International Journal of Molecular Sciences Article Role of Autophagy in Von Willebrand Factor Secretion by Endothelial Cells and in the In Vivo Thrombin-Antithrombin Complex Formation Promoted by the HIV-1 Matrix Protein p17 Antonella Bugatti 1,† , Stefania Marsico 2,† , Pietro Mazzuca 1 , Kai Schulze 3 , Thomas Ebensen 3 , Cinzia Giagulli 1 , Esther Peña 4 , Lina Badimón 4 , Mark Slevin 5 , Arnaldo Caruso 1 , Carlos A. Guzman 3 and Francesca Caccuri 1, * 1 Department of Molecular and Translational Medicine, Section of Microbiology, University of Brescia Medical School, 25123 Brescia, Italy; antonella.bugatti@unibs.it (A.B.); p.mazzuca@unibs.it (P.M.); cinzia.giagulli@unibs.it (C.G.); arnaldo.caruso@unibs.it (A.C.) 2 Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036 Arcavacata di Rende, Italy; stefania.marsico@unical.it 3 Helmholtz Center for Infection Research (HZI), Department of Vaccinology and Applied Microbiology, 38124 Braunschweig, Germany; kai.schulze@helmholtz-hzi.de (K.S.); thomas.ebensen@helmholtz-hzi.de (T.E.); CarlosAlberto.Guzman@helmholtz-hzi.de (C.A.G.) 4 Cardiovascular Program ICCC, CiberCV, Hospital de la Santa Creu i Sant Pau, 08041 Barcelona, Spain; epena@santpau.cat (E.P.); lbadimon@santpau.cat (L.B.) 5 John Dalton Building, School of Healthcare Science, Manchester Metropolitan University, Manchester M1 5GD, UK; m.a.slevin@mmu.ac.uk * Correspondence: francesca.caccuri@unibs.it † These authors have contributed equally to this work.  Received: 22 November 2019; Accepted: 11 March 2020; Published: 16 March 2020  Abstract: Although the advent of combined antiretroviral therapy has substantially improved the survival of HIV-1-infected individuals, non-AIDS-related diseases are becoming increasingly prevalent in HIV-1-infected patients. Persistent abnormalities in coagulation appear to contribute to excess risk for a broad spectrum of non-AIDS defining complications. Alterations in coagulation biology in the context of HIV infection seem to be largely a consequence of a chronically inflammatory microenvironment leading to endothelial cell (EC) dysfunction. A possible direct role of HIV-1 proteins in sustaining EC dysfunction has been postulated but not yet investigated. The HIV-1 matrix protein p17 (p17) is secreted from HIV-1-infected cells and is known to sustain inflammatory processes by activating ECs. The aim of this study was to investigate the possibility that p17-driven stimulation of human ECs is associated with increased production of critical coagulation factors. Here we show the involvement of autophagy in the p17-induced accumulation and secretion of von Willebrand factor (vWF) by ECs. In vivo experiments confirmed the capability of p17 to exert a potent pro-coagulant activity soon after its intravenous administration. Keywords: HIV-1; p17 matrix protein; coagulation; thrombin-antithrombin complex; AIDS-related diseases; autophagy 1. Introduction Autophagy has been largely known as a degradative process serving a pro-survival, metabolite-generating role. Over the last few decades, a continuous growing body of evidence points to a function of autophagy in secretion of different molecules under normal and pathological Int. J. Mol. Sci. 2020, 21, 2022; doi:10.3390/ijms21062022 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 2022 2 of 16 conditions. Enhancement or down-modulation of the autophagic molecular machinery is followed by alterations in secretion. Autophagy plays a critical role in regulating unconventional [1–3] and conventional [4] secretory pathways, whereas a growing number of studies have also recently suggested a connection between autophagy and secretion of intracellular granules [5,6]. Von Willebrand factor (vWF) is a glycoprotein stored in specific secretory granules known as Weibel-Palade bodies (WPBs) [7,8]. This molecule is contained mainly in endothelial cells (ECs) and once secreted it multimerizes and assembles into long strings which tether to the underlying connective tissue, playing a crucial role in platelet trapping to maintain normal homeostasis [9]. Reduced secretion of vWF results in bleeding disorders [10], whereas increased release of vWF following EC dysfunction is known to contribute to thrombotic disorders [11,12]. Autophagy has been recently shown to regulate EC processing, maturation and secretion of vWF [13]. Inhibition of the autophagic flux in ECs with chloroquine resulted in a marked down-modulation of vWF secretion, and chloroquine administration in mice resulted in an increased bleeding time [13]. These findings demonstrated, for the first time, that transient pharmacological inhibition of autophagic flux may be a useful strategy to prevent thrombotic events [13]. Combined antiretroviral therapy (cART) has dramatically improved the survival of human immunodeficiency virus type 1-infected (HIV+ ) patients. With control of HIV-1 replication, serious non-AIDS-related diseases are now the dominant cause of morbidity and mortality in cART-treated populations [14]. Particularly, cardiovascular diseases have emerged as a clinically significant issue [15]. Several reports indicate that HIV+ patients have an increased risk of venous thrombosis [16] and a greater risk for myocardial infarction [15] than uninfected control individuals. Abnormal levels of coagulation markers are observed in HIV+ individuals [17]. Mainly, elevated levels of vWF have been reported in patients with HIV-1 disease compared to levels in control individuals [18], whilst the administration of cART improves but does not fully normalize the level of this pro-coagulant molecule [19]. The precise mechanisms driving a state of activation of coagulation in HIV+ patients are not entirely clear. The evidence that successfully cART-treated patients with negative viremia display a hypercoagulable state suggests diverse pathogenic mechanisms at work in addition to the direct effects of the HIV-1 itself. HIV-1-encoded proteins are expressed in HIV+ patients in the absence of viremia and they induce a chronically inflamed microenvironment leading to EC dysfunction [20]. In particular, the viral matrix protein p17 (p17) is detected in the blood [21] and accumulates in different tissues and organs [22–24], even in patients under successful cART [25,26]. This is not surprising since latently HIV-1-infected resting T cells are capable of producing gag proteins in a model of HIV-1 latency [27]. Moreover, recent data show that p17 is continuously released into the extracellular space from HIV-1-infected cells even in the absence of viral protease, following its cellular aspartyl protease-dependent cleavage from the gag precursor protein [28]. Extracellularly, p17 plays a critical role in the immune cell-mediated inflammatory processes [29–31] and it is known to activate ECs and promote a potent angiogenic activity [26,32]. Interestingly, we demonstrated that angiogenesis induced by p17 is partly supported by the release of ET-1 and by activation of the ET-1/ET-1 B receptor axis [32]. ET-1 secretion from ECs upon p17 stimulation was found to rely on mechanisms of conventional secretory pathways regulated by autophagy both in vitro and in vivo [33]. In this study, we provide evidence that the p17-driven activation of human ECs is associated with an increased cytoplasmic accumulation and secretion of vWF following activation of the autophagy pathway. Moreover, the intravenous (i.v.) injection of p17 promotes a pro-coagulant state in vivo, which does not occur in autophagy-deficient animals. These findings offer a new way of thinking about the possible cause of increased risk for coagulopathy in people living with HIV-1 and suggest autophagy as a specific target for treating or preventing coagulation disorders. Int. J. Mol. Sci. 2020, 21, 2022 3 of 16 2. Results 2.1. The HIV-1 Matrix Protein p17 Induces vWF Cytoplasmic Accumulation in ECs Int. J. Mol. Sci. 2019, 20, x 3 of 16 In order to understand the role of p17 in vWF accumulation and secretion, a mCherry-vWF- expressingInplasmid order to was understand used tothe role of p17 transfect humanin vWF accumulation umbilical and secretion, vein endothelial a mCherry-vWF- cells (HUVECs) and expressing plasmid was used to transfect human umbilical vein endothelial monitor vWF accumulation in WPBs by the classical red bright cigar-shaped appearance cells (HUVECs) in andthe monitor vWF accumulation in WPBs by the classical red bright cigar-shaped appearance in the cytoplasm [13]. Nucleofected HUVECs were then cultured under normal or serum-deprived conditions cytoplasm [13]. Nucleofected HUVECs were then cultured under normal or serum-deprived in the presence or absence of p17 (Figure 1A). Under normal culture conditions, p17 did not increase conditions in the presence or absence of p17 (Figure 1A). Under normal culture conditions, p17 did WPBs accumulation of vWF as compared to untreated (NT) cells or to cells treated with the irrelevant not increase WPBs accumulation of vWF as compared to untreated (NT) cells or to cells treated with protein theGST or the HIV-1 irrelevant proteincapsid GST orprotein the HIV-1p24capsid (p24) (Figure protein 1A). In contrast, p24 (p24) (Figure serum 1A). Instarved contrast,HUVECs serum showed an increased accumulation of vWF in response to p17 stimulation starved HUVECs showed an increased accumulation of vWF in response to p17 stimulationas compared to NT cells as or to cells treated compared withcells to NT GST or or to p24 cells (Figure 1A and treated with GSTSupplementary or p24 (Figure 1A Figure S1). The effectFigure and Supplementary of p171).on vWF accumulation observed The effect of p17 on vWF in macrovascular ECs was in accumulation observed also confirmed in ECs macrovascular microvascular ECs usinginthe was also confirmed human lung microvascular microvascular ECs usingendothelial the human lungcells microvascular (HMVEC-Ls)endothelial model (Figure 1B). cells (HMVEC-Ls) model (Figure 1B). 1. The FigureFigure HIV-1 1. The HIV-1matrix matrixprotein p17induces protein p17 inducesvonvon Willebrand Willebrand factorfactor (vWF) accumulation (vWF) accumulation in Weibel-in Weibel-Palade bodies (WPBs) under serum deprivation. HUVECs (A) and Palade bodies (WPBs) under serum deprivation. HUVECs (A) and HMVEC-Ls (B) were nucleofected HMVEC-Ls (B) were nucleofected with a mCherry-vWF-expressing plasmid and 24 h after nucleofection cells were starved or not starved with a mCherry-vWF-expressing plasmid and 24 h after nucleofection cells were for 16 or nothfor and16 h and then then stimulated stimulated in the in the presence presence or absence orng/mL of 10 absence of 10p24 of GST, ng/mL or p17of in GST, p24medium. complete or p17 in complete The medium. The vWF images display images display signals in redvWF signals and cell nucleiininred and blue. cellbar, Scale nuclei in blue. 10 μm. Scale bar, Red-positive 10 µm. punctate structures Red-positive were counted punctate in order structures wereto quantify countedthe levels of in order to WPBs. quantifyNTthe indicates levelsnot of treated WPBs. cells. Values NT indicates reported not treated for Values cells. vWF positive structures reported for vWF arepositive the meanstructures ± SD of 3 independent are the mean experiments ± SD of 3 with similar independent results. with experiments Statistical analysis similar wasStatistical results. performedanalysis by one-way was ANOVA, performed andbythe Bonferroni one-way post-test ANOVA, andwasthe used to compare data (***p < 0.001). Bonferroni post-test was used to compare data (*** p < 0.001). Int. J. Mol. Sci. 2020, 21, 2022 4 of 16 Int. J. Mol. Sci. 2019, 20, x 4 of 16 TheThe effect of p17 effect was of p17 abrogated was abrogatedbybypreincubating preincubating thethemedium mediumcontaining containing p17 p17 with with the the p17p17 mAbmAb MBS-3, thus confirming the specificity of p17 activity both in HUVEC (Figure 2A) and in MBS-3, thus confirming the specificity of p17 activity both in HUVEC (Figure 2A) and in HMVEC-Ls HMVEC-Ls (Figure 2B). 2B). (Figure Altogether, our data Altogether, our demonstrate that cytoplasmic data demonstrate vWF accumulation that cytoplasmic upon p17 vWF accumulation upontreatment p17 treatment is specific andisdepends specific and depends onof on activation activation a cellularofstress a cellular stress pathway. pathway. Figure 2. vWF accumulation in WPBs is specifically induced by p17. mCherry-vWF nucleofected Figure(A) HUVECs 2. vWF accumulation(B) and HMVEC-Ls inwere WPBs is specifically serum starved forinduced by p17. 16 h and thenmCherry-vWF treated with 10nucleofected ng/mL of GST, HUVECs (A) and HMVEC-Ls (B) were serum starved for 16 h and then treated with 10 ng/mL of GST, p24 or p17 in complete medium. When indicated, cells were stimulated with p17 (10 ng/mL) after p24 or p17 in complete medium. When indicated, cells were stimulated with p17 (10 ng/mL) after preincubation of the viral protein with 1 µg/mL of mAb to p17 (MBS-3) or unrelated control mAb preincubation of the viral protein with 1 µ g/mL of mAb to p17 (MBS-3) or unrelated control mAb (Ctrl mAb) for 30 min at 37 ◦ C. The images display vWF signals in red and cell nuclei in blue. Scale (Ctrl mAb) for 30 min at 37 °C. The images display vWF signals in red and cell nuclei in blue. Scale bar, 10 µm. Red-positive punctate structures were counted in order to quantify the levels of WPBs. bar, 10 μm. Red-positive punctate structures were counted in order to quantify the levels of WPBs. NT NT indicates notnot indicates treated cells. treated cells.Values Valuesreported for vWF reported for vWFpositive positivestructures structures areare thethe mean mean ± SD± of SD3 of 3 independent experiments with similar results. Statistical analysis was performed by one-way ANOVA, and the Bonferroni post-test was used to compare data (*** p < 0.001). Int. J. Mol. Sci. 2020, 21, 2022 5 of 16 2.2. Autophagy Controls the Storage and Secretion of vWF Upon p17 Stimulation In addition to the well-known mechanism of vWF secretion, which is dependent on the classical endoplasmic reticulum (ER)–Golgi complex pathway, autophagy plays a critical role in the biology of WPBs and regulates the in vitro and in vivo release of vWF [13]. Recently, p17-mediated lymphangiogenesis was found to depend on secretory autophagy pathway and secretion of ET-1 by ECs [32,33]. Thus, we tested the ability of p17 to promote vWF storage and secretion from endothelium and the involvement of autophagy in its activity. We used 3-MA, a synthetic and cell-permeable autophagic sequestration blocker [34] to pharmacologically inhibit autophagy. Nucleofected HUVECs with a mCherry-vWF-expressing plasmid were serum starved for 16 h in the presence or absence of 3-MA and then incubated or not with p17. Inhibition of autophagy with 3-MA significantly decreased the vWF cytoplasmic storage induced by p17 stimulation (Figure 3A). To further confirm the involvement of autophagy in the p17 capability to promote vWF accumulation in WPBs, we used siRNA to inhibit the expression of Beclin-1 (siBeclin), a coiled-coil protein involved in the regulation of autophagy in mammalian cells [35]. HUVECs were co-nucleofected with the mCherry-vWF-expressing plasmid and siBeclin. SiScramble was used as a negative control. Then, 24 h after nucleofection, cells were serum starved for 16 h and then stimulated or not with p17. As expected, inhibition of autophagy by Beclin-1 gene silencing significantly decreased the p17-triggered vWF accumulation in WPBs (Figure 3A). To better characterize the capability of p17 to trigger secretion of constitutively produced vWF, we performed a kinetic study by collecting supernatants of serum-starved HUVECs for 16 h in the presence or absence of 3-MA that were then cultured for 15 and 30 min in complete medium containing or not p17. Kinetic studies were also run using serum-starved HUVECs silenced or not with siBeclin 24 h before serum starvation. As shown in Figure 3B, p17 induced a significant increase of vWF secretion at 15 and 30 min of HUVECs stimulation, as compared to untreated cells. At the same time, p17-triggered vWF secretion was significantly inhibited by silencing of Beclin-1 or by pretreatment of HUVECs with 3-MA. Int. Int. J.J. Mol. Mol. Sci. 2019, 21, Sci. 2020, 20, 2022 x 66 of of 16 16 Figure 3. Inhibition of autophagy decreases p17-mediated release of vWF. (A) HUVECs were nucleofected with a mCherry-vWF-expressing plasmid and, when indicated, in combination with Figure 3. Inhibition of autophagy decreases p17-mediated release of vWF. (A) HUVECs were siBeclin-1 or siScramble. Twenty-four h after nucleofection, cells were serum starved for 16 h and nucleofected with a mCherry-vWF-expressing plasmid and, when indicated, in combination with then stimulated or not with 10 ng/mL of p17 in complete medium. When indicated, HUVECs were siBeclin-1 or siScramble. Twenty-four h after nucleofection, cells were serum starved for 16 h and then serum starved for 16 h in the presence or absence of 3-MA (5 mM). The images display vWF signals stimulated or not with 10 ng/mL of p17 in complete medium. When indicated, HUVECs were serum in red and cell nuclei in blue. Scale bar, 10 µm. Red-positive punctate structures were counted in starved for 16 h in the presence or absence of 3-MA (5 mM). The images display vWF signals in red order to quantify the levels of WPBs. NT indicates not treated cells. Values reported for vWF positive and cell nuclei in blue. Scale bar, 10 μm. Red-positive punctate structures were counted in order to structures are the mean ± SD of 3 independent experiments with similar results. Statistical analysis was quantify the levels of WPBs. NT indicates not treated cells. Values reported for vWF positive performed by one-way ANOVA, and the Bonferroni post-test was used to compare data (** p < 0.01, structures are the mean ± SD of 3 independent experiments with similar results. Statistical analysis *** p < 0.001). (B) HUVECs were serum starved for 16 h in the presence or absence of 3-MA (5 mM). was performed by one-way ANOVA, and the Bonferroni post-test was used to compare data (**p < When indicated, cells were nucleofected with siBeclin-1 or siScramble and serum starved 24 h after 0.01, ***p < 0.001). (B) HUVECs were serum starved for 16 h in the presence or absence of 3-MA (5 nucleofection. After serum starvation, cells were cultured in complete medium containing or not 10 mM). When indicated, cells were nucleofected with siBeclin-1 or siScramble and serum starved 24 h ng/mL of p17. Supernatants were collected at 15 and 30 min of culture and analyzed for the presence of after nucleofection. After serum starvation, cells were cultured in complete medium containing or not vWF by a standard quantitative ELISA. Bars represent the mean ± SD of triplicate samples. Statistical 10 ng/mL of p17. Supernatants were collected at 15 and 30 min of culture and analyzed for the analysis was performed by one-way ANOVA, and Bonferroni post-test was used to compare data presence of vWF by a standard quantitative ELISA. Bars represent the mean ± SD of triplicate samples. (** p < 0.01, *** p < 0.001). NT indicates not treated cells. Statistical analysis was performed by one-way ANOVA, and Bonferroni post-test was used to compare The role data (**p < 0.01, ***p of autophagy < 0.001). in the NT indicates intracellular vWF notaccumulation treated cells. and its secretion following p17 stimulation was further confirmed in HMVEC-Ls (Figure 4A,B). These data demonstrate that vWF The in secretion role of autophagy p17-treated ECs isindependent the intracellular vWF accumulation on the activation and its secretion of an autophagy-based following p17 pathway. stimulation was further confirmed in HMVEC-Ls (Figure 4A,B). These data demonstrate that vWF secretion in p17-treated ECs is dependent on the activation of an autophagy-based pathway. Int. J. Mol. Sci. 2020, 21, 2022 7 of 16 Int. J. Mol. Sci. 2019, 20, x 7 of 16 Figure4.4.Inhibition Figure Inhibitionof autophagy of autophagy decreases p17-mediated decreases release of p17-mediated vWF inof release HMVEC-Ls. (A) HMVEC-Ls vWF in HMVEC-Ls. (A) were nucleofected with a mCherry-vWF plasmid and, when indicated, in HMVEC-Ls were nucleofected with a mCherry-vWF plasmid and, when indicated, in combination combination with siBeclin-1 or irrelevant with (siScramble) siBeclin-1 siRNAs. or irrelevant Twenty-four (siScramble) siRNAs.h after nucleofection, Twenty-four cells h after were serum cells nucleofection, starvedwereforserum 16 h and then stimulated or not with 10 ng/mL of p17 in complete medium. starved for 16 h and then stimulated or not with 10 ng/mL of p17 in complete medium. WhenWhen indicated, HMVEC-Ls were serumHMVEC-Ls indicated, starved forwere16 h serum in the presence starved for or 16 absence h in theof presence 3-MA (5 or mM). The images absence of 3-MAdisplay (5 mM). vWF The signals imagesindisplay red andvWFcell nuclei signalsininblue. Scalecell red and bar,nuclei 10 µm.inRed-positive blue. Scale bar,punctate 10 μm.structures were counted Red-positive punctate in order to quantify structures the levels were counted of WPBs. in order NT indicates to quantify not of the levels treated WPBs. cells. Values reported NT indicates for vWF not treated positive cells. Values structures are the mean ± SD of 3 independent experiments with similar results. reported for vWF positive structures are the mean ± SD of 3 independent experiments with similarStatistical analysis was performed by one-way ANOVA, and the Bonferroni post-test was used to compare results. Statistical analysis was performed by one-way ANOVA, and the Bonferroni post-test was data (*** p < 0.001). (B) HMVEC-Ls were serum starved for 16 h in the presence or absence of 3-MA (5 mM). When indicated, used to compare data (***p < 0.001). (B) HMVEC-Ls were serum starved for 16 h in the presence or cells were nucleofected with siBeclin-1 or irrelevant (siScramble) siRNAs and 24 h after nucleofection absence of 3-MA (5 mM). When indicated, cells were nucleofected with siBeclin-1 or irrelevant were serum starved. After serum starvation, cells were cultured in complete medium containing or (siScramble) siRNAs and 24 h after nucleofection were serum starved. After serum starvation, cells not 10 ng/mL of p17. Supernatants were collected at 15 and 30 min of culture and analyzed for the were cultured in complete medium containing or not 10 ng/mL of p17. Supernatants were collected presence of vWF by a standard quantitative ELISA. Bars represent the mean ± SD of triplicate samples. at 15 and 30 min of culture and analyzed for the presence of vWF by a standard quantitative ELISA. Statistical analysis was performed by one-way ANOVA, and Bonferroni post-test was used to compare Bars represent the mean ± SD of triplicate samples. Statistical analysis was performed by one-way data (*** p < 0.001). NT indicates not treated cells. ANOVA, and Bonferroni post-test was used to compare data (***p < 0.001). NT indicates not treated cells. 2.3. Mechanistic Insight in the p17-Triggered Processing of vWF The secretion 2.3. Mechanistic of proteins Insight by the autophagy-based in the p17-Triggered process Processing of vWF without entering the classical ER–Golgi complex pathway is now established in eukaryotic cells [1,3,36]. Hence, to better understand the role ofThe secretion in autophagy of p17-mediated proteins by the autophagy-based vWF secretion fromprocess HUVECs, without enteringtwo we silenced thecrucial classical ER– genes Golgi complex involved pathwaynamely in autophagy, is now GRASP55 established[3,36] in eukaryotic and Rab8a cells [3].[1,3,36]. Hence, Silencing to better of each understand of these genes in the role of autophagy in p17-mediated vWF secretion from HUVECs, we silenced serum-starved HUVECs was sufficient to reduce WBPs storage of vWF promoted by p17 stimulation two crucial genes involved (Figure in As 5A). autophagy, expected,namely GRASP55 no effect on vWF[3,36] and Rab8a accumulation was[3].observed Silencinginofsilenced each ofGRASP55 these genesandin serum-starved HUVECs was sufficient to reduce WBPs storage of vWF promoted Rab8a HUVECs in the absence of p17 stimulation. Since the autophagy-driven secretion of proteins by p17 stimulation (Figure and 5A). As cytokines expected, requires no effect cathepsin-β on vWF[3], activation accumulation was observed we pharmacologically in silenced inhibited GRASP55 cathepsin-β and through Rab8a HUVECs in the absence of p17 stimulation. Since the autophagy-driven secretion of proteins and cytokines requires cathepsin-β activation [3], we pharmacologically inhibited cathepsin-β Int. J. Mol. Sci. 2019, 20, x 8 of 16 Int. J. Mol. Sci. 2020, 21, 2022 8 of 16 through the specific inhibitor CA-074Me. This blockage normalized p17-driven intracellular accumulation of vWF. Moreover, inhibition of GRASP55, Rab8a and cathepsin-β activity resulted in the specific inhibitor CA-074Me. This blockage normalized p17-driven intracellular accumulation of suppression of p17-induced vWF secretion (Figure 5B). vWF. Moreover, inhibition of GRASP55, Rab8a and cathepsin-β activity resulted in suppression of Altogether, our data support the key role played by autophagy in the p17-triggered vWF p17-induced vWF secretion (Figure 5B). secretion. Figure 5. Mechanistic insights into the vWF secretory activity of p17. (A) HUVECs were nucleofected Figure 5. Mechanistic insights into the vWF secretory activity of p17. (A) HUVECs were nucleofected with a mCherry-vWF-expressing plasmid in combination or not with siGRASP55, siRab8a or siScramble. with a mCherry-vWF-expressing plasmid in combination or not with siGRASP55, siRab8a or Twenty-four h after nucleofection, cells were serum starved for 16 h and then treated with 10 ng/mL of siScramble. Twenty-four h after nucleofection, cells were serum starved for 16 h and then treated with p17. When reported, HUVECs were serum starved in the presence or absence of 50 µM of CA-074Me 10 ng/mL of p17. When reported, HUVECs were serum starved in the presence or absence of 50 µ M (cathepsin-β inhibitor) and then stimulated or not with 10 ng/mL of p17. The images display vWF of CA-074Me (cathepsin-β inhibitor) and then stimulated or not with 10 ng/mL of p17. The images signals in red and cell nuclei in blue. Scale bar, 10 µm. Red-positive punctate structures were counted display vWF signals in red and cell nuclei in blue. Scale bar, 10 μm. Red-positive punctate structures in order to quantify the levels of WPBs. NT indicates not treated cells. Values reported for vWF positive were counted in order to quantify the levels of WPBs. NT indicates not treated cells. Values reported structures are the mean ± SD of 3 independent experiments with similar results. Statistical analysis was for vWF positive structures are the mean ± SD of 3 independent experiments with similar results. performed by one-way ANOVA, and the Bonferroni post-test was used to compare data (** p < 0.01, Statistical analysis was performed by one-way ANOVA, and the Bonferroni post-test was used to *** p < 0.001). (B) HUVECs were serum starved in the presence or absence of 50 µM of CA-074Me. compare data (**p < 0.01, ***p < 0.001). (B) HUVECs were serum starved in the presence or absence of In some experiments, HUVECs were nucleofected with siScramble, siGRASP55 or siRab8a and 24 h 50 μM of CA-074Me. In some experiments, HUVECs were nucleofected with siScramble, siGRASP55 after nucleofection, cells were serum starved for 16 h. After serum starvation, HUVECs were cultured in or siRab8a and 24 h after nucleofection, cells were serum starved for 16 h. After serum starvation, complete medium containing or not 10 ng/mL of p17. After 15 and 30 min of culture, supernatants were HUVECs were cultured in complete medium containing or not 10 ng/mL of p17. After 15 and 30 min collected and analyzed by a standard quantitative ELISA. Bars represent the mean ± SD of triplicate of culture, supernatants were collected and analyzed by a standard quantitative ELISA. Bars represent samples. Statistical analysis was performed by one-way ANOVA, and the Bonferroni post-test was the mean used ± SD ofdata to compare triplicate (* p