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 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.

Characterization of Platelet Alpha-Granule Dynamics

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 327-327
Author(s):  
Ngozi A Wilkins ◽  
Brian Storrie ◽  
Jeffrey A Kamykowski
Keyword(s):  
Electron Microscopy ◽  
Confocal Microscopy ◽  
Guinea Pig ◽  
Von Willebrand Factor ◽  
Electron Tomography ◽  
Human Platelets ◽  
Dense Granules ◽  
Early Endosomes ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 327 Background: Platelets, anucleated cells that play a critical role in blood clotting, store proteins and small molecules in alpha-granules and dense granules, respectively, for secretion. Alpha-granules contain several proteins including von Willebrand factor and fibrinogen and dense granules contain serotonin. Rab4, a marker for the early endosomes has been implicated in regulating alpha granule secretions (Sirakawa et al, 2010). Previous fluorescence microscopy mapping of alpha-granule protein distributions suggested that there are either two different alpha-granule types or subdomains within a single granule population (Storrie and Seghal, 2007; Italiano et al, 2008). More recent work based on electron tomography (Kamykowski et al, manuscript in preparation) indicates that human platelets are comprised of one alpha granule population. We hypothesized that there was a single population of alpha-granules in which all fibrinogen is similarly compartmentalized. Hence, fibrinogen endocytocized by guinea pig megakaryocytes and platelets in vivo at 4 h (short label) and 24 h (long label) would map to the same location. Aims: We carried out several experiments to form a basis for future high-resolution (5 nm) electron tomography to establish packaging of HRP-conjugated fibrinogen or nanogold conjugated fibrinogen into platelet alpha-granules. (a) Using PD-10 columns, we prepared Cy3 conjugated fibrinogen. Using an in vivo guinea pig model to test the ability of guinea pig platelets to take up fluorescently labeled fibrinogen, we injected 10 mg/ml of Cy3 conjugated fibrinogen (short label, 4 h) and 10 mg/ml of commercially purchased AlexaFluor 488 conjugated fibrinogen (long label, 28 h) into guinea pigs. Platelets were then fixed, purified and confocal microscopy performed. (b) Using triple immunofluorescence, serotonin antibody was applied to fixed and purified resting state human and guinea pig platelets and immunofluorescence microscopy was performed to provide whole platelet information on the staining pattern of the dense granules in comparison to the alpha-granules and early endosomes. (c) Preliminary Electron Microscopy fixation conditions were also tested on guinea pig platelets. Results: For the uptake experiment, spinning-disk confocal microscopy was used to collect full platelet volume image stacks which were then deconvolved, pixel shift corrected for red and green channels and analyzed. Overlap of green and red fibrinogen conjugates was observed where the fluorescently tagged fibrinogens were taken up by structures presumed to be alpha-granules. For the triple labeling experiments, the distribution of serotonin, Rab4 and von Willebrand factor was observed in resting state platelets. Using spinning-disk confocal microscopy, full platelet volume image stacks were collected, deconvolved, pixel shift corrected for red, far red and green channels and analyzed. Serotonin antibody gave an abundant punctate staining pattern in both the triple-labeled human and guinea pig platelets. In both the human platelets and the guinea pig platelets, the serotonin positive punctate granules, presumed to be dense granules, had a more similar pattern to the von Willebrand factor positive punctate alpha granules, than to the Rab4 positive punctate granules, presumed to be the early endosomes. The triple label results were unexpected because previous electron microscopy studies have indicated that the dense granules in human platelets are fewer in number than the alpha-granules and fewer than the corresponding dense granules in guinea pig platelets. Results of the electron microscopy preparations are pending. Conclusions: Our results indicate that the guinea pig model, while its platelets are a much smaller size than human platelets, is a good system for loading alpha-granules with labeled proteins for electron tomography. The serotonin distribution results together with previous electron tomography also raise the question as to whether dense granules could be a specialized form of the alpha-granules. A summary of this research will be presented at the Promoting Minorities in Hematology event during the 2010 ASH meeting. Disclosures: No relevant conflicts of interest to declare.

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.

Imaging of Von Willebrand Factor Remodeling Upon Secretion From Vascular Endothelial Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 263-263
Author(s):  
Marjon J Mourik ◽  
Karine M Valentijn ◽  
Jack A Valentijn ◽  
Jan Voorberg ◽  
Abraham J Koster ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Light And Electron Microscopy ◽  
Live Cell ◽  
String Formation ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 263 In response to vascular injury, endothelial cells rapidly secrete high molecular weight multimers of the coagulation protein Von Willebrand factor (VWF). Once expelled from the cells, VWF unfurls in long strings that bind platelets from the bloodstream to induce primary hemostasis. VWF secreted upon stimulation is released from specialized storage compartments called Weibel Palade bodies (WPB) which have a typical rod or cigar shape. They emerge from the Trans Golgi network in a process driven by the formation of helical tubules consisting of VWF multimers and the VWF propeptide. When WPBs undergo exocytosis and release VWF, rapid structural changes occur which eventually result in platelet capturing VWF strings. It has been postulated that the tubular storage of VWF in WPBs is required for sufficient unfolding of the protein during string formation as agents disrupting the VWF tubules were shown to result in less strings. Recently we described a novel structure involved in VWF exocytosis which is formed only upon stimulation. We refer to this structure as a “secretory pod” as it seemed to derive from multiple WPBs and was identified as a VWF release site where strings seemed to be formed. By transmission electron microscopy (TEM) we identified this structure to be a membrane-delimited organelle containing filamentous material resembling unfurled VWF. The VWF tubules as seen in WPBs are absent in secretory pods suggesting that tubular packaging of VWF is not essential for sufficient release and string formation. To study the formation of secretory pods and the subsequent release and remodeling of VWF, several imaging techniques were used such as live-cell imaging and correlative light and electron microscopy. We expressed propeptide-EGFP in endothelial cells to label the WPBs and stimulated them with PMA. By live-cell imaging we visualized the exocytotic events. We observed, apart from single WPB exocytosis, the formation of secretory pods which occurred by the coalescence of several WPBs. In some cases the individual WPBs rounded up first, before they joined into one round structure while in other cases the coalescence event seemed to happen at once. After coalescence, fusion with the plasma membrane occurred to release the pooled VWF which resulted in the disappearance of the fluorescent signal as the propeptide rapidly diffused into the extracellular medium. How the secreted VWF is remodeled after secretion into VWF strings was studied by correlative light and electron microscopy. We correlated confocal pictures of stimulated endothelial cells, which were stained with VWF specific fluorescent antibodies, to consecutive TEM sections. We found that fluorescently labeled VWF dots that were connected to strings, correlated to secretory pods but also to globular mass of secreted VWF. Interestingly, when we analyzed consecutive EM sections, the globular masses were found to originate from the secretory pods. From the globular masses we also observed deriving strings indicating that once VWF is expelled, remodeling occurs independently from secretion. We hypothesize that fluid flow remodels the secreted globular VWF mass into strings. To study this we stimulated endothelial cells under flow. The intracellular VWF pool in the WPBs was labeled green by transient expression of propeptide-EGFP and the secreted VWF was labeled red with strongly diluted red fluorescent VWF specific antibodies in the perfusate. Using live-cell imaging we observed that upon fusion of EGFP labeled WPBs, the green signal transformed into a red signal revealing dots of labeled secreted VWF. These dots rolled, in the direction of the flow, to the edge of the cell where they aggregated and only then formed strings. In non-transfected cells we performed similar experiments and there we observed the same pattern, confirming even more the VWF aggregation and string formation at the edges of the cell. In conclusion, we demonstrated that several WPBs can fuse with each other to form secretory pods and that VWF is secreted as a globular mass of protein. From these globular masses strings originated indicating that string formation occurs independently from the mechanism of secretion in which the tubular packaging of VWF in WPBs does not seem to be of importance. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Eccentric localization of von Willebrand factor in an internal structure of platelet alpha-granule resembling that of Weibel-Palade bodies

Blood ◽  
1985 ◽  
Vol 66 (3) ◽  
pp. 710-713 ◽  
Author(s):  
EM Cramer ◽  
D Meyer ◽  
R le Menn ◽  
J Breton-Gorius
Keyword(s):  
Electron Microscopy ◽  
Endothelial Cells ◽  
Monoclonal Antibodies ◽  
Distribution Pattern ◽  
Von Willebrand Factor ◽  
Polyclonal Antibody ◽  
Tubular Structures ◽  
Immunogold Staining ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Immunogold staining was used to study the ultrastructural distribution of von Willebrand factor (vWF) in unstimulated platelets. vWF was detected in the alpha-granules with a specific eccentric distribution pattern opposite the nucleoids. Similar findings were obtained with a polyclonal antibody or a pool of monoclonal antibodies to human vWF. This labeling coincided with the presence of tubular structures located at the periphery of the alpha-granules. These structures were better visualized on platelets treated for standard electron microscopy: they formed a group of one to four tubules ranging from 200 A to 250 A in diameter. They closely resembled the internal tubular structures found in Weibel-Palade bodies, which are the storage organelles of vWF in endothelial cells.

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.

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.

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