Fibrinogen is required for maintenance of platelet intracellular and cell-surface P-selectin expression

Blood ◽  
2009 ◽  
Vol 114 (2) ◽  
pp. 425-436 ◽  
Author(s):  
Hong Yang ◽  
Sean Lang ◽  
Zhimin Zhai ◽  
Ling Li ◽  
Walter H. A. Kahr ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Human Fibrinogen ◽  
C Terminus ◽  
Β3 Integrin ◽  
Von Willebrand ◽  
Selectin Binding ◽  
Willebrand Factor ◽  
Deficient Mice ◽  
Granule Release

Abstract Platelet P-selectin plays important roles in inflammation and contributes to thrombosis and hemostasis. Although it has been reported that von Willebrand factor (VWF) affects P-selectin expression on endothelial cells, little information is available regarding regulation of platelet P-selectin expression. Here, we first observed that P-selectin expression was significantly decreased on platelets of fibrinogen and VWF double-deficient mice. Subsequently, we identified this was due to fibrinogen deficiency. Impaired P-selectin expression on fibrinogen-deficient platelets was further confirmed in human hypofibrinogenemic patients. We demonstrated that this impairment is unlikely due to excessive P-selectin shedding, deficient fibrinogen-mediated cell surface P-selectin binding, or impaired platelet granule release, but rather is due to decreased platelet P-selectin content. Fibrinogen transfusion completely recovered this impairment in fibrinogen-deficient (Fg−/−) mice, and engagement of the C-terminus of the fibrinogen γ chain with β3 integrin was required for this process. Furthermore, Fg−/− platelets significantly increased P-selectin expression following transfusion into β3 integrin–deficient mice and when cultured with fibrinogen. These data suggest fibrinogen may play important roles in inflammation, thrombosis, and hemostasis via enhancement of platelet P-selectin expression. Since human fibrinogen levels vary significantly in normal and diseased populations, P-selectin as an activation marker on platelets should be used with caution.

Engagement of Fibrinogen and β3 Integrin Is Required for Maintenance of Platelet Intracellular and Cell Surface P-Selectin Expression

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2868-2868
Author(s):  
Hong Yang ◽  
Sean Lang ◽  
Zhimin Zhai ◽  
Christopher M. Spring ◽  
Adili Reheman ◽  
...  
Keyword(s):  
Cell Surface ◽  
Human Platelets ◽  
Human Patient ◽  
Platelet Factor ◽  
C Terminus ◽  
Significant Difference ◽  
Β3 Integrin ◽  
Von Willebrand ◽  
Selectin Binding ◽  
Deficient Mice

Abstract Platelet P-selectin plays important roles in inflammation and contributes to thrombosis and hemostasis. While it has been reported that von Willebrand factor (VWF) affects P-selectin expression on endothelial cells, little information is available regarding regulation of platelet P-selectin expression. Here, we first observed that P-selectin expression was significantly decreased on platelets of fibrinogen and VWF double deficient mice. Subsequently, we identified this was due to fibrinogen, but not VWF, deficiency. The impaired P-selectin expression on fibrinogen-deficient platelets was further confirmed in a human patient with severe hypofibrinogenemia and his heterozygous parents. We demonstrated that this impairment is unlikely due to excessive P-selectin shedding, deficient fibrinogen-mediated cell surface P-selectin binding, or impaired platelet granule release, but rather is due to decreased platelet P-selectin content. This effect seems to be specific for P-selectin, as other alpha-granule proteins (e.g. thrombospondin-1, vitronectin, platelet factor 4) were not decreased in fibrinogen-deficient mouse or human platelets. We found that fibrinogen transfusion recovered the P-selectin expression in fibrinogen-deficient mice to >80% of the normal level within 48 hours, and four days post-transfusion, the P-selectin levels were nearly completely recovered. Furthermore, engagement of the C-terminus of the fibrinogen γ chain with β3 integrin was required for this process since a similar impairment of P-selectin expression was also observed in fibrinogen γΔ5 mice and β3 integrin-deficient mice. To determine whether fibrinogen affects the biogenesis of platelet alpha-granules, platelets were examined via electron microscopy. No statistically significant difference in the number of platelet alpha-granules was observed in fibrinogen-deficient or β3 integrin-deficient mice compared to controls (P>0.05). These data suggest fibrinogen may play important roles in inflammation, including immune-mediated inflammation, thrombosis and hemostasis via enhancement of platelet P-selectin expression. Furthermore, there are 2–3 fold variations in human plasma fibrinogen levels in healthy populations, and there are patients with hypo- and afibrinogenemia, hyperfibrinogenemia, and Glanzmann’s Thrombasthenia (β3 integrin deficiency). Our data suggest that platelet P-selectin expression can be affected by all these conditions. Therefore, P-selectin as a platelet activation marker should be used with caution.

P-selectin binds to the D′-D3 domains of von Willebrand factor in Weibel-Palade bodies

Blood ◽  
2006 ◽  
Vol 107 (10) ◽  
pp. 3922-3924 ◽  
Author(s):  
Grégoire Michaux ◽  
Timothy J. Pullen ◽  
Sandra L. Haberichter ◽  
Daniel F. Cutler
Keyword(s):  
Endothelial Cells ◽  
Membrane Protein ◽  
Cell Surface ◽  
Von Willebrand Factor ◽  
Integral Membrane Protein ◽  
Hek293 Cells ◽  
Binding Partner ◽  
Lumenal Domain ◽  
Von Willebrand ◽  
Willebrand Factor

It has recently been shown that the ultralarge platelet–recruiting von Willebrand factor (VWF) strings formed immediately at exocytosis from endothelial cells may be anchored to the cell surface by interaction with the integral membrane protein P-selectin. This finding of a new binding partner for VWF immediately prompts the question which domains of VWF bind to P-selectin. We have exploited the fact that VWF expression in HEK293 cells triggers the formation of Weibel-Palade body–like structures that can recruit P-selectin. A suitably modified version of this assay using coexpressed truncations of VWF, together with P-selectin variants in HEK293 cells, allowed us to determine which domains of VWF would recruit P-selectin within a physiologically appropriate intracellular environment. Confirming the results of such a cellular assay by conventional coimmunoprecipitation, we concluded that the lumenal domain of P-selectin interacts with the D′-D3 domains of VWF.

Von Willebrand Factor (VWF) Regulates Angiogenesis: Studies in Blood-Derived Endothelial Progenitor Cells From Von Willebrand's Disease Patients and VWF-Deficient Mice

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5311-5311
Author(s):  
Richard D Starke ◽  
Koralia Paschalaki ◽  
Francesco Ferraro ◽  
Thomas A J McKinnon ◽  
Nicola H Dryden ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Vascular Network ◽  
Vascular Density ◽  
Type I ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
In Vitro Angiogenesis ◽  
Deficient Mice

Abstract Abstract 5311 Dysregulation of angiogenesis is implicated in many diseases. Von Willebrand factor (VWF), a large plasma glycoprotein essential for normal haemostasis is synthesized by endothelial cells (EC) and megakaryocytes. Raised VWF plasma levels are a risk factor for arterial thrombosis, whilst deficiency of VWF causes Von Willebrand disease (VWD), the most common congenital bleeding disorder in man. VWD can be associated with angiodysplasia, vascular malformations linked to defective angiogenesis which are responsible for intractable bleeding. We recently showed that VWF is involved in angiogenesis. Inhibition of VWF expression in human umbilical vein EC (HUVEC) with specific siRNA resulted in increased in vitro angiogenesis on Matrigel, proliferation and migration. Mechanism studies implicated the endothelial VWF receptor, integrin αvβ3 and the angiogenesis regulator angiopoietin-2. The findings were confirmed in EC from VWD patients and in VWF-deficient mice. Blood outgrowth endothelial cells (BOEC) isolated from peripheral blood of patients with VWD showed decreased VWF release, compared to control BOEC, and increased in vitro angiogenesis, migration and proliferation, similar to what observed with VWF siRNA-treated HUVEC. In vivo studies using the matrigel model and imaging of blood vessels in the ear showed increased angiogenesis and vascular network in VWF-deficient mice compared to controls. Recent studies in patients BOEC and in VWF deficient mice provide new insight into the complexity of this phenotype. We have used the mouse model of post-natal angiogenesis in the retina to carry out detailed analysis of angiogenic networks in the VWF-deficient mouse, and found increased vascular density and defective vascular network. Moreover, using BOEC from patients with type I and type II VWD, we have studied VWF intracellular distribution by immunofluorescence confocal analysis and found patterns of expression that point to a variety of defects in synthesis, storage and secretion. These studies define a new function for VWF, which may have clinical implications for VWD and for patients at risk of CV disease. Moreover, studies with BOEC from VWF patients provide a novel understanding of the physiopathology of this disease. Disclosures: No relevant conflicts of interest to declare.

Interaction of ADAMTS13 with the Endothelial Cell Surface.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3710-3710 ◽  
Author(s):  
Anthony Vomund ◽  
Elaine M. Majerus
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Surface ◽  
Plasma Concentrations ◽  
Fluorescein Isothiocyanate ◽  
Endothelial Cell Surface ◽  
Sds Page ◽  
Carboxyl Terminal ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract ADAMTS13 proteolysis of von Willebrand Factor (VWF) generates smaller multimers that are less likely to promote blood clotting. Deficiency of ADAMTS13 leads to thrombotic thrombocytopenic purpura, a frequently fatal disease, characterized by microangiopathic hemolytic anemia and thrombocytopenia. ADAMTS13 has a characteristic domain structure that includes metalloprotease and disintegrin domains, a thrombospondin type 1 repeat (TSR), cysteine-rich and spacer domains, 7 additional TSRs, and 2 carboxyl-terminal CUB domains. The ADAMTS13 substrate, VWF, is synthesized in endothelial cells and forms large multimers within the cell. These large multimers are secreted and adhere to the endothelial cell surface where they can bind platelets flowing in blood leading to thrombosis. ADAMTS13 has been shown to cleave VWF on the surface of endothelial cells, but it is unclear if ADAMTS13 also interacts with the endothelial cell surface. We have used iodinated ADAMTS13, fluorescence-activated cell sorting (FACS), and biochemical analysis using flow conditions to demonstrate that ADAMTS13 does interact with the endothelial cell surface. Iodinated ADAMTS13 bound the endothelial cell surface at 4oC. This binding was specific since the binding was inhibited in the presence of 40-fold excess unlabeled ADAMTS13. Binding of ADAMTS13 to the cell surface was time-dependent with maximal binding occurring within two hours. The binding was also reversible; the half-time for dissociation was four hours. Binding was inhibited by heparin but not by dextran sulfate. The Kd of binding to endothelial cells was 75 nM (range 40–100 nM). FACS analysis also demonstrated binding of ADAMTS13 to endothelial cells. A fluorescein isothiocyanate labeled anti-epitope antibody bound to endothelial cells in the presence but not the absence of ADAMTS13. A polyclonal antibody to VWF inhibited binding of ADAMTS13 to VWF, but this antibody did not affect binding of ADAMTS13 to endothelial cells, suggesting that ADAMTS13 interacts with endothelial cells independently of VWF. Studies with C-terminal truncation constructs of ADAMTS13 indicated that the carboxyl-terminal TSRs are important for binding since constructs terminating with the metalloprotease domain, the first TSR, or the sixth TSR failed to compete with full-length ADAMTS13 for binding to endothelial cells, but constructs terminating with either the seventh or eighth TSR did compete for binding. Lastly, recombinant ADAMTS13 was found to be associated with endothelial cells in flow experiments. Endothelial cells were perfused with medium containing plasma concentrations of ADAMTS13 (1 μg/ml) at 10 dynes/cm2. After perfusion, the endothelial cells were washed and bound ADAMTS13 was identified from whole cell lysates through SDS-PAGE and immunoblotting with an anti-V5 epitope antibody. ADAMTS13 was found associated with endothelial cells after perfusion. Binding of ADAMTS13 to the endothelial cells prior to perfusion led to enhanced proteolysis of VWF as compared to addition of ADAMTS13 during perfusion only. This suggests that the interaction of ADAMTS13 with endothelial cells is important since it enhances the cleavage of VWF as compared to that of ADAMTS13 in solution.

scholarly journals Integrin αvβ3 on human endothelial cells binds von Willebrand factor strings under fluid shear stress

Blood ◽  
2009 ◽  
Vol 113 (7) ◽  
pp. 1589-1597 ◽  
Author(s):  
Jing Huang ◽  
Robyn Roth ◽  
John E. Heuser ◽  
J. Evan Sadler
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Von Willebrand Factor ◽  
Fluid Shear Stress ◽  
Immunofluorescence Microscopy ◽  
Integrin Αvβ3 ◽  
Platelet Glycoprotein ◽  
Human Endothelial Cells ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Acutely secreted von Willebrand factor (VWF) multimers adhere to endothelial cells, support platelet adhesion, and may induce microvascular thrombosis. Immunofluorescence microscopy of live human umbilical vein endothelial cells showed that VWF multimers rapidly formed strings several hundred micrometers long on the cell surface after stimulation with histamine. Unexpectedly, only a subset of VWF strings supported platelet binding, which depended on platelet glycoprotein Ib. Electron microscopy showed that VWF strings often consisted of bundles and networks of VWF multimers, and each string was tethered to the cell surface by a limited number of sites. Several approaches implicated P-selectin and integrin αvβ3 in anchoring VWF strings. An RGDS peptide or a function-blocking antibody to integrin αvβ3 reduced the number of VWF strings formed. In addition, integrin αv decorated the VWF strings by immunofluorescence microscopy. Furthermore, lentiviral transduction of shRNA against the αv subunit reduced the expression of cell-surface integrin αvβ3 and impaired the ability of endothelial cells to retain VWF strings. Soluble P-selectin reduced the number of platelet-decorated VWF strings in the absence of Ca2+ and Mg2+ but had no effect in the presence of these cations. These results indicate that VWF strings bind specifically to integrin αvβ3 on human endothelial cells.

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.

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.

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