3.1. vWF as a surrogate marker of endothelial dysfunction

Association between vascular dysfunction and increased vWF levels is well established. The first study found a correlation between factor VIII antigen (another name for vWF) and kidney vascular damage in the context of glomerulonephritis in the 1970s [118]. Since this time, there have been many reports of inflammatory diseases associated with elevated vWF, that is, in acute systemic inflammation disorders such as sepsis, acute respiratory distress syndrome or systemic inflammatory response syndrome [73], T2DM [119] and TTP [120].

Upon activation of ECs, vWF acts as an acute phase reactant and correlates with serum C-reactive protein (CRP) level, another acute phase reactant [121, 122]. Other hemostatic proteins also behave in this way (i.e. factor VIII, fibrinogen and plasminogen inhibitor 1). Active vWF corresponds to the form of vWF required for platelet receptor GpIb binding [123–125]. 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 septic shock while patients were mechanically ventilated [131].

#### 3.2. vWF/ADAMTS13 axis in vascular health and disease

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].

156 Endothelial Dysfunction - Old Concepts and New Challenges

3. Dynamics of vWF reactivity in acute diseases

3.1. vWF as a surrogate marker of endothelial dysfunction

systemic inflammatory response syndrome [73], T2DM [119] and TTP [120].

Association between vascular dysfunction and increased vWF levels is well established. The first study found a correlation between factor VIII antigen (another name for vWF) and kidney vascular damage in the context of glomerulonephritis in the 1970s [118]. Since this time, there have been many reports of inflammatory diseases associated with elevated vWF, that is, in acute systemic inflammation disorders such as sepsis, acute respiratory distress syndrome or

Upon activation of ECs, vWF acts as an acute phase reactant and correlates with serum C-reactive protein (CRP) level, another acute phase reactant [121, 122]. Other hemostatic proteins also behave in this way (i.e. factor VIII, fibrinogen and plasminogen inhibitor 1). Active vWF corresponds to the form of vWF required for platelet receptor GpIb binding [123–125].

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 of the storage conformation [132].

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 [25] and, when shear stress is above 30.000 s<sup>1</sup> , factor VIII is released from its carrier protein to 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].

The last biosynthetic step required for the formation of normal vWF is the limited proteolysis of the multimeric array by the circulating metalloprotease ADAMTS13 [126, 135]. ADAMTS-13 is produced mainly by the hepatic stellate cells [148]. Secondarily, the enzyme is synthesize in other cell type (ECs, podocytes, platelets and glial cells), but the physiological importance of these other sources was not yet established [149]. ADAMTS-13 plasma concentration is in the range of 0.7–1.4 μg/mL [150]. Interestingly, ADAMTS-13 is secreted into the circulation as an active enzyme and has a plasma half-life of approximately 2–4 days [151]. The mechanism of ADAMTS-13 clearance is not completely elucidated. It has been suggested that the hepatic asialoglycoprotein receptor could be involved in ADAMTS-13 clearance [152]. It is believed that newly released multimeric forms of vWF become tethered to the EC surface through the interaction with P-selectin [153, 154] and become partially unfolded [16], allowing for ADAMTS13 cleavage of the multimeric arrays [136]. The physiological proteolytic processing of vWF by ADAMTS13 occurs between Tyr1605-Met1606 in the central A2 domain of vWF, and is facilitated by partially unfolding vWF by flow conditions in the microcirculation [124, 126, 155–162].

damage [171]. vWF promotes intracerebral thrombo-inflammatory response in the context of acute stroke [167, 172–175] and the vWF/ADAMTS13 axis was found to be involved in acute and chronic ischemic cerebrovascular events in patients [175]. vWF-deficient mice are protected from ischemic stroke [174]. It is well known that circulating vWF originates primarily from the endothelium, with a minor contribution from platelets [20]. Our current understanding of the role of different pools of vWF in the pathophysiology of acute stroke is based on mouse models [176]. EC-vWF mice are chimeric mice that express vWF only in ECs. Irradiated mice transplanted with bone marrow from vWF/ mice to repopulate myeloid cells lack vWF in platelets but express vWF in ECs. Plt-mice are chimeric vWF/ mice transplanted with normal bone marrow, and therefore have vWF in platelets [176]. The wildtype (WT), vWF/, Plt-vWF and EC-vWF mice were subjected to 1 h ischemia followed by 23 h reperfusion and the outcome was compared [176]. The infarct volume and neurological outcome were comparable in WT and EC-vWF mice and decreased in vWF/ mice. The stroke outcome in EC-vWF mice was documented using triphenyl-tetrazolium chloride-stained serial brain sections after 1 h transient middle cerebral artery occlusion. Also, it was observed in EC-VWF mice that there was a reduced local cerebral blood flow as assessed by Doppler flowmeter and a higher rate of thrombus formation in comparison to vWF/ mice assessed by intravital microscopy. Stroke, infarction, vascular spasm and iatrogenic thrombotic events cause ischemia, which has a dramatic impact on vital organs. Post-ischemic reperfusion changes the pH, and extracellular alkalinization stimulates the physiological mechanisms that directly promote EC activation and can double vWF release in a [Ca2+]-dependent manner

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the perfusion media) [171] might prove to be a promising new way of inhibiting EC activation upon reperfusion injury. Taken together, EC-vWF seems to be the primary determinant of vWF-dependent ischemic stroke [171, 176], while platelet vWF plays a minor role [20, 176]. The ability to control vWF secretion from dysfunctional endothelium might be a valuable

Apart from its primary role in hemostasis, vWF has a dual role in the pathophysiology of severe sepsis. On the one hand, vWF is involved in host-defense with a possible initial protective role in preventing complement activation [121]. In addition to its classical role in platelet binding, the vWF A1 domain also plays a role in vWF-dependent inflammatory responses. vWF and platelets promote leukocyte diapedesis, downstream of leukocyte tethering, rolling and adhesion in a mouse model of thioglycollate-induced peritonitis [177]. Polymorphonuclear leukocytes seem to directly interact with vWF via P-selectin glycoprotein ligand-1 and β2-integrins [178] As shown in mice in which inflammation was provoked by two different methods, [27] vWF-regulated leukocyte recruitment can be blocked by administration of a blocking anti-vWF llama nanobody, KB-VWF-006, which has picomolar affinity for the vWF

On the other hand, sepsis is not only a systemic inflammatory condition, it is also a state of dysfunctional endothelium and coagulation. One of the early on signs of systemic endothelial

/H<sup>+</sup> exchange subtype I pump inhibitor (applied in

[171]. Delaying the pH recovery with a Na+

therapeutic target in stroke prone patients.

3.4. vWF role in sepsis

A1 domain [27].

Too little cleavage of the newly released vWF produces a tendency for microvascular thrombotic occlusion, as in TTP [120], and too much cleavage by ADAMTS13 results in type 2 like vWD and a bleeding phenotype [38]. Cleavage is dramatically increased during systemic inflammation [45] and other prothrombotic conditions, and there is an inverse relationship between plasma levels of vWF and ADAMTS-13; under high shear stress/inflammatory conditions, the circulating vWF can acquire autoimmune resistance to proteolysis by ADAMTS-13 or ADAMTS-13 is quickly exhausted as there is more vWF to cleave than available cleaving enzyme for consumption [45]. Additionally, a decrease in the vWF-inactivating protease ADAMTS13 is dependent on the severity of the disease and organ dysfunction [163].

In mice models, vWF-deficiency abrogates the ADAMTS13-deficient prothrombotic state [164] and endotoxemia-induced thrombosis in mice [165], consistent with the observation that vWFdeficient mice subjected to the polymicrobial model of sepsis exhibit increased survival [29]. It has been recently reported that neutrophil-derived small peptides or human neutrophil peptides (HNPs) also known as α-defensins can inhibit cleavage of vWF by ADAMTS13 [166]; these peptides [167] have sequence similarities with the ADAMTS-13 spacer domain RRY motif and can bind to the cleavage site for ADAMTS-13 on the vWF A2 domain. It was shown that HNPs levels are high in TTP patients [166]. Pro-inflammatory cytokine IL-6 limits ADAMTS-13 function [168, 169], while IL-8 and TNF-α stimulate further the release of vWF from WPBs [168].

In conclusion, excessive levels of the highly prothrombotic and multimeric form of vWF and/or ADAMTS13 deficiency constitute a unifying pathologic mechanism linking inflammation to thrombosis [170].

#### 3.3. vWF role in ischemic stroke

Stroke remains a major health concern and a leading cause of death in the adult population. As a result of intracerebral thrombosis, cerebral ischemia/reperfusion injury causes brain tissue damage [171]. vWF promotes intracerebral thrombo-inflammatory response in the context of acute stroke [167, 172–175] and the vWF/ADAMTS13 axis was found to be involved in acute and chronic ischemic cerebrovascular events in patients [175]. vWF-deficient mice are protected from ischemic stroke [174]. It is well known that circulating vWF originates primarily from the endothelium, with a minor contribution from platelets [20]. Our current understanding of the role of different pools of vWF in the pathophysiology of acute stroke is based on mouse models [176]. EC-vWF mice are chimeric mice that express vWF only in ECs. Irradiated mice transplanted with bone marrow from vWF/ mice to repopulate myeloid cells lack vWF in platelets but express vWF in ECs. Plt-mice are chimeric vWF/ mice transplanted with normal bone marrow, and therefore have vWF in platelets [176]. The wildtype (WT), vWF/, Plt-vWF and EC-vWF mice were subjected to 1 h ischemia followed by 23 h reperfusion and the outcome was compared [176]. The infarct volume and neurological outcome were comparable in WT and EC-vWF mice and decreased in vWF/ mice. The stroke outcome in EC-vWF mice was documented using triphenyl-tetrazolium chloride-stained serial brain sections after 1 h transient middle cerebral artery occlusion. Also, it was observed in EC-VWF mice that there was a reduced local cerebral blood flow as assessed by Doppler flowmeter and a higher rate of thrombus formation in comparison to vWF/ mice assessed by intravital microscopy. Stroke, infarction, vascular spasm and iatrogenic thrombotic events cause ischemia, which has a dramatic impact on vital organs. Post-ischemic reperfusion changes the pH, and extracellular alkalinization stimulates the physiological mechanisms that directly promote EC activation and can double vWF release in a [Ca2+]-dependent manner [171]. Delaying the pH recovery with a Na+ /H<sup>+</sup> exchange subtype I pump inhibitor (applied in the perfusion media) [171] might prove to be a promising new way of inhibiting EC activation upon reperfusion injury. Taken together, EC-vWF seems to be the primary determinant of vWF-dependent ischemic stroke [171, 176], while platelet vWF plays a minor role [20, 176]. The ability to control vWF secretion from dysfunctional endothelium might be a valuable therapeutic target in stroke prone patients.

#### 3.4. vWF role in sepsis

The last biosynthetic step required for the formation of normal vWF is the limited proteolysis of the multimeric array by the circulating metalloprotease ADAMTS13 [126, 135]. ADAMTS-13 is produced mainly by the hepatic stellate cells [148]. Secondarily, the enzyme is synthesize in other cell type (ECs, podocytes, platelets and glial cells), but the physiological importance of these other sources was not yet established [149]. ADAMTS-13 plasma concentration is in the range of 0.7–1.4 μg/mL [150]. Interestingly, ADAMTS-13 is secreted into the circulation as an active enzyme and has a plasma half-life of approximately 2–4 days [151]. The mechanism of ADAMTS-13 clearance is not completely elucidated. It has been suggested that the hepatic asialoglycoprotein receptor could be involved in ADAMTS-13 clearance [152]. It is believed that newly released multimeric forms of vWF become tethered to the EC surface through the interaction with P-selectin [153, 154] and become partially unfolded [16], allowing for ADAMTS13 cleavage of the multimeric arrays [136]. The physiological proteolytic processing of vWF by ADAMTS13 occurs between Tyr1605-Met1606 in the central A2 domain of vWF, and is facilitated by partially unfolding vWF by flow conditions in the microcirculation [124,

Too little cleavage of the newly released vWF produces a tendency for microvascular thrombotic occlusion, as in TTP [120], and too much cleavage by ADAMTS13 results in type 2 like vWD and a bleeding phenotype [38]. Cleavage is dramatically increased during systemic inflammation [45] and other prothrombotic conditions, and there is an inverse relationship between plasma levels of vWF and ADAMTS-13; under high shear stress/inflammatory conditions, the circulating vWF can acquire autoimmune resistance to proteolysis by ADAMTS-13 or ADAMTS-13 is quickly exhausted as there is more vWF to cleave than available cleaving enzyme for consumption [45]. Additionally, a decrease in the vWF-inactivating protease

ADAMTS13 is dependent on the severity of the disease and organ dysfunction [163].

In mice models, vWF-deficiency abrogates the ADAMTS13-deficient prothrombotic state [164] and endotoxemia-induced thrombosis in mice [165], consistent with the observation that vWFdeficient mice subjected to the polymicrobial model of sepsis exhibit increased survival [29]. It has been recently reported that neutrophil-derived small peptides or human neutrophil peptides (HNPs) also known as α-defensins can inhibit cleavage of vWF by ADAMTS13 [166]; these peptides [167] have sequence similarities with the ADAMTS-13 spacer domain RRY motif and can bind to the cleavage site for ADAMTS-13 on the vWF A2 domain. It was shown that HNPs levels are high in TTP patients [166]. Pro-inflammatory cytokine IL-6 limits ADAMTS-13 function [168, 169], while IL-8 and TNF-α stimulate further the release of vWF

In conclusion, excessive levels of the highly prothrombotic and multimeric form of vWF and/or ADAMTS13 deficiency constitute a unifying pathologic mechanism linking inflammation to

Stroke remains a major health concern and a leading cause of death in the adult population. As a result of intracerebral thrombosis, cerebral ischemia/reperfusion injury causes brain tissue

126, 155–162].

158 Endothelial Dysfunction - Old Concepts and New Challenges

from WPBs [168].

thrombosis [170].

3.3. vWF role in ischemic stroke

Apart from its primary role in hemostasis, vWF has a dual role in the pathophysiology of severe sepsis. On the one hand, vWF is involved in host-defense with a possible initial protective role in preventing complement activation [121]. In addition to its classical role in platelet binding, the vWF A1 domain also plays a role in vWF-dependent inflammatory responses. vWF and platelets promote leukocyte diapedesis, downstream of leukocyte tethering, rolling and adhesion in a mouse model of thioglycollate-induced peritonitis [177]. Polymorphonuclear leukocytes seem to directly interact with vWF via P-selectin glycoprotein ligand-1 and β2-integrins [178] As shown in mice in which inflammation was provoked by two different methods, [27] vWF-regulated leukocyte recruitment can be blocked by administration of a blocking anti-vWF llama nanobody, KB-VWF-006, which has picomolar affinity for the vWF A1 domain [27].

On the other hand, sepsis is not only a systemic inflammatory condition, it is also a state of dysfunctional endothelium and coagulation. One of the early on signs of systemic endothelial activation is sepsis-induced elevation of plasma vWF levels [121, 129, 163, 179]. Sepsis can promote DIC, which is common in critically ill patients. When unfolded, highly multimeric sepsis-induced vWF interacts with platelets [26] and generates small clots in the microvasculature [45, 124]. Microvascular thrombosis appears to be a major pathological mechanism in sepsis pathology resulting in multi-organ dysfunction syndrome (MODS) [124]. Hence, sepsisinduced vWF secretion in excess in DIC and MODS are inevitably linked. In addition, sepsisinduced ultra-large high molecular weight multimeric vWF permits complement activation, [121] leading to a positive feedback cycle of inflammation and thrombosis.

vWF antagonism, illustrating how they might become an option to overcome current limita-

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Most of the studies involving anti-vWF antibodies were conducted in animal models. Monoclonal antibodies have been widely tested in murine, monkey and rabbit models of cardiovascular disease, including coronary thrombosis, stroke and in-stent stenosis: GPG-290, 6B4-Fab, AJW200, 82D6A6 and SZ-123 [188, 189]. These are all agents with antithrombotic and antiinflammatory effects which do not prolong bleeding time and do not provoke thrombocytopenia. However, to date, none of these have made it into clinical trials. Hillgruber and colleagues recently published a study that found a massive accumulation of vWF in skin biopsies of patients suffering from leukocytoclastic vasculitis, an immune complex (IC)-mediated vasculitis (ICV) frequently encountered in dermatology that is caused by IC precipitation in the vessel wall followed by recruitment of neutrophils [28]. These results were confirmed in a murine model of vasculitis [28], and importantly, they identified ICs as possible vWF secretagogues. Of note, polyclonal anti-vWF blocking antibodies had an anti-inflammatory effect, reducing leukocyte recruitment and edema formation in a murine model of vascular inflammation [28]. Although the physiological implications of elevated vWF levels (increasing up to 400% of the normal level in various conditions [190, 191]) require additional investigation, it is currently believed that plasma vWF level is a surrogate marker of increased risk of vascular complications in septic patients and in those with vascular disease, which makes vWF antagonism a

Aptamers and nanobodies have been demonstrated to directly interfere with the vWF pathway, suggesting their powerful antithrombotic properties and acceptable level of safety may prove to be useful strategies. Aptamers are oligonucleotides that have similar characteristics with monoclonal antibodies and with small molecules. ARC1779 is an aptamer that binds to the A1 domain of vWF, blocking its interaction with GpIb and thereby inhibiting vWFdependent platelet function. Clinical trials with ARC1779 were conducted and completed, but until truly meaningful clinical endpoints can be obtained that indicate effective inhibition of thrombosis without increasing bleeding, the utility of aptamers will remain unclear. Before the clinical trial was prematurely closed by the sponsor, the clinical and laboratory data after 14 days of dosing in nine patients with TTP aged 18–75 were very promising, and the authors optimistically proposed the study continuation (www.clinicaltrials.com; [192]) suggesting fur-

Nanobodies have the advantage of being highly specific therapeutic agents. Caplacizumab (ALX-0081) is an anti-vWF humanized nanobody that selectively targets vWF A1 domain, locking platelets launching on VWF A1 domain via interaction with their receptor GPIb. TITAN phase II trial concluded that caplacizumab administration in 75 patients with acquired TTP lead to a rapid resolution of the acute TTP episode, but it was adversely seconded by mild

Unlike the vWF aptamer or antibodies, a pharmacological inhibitor of vWF secretion would have the advantage of acting from the inside of ECs, thereby limiting the amount of vWF available to support binding of platelets and thrombus formation. Studies thus far from our group suggest inhibition of Gα12-dependent activation of vWF secretion from WPBs may in

promising therapeutic target in thrombosis and inflammation.

tions of antithrombotic therapy.

ther development may be in order.

bleeding when compared to placebo group [193–195].

In murine model of sepsis, mice subjected to cecal ligation and puncture (CLP) have increased circulating levels of vWF that promote a procoagulant phenotype and poor outcome [29]. vWF-deficient mice subjected to the CLP model of sepsis exhibit increased survival [180]. For the purpose of testing whether Gα12 modulates vWF secretion in sepsis pathological setting, we subjected Gα12/ mice and WT control mice to the CLP model of polymicrobial sepsis and monitored survival [181]. Our preliminary results indicate that 80% of the Gα12/ mice survived, while all WT mice succumbed in the first 96 h [182]. To further determine whether decreased vWF was responsible for the protective effect of G12 deficiency, we restored circulating vWF level in Gα12/ mice, by intravenous administration of purified vWF [10], to the level observed in WT mice. This procedure resulted in the loss of the protective effect of G12 deletion [182]. Furthermore, we employed a pharmacological G12 inhibition approach to prove G12 involvement in sepsis-induced vWF secretion [183]. We hypothesized that a synthetic peptide derived from Gα12 would inhibit α-SNAP-dependent WPB priming and fusion with the plasma membrane [89, 182], thereby inhibiting vWF secretion from activated ECs, and reducing the risk of thrombotic microangiopathy during sepsis [182].

#### 3.5. vWF role in other inflammatory diseases

Transient elevations of plasma vWF level were also observed after epinephrine infusion [184], and chronic elevation of circulating vWF (2–3-fold) was reported in hyperthyroidism, which is thought to be due to β-adrenergic receptor stimulation. In contrast, hypothyroidism is associated with a 15% decrease in vWF level, although this is reversible with appropriate treatment [185].

vWF secretion from ECs might play a role in sickle cell disease pathogenesis [186, 187]. Erythrocytes bind specifically to vWF [186] and stasis, or the reduction of blood flow and shear, promotes binding of erythrocytes to vWF which form fibrin-rich regions in venous thrombi [186].
