VWF other roles than hemostasis. Summary 1: VWF & hemostasis synthesis 11/4/16. Structure/function relationship & functions kDa.

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1 VWF other roles than hemostasis Len$ng PJ, Casari C et al JTH 2012 Summary 1: VWF & hemostasis synthesis Structure/function relationship & functions (HMWM) kDa multimerization propeptide FVIII GPIb collagen collagen αiibβ3 dimerization (LMWM) 500KDa(dimers) 1

2 VWF other roles than hemostasis Len$ng PJ, Casari C et al JTH 2012 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 2

3 VWF and angiogenesis 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 Starke RD et al Blood 2011 VWF and angiogenesis 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. Starke RD et al Blood

4 How does VWF control angiogenesis? Ø Extracellular pathway Ø Intracellular pathway Randi AM & Laffan MA JTH 2016 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 4

5 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. 5

6 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 6

7 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$ng PJ, Casari C et al JTH

8 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 β 2 -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 8

9 the immune cell receptor Siglec-5 has been identified as a receptor for VWF HEK293 HEK293-Siglec-5 DUOLINK Monocyte + PMA no VWF Pegon J, Casari C et al Haematologica 2012 Monocyte + PMA + VWF To visualize proteins that colocalize (<40 nm) hbp:// Two primary anmbodies raised in different species recognize the target anmgen or anmgens of interest. Species-specific secondary anmbodies, called PLA probes, each with a unique short DNA strand abached to it, bind to the primary anmbodies. When the PLA probes are in close proximity (<40 nm), the DNA strands can interact through a subsequent addimon of two other connecmng DNA oligonucleomdes. A^er joining of the two added oligonucleomdes by enzymamc ligamon, they are amplified via rolling circle amplificamon using a polymerase. Following amplificamon the amplicons (several-hundred fold) are visualized by hybridizamon to labeled complementary oligonucleomde probes. The resulmng high concentramon of fluorescent probes 9

10 hbp:// Ø platelet-decorated VWF strings at the surface of EC efficiently recruit leukocytes, even under conditions of high shear stress Platelets adhered to endothelial cell-bound ultra-large von Willebrand factor strings support leukocyte tethering and rolling under high shear stress No ADAMTS13 No platelets Bernardo A et al JTH 2005 Journal of Thrombosis and Haemostasis Volume 3, Issue 3, pages , 3 FEB 2005 DOI: /j x hbp://onlinelibrary.wiley.com/doi/ /j x/full#f5 10

11 Ø platelet-decorated VWF strings at the surface of EC efficiently recruit leukocytes, even under conditions of high shear stress Platelets adhered to endothelial cell-bound ultra-large von Willebrand factor strings support leukocyte tethering and rolling under high shear stress with ADAMTS13 Bernardo A et al JTH 2005 Journal of Thrombosis and Haemostasis Volume 3, Issue 3, pages , 3 FEB 2005 DOI: /j x hbp://onlinelibrary.wiley.com/doi/ /j x/full#f6 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. 11

12 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) Chauhan AK et al JEM

13 How can the proinflammatory activity of VWF be counteracted? stroke model: ADAMTS-13 regulates VWFdependent inflammatory responses > ADAMTS-13 deficiency increases susceptibility to focal cerebral ischemia Because of VWF proinflammatory response Zhao BQ et al Blood 2009 How can the proinflammatory activity of VWF be counteracted? stroke model: ADAMTS-13 regulates VWFdependent inflammatory responses > ADAMTS-13 deficiency increases susceptibility to focal cerebral ischemia intravenous administration of recombinant ADAMTS-13 in wildtype mice markedly reduced infarction volume Because of VWF proinflammatory response Zhao BQ et al Blood

14 How can the proinflammatory activity of VWF be counteracted? stroke model: ADAMTS-13 regulates VWFdependent inflammatory responses > ADAMTS-13 deficiency increases susceptibility to focal cerebral ischemia intravenous administration of recombinant ADAMTS-13 in wildtype mice markedly reduced infarction volume These Because results of VWF proinflammatory suggest that response proteolytic degradation of VWF by ADAMTS-13 downregulates the proinflammatory potential of VWF. Zhao BQ et al Blood 2009 VWF other roles than hemostasis Len$ng PJ, Casari C et al JTH