scholarly journals Rab27a and MyRIP regulate the amount and multimeric state of VWF released from endothelial cells

Blood ◽  
2009 ◽  
Vol 113 (20) ◽  
pp. 5010-5018 ◽  
Author(s):  
Thomas D. Nightingale ◽  
Krupa Pattni ◽  
Alistair N. Hume ◽  
Miguel C. Seabra ◽  
Daniel F. Cutler
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Crucial Role ◽  
Shear Force ◽  
Small Gtpases ◽  
Cargo Protein ◽  
Platelet Binding ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Rab Effector

Endothelial cells contain cigar-shaped secretory organelles called Weibel-Palade bodies (WPBs) that play a crucial role in both hemostasis and the initiation of inflammation. The major cargo protein of WPBs is von Willebrand factor (VWF). In unstimulated cells, this protein is stored in a highly multimerized state coiled into protein tubules, but after secretagogue stimulation and exocytosis it unfurls, under shear force, as long platelet-binding strings. Small GTPases of the Rab family play a key role in organelle function. Using siRNA depletion in primary endothelial cells, we have identified a role for the WPB-associated Rab27a and its effector MyRIP. Both these proteins are present on only mature WPBs, and this rab/effector complex appears to anchor these WPBs to peripheral actin. Depletion of either the Rab or its effector results in a loss of peripheral WPB localization, and this destabilization is coupled with an increase in both basal and stimulated secretion. The VWF released from Rab27a-depleted cells is less multimerized, and the VWF strings seen under flow are shorter. Our results indicate that this Rab/effector complex controls peripheral distribution and prevents release of incompletely processed WPB content.

scholarly journals von Willebrand factor released from Weibel-Palade bodies binds more avidly to extracellular matrix than that secreted constitutively

Blood ◽  
1987 ◽  
Vol 69 (5) ◽  
pp. 1531-1534 ◽  
Author(s):  
LA Sporn ◽  
VJ Marder ◽  
DD Wagner
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Immunofluorescent Staining ◽  
Cultured Endothelial Cells ◽  
Human Foreskin Fibroblasts ◽  
Platelet Binding ◽  
The Matrix ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Large multimers of von Willebrand factor (vWf) are released from the Weibel-Palade bodies of cultured endothelial cells following treatment with a secretagogue (Sporn et al, Cell 46:185, 1986). These multimers were shown by immunofluorescent staining to bind more extensively to the extracellular matrix of human foreskin fibroblasts than constitutively secreted vWf, which is composed predominantly of dimeric molecules. Increased binding of A23187-released vWf was not due to another component present in the releasate, since releasate from which vWf was adsorbed, when added together with constitutively secreted vWf, did not promote binding. When iodinated plasma vWf was overlaid onto the fibroblasts, the large forms bound preferentially to the matrix. These results indicated that the enhanced binding of the vWf released from the Weibel-Palade bodies was likely due to its large multimeric size. It appears that multivalency is an important component of vWf interaction with the extracellular matrix, just as has been shown for vWf interaction with platelets. The pool of vWf contained within the Weibel-Palade bodies, therefore, is not only especially suited for platelet binding, but also for interaction with the extracellular matrix.

scholarly journals Anti-β2-GPI Antibodies Induce Von Willebrand Factor Release, Modulate Platelet Binding, and Affect VWF Proteolysis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1444-1444
Author(s):  
Christopher J. Ng ◽  
Keith R. McCrae ◽  
Junmei Chen ◽  
Michael Wang ◽  
Marilyn J. Manco-Johnson ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Antiphospholipid Syndrome ◽  
Von Willebrand Factor ◽  
Image Acquisition ◽  
Type I ◽  
Adamts13 Activity ◽  
Normal Plasma ◽  
Platelet Binding ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Background: The antiphospholipid syndrome (APS) is characterized by predisposition to thrombosis. The cause for this pathology is poorly understood but is likely multifactorial, involving activation of blood cells and vasculature. The role that anti-β2-GPI antibodies play in von Willebrand factor (VWF) release from endothelial cells, VWF-platelet binding, and VWF cleavage by ADAMTS13 has not been well characterized in APS. We decided to study the effect of these antibodies on expressed ultra large VWF strings (ULVWF strings) that bind platelets (VWF-PLT strings) under flow to better understand platelet–VWF binding and ADAMTS13 regulation in APS. Hypothesis: We hypothesized that Anti-β2-GPI antibodies could induce VWF release from endothelial cells and modulate VWF’s prothombotic effect through alterations in VWF-Platelet binding and VWF cleavage by ADAMTS13. Methods: Human umbilical vein endothelial cells were seeded in 96-well plates/flow chambers prepared with a collagen Type I substrate for static/flow experiments, respectively. Static assays: Cells were incubated for 1 hr with Anti-β2-GPI or control antibodies and the conditioned media was assayed for VWF by ELISA, normalized to normal plasma. Flow Assay Analysis: After stimulation with agonist and perfusion with a platelet suspension, platelets bound to ULVWF in a string pattern were quantified via brightfield microscopy. Images of chambers were captured and VWF-PLT string-units (defined as a string length of 25μM) per slide were quantified. To minimize bias, image acquisition was standardized and the investigator was blinded at time of image acquisition/analysis. β2-GPI Flow assays: Endothelial cells in flow chambers were stimulated with 50ng/mL of phorbol myristate acetate (PMA), and a solution of fixed platelets with β2-GPI or β2-GPI+Anti-β2-GPI were perfused prior to image acquisition. ADAMTS13 assays: After stimulation with 25ng/mL PMA and perfusion with fixed platelets, images were acquired. Then control/patient plasma was perfused over formed strings. Images taken after plasma perfusion were quantified and compared to images prior to plasma perfusion. Data are shown as mean +/- SEM, and significance was determined as p<0.05 by student’s t-test or Mann-Whitney U Test, when appropriate. Results: Static Assays: Compared to control human IgG (8.28 +/- 3.34 mU/mL), VWF release was increased in the presence of two patient-derived Anti-β2-GPI antibodies, APS25-6 Anti-β2-GPI, 35.73 +/- 7.83 mU/mL (P = 0.008) and APS203-2 Anti-β2-GPI, 34.08 +/- 7.119 mU/mL (P = 0.039). As compared to control rabbit IgG (15.80 +/- 7.12 mU/mL), a rabbit polyclonal Anti-β2-GPI antibody, R24-6, also demonstrated increased soluble VWF (43.16 +/- 9.60 mU/mL, P = 0.013) release. β2GPI Flow Assays:The presence of β2GPI (2µM) reduced String-unit formation from 50.10 +/-5.57 Sting-units/image to 20.98 +/- 2.05 String Units/image (P < 0.0001) as compared to buffer. Addition of goat Anti-β2-GPI antibody (1µM) increased the VWF-PLT string observed as compared to β2GPI (2µM), 30.09 +/- 1.83 String Units to 20.98 +/- 2.05 String Units (P = 0.012) indicating that an Anti-β2-GPI antibody partially reverses the effect of β2GPI on reducing VWF-PLT string formation. ADAMTS13 Assay:Compared to pooled normal plasma (ADAMTS13 Activity 100%) (4.57 +/- 0.60 String Units/image cleaved), there was a significant decrease in the amount of string units/image cleaved in two APS plasmas with Anti-β2-GPI antibodies, APS232-9 (-0.23 +/- 0.98, P = 0.0003) and APS227-9 (2.23 +/- 0.73, P = 0.0009). ADAMTS13 Activity of patient plasma was 98.37% and 83.97%, respectively. These results suggest an inhibitory role of APS plasma on the cleavage of ULVWF strings. Conclusions: Anti-β2-GPI antibodies and antiphospholipid syndrome plasma may contribute to the prothrombotic phenotype observed in APS by three mechanisms: 1) the increased release of VWF from endothelial cells after incubation with Anti-β2-GPI, 2) increased platelet binding to ULVWF strings likely mediated by interfering with β2GPI’s known inhibition of Gp1bα VWF-platelet binding, and 3) a reduced ability to cleave VWF-PLT strings by APS plasma, suggestive of ADAMTS13 inhibition that does not correlate with ADAMTS13 activity. Taken together, our results suggest that VWF and its modulation may contribute to the prothrombotic phenotype observed in the antiphospholipid syndrome. Disclosures No relevant conflicts of interest to declare.

von Willebrand factor released from Weibel-Palade bodies binds more avidly to extracellular matrix than that secreted constitutively

Blood ◽  
1987 ◽  
Vol 69 (5) ◽  
pp. 1531-1534 ◽  
Author(s):  
LA Sporn ◽  
VJ Marder ◽  
DD Wagner
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Immunofluorescent Staining ◽  
Cultured Endothelial Cells ◽  
Human Foreskin Fibroblasts ◽  
Platelet Binding ◽  
The Matrix ◽  
Von Willebrand ◽  
Willebrand Factor

Large multimers of von Willebrand factor (vWf) are released from the Weibel-Palade bodies of cultured endothelial cells following treatment with a secretagogue (Sporn et al, Cell 46:185, 1986). These multimers were shown by immunofluorescent staining to bind more extensively to the extracellular matrix of human foreskin fibroblasts than constitutively secreted vWf, which is composed predominantly of dimeric molecules. Increased binding of A23187-released vWf was not due to another component present in the releasate, since releasate from which vWf was adsorbed, when added together with constitutively secreted vWf, did not promote binding. When iodinated plasma vWf was overlaid onto the fibroblasts, the large forms bound preferentially to the matrix. These results indicated that the enhanced binding of the vWf released from the Weibel-Palade bodies was likely due to its large multimeric size. It appears that multivalency is an important component of vWf interaction with the extracellular matrix, just as has been shown for vWf interaction with platelets. The pool of vWf contained within the Weibel-Palade bodies, therefore, is not only especially suited for platelet binding, but also for interaction with the extracellular matrix.

Transition from Non-Platelet-Binding to Platelet-Binding Conformation of Von Willebrand Factor Occurs upon Exocytosis

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3917-3917
Author(s):  
Evelyn Groot ◽  
Rob Fynheer ◽  
Silvie AE Sebastian ◽  
Peter J Lenting ◽  
Philip G De Groot
Keyword(s):  
Monoclonal Antibody ◽  
Endothelial Cells ◽  
Vascular Injury ◽  
Von Willebrand Factor ◽  
Binding Conformation ◽  
Platelet Binding ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Adhesion Of Platelets

Abstract Introduction Von Willebrand factor (VWF) is a large multimeric glycoprotein that contributes to platelet recruitment at sites of vascular injury. VWF is mainly produced in endothelial cells from where it is secreted directly into the circulation or stored in the rod-shaped organelles called Weibel-Palade bodies. VWF present in the circulation does not bind to platelets. Stimulated endothelial cells secrete VWF that has the capacity to spontaneously interact with platelets. Conversion of the platelet-binding conformation of secreted VWF into the non-binding conformation of plasma VWF involves proteolytic processing by the metalloprotease ADAMTS13. At sites of vascular injury, binding of VWF to the exposed subendothelial collagen induces a conformational change in VWF allowing a strong interaction with the platelet receptor glycoprotein (Gp)Ibα. Undesired secretion of active VWF may also occur in several pathological conditions. One example is von Willebrand disease type 2B (VWD2B), where a gain of function mutation in the VWF/A1 domain induces a permanent platelet-binding state in the VWF molecule. As a consequence, VWF can spontaneously interact with platelets in the circulation, leading to thrombocytopenia, a hallmark of VWD2B. Objective The aim of this study was to investigate whether VWF present in the Weibel-Palade bodies of endothelial cells is stored in a platelet-binding conformation. Methods Immunofluorescence experiments were performed on wildtype and VWD2B endothelial cells. Monoclonal antibody AU/VWF-a11 is directed against the VWF/A1 domain and recognizes VWF only when it is in its GpIb-binding conformation. Monoclonal antibody AU/VWF-C37H is directed against the VWF/A3 domain and recognizes both the platelet-binding and the non-platelet- binding conformation. Experiments were performed on cultured endothelial cells to study the conformation of VWF in the Weibel-Palade bodies. To study the conformation of secreted VWF, stimulated endothelial cells were perfused with washed platelets. Results AU/VWF-C37H fluorescence was observed in the Weibel-Palade bodies of both wildtype and VWD2B endothelial cells, whereas AU/VWF-a11 fluorescence was only detected in the Weibel-Palade bodies of the VWD2B cells. Perfusion of washed platelets over wildtype and VWD2B endothelial cells resulted in adhesion of platelets to thin strings of secreted VWF. These strings stained positive for both AU/VWF-C37H and AU/VWF-a11. Of note, significantly more platelets adhered to VWF secreted from VWD2B than from wildtype endothelial cells. This hyperactive VWD2B-like platelet adhesion pattern could be mimicked by wildtype endothelial cells upon perfusion with platelets that were mixed with ristocetin. Conclusions VWF stored within the Weibel-Palade bodies of endothelial cells does not possess platelet-binding capacities. Upon secretion, VWF undergoes a conformational change that allows the adhesion of platelets. The presence of ADAMTS13 is necessary to prevent the release of platelet-binding VWF in the circulation. In the absence of ADAMTS13, secreted VWF does not expose all its GpIb-binding sites as more platelets adhere to ristocetin activated VWF-strings or strings released from VWD2B endothelial cells.

Co-Trafficking of Coagulation Factor VIII with Von Willebrand Factor Alters the Macromolecular Structure Inside Secretory Weibel-Palade Bodies

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 325-325
Author(s):  
Eveline Bouwens ◽  
Marjon Mourik ◽  
Maartje van den Biggelaar ◽  
Jan Voorberg ◽  
Karine Valentijn ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Conflicts Of Interest ◽  
Coagulation Factor ◽  
Live Cell ◽  
Confocal Imaging ◽  
Macromolecular Structure ◽  
Platelet Binding ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 325 The liver is generally recognized as the major site of coagulation factor (F)VIII synthesis. However, there is now increasing evidence that FVIII can also be synthesized in specific endothelial cells where it is stored with its natural carrier protein von Willebrand factor (VWF) in the Weibel-Palade bodies (WPBs). WPBs have a typical cigar-shaped appearance that most likely originates from the macromolecular organization of VWF multimers into tubules. The tubular storage of VWF is thought to be essential for orderly secretion of VWF strings during activation of endothelial cells. Recently we have shown that expression of FVIII with VWF changes the WPB morphology to spherical vesicles. This finding suggests alterations in the biochemical properties of stored VWF. We now studied in detail the effect of FVIII co-expression on the VWF molecule using a combination of innovative techniques, including correlative light-electron microscopy (CLEM), and live-cell fluorescence microscopy under flow conditions. Analysis of human blood outgrowth endothelial cells (BOECs) expressing human B-domain deleted FVIII-GFP by CLEM revealed that FVIII containing WPBs were electron-dense, spherical structures. These structures contained disorganized short VWF tubules, which was confirmed in 3D by electron tomography. Double immunogold labelling with VWF and GFP antibodies showed that the spherical FVIII containing structures were always positive for VWF. These observations imply that FVIII blocks the expansion of VWF tubules, possibly by binding to the N-terminal VWF domains. As the N-terminal domains are also implicated in the formation of multimers, we therefore investigated whether FVIII affects VWF multimer size. Indeed, multimer analysis showed that VWF secreted by FVIII-GFP transduced BOECs was multimerized to a lesser extent when compared to VWF secreted by non-transduced BOECs. The combined absence of high molecular weight (HMW) VWF multimers and long VWF tubules made us question whether these cells could still release ultra-large VWF (UL-VWF) strings. UL-VWF strings play a key role in bleeding arrest, as platelets adhere to the released VWF string which ultimately leads to the formation of a platelet plug. We examined the release of UL-VWF strings under shear stress from BOECs expressing FVIII-GFP employing live-cell confocal imaging. This technique allowed us to follow FVIII release during exocytosis of WPBs in real-time as well. When we stimulated FVIII-transduced BOECs with histamine, these cells were equally able to release VWF strings as non-transduced BOECs. Although spherical WPBs lacked long VWF tubules and did not secrete HMW multimers, released VWF strings were of similar length as strings secreted by non-transduced BOECs. Surprisingly, released VWF strings were completely covered with FVIII which remained attached to the strings throughout the whole experiment. Another remarkable observation was that platelet binding to the FVIII-covered VWF strings was almost completely absent. We hypothesize that FVIII either shields the A1 domain for platelet binding or causes a conformational change in the VWF strings that prevents platelets from binding to the strings. Our results demonstrate that FVIII co-trafficking with VWF has a major impact on properties of VWF as it reduces the degree of multimerization, shortens tubules and prevents platelets from adhering to strings. This leads us to the conclusion that the macromolecular structure of VWF is considerably altered when FVIII is present in WPBs. Disclosures: No relevant conflicts of interest to declare.

Factor VIII alters tubular organization and functional properties of von Willebrand factor stored in Weibel-Palade bodies

Blood ◽  
2011 ◽  
Vol 118 (22) ◽  
pp. 5947-5956 ◽  
Author(s):  
Eveline A. M. Bouwens ◽  
Marjon J. Mourik ◽  
Maartje van den Biggelaar ◽  
Jeroen C. J. Eikenboom ◽  
Jan Voorberg ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Endothelial Cells ◽  
Confocal Microscopy ◽  
Factor Viii ◽  
Functional Properties ◽  
Von Willebrand Factor ◽  
Equal Length ◽  
Platelet Binding ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract In endothelial cells, von Willebrand factor (VWF) multimers are packaged into tubules that direct biogenesis of elongated Weibel-Palade bodies (WPBs). WPB release results in unfurling of VWF tubules and assembly into strings that serve to recruit platelets. By confocal microscopy, we have previously observed a rounded morphology of WPBs in blood outgrowth endothelial cells transduced to express factor VIII (FVIII). Using correlative light-electron microscopy and tomography, we now demonstrate that FVIII-containing WPBs have disorganized, short VWF tubules. Whereas normal FVIII and FVIII Y1680F interfered with formation of ultra-large VWF multimers, release of the WPBs resulted in VWF strings of equal length as those from nontransduced blood outgrowth endothelial cells. After release, both WPB-derived FVIII and FVIII Y1680F remained bound to VWF strings, which however had largely lost their ability to recruit platelets. Strings from nontransduced cells, however, were capable of simultaneously recruiting exogenous FVIII and platelets. These findings suggest that the interaction of FVIII with VWF during WPB formation is independent of Y1680, is maintained after WPB release in FVIII-covered VWF strings, and impairs recruitment of platelets. Apparently, intra-cellular and extracellular assembly of FVIII-VWF complex involves distinct mechanisms, which differ with regard to their implications for platelet binding to released VWF strings.

scholarly journals Formation of platelet-binding von Willebrand factor strings on non-endothelial cells

2012 ◽  
Vol 10 (10) ◽  
pp. 2168-2178 ◽  
Author(s):  
J. W. WANG ◽  
J. A. VALENTIJN ◽  
K. M. VALENTIJN ◽  
B. S. DRAGT ◽  
J. VOORBERG ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Platelet Binding ◽  
Von Willebrand ◽  
Willebrand Factor

Requirement for Both D Domains of the Propolypeptide in von Willebrand Factor Muitimerization and Storage

1993 ◽  
Vol 70 (06) ◽  
pp. 1053-1057 ◽  
Author(s):  
Agnès M Journet ◽  
Simin Saffaripour ◽  
Denisa D Wagner
Keyword(s):  
Endothelial Cells ◽  
Endoplasmic Reticulum ◽  
Von Willebrand Factor ◽  
Deletion Mutants ◽  
Relative Importance ◽  
D2 Domain ◽  
Constitutive Secretion ◽  
Von Willebrand ◽  
And Storage ◽  
Willebrand Factor

SummaryBiosynthesis of the adhesive glycoprotein von Willebrand factor (vWf) by endothelial cells results in constitutive secretion of small multimers and storage of the largest multimers in rodshaped granules called Weibel-Palade bodies. This pattern is reproduced by expression of pro-vWf in heterologous cells with a regulated pathway of secretion, that store the recombinant protein in similar elongated granules. In these cells, deletion of the vWf prosequence prevents vWf storage. The prosequence, composed of two homologous domains (D1 and D2), actively participates in vWf multimer formation as well. We expressed deletion mutants lacking either the D1 domain (D2vWf) or the D2 domain (D1vWf) in various cell lines to analyze the relative importance of each domain in vWf muitimerization and storage. Both proteins were secreted efficiently without being retained in the endoplasmic reticulum. Despite this, neither multimerized past the dimer stage and they were not stored. We conclude that several segments of the prosequence are jointly involved in vWf muitimerization and storage.

Adverse Influence of Cigarette Smoking on the Endothelium

1993 ◽  
Vol 70 (04) ◽  
pp. 707-711 ◽  
Author(s):  
Andrew D Blann ◽  
Charles N McCollum
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Tobacco Smoke ◽  
Lipid Peroxides ◽  
Short Exposure ◽  
Acute Effects ◽  
Adverse Influence ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryThe effect of smoking on the blood vessel intima was examined by comparing indices of endothelial activity in serum from smokers with that from non-smokers. Serum from smokers contained higher levels of von Willebrand factor (p <0.01), the smoking markers cotinine (p <0.02) and thiocyanate (p <0.01), and was more cytotoxic to endothelial cells in vitro (p <0.02) than serum from non-smokers. The acute effects of smoking two unfiltered medium tar cigarettes was to briefly increase von Willebrand factor (p <0.001) and cytotoxicity of serum to endothelial cells in vitro (p <0.005), but lipid peroxides or thiocyanate were not increased by this short exposure to tobacco smoke. Although there were correlations between von Willebrand factor and smokers consumption of cigarettes (r = 0.28, p <0.02), number of years smoking (r = 0.41, p <0.001) and cotinine (r = 0.45, p <0.01), the tissue culture of endothelial cells with physiological levels of thiocyanate or nicotine suggested that these two smoking markers were not cytotoxic. They are therefore unlikely to be directly responsible for increased von Willebrand factor in the serum of smokers. We suggest that smoking exerts a deleterious influence on the endothelium and that the mechanism is complex.

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