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VWF other roles than hemostasis

Len$ngPJ,CasariCetalJTH2012

Summary 1: VWF & hemostasis

GPIb collagen collagen αIIbβ3 FVIII

multimerization dimerization

propeptide

synthesis

Structure/function relationship & functions

20.000kDa

500KDa(dimers) (LMWM)

(HMWM)

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VWF other roles than hemostasis

Len$ngPJ,CasariCetalJTH2012

Vascular abnormalities in patients with VWD or AVWS Ø Up to 20% of patients with VWD present with gastrointestinal

(GI) bleeding particularly common in absence of HMWMs

Ø  GI bleeding has been linked to the presence of angiodysplasia (small vascular malformation of the gut)

Ø  Angiodysplastic lesions are thought to develop due to dysregulated angiogenesis, leading to the production of fragile vessels prone to bleeding

Ø Vascular malformations outside the GI track have also been reported in patients with VWD

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VWF and angiogenesis

StarkeRDetalBlood2011

Ø  Inhibition of VWF expression in human umbilical vein EC (HUVEC) using siRNA resulted in increased proliferation, migration and in vitro angiogenesis

HUVEC ± VWF-siRNA ± VWF on matrigel (in absence of growth factors) Capillary network formation was observed and quantify measuring total tube length

VWF and angiogenesis

StarkeRDetalBlood2011

Ø  In vivo, angiogenesis and vascular density were found to be increased in the VWF deficient mouse, (in several physiological and pathological models)

Blood vessels (anti-α-smooth muscle actin-Cy3) in ears from CTR or VWF-ko mice Ø  the constitutive vascular

network in VWF-ko mice is increased

Ø  These data suggest that VWF is involved in the control of vascular development.

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How does VWF control angiogenesis?

RandiAM&LaffanMAJTH2016

Ø  Extracellular pathway

Ø  Intracellular pathway

How does VWF control angiogenesis? Extracellular pathway: •  VWF binds integrin αVβ3 on EC

•  Pharmacological inhibition of αVβ3 inhibits angiogenesis •  Genetic deficiency of β3 enhances angiogenesis

•  αVβ3 is associated with VEGFR-2 signaling •  Lack of β3 > é VEGFR-2 signaling > immature and fragile blood vessels

(similar to angiodysplastic lesions) •  Lack of VWF > é VEGFR-2 signaling in EC (+ proliferation and

migration) > suggesting that VWF may control angiogenesis by inhibiting VEGFR-2 signaling

•  In vascular smooth muscle cells the interaction of VWF with αVβ3 has been shown to affect arterial maturation

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How does VWF control angiogenesis? Extracellular pathway: •  VWF binds integrin αVβ3 on EC

•  Pharmacological inhibition of αVβ3 inhibits angiogenesis •  Genetic deficiency of β3 enhances angiogenesis

•  αVβ3 is associated with VEGFR-2 signaling •  Lack of β3 > é VEGFR-2 signaling > immature and fragile blood vessels

(similar to angiodysplastic lesions) •  Lack of VWF > é VEGFR-2 signaling in EC (+ proliferation and

migration) > suggesting that VWF may control angiogenesis by inhibiting VEGFR-2 signaling

•  In vascular smooth muscle cells the interaction of VWF with αVβ3 has been shown to affect arterial maturation

How does VWF control angiogenesis? Intracellular pathway: •  VWF is stored in WPBs, which also contain vasoactive

molecules such as Angiopoietin-2 (Ang-2) •  Ang-2 can act to destabilize blood vessels •  and synergize with VEGFR-2 to promote angiogenesis

•  Inhibition of VWF expression > increases (synthesis and) release of Ang-2

•  Whether Ang-2 has a role in the increased, disrupted

angiogenesis and in angiodysplasia, associated with lack of VWF, is not know.

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How does VWF control angiogenesis? Intracellular pathway: •  VWF is stored in WPBs, which also contain vasoactive

molecules such as Angiopoietin-2 (Ang-2) •  Ang-2 can act to destabilize blood vessels •  and synergize with VEGFR-2 to promote angiogenesis

•  Inhibition of VWF expression > increases (synthesis and) release of Ang-2

•  Whether Ang-2 has a role in the increased, disrupted

angiogenesis and in angiodysplasia, associated with lack of VWF, is not know.

Ø All these data suggest that VWF is required for physiological angiogenesis possibly acting at multiple stages of blood vessel development.

von Willebrand Disease (VWD)

Acquired von Willebrand Syndrome

(AVWS)

Ø One of the most frequent bleeding disorder in humans

Ø Caused by congenital decrease or dysfunction of VWF

Ø Due to dysfunction or degradation of VWF, often in association with myeloproliferative and malignant disorders, aortic valve stenosis or left ventricular assist devices

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VWF Ø VWF is synthetized by EC and stored in Weibel-Palade bodies

(WPB)

Ø  The formation WPB is dependent on the synthesis of VWF

Ø WPB also contain vasoactive molecules, which can bind to VWF >>> raising the possibility that VWF directs and regulates their action after release (with consequent effect on angiogenesis)

VWF other roles than hemostasis

Len$ngPJ,CasariCetalJTH2012

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VWF & inflammation Ø  Indirect link: VWF directs P-selectin to WPBs, which is

important for its regulated exposure on activated EC

•  Deficiency of VWF provokes impaired P-selectin surface expression and subsequent defects in leukocyte recruitment in the early phases of inflammation

VWF & inflammation Ø  Indirect link: VWF directs P-selectin to WPBs, which is

important for its regulated exposure on activated EC

•  Deficiency of VWF provokes impaired P-selectin surface expression and subsequent defects in leukocyte recruitment in the early phases of inflammation

Ø VWF may actively participate in the inflammatory response

•  VWF can function as an adhesive surface for leukocytes via interactions with P-selectin, GP ligand-1 and β2-integrins

•  the immune cell receptor Siglec-5 has also been identified as a receptor for VWF *

•  platelet-decorated VWF strings at the surface of EC efficiently recruit leukocytes, even under conditions of high shear stress *

•  finally, VWF–platelet complexes are critical to optimal extravasation of leukocytes

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PegonJ,CasariCetalHaematologica2012

HEK293 HEK293-Siglec-5

Monocyte+PMAnoVWF

Monocyte+PMA+VWF

DUOLINK

To visualize proteins that colocalize (<40 nm)

the immune cell receptor Siglec-5 has been identified as a receptor for VWF

hBp://www.sigmaaldrich.com

•  TwoprimaryanMbodiesraisedindifferentspeciesrecognizethetargetanMgenoranMgensofinterest.Species-specificsecondaryanMbodies,calledPLAprobes,eachwithauniqueshortDNAstrandaBachedtoit,bindtotheprimaryanMbodies.WhenthePLAprobesareincloseproximity(<40nm),theDNAstrandscaninteractthroughasubsequentaddiMonoftwootherconnecMngDNAoligonucleoMdes.A^erjoiningofthetwoaddedoligonucleoMdesbyenzymaMcligaMon,theyareamplifiedviarollingcircleamplificaMonusingapolymerase.FollowingamplificaMontheamplicons(several-hundredfold)arevisualizedbyhybridizaMontolabeledcomplementaryoligonucleoMdeprobes.TheresulMnghighconcentraMonoffluorescentprobes

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hBp://www.abnova.com/products/products_detail.asp?catalog_id=DP0031

Plateletsadheredtoendothelialcell-boundultra-largevonWillebrandfactorstringssupportleukocytetetheringandrollingunderhighshearstress

JournalofThrombosisandHaemostasisVolume3,Issue3,pages562-570,3FEB2005DOI:10.1111/j.1538-7836.2005.01122.xhBp://onlinelibrary.wiley.com/doi/10.1111/j.1538-7836.2005.01122.x/full#f5

No ADAMTS13

BernardoAetalJTH2005

Ø  platelet-decorated VWF strings at the surface of EC efficiently recruit leukocytes, even under conditions of high shear stress

No platelets

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Plateletsadheredtoendothelialcell-boundultra-largevonWillebrandfactorstringssupportleukocytetetheringandrollingunderhighshearstress

JournalofThrombosisandHaemostasisVolume3,Issue3,pages562-570,3FEB2005DOI:10.1111/j.1538-7836.2005.01122.xhBp://onlinelibrary.wiley.com/doi/10.1111/j.1538-7836.2005.01122.x/full#f6

with ADAMTS13

BernardoAetalJTH2005

Ø  platelet-decorated VWF strings at the surface of EC efficiently recruit leukocytes, even under conditions of high shear stress

VWF & inflammation

Importantly: Ø  our knowledge of the physiologic relevance of VWF in

inflammatory processes is primarily derived from animal studies using different models for inflammation (such as atherosclerosis, wound healing, experimental allergic encephalomyelitis, cytokine-induced meningitis, and stroke)

Ø  In patients, this connection is less well established, which

is probably attributable to the multifactorial nature of these inflammatory conditions.

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VWF & inflammation Ø  how the contribution of VWF to the inflammatory process might be regulated?

•  secretion of VWF from EC and/or platelets allows the protein to participate in a timely manner in the inflammatory response process

How can the proinflammatory activity of VWF be counteracted?

ADAMTS-13 deficiencly is associated with: •  increased leukocyte rolling on

unstimulated veins •  and increased leukocyte

adhesion in inflamed veins, but only when VWF is present in experimental models of atherosclerosis (in which lack of ADAMTS-13 exaggerates the VWF–platelet dependent inflammatory response by increasing leukocyte recruitment to lesion sites)

ChauhanAKetalJEM2008

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stroke model: •  ADAMTS-13 regulates VWF-

dependent inflammatory responses > ADAMTS-13 deficiency increases susceptibility to focal cerebral ischemia

How can the proinflammatory activity of VWF be counteracted?

BecauseofVWFproinflammatoryresponse

ZhaoBQetalBlood2009

stroke model: •  ADAMTS-13 regulates VWF-

dependent inflammatory responses > ADAMTS-13 deficiency increases susceptibility to focal cerebral ischemia

•  intravenous administration of recombinant ADAMTS-13 in wild-type mice markedly reduced infarction volume

How can the proinflammatory activity of VWF be counteracted?

BecauseofVWFproinflammatoryresponse

ZhaoBQetalBlood2009

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stroke model: •  ADAMTS-13 regulates VWF-

dependent inflammatory responses > ADAMTS-13 deficiency increases susceptibility to focal cerebral ischemia

•  intravenous administration of recombinant ADAMTS-13 in wild-type mice markedly reduced infarction volume

How can the proinflammatory activity of VWF be counteracted?

BecauseofVWFproinflammatoryresponse

ZhaoBQetalBlood2009

These results suggest that proteolytic degradation of VWF by ADAMTS-13 downregulates the proinflammatory potential of VWF.

VWF other roles than hemostasis

Len$ngPJ,CasariCetalJTH2012