DIVERGENT PATES OF VON WILLEBRAND FACTOR AND ITS PROPOLYPEPTIDE (VON WILLEBRAND ANTIGEN II) AFTER SECRETION FROM ENDOTHELIAL CELLS

1987 ◽  
Author(s):  
D D Wagner ◽  
P J Fay ◽  
L A Sporn ◽  
S Sinha ◽  
S O Lawrence ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Culture Medium ◽  
Platelet Adhesion ◽  
Human Foreskin Fibroblasts ◽  
Covalently Linked ◽  
Von Willebrand ◽  
Willebrand Factor

The intracellular site of cleavage of pro-von Willebrand factor subunit and the subsequent fate of the propolypeptide (von Willebrand antigen II) and of the mature von Willebrand factor (vWf) were investigated. Both the propolypeptide, which was found to be a homodimer of non-covalently linked subunits, and mature vWf were released from Weibel-Palade bodies of endothelial cells following stimulation with secretagogues. The stoichiometry of the two proteins in the releasate was essentially equimolar. This indicates that vWf and the propolypeptide were packaged into the Weibel-Palade bodies as one unit, pro-vWf, and that the proteolytic cleavage of pro-vWf is likely to be a post-Golgi event. The association of prosequences into dimers provides support for their hypothetical role in the multimerization process. After secretion, the two proteins were distributed differently, as based on the following observations. The propolypeptide did not associate with vWf in the culture medium, did not co-distribute with vWf in the extracellular "patches of release" on stimulated endothelial cells, and was not detected in the endothelial cell extracellular matrix, which did contain vWf. Additionally, in contrast to vWf, the propolypeptide did not bind to matrix of human foreskin fibroblasts. Since the propolypeptide does not associate with vWf and does not interact witji extracellular matrices in vitro, it is highly unlikely that it would promote platelet adhesion to subendothelium in vivo.

Epinephrine Induces von Willebrand Factor Release from Cultured Endothelial Cells: Involvement of Cyclic AMP-dependent Signalling in Exocytosis

1997 ◽  
Vol 77 (06) ◽  
pp. 1182-1188 ◽  
Author(s):  
Ulrich M Vischer ◽  
Claes B Wollheinn
Keyword(s):  
Endothelial Cells ◽  
Adenylate Cyclase ◽  
Von Willebrand Factor ◽  
Umbilical Vein ◽  
Free Calcium ◽  
Camp Content ◽  
Von Willebrand ◽  
Willebrand Factor

Summaryvon Willebrand factor (vWf) is released from endothelial cell storage granules after stimulation with thrombin, histamine and several other agents that induce an increase in cytosolic free calcium ([Ca2+]i). In vivo, epinephrine and the vasopressin analog DDAVP increase vWf plasma levels, although they are thought not to induce vWf release from endothelial cells in vitro. Since these agents act via a cAMP-dependent pathway in responsive cells, we examined the role of cAMP in vWf secretion from cultured human umbilical vein endothelial cells. vWf release increased by 50% in response to forskolin, which activates adenylate cyclase. The response to forskolin was much stronger when cAMP degradation was blocked with IBMX, an inhibitor of phosphodiesterases (+200%), whereas IBMX alone had no effect. vWf release could also be induced by the cAMP analogs dibutyryl-cAMP (+40%) and 8-bromo-cAMP (+25%); although their effect was weak, they clearly potentiated the response to thrombin. Epinephrine (together with IBMX) caused a small, dose-dependent increase in vWf release, maximal at 10-6 M (+50%), and also potentiated the response to thrombin. This effect is mediated by adenylate cyclase-coupled β-adrenergic receptors, since it is inhibited by propranolol and mimicked by isoproterenol. In contrast to thrombin, neither forskolin nor epinephrine caused an increase in [Ca2+]j as measured by fura-2 fluorescence. In addition, the effects of forskolin and thrombin were additive, suggesting that they act through distinct signaling pathways. We found a close correlation between cellular cAMP content and vWf release after stimulation with epinephrine and forskolin. These results demonstrate that cAMP-dependent signaling events are involved in the control of exocytosis from endothelial cells (an effect not mediated by an increase in [Ca2+]i) and provide an explanation for epinephrine-induced vWf release.

scholarly journals The functions of the A1A2A3 domains in von Willebrand factor include multimerin 1 binding

2016 ◽  
Vol 116 (07) ◽  
pp. 87-95 ◽  
Author(s):  
D'Andra Parker ◽  
Subia Tasneem ◽  
Richard Farndale ◽  
Dominique Bihan ◽  
J. Sadler ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Structural Features ◽  
High Shear ◽  
The Individual ◽  
Static Conditions ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryMultimerin 1 (MMRN1) is a massive, homopolymeric protein that is stored in platelets and endothelial cells for activation-induced release. In vitro, MMRN1 binds to the outer surfaces of activated platelets and endothelial cells, the extracellular matrix (including collagen) and von Willebrand factor (VWF) to support platelet adhesive functions. VWF associates with MMRN1 at high shear, not static conditions, suggesting that shear exposes cryptic sites within VWF that support MMRN1 binding. Modified ELISA and surface plasmon resonance were used to study the structural features of VWF that support MMRN1 binding, and determine the affinities for VWF-MMRN1 binding. High shear microfluidic platelet adhesion assays determined the functional consequences for VWF-MMRN1 binding. VWF binding to MMRN1 was enhanced by shear exposure and ristocetin, and required VWF A1A2A3 region, specifically the A1 and A3 domains. VWF A1A2A3 bound to MMRN1 with a physiologically relevant binding affinity (KD: 2.0 ± 0.4 nM), whereas the individual VWF A1 (KD: 39.3 ± 7.7 nM) and A3 domains (KD: 229 ± 114 nM) bound to MMRN1 with lower affinities. VWF A1A2A3 was also sufficient to support the adhesion of resting platelets to MMRN1 at high shear, by a mechanism dependent on VWF-GPIbD binding. Our study provides new information on the molecular basis of MMRN1 binding to VWF, and its role in supporting platelet adhesion at high shear. We propose that at sites of vessel injury, MMRN1 that is released following activation of platelets and endothelial cells, binds to VWF A1A2A3 region to support platelet adhesion at arterial shear rates.

scholarly journals Rickettsia rickettsii infection of cultured endothelial cells induces release of large von Willebrand factor multimers from Weibel-Palade bodies

Blood ◽  
1991 ◽  
Vol 78 (10) ◽  
pp. 2595-2602 ◽  
Author(s):  
LA Sporn ◽  
RJ Shi ◽  
SO Lawrence ◽  
DJ Silverman ◽  
VJ Marder
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Culture Medium ◽  
Spotted Fever ◽  
Clinical Manifestations ◽  
Tissue Ischemia ◽  
Rickettsia Rickettsii ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract The clinical manifestations of Rocky Mountain spotted fever (RMSF) result from Rickettsia rickettsii (R rickettsii) infection of endothelial cells and are mediated by pathologic changes localized to the vessel, including in situ thrombosis and tissue ischemia. This study uses in vitro infection of cultured human umbilical vein endothelial cells with R rickettsii to test the hypothesis that such infection induces von Willebrand factor (vWF) release from Weibel- Palade bodies, a process that could contribute to thrombotic changes. At 24 hours postinfection, there was an increase in metabolically prelabeled large multimers of vWF in the culture medium, with a concomitant decrease of these forms in the cell lysate samples. This release reaction was specific for the large multimer pool of vWF, localized to Weibel-Palade bodies, because no change in the distribution of dimeric forms between cells and culture medium was detected. Double-label immunofluorescence staining showed an inverse correlation between the number of R rickettsii and the number of Weibel- Palade bodies in infected cells. Cell lysis was minimal at 24 hours postinfection, as no detectable intracellular precursor forms (molecular weight 260,000) of vWF were released into the culture medium, there was no decrease in cell viability as measured by trypan blue exclusion, and no increase in 51Cr-release into the culture medium was observed when compared with uninfected controls. Release was likely a direct effect of the intracellular presence of the organism, rather than due to a noxious soluble factor such as endotoxin, because culture medium conditioned by infected endothelial cells was ineffective at inducing release in uninfected endothelial cell cultures. In summary, in vitro infection of endothelial cells by R rickettsii induces release of Weibel-Palade body contents, a process that may contribute to the pathogenesis of RMSF.

VON WILLEBRAND FACTOR RELEASED FROM WEIBEL-PALADE BODIES BINDS MORE AVIDLY TO EXTRACELLULAR MATRIX THAN THAT SECRETED CONSTITUTIVELY

1987 ◽  
Author(s):  
L A Sporn ◽  
V J Marder ◽  
D D Wagner
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Starting Material ◽  
Human Foreskin Fibroblasts ◽  
Vitro Model ◽  
Trace Of Matrix ◽  
Von Willebrand ◽  
Willebrand Factor

Large multimers of von Willebrand factor (vWf) are released from the Weibel-Palade bodies (WPB) of cultured endothelial cells following treatment with a secretagogue, whereas predominantly dimeric forms are secreted constitutively. These two pools of vWf were used to compare binding of the various multimeric forms of vWf to the extracellular matrix (ECM), the in vitro model of the basement membrane. The released multimers and an equal number of subunits of constitutively secreted vWf were placed, for 72 hours, on cultures of human foreskin fibroblasts (HFF) grown on glass coverslips, then fixed and stained by fluorescence using anti-vWf antiserum. Constitutively secreted vWf produced only a trace of matrix decoration, whereas the released large multimers bound more extensively. In order to determine if increased binding of released vWf was due to the presence of another component in the releasate, releasate from which vWf was adsorbed was combined with constitutively secreted vWf, and this mixture was overlaid onto HFF. The presence of the adsorbed releasate did not promote binding of constitutively secreted vWf. Therefore, it appears that the enhanced binding observed was due to the large multimeric size of vWf stored in the WPB. To further substantiate this, iodinated plasma vWf which was presumably constitutively secreted from endothelial cells was overlaid on.to HFF for 72 hours, labeled vWf was removed, and cells were washed extensively and lysed. Samples of iodinated plasma vWf (starting material) and cell lysates were e1ectrophoresed, non-reduced on an agarose gel. Densitometric scans of starting material and of bound vWf revealed that the large multimeric forms bound preferentially. It appears that multivalency is likely an important property in vWf interaction with the ECM, just as has been shown for vWf interaction with platelets. The pool of vWf contained within the WPBs, therefore, is not only especially suited for platelet interaction, but also for interaction with the ECM.

scholarly journals Unwinding the von Willebrand factor strings puzzle

Blood ◽  
2013 ◽  
Vol 121 (2) ◽  
pp. 270-277 ◽  
Author(s):  
Karen De Ceunynck ◽  
Simon F. De Meyer ◽  
Karen Vanhoorelbeke
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Cleavage Sites ◽  
Normal Functional ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract von Willebrand factor (VWF) is amongst others synthesized by endothelial cells and stored as ultra-large (UL) VWF multimers in Weibel-Palade bodies. Although UL-VWF is proteolysed by ADAMTS13 (a disintegrin-like and metalloprotease domain with thrombospondin type-1 motif, number 13) on secretion from endothelial cells, in vitro experiments in the absence of ADAMTS13 have demonstrated that a proportion of these UL-VWF multimers remain anchored to the activated endothelium. These multimers unravel, bind platelets, and wave in the direction of the flow. These so-called VWF “strings” have also been visualized in vivo, lining the lumen of activated mesenteric veins of Adamts13−/− mice. Various studies have demonstrated the extraordinary length of these VWF strings, the availability of their platelet binding and ADAMTS13 cleavage sites, and the possible nature of their endothelial attachment. VWF strings are also capable of tethering leukocytes and parasite-infected red blood cells. However, the majority of studies have been performed in the absence of ADAMTS13, a condition only experienced in thrombotic thrombocytopenic purpura. A normal functional role of VWF strings in healthy persons or in other disease pathologies remains unclear. In this review, we discuss some of the puzzling characteristics of VWF strings, and we debate whether the properties of VWF strings in the absence of ADAMTS13 might be relevant for understanding (patho)physiologic mechanisms.

scholarly journals EPAC regulates von Willebrand factor secretion from endothelial cells in a PI3K/eNOS-dependent manner during inflammation

2020 ◽  
Author(s):  
Jie Xiao ◽  
Ben Zhang ◽  
Zhengchen Su ◽  
Yakun Liu ◽  
Thomas R. Shelite ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Umbilical Vein ◽  
Null Mouse ◽  
Intracellular Camp ◽  
Dependent Manner ◽  
Von Willebrand ◽  
Willebrand Factor

AbstractCoagulopathy is associated with both inflammation and infection, including infection with the novel SARS-CoV-2 (COVID-19). Endothelial cells (ECs) fine tune hemostasis via cAMP-mediated secretion of von Willebrand factor (vWF), which promote the process of clot formation. The exchange protein directly activated by cAMP (EPAC) is a ubiquitously expressed intracellular cAMP receptor that plays a key role in stabilizing ECs and suppressing inflammation. To assess whether EPAC could regulate vWF release during inflammation, we utilized our EPAC1-null mouse model and revealed an increased secretion of vWF in endotoxemic mice in the absence of the EPAC1 gene. Pharmacological inhibition of EPAC1 in vitro mimicked the EPAC1−/− phenotype. EPAC1 regulated TNFα-triggered vWF secretion from human umbilical vein endothelial cells (HUVECs) in a phosphoinositide 3-kinases (PI3K)/endothelial nitric oxide synthase (eNOS)-dependent manner. Furthermore, EPAC1 activation reduced inflammation-triggered vWF release, both in vivo and in vitro. Our data delineate a novel regulatory role of EPAC1 in vWF secretion and shed light on potential development of new strategies to controlling thrombosis during inflammation.Key PointPI3K/eNOS pathway-mediated, inflammation-triggered vWF secretion is the target of the pharmacological manipulation of the cAMP-EPAC system.

scholarly journals Rickettsia rickettsii infection of cultured endothelial cells induces release of large von Willebrand factor multimers from Weibel-Palade bodies

Blood ◽  
1991 ◽  
Vol 78 (10) ◽  
pp. 2595-2602
Author(s):  
LA Sporn ◽  
RJ Shi ◽  
SO Lawrence ◽  
DJ Silverman ◽  
VJ Marder
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Culture Medium ◽  
Spotted Fever ◽  
Clinical Manifestations ◽  
Tissue Ischemia ◽  
Rickettsia Rickettsii ◽  
Von Willebrand ◽  
Willebrand Factor

The clinical manifestations of Rocky Mountain spotted fever (RMSF) result from Rickettsia rickettsii (R rickettsii) infection of endothelial cells and are mediated by pathologic changes localized to the vessel, including in situ thrombosis and tissue ischemia. This study uses in vitro infection of cultured human umbilical vein endothelial cells with R rickettsii to test the hypothesis that such infection induces von Willebrand factor (vWF) release from Weibel- Palade bodies, a process that could contribute to thrombotic changes. At 24 hours postinfection, there was an increase in metabolically prelabeled large multimers of vWF in the culture medium, with a concomitant decrease of these forms in the cell lysate samples. This release reaction was specific for the large multimer pool of vWF, localized to Weibel-Palade bodies, because no change in the distribution of dimeric forms between cells and culture medium was detected. Double-label immunofluorescence staining showed an inverse correlation between the number of R rickettsii and the number of Weibel- Palade bodies in infected cells. Cell lysis was minimal at 24 hours postinfection, as no detectable intracellular precursor forms (molecular weight 260,000) of vWF were released into the culture medium, there was no decrease in cell viability as measured by trypan blue exclusion, and no increase in 51Cr-release into the culture medium was observed when compared with uninfected controls. Release was likely a direct effect of the intracellular presence of the organism, rather than due to a noxious soluble factor such as endotoxin, because culture medium conditioned by infected endothelial cells was ineffective at inducing release in uninfected endothelial cell cultures. In summary, in vitro infection of endothelial cells by R rickettsii induces release of Weibel-Palade body contents, a process that may contribute to the pathogenesis of RMSF.

The Effect of von Willebrand Factor (VWF) on Site-Specific Factor VIII (FVIII) Expression in Hemophilia A Mice.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 764-764
Author(s):  
Qizhen Shi ◽  
Scot A. Fahs ◽  
Erin L. Kuether ◽  
Robert R. Montgomery
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Double Knockout ◽  
Site Specific ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract von Willebrand factor (VWF) is a carrier protein for factor VIII (FVIII) and protects plasma FVIII from protease degradation. Our laboratory has had a longstanding interest in the association of FVIII with VWF both in vitro and in vivo. Our in vitro studies have demonstrated that FVIII stores together with VWF in both endothelial cells and megakaryocytes if FVIII is made in these cells. Furthermore, we demonstrated that FVIII and VWF are both releasable by agonist stimulation. To investigate the association of VWF and FVIII in vivo, we generated two lines of transgenic mice that express FVIII either in endothelial cells or in platelets using either the endothelial cell-specific Tie2 promoter or the platelet-specific αIIb promoter, respectively. When the platelet-specific FVIII (2bF8) transgene is bred into the FVIIInull mouse, FVIII can only be detected in platelets, with a level of 0.76 ± 0.27 mU/108 platelets in heterozygous and 1.53 ± 0.14 mU/108 platelets in homozygous 2bF8 mice. When the endothelial cell-specific FVIII (Tie2F8) transgene is bred into the FVIIInull mouse, homozygous Tie2F8 mice maintained normal plasma FVIII levels (1.15 ± 0.16 U/ml) and 50% levels in heterozygous mice (0.56 ± 0.16 U/ml). Both 2bF8trans and Tie2F8trans phenotypes effectively abrogate the bleeding diathesis in hemophilic mice. When 2bF8 transgene was bred into a FVIII and VWF double knockout background, the level of platelet-FVIII significantly decreased, but this platelet-derived FVIII was still stored in a-granules and still maintained clinical efficacy. In contrast, when the Tie2F8 transgene was bred into the double knockout background, plasma FVIII dropped to undetectable levels. This is in contrast to the situation in VWFnull mice in which normal endogenous murine FVIII is synthesized with about 10% of normal FVIII activity persisting in plasma. This could be due to a difference in survival between human FVIII and murine FVIII. All Tie2F8trans/FVIIInullVWFnull mice (n=15) survived tail clipping even though there is no FVIII:C detected in the plasma. To investigate the effect of murine VWF on the levels of plasma FVIII, plasma from FVIIInull mice was infused into Tie2F8trans/FVIIInullVWFnull mice to restore VWF levels to 25% of normal. As expected, the endothelial cell-derived plasma FVIII was stabilized by the infused VWF and was detected within 1 hour after infusion, with a peak (25% level) at 4 hours. The level of plasma FVIII at 24 hours was still about 20% of normal while the level of remaining VWF was only 5% of normal. These results demonstrate that VWF is important for site-specific FVIII expression. Co-expression with VWF in platelets is important for optimal platelet-specific FVIII expression and endothelial cell-derived plasma FVIII is VWF-dependent.

Abnormal Structure of von Willebrand Factor in Myeloproliferative Syndrome Is Associated to Either Thrombotic or Bleeding Diathesis

1987 ◽  
Vol 58 (02) ◽  
pp. 753-757 ◽  
Author(s):  
M F López-Fernández ◽  
C López-Berges ◽  
R Martín ◽  
A Pardo ◽  
F J Ramos ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Proteinase Inhibitors ◽  
Relative Proportion ◽  
Variable Degree ◽  
Bleeding Diathesis ◽  
Myeloproliferative Syndrome ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryThe multimeric and subunit patterns of plasma von Willebrand factor (vWF) were analyzed in eight patients with myeloproliferative syndrome (MS) in order to investigate the possible existence of heterogeneity in the “in vivo” proteolytic cleavage of the protein, previously observed in this entity. Six patients lacked large vWF multimers, five of them having normal bleeding times (BT) and clinically documented episodes of thrombotic origin, whereas one patient had long BT and bleeding symptoms. Seven patients showed a relative increase in the 176 kDa subunit fragment while the 189 kDa polypeptide was increased in only one. In addition, another patient (and prior to any therapy) showed the presence of a new fragment of approximately 95 kDa which disappeared after Busulfan therapy. The collection of blood from these patients with proteinase inhibitors did not correct the abnormalities.The infusion of DDAVP to two patients with abnormal vWF was accompanied by: the appearance of larger vWF multimers which disappeared rapidly from plasma; an increase in the relative proportion of the satellite bands of each multimer and a further increase of the 176 kDa fragment. These data point to some heterogeneity in the vWF abnormality present in MS which may be related in part to a variable degree of proteolysis of vWF occurring “in vivo” rather than “in vitro”, and which may be associated to either a thrombotic or a bleeding diathesis. They also suggest that despite the presence of abnormal, already proteolyzed vWF, DDAVP-enhanced proteolysis occurs in MS to a similar extent to what is described in normal individuals.

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