several pathological conditions are associated with a disturbed balance in vWF activation and inactivation kinetics and thereby increased levels of active vWF and thrombotic complications [126]. The same active vWF assay revealed that levels of circulating active vWF increased approximately twofold in patients with acquired and congenital TTP [123, 126]. More and more evidence indicates that vWF is a biomarker of EC activation, but there are numerous discrepancies among the various clinical studies [127, 128]. In a more recent effort to advance the use of plasma vWF as a clinical marker of vascular inflammation, Hyseni et al. measured plasma concentrations of active vWF in a cohort of 275 patients with systemic inflammatory response syndrome [45]. They reported that patients with an elevated level of active vWF on admission had a twofold higher mortality rate [45]. In contrast, despite strongly elevated vWF levels, no predictions of mortality could be obtained based on total vWF [45]. Elevated active vWF is thus now regarded as an independent biomarker of poor outcome in patients with acute lung injury [129]. Mechanical ventilation is necessary to support the critical ill, but it also exacerbates injury through mechanical stress-activated signaling pathways, therefore it is expected to affect the disease outcome [130]. Consistent with these findings, an earlier 28-day study of 50 patients van der Heijden et al. reported that high vWF levels correlated with pulmonary compliance [Vt/(Pplat –PEEP)], where Vt = tidal volume, Pplat = plateau pressure, PEEP = positive end-expiratory pressure throughout the course of

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In order to fulfill its functions, vWF remodels in a few distinct ways [16, 132–136]. In ECs, vWF forms tubular structures inside acidic WPBs secretory granules [137]. The switch that converts highly packed vWF tubules into ultra-large vWF strings in the blood stream is critically important but poorly understood. Recently, more insight has been gained into the mechanism of rapid transition from tightly packed vWF tubules into intraendothelial granules to vWF strings that function at physiological pH. It is likely that distal to the fused end of the WPB, alkalinisation induces a rapid conformational change in the structure of vWF, which propagates causing vWF to unfurl in a concerted manner at the site of secretion, resulting in the loss

The highly multimeric, elongated form of vWF is not present in healthy plasma, but it is found in various pathological settings. This observation can be explained by the fact that vWF senses shear forces and remodels accordingly [134]. Atomic force micrographs have demonstrated at the single molecule level that under static conditions, vWF assumes a globular conformation, whereas, under high shear flow, vWF turns into an extended chain format [16] that forms ultra-large strings to which platelets bind to initiate clot formation at sites of vascular damage

provide factor VIII to the coagulation cascade [138]. We now realize that, while ultra-large MW (molecular weight) vWF is essential for the normal hemostasis, this multimeric array should not become too large because it alters the thrombotic propensity [15, 16, 133, 134, 138–147].

, factor VIII is released from its carrier protein to

septic shock while patients were mechanically ventilated [131].

3.2. vWF/ADAMTS13 axis in vascular health and disease

of the storage conformation [132].

[25] and, when shear stress is above 30.000 s<sup>1</sup>
