Role of the Leucine-Rich Domain of Platelet GPIbα in Correct Post-translational Processing – The Nancy I Bernard-Soulier Mutation Expressed on CHO Cells

2000 ◽  
Vol 84 (07) ◽  
pp. 104-111 ◽  
Author(s):  
Philippe Ulsemer ◽  
Marie-Jeanne Baas ◽  
Agnès Schwartz ◽  
Catherine Ravanat ◽  
Marie-Elisabeth Briquel ◽  
...  
Keyword(s):  
Cho Cells ◽  
Surface Expression ◽  
Rich Domain ◽  
Coated Surface ◽  
Natural Mutation ◽  
Static Conditions ◽  
And Function ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryThe mechanisms governing the biosynthesis and surface expression of platelet adhesive receptors on parent megakaryocytes are as yet poorly understood. In particular, the assembly and processing of the multisubunit glycoprotein (GP) Ib-IX-V complex, a receptor for von Willebrand factor (vWf) is not fully understood. In the present work, these questions were addressed by reproducing a natural mutation of GPIbα found in a variant case of Bernard-Soulier syndrome (Nancy I), due to the deletion of leucine 179 in the seventh leucine-rich repeat of the polypeptide. Wild type and mutated GPIbα were transfected into CHO cells expressing GPIbβ and GPIX. Flow cytometry showed surface expression of the three subunits of both GPIb-IX complexes, but GPIbαΔLeu was present at lower levels (20-40%) and was recognized only by a sub class of monoclonal antibodies which epitopes were not modified by the mutation. These properties reproduce the defect found in the patient’s platelets, demonstrating the causative nature of the mutation and validate the use of the CHO cells model. Biochemical studies were performed in an attempt to elucidate the mechanism of the conformational change of GPIbαΔLeu. They unexpectedly revealed a major glycosylation deficiency of the mutated GPIbα leading to a 40% decrease in molecular weight. The other two subunits of the complex were however normal and present at the plasma membrane. The deletion led to complete functional deficiency with lack of vWf binding of CHOαΔLeu transfected cells in the presence of botrocetin and defective adhesion to a vWf coated surface under static conditions. Finally, in contrast to normal CHOαβIX cells, which displayed rolling and deceleration when perfused over a vWf surface, CHOαΔLeuβIX cells were unable to roll over or attach to a vWf substratum. These results show that the integrity of the leucine-rich region of GPIbα is essential for normal processing and function of the GPIb-IX complex. In addition, these results obtained in a cellular system supported the suspected role of the macroglycopeptide region of GPIbα in maintaining a suitable conformation of this multisubunit receptor to perform its adhesive function.

Complete Removal of N-Linked Glycans from Platelet GPIbα Impairs Platelet Agglutination and von Willebrand Factor Binding In Vitro.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1535-1535
Author(s):  
Suzana M. Zorca ◽  
Emma C. Josefsson ◽  
Viktoria Rumjantseva ◽  
John H. Hartwig ◽  
Karin M. Hoffmeister
Keyword(s):  
Flow Cytometry ◽  
Von Willebrand Factor ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Surface Expression ◽  
Lectin Domain ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract We previously reported that the lectin domain of the αMβ2 receptor on macrophages mediates the rapid clearance of transfused washed murine platelets which have been refrigerated for 2 hrs in the absence of plasma. The clearance is mediated by the recognition of exposed βN-acetylglucosamine (β-GlcNAc) residues on N-linked glycans of clustered platelet GPIbα molecules. Covering the exposed β-GlcNAc residues on GPIbα N-linked glycans via galactosylation prevents the clearance of chilled murine platelets from the circulation. The role of N-linked glycans in platelet function and survival is unclear. To dissect the role of N-linked glycosylation of GPIbα on the binding of von Willebrand factor (vWf), we use human platelets and Chinese Hamster Ovary (CHO) cells, stably expressing human GPIbα/βand GPIX. Deglycosylation of platelet GPIbα N-liked glycans was achieved using the enzyme peptide-N-glycosidase F (PGNaseF), specific for complex N-linked glycans. In agglutination assays using platelets incubated with and without PNGaseF for 16hrs at 37°C, we observed 30-40 % less agglutination in response to ristocetin for platelets depleted of N-linked glycans with PNGaseF. Additionally, a 30 % reduction in vWf binding to PNGaseF-treated platelets compared with control platelets was measured by flow cytometry, using a FITC-conjugated mAb that detects surface-bound vWf. In CHO cells, GPIbα N-linked oligosaccharides were manipulated by adding swainsonine or tunicamycin, two inhibitors of N-linked oligosaccharide synthesis in the Golgi. vWf binding to platelets or to CHO cells was studied by aggregometry or by light microscopy to establish the fraction of CHO-cell aggregates. As was the case with platelets, vWf-dependent aggregation of CHO cells expressing GPIb-IX decreased three fold in response to botrocetin, but only following complete N-linked glycans depletion with tunicamycin. In contrast, partial N-linked carbohydrate modification with swainsonine did not significantly alter aggregate formation in CHO- cells expressing GPIb-IX. Complete inhibition of N-linked glycosylation decreased botrocetin-induced vWf binding to CHO- cells expressing GPIb-IX by ~50%, as determined by flow cytometry. No change was observed following swainsonine treatment. Surface expression of GP1bα remained unchanged after both tunicamycin and swainsonine treatment, and with PGNaseF treatment of platelets. These results confirm that 1) N-linked glycans are not required for GPIbα surface expression, and 2) indicate that N-linked glycans likely play a role in vWf binding to platelet GPIbα.

Von Willebrand disease and Weibel-Palade bodies

2010 ◽  
Vol 30 (03) ◽  
pp. 150-155 ◽  
Author(s):  
J. W. Wang ◽  
J. Eikenboom
Keyword(s):  
Platelet Adhesion ◽  
Coagulation Factor ◽  
Von Willebrand Disease ◽  
Inflammatory Factors ◽  
Limited Data ◽  
Storage Organelle ◽  
And Function ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryVon Willebrand factor (VWF) is a pivotal haemostatic protein mediating platelet adhesion to injured endothelium and carrying coagulation factor VIII (FVIII) in the circulation to protect it from premature clearance. Apart from the roles in haemostasis, VWF drives the formation of the endothelial cell specific Weibel-Palade bodies (WPBs), which serve as a regulated storage of VWF and other thrombotic and inflammatory factors. Defects in VWF could lead to the bleeding disorder von Willebrand disease (VWD).Extensive studies have shown that several mutations identified in VWD patients cause an intracellular retention of VWF. However, the effects of such mutations on the formation and function of its storage organelle are largely unknown. This review gives an overview on the role of VWF in WPB biogenesis and summarizes the limited data on the WPBs formed by VWD-causing mutant VWF.

Baboon Fibrinogen Adsorption and Platelet Adhesion to Polymeric Materials

1991 ◽  
Vol 65 (05) ◽  
pp. 608-617 ◽  
Author(s):  
Joseph A Chinn ◽  
Thomas A Horbett ◽  
Buddy D Ratner
Keyword(s):  
Platelet Adhesion ◽  
Coagulation Factor ◽  
Polymeric Materials ◽  
Platelet Suspension ◽  
Perfusion Chamber ◽  
Static Conditions ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Fibrinogen Adsorption

SummaryThe role of fibrinogen in mediating platelet adhesion to polymers exposed to blood plasma was studied by comparison of the effect of plasma dilution on fibrinogen adsorption and platelet adhesion, and by the use of coagulation factor deficient plasmas. Polyetherurethane substrates were first preadsorbed with dilute plasma, then contacted with washed platelets suspended in a modified, apyrase containing Tyrode’s buffer. Platelet adhesion was studied under static conditions in Multiwell dishes, and also under shearing conditions using a parallel plate perfusion chamber. Fibrinogen adsorption and platelet adhesion were measured using 125I radiolabeled baboon fibrinogen and min radiolabeled baboon platelets, respectively. Surfaces were characterized by electron spectroscopy for chemical analysis (ESCA).When fibrinogen adsorption to Biomer was measured after 2 h contact with a series of dilute plasma solutions under static conditions, a peak in adsorption was observed from 0.26% plasma, i.e., adsorption was greater from 0.26% plasma than from either more or less dilute plasma. A peak in subsequent platelet adhesion to the plasma preadsorbed surfaces, measured after 2 h static incubation with washed platelets, was also observed but occurred on Biomer preadsorbed with 1.0% plasma.When fibrinogen adsorption was measured after 5 min contact under shearing conditions, the fibrinogen adsorption peak occurred on surfaces that had been exposed to 1.0% plasma. A peak in platelet adhesion to these preadsorbed surfaces, measured after 5 min contact with the platelet suspensions under shearing conditions, was observed on Biomer preadsorbed with 0.1% plasma. Shifts between the positions of the peaks in protein adsorption and platelet adhesion occurred on other polymers tested as well.Platelet adhesion was almost completely inhibited when baboon and human plasmas lacking fibrinogen (i. e., serum, heat defibrinogenated plasma, and congenitally afibrinogénémie plasma) were used. Platelet adhesion was restored to near normal when exogenous fibrinogen was added to fibrinogen deficient plasmas. Adhesion was also inhibited completely when a monoclonal antibody directed against the glycoprotein IIb/IIIa complex was added to the platelet suspension. Platelet adhesion to surfaces preadsorbed to von Willebrand factor deficient plasma was the same as to surfaces preadsorbed with normal plasma.While it appears that surface bound fibrinogen does mediate the initial attachment of platelets to Biomer, the observation that the fibrinogen adsorption and platelet adhesion maxima do not coincide exactly also suggests that the degree of subsequent platelet adhesion is dictated not only by the amount of surface bound fibrinogen but also by its conformation.

Role of glycoprotein Ibα mobility in platelet function

2010 ◽  
Vol 103 (05) ◽  
pp. 1033-1043 ◽  
Author(s):  
Dianne van der Wal ◽  
Sandra Verhoef ◽  
Roger Schutgens ◽  
Marjolein Peters ◽  
Yaping Wu ◽  
...  
Keyword(s):  
Amino Acids ◽  
Thromboxane A2 ◽  
Membrane Skeleton ◽  
Platelet Destruction ◽  
Coated Surface ◽  
Von Willebrand ◽  
Induced Aggregation ◽  
Willebrand Factor ◽  
Dependent Mechanism

SummaryIncubation at 0°C is known to expose β-N-acetyl-D-glucosamine residues on glycoprotein (GP) Ibα inducing receptor clustering and αMβ2-mediated platelet destruction by macrophages. Here we show that incubation at 0/37°C (4 hours at 0°C, followed by 1 hour at 37°C to mimic cold-storage and post-transfusion conditions) triggers a conformational change in the N-terminal flank (NTF, amino acids, aa 1–35) but not in aa 36–282 of GPIbα as detected by antibody binding. Addition of the sugar N-acetyl-D-glucosamine (GN) inhibits responses induced by 0/37°C. Incubation at 0°C shifts GPIbα from the membrane skeleton to the cytoskeleton. Different GPIbα conformations have little effect on VWF/ristocetin-induced aggregation, but arrest of NTF change by GN interferes with agglutination and spreading on a VWF-coated surface under flow. Strikingly, incubation at 0/37°C initiates thromboxane A2 formation through a von Willebrand factor (VWF)-independent and GPIbα-dependent mechanism, as confirmed in VWF- and GPIbαﺹdeficient platelets. We conclude that the NTF change induced by 0/37°C incubation reflects clustering of GPIbα supports VWF/ristocetin-induced agglutination and spreading and is sufficient to initiate platelet activation in the absence of VWF.

scholarly journals A novel binding site for ADAMTS13 constitutively exposed on the surface of globular VWF

Blood ◽  
2009 ◽  
Vol 114 (13) ◽  
pp. 2819-2828 ◽  
Author(s):  
Sara Zanardelli ◽  
Alain C. K. Chion ◽  
Evelyn Groot ◽  
Peter J. Lenting ◽  
Thomas A. J. McKinnon ◽  
...  
Keyword(s):  
Surface Plasmon Resonance ◽  
Binding Site ◽  
Surface Plasmon ◽  
Binding Assay ◽  
Initial Step ◽  
Static Conditions ◽  
Von Willebrand ◽  
A2 Domain ◽  
Willebrand Factor

AbstractADAMTS13 metalloprotease regulates the multimeric size of von Willebrand factor (VWF) by cleaving the Tyr1605-Met1606 bond in the VWF A2 domain. The mechanisms of VWF recognition by ADAMTS13 have yet to be fully resolved. Most studies have focused on the role of exosites within the VWF A2 domain, involved in interaction with the ADAMTS13 spacer domain. In the present study, we expressed different C-terminal domain VWF fragments and evaluated their binding to ADAMTS13 and its truncated mutants, MDTCS and del(TSP5-CUB). Using plate binding assay and surface plasmon resonance, we identified a novel ADAMTS13 binding site (KD ∼ 86 nM) in the region of VWF spanning residues 1874 to 2813, which includes the VWF D4 domain and that interacts with the C-terminal domains of ADAMTS13. We show that the interaction occurs even when VWF is in static conditions, assumed to be globular and where the VWF A2 domain is hidden. We demonstrate that C-terminal VWF fragments, as well as an antibody specifically directed toward the VWF D4 domain, inhibit VWF proteolysis by ADAMTS13 under shear conditions. We propose that this novel VWF C-terminal binding site may participate as the initial step of a multistep interaction ultimately leading to proteolysis of VWF by ADAMTS13.

Effects of microgravity and hypergravity on platelet functions

2009 ◽  
Vol 101 (05) ◽  
pp. 902-910 ◽  
Author(s):  
Yuedan Wang ◽  
Rong Yan ◽  
Quanwei Shi ◽  
Zhicheng Wang ◽  
Yanhong Yuan ◽  
...  
Keyword(s):  
Simulated Microgravity ◽  
Surface Expression ◽  
Gravity Change ◽  
Thrombotic Diseases ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
The Brain ◽  
Platelet Functions

SummaryMany serious thrombotic and haemorrhagic diseases or fatalities have been documented in human being exposed to micro-gravity or hypergravity environments, such as crewmen in space, roller coaster riders, and aircrew subjected to high-G training. Some possible related organs have been examined to explore the mechanisms underlying these gravity change-related diseases. However, the role of platelets which are the primary players in both thrombosis and haemostasis is unknown. Here we show that platelet aggregation induced by ristocetin or collagen and platelet adhesion to von Willebrand factor (VWF) were significantly decreased after platelets were exposed to simulated microgravity. Conversely, these platelet functions were increased after platelets were exposed to hypergravity. The tail bleeding time in vivo was significantly shortened in mice exposed to high-G force, whereas, was prolonged in hindlimb unloaded mice. Furthermore, three of 23 mice died after 15 minutes of –8 Gx stress. Platelet thrombi disseminated in the heart ventricle and blood vessels in the brain, lung, and heart from the dead mice. Finally, glycoprotein (GP) Ibα surface expression and its association with the cytoskeleton were significantly decreased in platelets exposed to simulated microgravity, and obviously increased in hypergravity-exposed platelets. These data indicate that the platelet functions are inhibited in microgravity environments, and activated under high-G conditions, suggesting a novel mechanism for gravity change-related haemorrhagic and thrombotic diseases. This mechanism has important implications for preventing and treating gravity change-related diseases, and also suggests that special attentions should be paid to human actions under different gravity conditions.

KINETIC BEHAVIOUR OF VON WILLEBRAND FACTOR AND FIBRONECTIN EXPLAINS DEPENDENCE OF PLATELET ADHESION ON PHYSICAL PARAMETERS

1987 ◽  
Author(s):  
Hans H F I van Breugel ◽  
Philip G de Groot ◽  
Jan J Sixma
Keyword(s):  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Physical Parameters ◽  
Flow Conditions ◽  
Binding Studies ◽  
Platelet Suspension ◽  
Coated Surface ◽  
Static Conditions ◽  
Von Willebrand ◽  
Willebrand Factor

To study the kinetics of the contribution of von Willebrand Factor (vWF) and fibronectin (FN) in platelet adhesion we developed a method with which we can perform binding studies of platelets to these purified proteins under static and flow conditions. Glass coverslips were incubated for one hour with vWF (50 (jg/ml) or FN (300 pg/ml) in saline and were perfused with washed platelets (resuspended in human albumin solution) in the flat perfusion chamber as developed by Sakariassen (J.Lab.Clin.Med. 102, 522-535, 1983). Static conditions were achieved by incubating the coated coverslips with the platelet suspension.In this system, adhesion of platelets to FN coated coverslips strongly decreased at shear rates above 300 /s. The adhesion to this surface could be inhibited with antibodies against platelet glycoprotein Ilbllla and against lb, under static and under flow conditions.Adhesion to vWF coated surfaces increased with increasing shear rate and ultimately reached a plateau at about 800 /s. Adhesion to a vWF coated surface could be totally inhibited by anti GP-Ib and only partially by GP-IIbllla.When after perfusion of a FN coated surface with platelets, the same surface was perfused with a platelet free perfusate, the coverage of platelets on this surface decreased. No decrease in platelet coverage was found when this experiment was performed with a vWF coated coverslip.From these results we conclude that platelets bind to FN at a high rate and with a low affinity, while they bind slowly but with a high affinity to vWF, probablyvia similar platelet receptors.

A Small Fragment of ADAMTS13 Enhances Cleavage of von Willebrand Factor by the Intact Enzyme.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1742-1742
Author(s):  
Elaine M. Majerus
Keyword(s):  
Conformational Change ◽  
Von Willebrand Factor ◽  
Additional Information ◽  
Rich Domain ◽  
Presence And Absence ◽  
Function Relationship ◽  
Static Conditions ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract ADAMTS13 regulates hemostasis by cleaving von Willebrand Factor (VWF) to smaller less thrombogenic fragments. Absence of ADAMTS13 activity leads to thrombotic thrombocytopenic purpura, an often fatal disease that is characterized by microangiopathic hemolytic anemia, thrombocytopenia and microvascular thrombosis especially in renal and central nervous system vasculature. ADAMTS13 like other ADAMTS family members has a characteristic domain structure consisting of a metalloprotease domain, a disintegrin domain, a thrombospondin type 1 repeat (TSR), a cysteine-rich domain, a spacer domain, 7 additional TSRs and 2 carboxyl terminal CUB domains. Regions in the spacer and C-terminal thrombospondin type 1 repeats (TSR) and CUB domains have been shown previously to be important for the interaction of ADAMTS13 and VWF. To further characterize the interaction of ADAMTS13 and VWF, we generated a construct consisting of the spacer domain and the second TSR of ADAMTS13. This construct is called ST2 for Spacer and Tsr2. Although many constructs of ADAMTS13 fail to be secreted because of poor folding, ST2 is folded since it is secreted from human embryonic kidney 293 cells. Previous work has shown that the spacer domain alone can bind VWF under static conditions, therefore ST2 was included in VWF cleaving assays. Unexpectedly, ST2 was found to enhance the cleavage of VWF by ADAMTS13 by approximately 10-fold in assays using either guanidine or urea-denatured VWF. To understand how ST2 enhanced the cleavage of VWF by ADAMTS13, interactions of ST2 with ADAMTS13 and VWF were studied. ST2 was found to specifically co-immunoprecipitate with VWF but not with ADAMTS13. A Kd calculated from these co-immunoprecipitation experiments was 8.7 nM and the stoichiometry of binding was approximately 1 mole/mole. One explanation for the enhanced cleavage of VWF by ADAMTS13 in the presence of ST2 is that ST2 may induce a conformational change in VWF that makes the cleavage site more accessible. To test this hypothesis, VWF in the presence and absence of ST2 was subjected to trypsin digestion at different concentrations of trypsin. Differential cleavage of VWF was seen in the presence and absence of ST2 at all concentrations of trypsin tested. This is consistent with induction of a conformational change in VWF by ST2. These studies provide additional information on the structure-function relationship of these two key proteins in the control of hemostasis.

Ser559 of Glycoprotein Ibα Is Phosphorylated by Protein Kinase A Which Plays Key Roles In Regulating Von Willebrand Factor Binding, Surface Expression and Signaling of Glycoprotein Ib-IX

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3192-3192
Author(s):  
Zhang Weilin ◽  
Liu Guanglei ◽  
Yuan Yanhong ◽  
Zhao Lili ◽  
Yan Rong ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Von Willebrand Factor ◽  
Cho Cells ◽  
Surface Expression ◽  
Filamin A ◽  
Binding Surface ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Kinase A

Abstract Abstract 3192 The interaction of glycoprotein (GP) Iba with von Willebrand factor (VWF) is regulated by the associations of 14-3-3ζ and filamin A with the cytoplasmic domain of GPIba. We reported recently that phosphorylation at Ser559 of GPIba is important for 14-3-3ζ binding, however, the protein kinase responsible for Ser559 phosphorylation remains unknown. Here we show that a peptide corresponding to the Arg557-Pro561 region of GPIba and purified GPIba could be phosphorylated at Ser559 by the catalytic subunit of protein kinase A (PKA). Ser559 phosphorylation was enhanced by PKA activators and reduced by PKA inhibitors in platelets and Chinese hamster ovary (CHO) cells expressing GPIb-IX. Furthermore, PKA inhibitor enhanced, however, reduced VWF binding to two kinds of CHO cells expressing GPIb-IX mutant replacing Ser559 of GPIba (S559A) or Ser166 of GPIbβ (S166A) with alanine, respectively. GPIb-IX association with filamin A and membrane expression were enhanced in S559A cells, whereas reduced in S166A cells. The peptide disrupting the interaction of 14-3-3ζ with Ser559 of GPIba inhibited platelet spreading on VWF matrix and GPIb-IX-VWF interaction-induced association of Src with GPIb-IX. These data indicate that Ser559 of GPIba is phosphorylated by PKA which plays key roles in regulating VWF binding, surface expression, and signaling of GPIb-IX. Disclosures: No relevant conflicts of interest to declare.

Thrombocytopenia and platelet hypoaggregation induced by Bothrops asper snake venom

2005 ◽  
Vol 94 (07) ◽  
pp. 123-131 ◽  
Author(s):  
Mónica Soto ◽  
Teresa Escalante ◽  
Gilbert D. Loría ◽  
Raghuvir Arni ◽  
José María Gutiérrez ◽  
...  
Keyword(s):  
Degradation Products ◽  
Ex Vivo ◽  
Serine Proteinase ◽  
Α2 Macroglobulin ◽  
Hemorrhagic Activity ◽  
Lectin Family ◽  
And Function ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryThrombocytopenia and platelet dysfunction occur in patients bitten by Bothrops sp snakes in Latin America. An experimental model was developed in mice to study the effects of B. asper venom in platelet numbers and function. Intravenous administration of this venom induces rapid and prominent thrombocytopenia and ex vivo platelet hypoaggregation. The drop in platelet numbers was primarily due to aspercetin, a protein of the C-type lectin family which induces von Willebrand factor-mediated platelet aggregation/agglutination. In addition, the effect of class P-III hemorrhagic metalloproteinases on the microvessel wall also contributes to thrombocytopenia since jararhagin, a P-III metalloproteinase, reduced platelet counts. Hypoaggregation was associated with the action of procoagulant and defibrin(ogen)ating proteinases jararacussin-I (a thrombin-like serine proteinase) and basparin A (a prothrombin activating metalloproteinase). At the doses which induced hypoaggregation, these enzymes caused defibrin(ogen)ation, increments in fibrin(ogen) degradation products and D-dimer and prolongation of the bleeding time. Incubation of B. asper venom with batimastat and α2-macroglobulin abrogated the hypoaggregating activity, confirming the role of venom proteinases in this effect. Neither aspercetin nor the defibrin(ogen)ating and hypoaggregating components induced hemorrhage upon intravenous injection. However, aspercetin, but not the thrombin-like or the prothrombin-activating proteinases, potentiated the hemorrhagic activity of two hemorrhagic metalloproteinases in the lungs.

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