Filamin A Binding Stabilizes Nascent Glycoprotein Ibα Trafficking and Thereby Enhances Its Surface Expression.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3656-3656
Author(s):  
Shuju Feng ◽  
Xin Lu ◽  
Michael H. Kroll
Keyword(s):  
Cho Cells ◽  
Protein Trafficking ◽  
Structural Changes ◽  
Extracellular Domain ◽  
Surface Expression ◽  
Cytoplasmic Domain ◽  
Filamin A ◽  
Von Willebrand ◽  
And Control

Abstract The glycoprotein (Gp) Ib-IX-V complex is essential for platelet function. The extracellular domain of GpIbα binds to von Willebrand factor (VWF) and rapidly effects shear-dependent platelet rolling, tether formation, adherence and aggregation onto freshly exposed subendothelium. Following VWF binding, GpIbα transduces proaggregatory signals through its cytoplasmic domain, and these signals may be modulated by the activity of GpIbβ and GpV. The expression of GpIbα, GpIbβ and GpIX in megakaryocytes also regulates terminal maturation and platelet release (its absence results in Bernard-Soulier syndrome) and there is human genetic and in vitro evidence that the cytoplasmic domain of GpIbα regulates megakaryocyte growth and surface expression of the complex. The cytoplasmic domain of GpIbα possesses a binding region for filamin A, which links GpIb-IX-V to the platelet cytoskeleton, and there is evidence that filamin A binding to GpIbα directs the surface expression of GpIb-IX in CHO cells. To investigate the mechanism of this effect, we examined GpIbα biosynthesis in CHO cells stably co-expressing wild-type or mutant GpIbα with GpIbβ, GpIX and filamin A. We observed that surface GpIbα expression as measured by flow cytometry is enhanced ~ 1 log-order in CHO cells co-expressing human filamin A. In comparison with CHO-GpIbαβIX cell lysates, lysates from CHO-GpIbαβIX-filamin A cells showed greater amounts of immature (~ 65 kDa), incompletely glycosylated (~ 80 kDa and ~ 90 kDa) and fully mature GpIbα (~ 120 kDa), but lesser amounts of the ~ 15 kDa C-terminal peptide released when the extracellular domain of GpIbα (glycocalycin) is cleaved by surface proteases. To determine if the effect of filamin A is due to its binding to GpIbα, we examined GpIbα biosynthesis using several mutants of GpIbα co-expressed with GpIbβ, GpIX and filamin A. When filamin A binding is eliminated by truncation of GpIbα at C-terminal residue 557 or by a deletion in GpIbα between amino acids 542–570, the decreased synthesis of mature GpIbα is accompanied by nearly complete elimination of all immunodetectable immature GpIbα and by increased immunodetectable C-terminal peptide. To corroborate these data and control for cell selection and non-specific secondary structural changes, we examined the expression of three cell lines expressing two additional mutants of GpIbα in CHO cells stably transfected with GpIbβ, GpIX and filamin A. The expression of GpIbα with a C-terminal deletion between residues 560 and 570 (which inhibits filamin A binding), but not GpIbα with alanine substitutions at C-terminal residues 557 through 559 (which converts the sequence RGS to AAA but doesn’t inhibit filamin binding), results in the near-elimination of immature GpIbα. These results suggest that GpIbα binding to filamin enhances the surface expression of GpIb-IX by directing nascent protein trafficking away from a degradative pathway and towards the golgi. This leads to increased glycosylation, which further stabilizes the complex by attenuating the susceptibility of the extracellular domain to proteolytic cleavage.

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.

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.

Filamin A Deficiency Leads to Premature Platelet Formation and Increased Clearance

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2027-2027
Author(s):  
Antonija Jurak Begonja ◽  
Sarah E. Weber ◽  
Karin M. Hoffmeister ◽  
John H. Hartwig ◽  
Herve Falet
Keyword(s):  
Conflicts Of Interest ◽  
Surface Expression ◽  
Signaling Proteins ◽  
Filamin A ◽  
Membrane Glycoproteins ◽  
Filament Networks ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Platelet Formation

Abstract Abstract 2027 Filamin A (FlnA) is a large cytoplasmic protein that crosslinks and stabilizes actin filament networks and links membrane glycoproteins and signaling proteins to the underlying cytoskeleton. FlnA loxP-PF4-Cre mice that specifically lack FlnA in their platelets and megakaryocytes (MKs) have a macrothrombocytopenia with increased tail bleeding time. FlnA KO platelets have decreased surface expression of the von Willebrand factor receptor (vWfR) and are cleared rapidly from the circulation of WT mice, indicating an inherent surface defect that leads to detection and removal. FlnA loxP-PF4-Cre mice have a marked increase in MK numbers in bone marrow and spleen. Detailed analysis of platelet production by FlnA KO MKs in vitro reveals an altered maturation program and defects in vWfR stability, compared to WT. Surprisingly, the surface expression of vWfR components (Ibα, Ibβ, IX and V) on FlnA KO MKs is normal or increased compared to WT MKs, although the total amount of GPIbα is decreased and GPIbα is not associated with the actin cytoskeleton. Analysis of the GPIbβ subunit shows increased degradation in FlnA KO MKs and platelets. Although FlnA KO and WT MK cultures contain comparable cell numbers, FlnA KO MKs more rapidly convert their proplatelets into large CD61+ platelet-sized particles. These findings suggest that aberrant platelet maturation by FlnA KO MKs results in enlarged platelets that are cleared rapidly because of altered vWfR expression. Disclosures: No relevant conflicts of interest to declare.

Serine 559 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 ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3520-3520
Author(s):  
Jie Zhang ◽  
Weilin Zhang ◽  
Guanglei Liu ◽  
Lili Zhao ◽  
Rong Yan ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cho Cells ◽  
Surface Expression ◽  
Filamin A ◽  
Binding Function ◽  
Binding Surface ◽  
Platelet Spreading ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Kinase A

Abstract Conformational changes in von Willebrand factor (VWF) exposed at the injured vessel wall initiate glycoprotein (GP) Ib-IX-dependent platelet adhesion, and simultaneously trigger signaling cascades leading to integrin αIIbβIII activation, platelet spreading, and thrombus formation. The intracellular signaling protein 14-3-3ζ and the membrane skeleton protein filamin A have been confirmed to interact with the cytoplasmic domain of GPIbα and play key roles in regulating the VWF binding function of GPIb-IX. Several phosphoserine containing motifs in GPIb-IX complex, such as Ser609 of GPIbα and Ser166 of GPIbβ, have been confirmed to bind to 14-3-3ζ and involved in the regulation of VWF binding function. We have recently identified a novel 14-3-3ζ binding site located in the filamin A binding domain of GPIbα at Ser559. Phosphorylation of GPIbα at Ser559 not only is important for 14-3-3ζ binding, but also plays a key role in the regulation of VWF binding function of GPIb-IX. However, the protein kinase responsible for Ser559 phosphorylation remains unknown. Here we show that a peptide corresponding to the Arg557-Pro561 region of GPIbα and purified GPIbα 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 VWF binding to the CHO cells expressing GPIb-IX mutant replacing Ser559 of GPIbα (S559A) with alanine, while reduced in the CHO cells of Ser166 of GPIbβ (S166A) with alanine. 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 GPIbα inhibited platelet spreading on VWF matrix and GPIb-IX-VWF interaction-induced association of Src with GPIb-IX. These data indicate that Ser559 of GPIbα 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.

Filamin A binding to the cytoplasmic tail of glycoprotein Ibα regulates von Willebrand factor–induced platelet activation

Blood ◽  
2003 ◽  
Vol 102 (6) ◽  
pp. 2122-2129 ◽  
Author(s):  
Shuju Feng ◽  
Julio C. Reséndiz ◽  
Xin Lu ◽  
Michael H. Kroll
Keyword(s):  
Shear Stress ◽  
Platelet Aggregation ◽  
Fusion Protein ◽  
Platelet Activation ◽  
Von Willebrand Factor ◽  
Cytoplasmic Tail ◽  
Cytoplasmic Domain ◽  
Filamin A ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract We examined the hypothesis that filamin A binding to the cytoplasmic tail of platelet glycoprotein Ibα (GpIbα) is regulated by pathologic shear stress and modulates von Willebrand factor (VWF)–induced platelet activation. To begin, we examined filamin binding to GpIbα in Chinese hamster ovary cells coexpressing mutant human GpIb-IX and wild-type human filamin A. We observed that many different deletions and truncations N-terminal to GpIbα's cytoplasmic domain residue 594 disrupted filamin A binding, but that binding was unaffected by 14 different point mutations in hydrophilic residues between amino acids 557 and 593. To try to narrow GpIbα's filamin A–binding domain, we next measured the effect of several cytoplasmic domain peptides on human filamin A binding to a GST-GpIbα cytoplasmic domain fusion protein. One peptide (residues 557-575; designated “A4 peptide”) inhibited filamin A binding to the GST-GpIbα cytoplasmic domain fusion protein and competed with GpIbα for binding to filamin A. When the A4 peptide was delivered to intact human platelets using a carrier peptide, we observed the dose-dependent inhibition of VWF-induced platelet aggregation in response to both ristocetin and shear stress. The effect of the A4 peptide on shear-induced platelet aggregation was accompanied by the attenuation of shear-induced filamin A binding to GpIbα and diminished shear-dependent protein tyrosine phosphorylation. These results suggest that shear-dependent VWF-induced platelet activation affects filamin A binding to GpIb-IX-V, and that filamin A binding to the cytoplasmic tail of GpIbα regulates proaggregatory tyrosine kinase signaling.

The Cytoplasmic Domain of Glycoprotein Ibβ Is Critical to Surface Trafficking of Glycoprotein Ib-IX Complex.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1531-1531
Author(s):  
Xi Mo ◽  
Jose A. Lopez ◽  
Renhao Li
Keyword(s):  
Cho Cells ◽  
Transmembrane Domain ◽  
Complex Structure ◽  
Chinese Hamster ◽  
Surface Expression ◽  
Cytoplasmic Domain ◽  
Expression Level ◽  
Platelet Glycoprotein ◽  
Cellular Expression

Abstract Platelet glycoprotein (GP) Ib-IX-V complex mediates platelet adhesion to the subendothelium of damaged vessel walls under high shear conditions. Delineating the assembly process of the GP Ib-IX complex will aid our understanding of the complex structure and shed light on the signaling mechanism underlying platelet activation. The GP Ib-IX complex comprises three polypeptides, GP Ibα, GP Ibβ and GP IX, and efficient surface expression of the complex in transfected Chinese hamster ovary (CHO) cells requires all of its three subunits, indicating that the assembly of the complex in the endoplasmic reticulum is required for its subsequent trafficking to the plasma membrane. We recently showed that the interaction between the transmembrane domain of GP Ibβ and the other subunits is critical to efficient surface expression of the GP Ib-IX complex. Here, we have explored the role of the Ibβ cytoplasmic domain in the complex assembly and surface expression. In CHO cells transiently expressing the mutant Ib-IX complex in which the Ibβ cytoplasmic domain was deleted or replaced entirely with the IX counterpart, neither cellular expression of GP Ibα nor surface expression of GP Ibα and IX was detected. In contrast, deletion and/or replacement of the Ibα or IX cytoplasmic domains did not affect significantly the assembly and surface expression of the receptor complex. Furthermore, deletion of the last six residues in the Ibβ cytoplasmic domain did not affect the cellular expression level of GP Ibα but significantly decreased its surface expression level in CHO cells. Intriguingly, further deletion of the Ibβ cytoplasmic domain that removed the binding site for 14-3-3ζ restored surface expression of the GP Ib-IX complex to a level comparable to the wild type construct. Overall, our results demonstrated an important role of the Ibβ cytoplasmic domain in both assembly and trafficking of the GP Ib-IX complex. Critical residues in the Ibβ cytoplasmic domain have been identified and further characterization is currently underway.

Deficiency of the VWF-Cleaving Protease ADAMTS13 Results in Increased Leukocyte Rolling and Adhesion in Mice.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 290-290 ◽  
Author(s):  
Anil K. Chauhan ◽  
Janka Kiucka ◽  
Alexander Brill ◽  
Meghan T. Walsh ◽  
Denisa D. Wagner
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Leukocyte Adhesion ◽  
Surface Expression ◽  
Leukocyte Rolling ◽  
Type I ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Adherent Leukocytes

Abstract von Willebrand factor (VWF) is synthesized in megakaryocytes and endothelial cells and stored in a-granules and Weibel-Palade bodies, respectively. VWF levels are elevated in both chronic and acute inflammation. ADAMTS13 (A D isintegrin-like A nd M etalloprotease with T hrombo s pondin type I repeats-13) is a metalloprotease that cleaves ultra large von Willebrand factor (ULVWF) multimers quickly after its release from endothelium. Recent studies have found that VWF promotes leukocyte adhesion in vitro and that ADAMTS13 activity is reduced in inflammation and sepsis. We hypothesized that by cleaving ULVWF multimers, ADAMTS13 not only inhibits thrombosis, but also attenuates leukocyte rolling and adhesion. Using intravital microscopy, we found more leukocyte rolling/min on the unstimulated veins in Adamts13-/- mice (Mean ± SE: 98 ± 16) compared to WT (Mean ± SE: 35 ± 6, P<0.001), n=18–20 from 10–11 mice per group. This process was dependent on VWF because the number of leukocytes rolling in Adamts13-/-/Vwf-/- veins was similar to that in Vwf-/-. Significantly increased soluble P-selectin and VWF concentrations were found in the plasma of Adamts13-/- compared to WT mice as quantitated by ELISA. In addition, endothelial P-selectin surface expression was increased in Adamts13-/- mice compared to WT. These results suggest elevated release of Weibel-Palade bodies in Adamts13-/- mice. Notably, circulating platelets were not activated in the absence of ADAMTS13. Upon stimulation of the mesentery with histamine, leukocyte rolling was slower in Adamts13-/- veins compared to WT. Furthermore, upon stimulation with the inflammatory cytokine TNF-alpha (i.v) 3.5 h prior to surgery, the number of leukocytes adhering/250 um was significantly increased in microvenules (diameter of 25–30 um) of Adamts13-/- mice (Mean ± SD: 21 ± 6) compared to WT (Mean ± SD: 12 ± 5, P<0.001), n=10–11 mice per group. This firm adhesion was also dependent on VWF because the number of adherent leukocytes in veins of Adamts13-/-/Vwf-/- was similar to Vwf-/-. Our studies indicate a crucial role for ADAMTS13 in preventing excessive spontaneous Weibel-Palade secretion and in attenuating leukocyte rolling and adhesion to ultra large VWF presented by endothelial cells during inflammation.

Inhibition of Calmodulin Triggers Apoptosis Events in Human Platelets.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3003-3003
Author(s):  
Zhicheng Wang ◽  
Suping Li ◽  
Guanglei Liu ◽  
Quanwei Shi ◽  
Rong Yan ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Conflicts Of Interest ◽  
Surface Expression ◽  
Eukaryotic Cell ◽  
Human Platelets ◽  
Platelet Apoptosis ◽  
Potent Antagonist ◽  
Von Willebrand

Abstract Abstract 3003 Poster Board II-980 Calmodulin (CaM) is a calcium-sensing protein ubiquitously expressed in every eukaryotic cell type regulating biological processes such as cell proliferation, vesicular fusion, fertilization and apoptosis. CaM antagonists induce apoptosis in various tumor models and inhibit tumor cell invasion and metastasis, thus some of which have been extensively used as anti-cancer agents. Tamoxifen (TMX), a potent antagonist of CaM, has been in the center of management of hormone-sensitive breast cancer, and also represents the best example of chemo-prevention to reduce the incidence of invasive breast cancer. Furthermore, TMX is potentially useful in treatment of other kinds of cancer. However, TMX has some severe side effects, one of which is thrombocytopenia. Up to now, the pathogenesis of thrombocytopenia still remains unclear. In platelets, CaM has been found to bind directly to cytoplasmic domains of several platelet receptors. Incubation of platelets with CaM antagonists impairs the receptors-related platelet function. However, it is still unclear whether CaM antagonists, especially TMX, induce platelet apoptosis. Here, we show that CaM antagonists TMX and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W7) dose-dependently induce apoptotic events in human platelets, including depolarization of mitochondrial inner transmembrane potential, caspase-3 activation, gelsolin cleavage and phosphatidylserine (PS) exposure. CaM antagonist did not incur platelet activation as detected by P-selectin surface expression and PAC-1 binding. However, ADP- and botrocetin-induced platelet aggregation and platelet adhesion and spreading on von Willebrand factor surface were significantly reduced in platelets pre-treated with CaM antagonist. Therefore, these findings indicate that CaM antagonists induce platelet apoptosis, which suggests a possible pathogenesis of thrombocytopenia in some patients treated with CaM antagonist drugs, and also may present as a novel mechanism for platelet clearance and dysfunction in vivo or in vitro. The elevation of the cytosolic Ca2+ level may involve in the regulation of CaM antagonist-induced platelet apoptosis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Snake Venom Botrocetin Induces GPIb-Independent Platelet Aggregation

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4203-4203
Author(s):  
Chuanbin Shen ◽  
Daniel Mackeigan ◽  
Guangheng Zhu ◽  
Miguel A. D. Neves ◽  
Wenjing Ma ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Cho Cells ◽  
Thrombus Formation ◽  
Extracellular Domain ◽  
Shear Stresses ◽  
Antithrombotic Drugs ◽  
Integrin Αiibβ3 ◽  
Von Willebrand ◽  
Induced Aggregation ◽  
Deficient Mice

Abstract Abstract Introduction：Snake venom-derived botrocetin facilitates von Willebrand factor (VWF) binding to GPIbα, and has been used clinically for the detection of von Willebrand disease (VWD) and GPIb-related disorders. Botrocetin has also been widely used experimentally for the development and characterization of potential antithrombotic drugs targeting the GPIb-VWF axis. Although compelling evidence suggests GPIb is responsible for botrocetin-induced VWF binding and platelet aggregation, some reports suggest that botrocetin could induce platelet aggregation in some Bernard-Soulier syndrome (BSS) patients who lack a functional GPIb complex. However, the alternative mechanism for botrocetin-induced BSS platelet aggregation and the receptor(s) mediating this action are unclear. Methods: Botrocetin was purified from the lyophilized venom of Bothrops jararaca using ion-exchange column chromatography. Light transmission aggregometry assay was performed using platelet-rich plasma (PRP) from human, wild type (WT) mice, GPIbα-deficient mice, αIIbβ3-deficient mice and VWF-deficient mice, or CHO cells stably transfected with αIIbβ3 integrin. O-sialoglycoprotein endopeptidase (OSGE) was used to cleave the N-terminal extracellular domain of GPIbα. The binding of botrocetin, VWF and fibrinogen to platelets from WT or the gene-deficient mice were measured by flow cytometry. Antibodies against GPIbα (SZ2, NIT A) and integrin αIIbβ3 (abciximab, JON/A, M1, PSI E1) were used to investigate the binding site of botrocetin. Perfusion chamber assay was used to measure thrombus formation under different shear stresses. Results: We discovered that botrocetin induced aggregation of human platelets lacking the N-terminal extracellular domain of GPIbα and platelets from GPIbα-deficient mice in the presence of VWF. This VWF-dependent, GPIbα-independent platelet aggregation induced by botrocetin was inhibited by αIIbβ3 antagonists. Botrocetin also induced aggregation of CHO cells stably transfected with αIIbβ3 in VWF-dependent manner. Further experiments with gel-filtered platelets showed that botrocetin competitively bound to the ligand-binding area exposed on αIIbβ3 and blocked fibrinogen and other ligands from binding to the active state of αIIbβ3 in the absence of VWF. Botrocetin inhibited platelet aggregation and thrombus formation in VWF-deficient mice. Conclusion: Integrin αIIbβ3 is the alternative receptor that mediates VWF-dependent, GPIb-independent platelet aggregation induced by botrocetin. However, via targeting αIIbβ3, botrocetin itself inhibits platelet aggregation in the absence of VWF. These results demonstrate versatility in the mechanism of botrocetin, which may provide snakes containing this toxin the adaptability necessary to aggregate platelets/thrombocytes of different prey or predators. Our data reveals a previously unknown role of botrocetin in the integrin-VWF interaction and also provides insight into developing new antithrombotic drugs that target the active conformation of integrin αIIbβ3. The target switching of botrocetin between GPIb-VWF and αIIbβ3-VWF may explain the possible misdiagnosis of the GPIb-related congenital disorders evaluated by botrocetin. Disclosures No relevant conflicts of interest to declare.

The Association of 14-3-3ζ and Filamin A with the Central Cytoplasmic Domain of GPIbα Involves in Regulation of the Von Willebrand Factor Binding and Signaling Functions of Platelet Glycoprotein Ib-IX.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 5073-5073
Author(s):  
Weilin Zhang ◽  
Yanhong Yuan ◽  
Lili Zhao ◽  
Yi Liao ◽  
Changgeng Ruan ◽  
...  
Keyword(s):  
Binding Site ◽  
Von Willebrand Factor ◽  
Intracellular Signaling ◽  
Cytoplasmic Domain ◽  
Platelet Glycoprotein ◽  
Filamin A ◽  
Glycoprotein Ib ◽  
Binding Function ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 5073 Introduction Conformational changes in von Willebrand factor (VWF) exposed at the injured vessel wall initiate platelet tethering and simultaneously trigger signaling cascades leading to platelet adhesion, spreading, and thrombus formation. Although the intracellular signaling protein 14-3-3ζ and the membrane skeleton protein filamin A have been confirmed to interact with the cytoplasmic domain of GPIbα and involve in the regulation of VWF binding function, whereas, the mechanisms still remain unclear. We have recently identified a novel 14-3-3ζ binding site with partial phosphorylation of Ser559 in resting platelets located in the cytoplasmic filamin A binding domain of GPIbα. Disruption of the central cytoplasmic 14-3-3ζ binding site of GPIbα did not abolish the interaction between 14-3-3ζ and GPIb-IX, whereas obviously diminished VWF binding function. Here we show that disruption of 14-3-3ζ binding to GPIbα by GPIbα mutants or cell permeable peptides enhances the association of filamin A with GPIb-IX and the expression of GPIb-IX in the plasma membrane. In contrast, disruption of 14-3-3ζ binding to GPIbβ, which would enhance the opportunity for 14-3-3ζ monomer to bind to GPIbα, reduces filamin A-GPIb-IX interaction and GPIb-IX membrane expression. Compared with a GPIb-IX mutant truncating the whole cytoplasmic domain of GPIbα (Δ551), GPIb-IX mutant retaining only the central 14-3-3ζ binding site of the cytoplasmic domain of GPIbα (Δ565) showed an enhanced cell adhesion to VWF under flow and ristocetin-induced VWF binding. Furthermore, the two cell permeable peptides, MP-P and MPαC, which had been confirmed to block the central 14-3-3ζ binding site of GPIbα (MP-P) or diminished the association of 14-3-3ζ with GPIb-IX complex (MPαC), obviously inhibited ristocetin induced wild-type GPIb-IX cell aggregation. In addition, MP-P and MPαC inhibited botrocetin-induced platelet spreading and lamellipodia formation on VWF matrix, and GPIb-IX-VWF interaction induced co-immunoprecipitation of Src with GPIb-IX. Taken together, these data indicate that the binding of 14-3-3ζ and filamin A to the central cytoplasmic domain of GPIbα involves in regulation of surface expression, VWF binding and signaling functions of glycoprotein Ib-IX. Disclosures No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document