The GPIbα intracellular tail – role in transducing VWF- and collagen/GPVI-mediated signaling

The GPIbT-VWF A1 domain interaction is essential for platelet tethering under high shear. Synergy between GPIbα and GPVI signaling machineries has been suggested previously, however its molecular mechanism remains unclear. We generated a novel GPIbα transgenic mouse (GpIbαΔsig/Δsig) by CRISPR-Cas9 technology to delete the last 24 residues of the GPIbα intracellular tail that harbors the 14-3-3 and phosphoinositide-3 kinase binding sites. GPIbαΔsig/Δsig platelets bound VWF normally under flow. However, they formed fewer filopodia on VWF/botrocetin in the presence of a oIIbI3 blocker, demonstrating that despite normal ligand binding, VWF-dependent signaling is diminished. Activation of GpIbαΔsig/Δsig platelets with ADP and thrombin was normal, but GpIbαΔsig/Δsig platelets stimulated with collagen-related-peptide (CRP) exhibited markedly decreased P-selectin exposure and eIIbI3 activation, suggesting a role for the GpIbaaintracellular tail in GPVI-mediated signaling. Consistent with this, while haemostasis was normal in GPIbαΔsig/Δsig mice, diminished tyrosine-phosphorylation, (particularly pSYK) was detected in CRP-stimulated GpIbαΔsig/Δsig platelets as well as reduced platelet spreading on CRP. Platelet responses to rhodocytin were also affected in GpIbαΔsig/Δsig platelets but to a lesser extent than those with CRP. GpIbαΔsig/Δsig platelets formed smaller aggregates than wild-type platelets on collagen-coated microchannels at low, medium and high shear. In response to both VWF and collagen binding, flow assays performed with plasma-free blood or in the presence of bIIbI3- or GPVI-blockers suggested reduced bIIbI3 activation contributes to the phenotype of the GpIbαΔsig/Δsig platelets. Together, these results reveal a new role for the intracellular tail of GPIbiiin transducing both VWF-GPIbGGand collagen-GPVI signaling events in platelets.

Download Full-text

VWF Interaction With Type IV Collagen Is Mediated Through Critical VWF A1 Domain Residues

Blood ◽

10.1182/blood.v122.21.29.29 ◽

2013 ◽

Vol 122 (21) ◽

pp. 29-29

Author(s):

Veronica H. Flood ◽

Abraham C. Schlauderaff ◽

Paula M. Jacobi ◽

Tricia L. Slobodianuk ◽

Robert R. Montgomery ◽

...

Keyword(s):

Binding Sites ◽

Type Iv Collagen ◽

Platelet Glycoprotein ◽

Collagen Binding ◽

Common Component ◽

Type Iv ◽

Static Conditions ◽

A1 Domain ◽

Von Willebrand

Abstract Von Willebrand factor (VWF) plays a key role in coagulation by tethering platelets to injured subendothelium via binding sites for platelet glycoprotein Ib and collagen. The binding sites for types I and III collagen in the VWF A3 domain are well characterized, and defects in this region have been implicated in von Willebrand disease (VWD). Additional collagens present in the vasculature may also be involved in interactions with VWF. A VWF A1 sequence variation, p.R1399H, has been associated with decreased binding to type VI collagen, but the clinical significance of this observation remains unclear. Type IV collagen is a common component of the basement membrane and as such may be an important ligand for VWF. While some VWD testing utilizes types I or III collagen, current clinical testing does not include collagen IV or VI. To characterize the role of the VWF A1 domain in VWF-type IV collagen interactions, we generated several A1 domain variant human and/or murine recombinant VWF (rVWF) constructs including R1399H and several type 2M VWD variants localized to the same region (S1387I, Q1402P, and an 11 amino acid deletion mutant encompassing amino acids 1392-1402). These constructs were then expressed in HEK 293T cells. To further assess the role of the A1 domain, scanning alanine mutagenesis (SAM) of residues 1387 through 1412 was conducted. VWF antigen levels (VWF:Ag), collagen binding with type III (VWF:CB3), IV (VWF:CB4), or VI (VWF:CB6) collagen were determined, and multimer distribution was assessed for all recombinant VWF variants. The role of R1399H in the context of human rVWF was characterized initially. Although VWF:Ag, VWF:CB3, and multimer distribution were normal for R1399H compared to wild-type (WT VWF), VWF:CB4 was undetectable. To examine this effect in a mouse model, the R1399H variant was expressed in the context of murine rVWF and collagen binding was determined. Similar to the human variant, murine R1399H rVWF demonstrated significantly reduced binding to murine type IV collagen, at only 7% of the binding seen with WT murine rVWF. In order to examine the behavior of R1399H under shear conditions, either WT or R1399H murine rVWF DNA was hydrodynamically injected into the tail veins of VWF -/- mice to induce expression of the proteins; blood was drawn from the vena cava 24 hours later and then examined on the VenaFlux flow apparatus. VWF expression levels and multimer distribution were similar for the R1399H- and WT-injected mice. Under static conditions, the murine plasma-derived R1399H demonstrated decreased VWF:CB4, at only 16% of the levels seen with WT VWF. No defect was seen in VWF:CB3. Furthermore, when binding to type IV collagen was assessed under flow conditions by VenaFlux, platelet adhesion was significantly decreased in mice expressing R1399H VWF as compared to mice expressing WT VWF. When examining other A1 domain variants, Q1402P and del1392-1402 demonstrated absent VWF:CB4 while S1387I demonstrated a significant reduction in VWF:CB4 compared to WT VWF. All SAM VWF A1 domain variants demonstrated normal expression, multimerization, and VWF:CB3. However, type IV collagen binding was absent for R1392A, R1395A, R1399A, and K1406A and was reduced to less than 50% of WT VWF for Q1402A, K1405A, and K1407A. These residues map to an outside face of the VWF A1 domain crystal structure, and are likely the critical residues for VWF binding to type IV collagen. Taken together, these data demonstrate that the type IV collagen binding site localizes to a specific region of the VWF A1 domain. Mutations in this region of VWF may be clinically significant due to a defect in the ability of VWF to attract platelets to exposed type IV collagen which may contribute to bleeding symptoms seen in VWD. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Mechanisms of Static and Shear-Induced Platelet Adhesion: Differences in the Adhesion Supported by MMRN1 Comparisons with Other β3 Integrin Ligands.

Blood ◽

10.1182/blood.v106.11.2659.2659 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2659-2659

Author(s):

Frederic Adam ◽

Shilun Zheng ◽

Aurelio V. Santos ◽

John G. Kelton ◽

Catherine P.M. Hayward

Keyword(s):

Ligand Binding ◽

Binding Sites ◽

Platelet Adhesion ◽

Platelet Glycoprotein ◽

High Shear ◽

Shear Rates ◽

Activated Platelets ◽

High Shear Rates ◽

Adhesive Interactions ◽

Adhesion Assays

Abstract Platelet adhesion and aggregation at the site of vascular injury are key events in hemostasis and thrombosis. These processes are supported by interactions between platelet glycoprotein (GP) receptors (including integrin αIIbβ3, GP Ib-IX-V and GPVI) and ligands that include von Willebrand factor (VWF), collagen, and fibrinogen (Fg). Recently, the polymeric protein multimerin 1 (MMRN1) was identified to bind β3 integrins. Normally, MMRN1 is sequestered within secretion granules of platelets, megakaryocytes, and endothelium until its release. In static adhesion assays, MMRN1 supports platelet adhesion to integrins αIIbβ3 and αvβ3 by an RGD-dependent mechanism. Study goals and methods: To further determine the mechanisms of MMRN1 binding to platelets, we investigated (i) the importance of platelet activation in MMRN1 binding to platelets, (ii) the ability of MMRN1 to support platelet adhesion compared to other adhesive ligands (Fg, VWF), and (iii) the role of β3 integrins in MMRN1 binding to platelets at low and high shear. Results: The binding of secreted platelet MMRN1 to thrombin activated platelets was significantly reduced by antibody inhibitors of ligand binding to αIIbβ3 and αvβ3. Thrombasthenic platelets (GT), deficient in αIIbβ3 and αvβ3 integrins, stored normal quantities of MMRN1 but after activation, they retained 34% less MMRN1 than normal platelets. Platelet adhesion experiments confirmed that αIIbβ3/αvβ3 and other binding sites supported MMRN1 binding to platelets. MMRN1 supported platelet adhesion at both low (150 s-1) and high (1500 s-1) shear rates. Like platelet adhesion to VWF, platelet adhesion to MMRN1 was greater at high shear rates. Platelet stimulation by agonists was essential to induce platelet binding to MMRN1 in static and shear adhesion assays, but not to induce platelet adhesion to Fg and vWF. While platelets activated by ADP and TRAP showed similar adhesion to Fg and VWF, TRAP induced more platelet adhesion to MMRN1 at high shear rates than ADP. Inhibitors of ligand binding to αIIbβ3 and αvβ3 had greater effects on high compared to low shear platelet adhesion to MMRN1. Conclusions: These data indicate interesting differences in the mechanisms that support platelet adhesion to MMRN1 and other β3 ligands, which could be important for molecular events in hemostasis and thrombosis. The activation-dependent binding of MMRN1 to platelets, augmented by high shear flow, may reflect the unique recognition properties of platelet β3 integrins and/or exposure of other binding sites (e.g. phosphatidylserine) on activated platelets that promote adhesive interactions with MMRN1.

Download Full-text

Ligand Binding Sites of Inducible Costimulator and High Avidity Mutants with Improved Function

Journal of Experimental Medicine ◽

10.1084/jem.20011607 ◽

2002 ◽

Vol 195 (8) ◽

pp. 1033-1041 ◽

Cited By ~ 22

Author(s):

Shengdian Wang ◽

Gefeng Zhu ◽

Koji Tamada ◽

Lieping Chen ◽

Jürgen Bajorath

Keyword(s):

T Cell ◽

Immune Responses ◽

Ligand Binding ◽

Binding Sites ◽

T Cell Response ◽

Surface Patch ◽

High Avidity ◽

Wild Type ◽

Ligand Interaction ◽

Inducible Costimulator

Interaction between inducible costimulator (ICOS) and its ligand is implicated in the induction of cell-mediated and humoral immune responses. However, the molecular details of this interaction are unknown. We report here a mutagenesis analysis of residues in ICOS that are critical for ligand binding. A three-dimensional model of the extracellular immunoglobulin-like domain of ICOS was used to map the residues conserved within the CD28 family. This analysis identified a surface patch containing the characteristic “PPP” sequence and is conserved in human and mouse ICOS. Mutations in this region of human ICOS reduce or abolish ligand binding. Our results suggest that the ligand binding site in ICOS maps to a region overlapping yet distinct from the CD80/CD86 binding sites in CD28 and cytotoxic T lymphocyte antigen (CTLA)-4. Thus, the analysis suggests that differences in ligand binding specificity between these related costimulatory molecules have evolved by utilization of overlapping regions with different patterns of conserved and nonconserved residues. Two site-specific mutants generated in the course of our studies bound ICOS ligand with higher avidity than wild-type ICOS. An S76E mutant protein of ICOS blocked T cell costimulatory function of ICOS ligand and inhibited T cell response to allogeneic antigens superior to wild-type ICOS. Our studies thus identified critical residues involving in ICOS receptor–ligand interaction and provide new modulators for immune responses.

Download Full-text

O-linked glycosylation of von Willebrand factor modulates the interaction with platelet receptor glycoprotein Ib under static and shear stress conditions

Blood ◽

10.1182/blood-2012-02-410050 ◽

2012 ◽

Vol 120 (1) ◽

pp. 214-222 ◽

Cited By ~ 38

Author(s):

Agata A. Nowak ◽

Kevin Canis ◽

Anne Riddell ◽

Michael A. Laffan ◽

Thomas A. J. McKinnon

Keyword(s):

Shear Stress ◽

Binding Sites ◽

Stress Conditions ◽

Collagen Binding ◽

Platelet Receptor ◽

A1 Domain ◽

Von Willebrand ◽

Willebrand Factor ◽

Double Cluster

AbstractWe have examined the effect of the O-linked glycan (OLG) structures of VWF on its interaction with the platelet receptor glycoprotein Ibα. The 10 OLGs were mutated individually and as clusters (Clus) on either and both sides of the A1 domain: Clus1 (N-terminal side), Clus2 (C-terminal side), and double cluster (DC), in both full-length-VWF and in a VWF construct spanning D′ to A3 domains. Mutations did not alter VWF secretion by HEK293T cells, multimeric structure, or static collagen binding. The T1255A, Clus1, and DC variants caused increased ristocetin-mediated GPIbα binding to VWF. Platelet translocation rate on OLG mutants was increased because of reduced numbers of GPIbα binding sites but without effect on bond lifetime. In contrast, OLG mutants mediated increased platelet capture on collagen under high shear stress that was associated with increased adhesion of these variants to the collagen under flow. These findings suggest that removal of OLGs increases the flexibility of the hinge linker region between the D3 and A1 domain, facilitating VWF unfolding by shear stress, thereby enhancing its ability to bind collagen and capture platelets. These data demonstrate an important functional role of VWF OLGs under shear stress conditions.

Download Full-text

Identification of FcγRIIa as the ITAM-bearing receptor mediating αIIbβ3 outside-in integrin signaling in human platelets

Blood ◽

10.1182/blood-2008-02-142125 ◽

2008 ◽

Vol 112 (7) ◽

pp. 2780-2786 ◽

Cited By ~ 119

Author(s):

Brian Boylan ◽

Cunji Gao ◽

Vipul Rathore ◽

Joan C. Gill ◽

Debra K. Newman ◽

...

Keyword(s):

Ligand Binding ◽

Tyrosine Phosphorylation ◽

Protein Tyrosine Kinase ◽

Cytoplasmic Domain ◽

Human Platelets ◽

Integrin Signaling ◽

Specific Monoclonal Antibody ◽

Fibrinogen Binding ◽

Platelet Spreading ◽

Activation Motif

AbstractImmunoreceptor tyrosine-based activation motif (ITAM)–containing proteins have recently been demonstrated in macrophages and neutrophils to be required for cell surface integrins to transmit activation signals into the cell. To identify ITAM-bearing proteins that mediate signaling via the platelet-specific integrin αIIbβ3, fibrinogen binding was induced by (1) allowing platelets to spread directly on immobilized fibrinogen, or (2) activating the PAR1 thrombin receptor on platelets in suspension. Both initiated strong, ligand binding–dependent tyrosine phosphorylation of the ITAM-bearing platelet Fc receptor, FcγRIIa, as well as downstream phosphorylation of the protein tyrosine kinase Syk and activation of phospholipase Cγ2 (PLCγ2). Addition of Fab fragments of an FcγRIIa-specific monoclonal antibody strongly inhibited platelet spreading on immobilized fibrinogen, as well as downstream tyrosine phosphorylation of FcγRIIa, Syk, and PLCγ2, and platelets from a patient whose platelets express reduced levels of FcγRIIa exhibited markedly reduced spreading on immobilized fibrinogen. Finally, fibrinogen binding–induced FcγRIIa phosphorylation did not occur in human platelets expressing a truncated β3 cytoplasmic domain. Taken together, these data suggest that ligand binding to platelet αIIbβ3 induces integrin cytoplasmic domain–dependent phosphorylation of FcγRIIa, which then enlists selected components of the immunoreceptor signaling cascade to transmit amplification signals into the cell.

Download Full-text

Ligand specificities of recombinant retinoic acid receptors RAR α and RAR β

Biochemical Journal ◽

10.1042/bj2720391 ◽

1990 ◽

Vol 272 (2) ◽

pp. 391-397 ◽

Cited By ~ 92

Author(s):

M Crettaz ◽

A Baron ◽

G Siegenthaler ◽

W Hunziker

Keyword(s):

Escherichia Coli ◽

Retinoic Acid ◽

Ligand Binding ◽

Binding Sites ◽

Retinoic Acid Receptors ◽

Wild Type ◽

Binding Characteristics ◽

Binding Domains ◽

Ligand Binding Sites ◽

End Group

Binding of retinoic acid (RA) to specific RA receptors alpha and beta (RAR alpha and RAR beta) was studied. Receptors were obtained in two ways: (1) full-length receptors were produced by transient expression of the respective human cDNAs in COS 1 cells; and (2) the ligand-binding domains of RAR alpha and RAR beta were produced in Escherichia coli. RA binding to the wild-type and truncated forms of the receptor was identical for both RAR alpha and RAR beta, indicating that the ligand-binding domains have retained the binding characteristics of the intact receptors. Furthermore, RA bound with the same affinity to both RAR alpha and RAR beta. Only retinoid analogues with an acidic end-group were able to actively bind to both receptors. On measuring the binding of various retinoids, we have found that the properties of the ligand-binding sites of RAR alpha and RAR beta were rather similar. Two retinoid analogues were capable of binding preferentially to either RAR alpha or RAR beta, suggesting that it may be possible to synthesize specific ligands for RAR alpha and RAR beta.

Download Full-text

Targeting the cryptic sites: NMR-based strategy to improve protein druggability by controlling the conformational equilibrium

Science Advances ◽

10.1126/sciadv.abd0480 ◽

2020 ◽

Vol 6 (40) ◽

pp. eabd0480

Author(s):

Yumiko Mizukoshi ◽

Koh Takeuchi ◽

Yuji Tokunaga ◽

Hitomi Matsuo ◽

Misaki Imai ◽

...

Keyword(s):

Ligand Binding ◽

Protein Interactions ◽

Binding Sites ◽

Drug Targets ◽

Conformational Equilibrium ◽

Wild Type ◽

Protein Protein Interactions ◽

Hit Rate ◽

Open Conformation ◽

Ligand Binding Sites

Cryptic ligand binding sites, which are not evident in the unligated structures, are beneficial in tackling with difficult but attractive drug targets, such as protein-protein interactions (PPIs). However, cryptic sites have thus far not been rationally pursued in the early stages of drug development. Here, we demonstrated by nuclear magnetic resonance that the cryptic site in Bcl-xL exists in a conformational equilibrium between the open and closed conformations under the unligated condition. While the fraction of the open conformation in the unligated wild-type Bcl-xL is estimated to be low, F143W mutation that is distal from the ligand binding site can substantially elevate the population. The F143W mutant showed a higher hit rate in a phage-display peptide screening, and the hit peptide bound to the cryptic site of the wild-type Bcl-xL. Therefore, by controlling the conformational equilibrium in the cryptic site, the opportunity to identify a PPI inhibitor could be improved.

Download Full-text

Reciprocal feedback regulation of insulin receptor and insulin receptor substrate tyrosine phosphorylation by phosphoinositide 3-kinase in primary adipocytes

Biochemical Journal ◽

10.1042/bj20020903 ◽

2002 ◽

Vol 368 (3) ◽

pp. 875-884 ◽

Cited By ~ 17

Author(s):

Ingeborg HERS ◽

Christopher J. BELL ◽

Alastair W. POOLE ◽

Donyang JIANG ◽

Richard M. DENTON ◽

...

Keyword(s):

Insulin Receptor ◽

Binding Site ◽

Tyrosine Phosphorylation ◽

Binding Sites ◽

Specific Binding ◽

Sh2 Domain ◽

Insulin Receptor Substrate ◽

Insulin Receptor Tyrosine Kinase ◽

Irs Proteins ◽

Phosphoinositide 3 Kinase

Signalling by the insulin receptor substrate (IRS) proteins is critically dependent on the tyrosine phosphorylation of specific binding sites that recruit Src homology 2 (SH2)-domain-containing proteins, such as the p85 subunit of phosphoinositide 3-kinase (PI 3-kinase), the tyrosine phosphatase SHP-2 and the adapter protein Grb2. Here we show that stimulation by insulin of freshly isolated primary adipocytes resulted in the expected rapid tyrosine phosphorylation of the insulin receptor, IRS-1 and IRS-3. Inhibition of PI 3-kinase enhanced the insulin-stimulated phosphorylation of IRS-1 on (i) Tyr612 and Tyr941 (p85 binding sites), concomitant with an increased association of the p85 subunit of PI 3-kinase; (ii) Tyr896 (a Grb2 binding site); and (iii) Tyr1229 (an SHP-2 binding site), although little or no binding of SHP-2 to IRS-1 was detectable under any conditions. In contrast, inhibition of PI 3-kinase led to a decrease in insulin-stimulated p85 binding to IRS-3, but had no effect on SHP-2 binding. Furthermore, insulin-induced insulin receptor tyrosine phosphorylation, phosphorylation of Tyr1158 and insulin receptor tyrosine kinase activity were all reduced by inhibition of PI 3-kinase at later time points (20min). The results demonstrate that, in primary adipocytes, PI 3-kinase feedback control of signalling by the insulin receptor and IRS proteins is multifaceted and reciprocal, illustrating the complexity of predicting the net flux of the insulin signal(s) through the IRS proteins.

Download Full-text

Platelet Activation and Aggregation Induced by Recombinant von Willebrand Factors Reproducing Four Type 2B von Willebrand Disease Missense Mutations

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614241 ◽

1998 ◽

Vol 79 (01) ◽

pp. 211-216 ◽

Cited By ~ 13

Author(s):

Lysiane Hilbert ◽

Claudine Mazurier ◽

Christophe de Romeuf

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Von Willebrand Disease ◽

Platelet Glycoprotein ◽

Missense Mutations ◽

Inhibit Platelet Aggregation ◽

Wild Type ◽

A1 Domain ◽

Von Willebrand ◽

Willebrand Factor

SummaryType 2B of von Willebrand disease (vWD) refers to qualitative variants with increased affinity of von Willebrand factor (vWF) for platelet glycoprotein Ib (GPIb). All the mutations responsible for type 2B vWD have been located in the A1 domain of vWF. In this study, various recombinant von Willebrand factors (rvWF) reproducing four type 2B vWD missense mutations were compared to wild-type rvWF (WT-rvWF) for their spontaneous binding to platelets and their capacity to induce platelet activation and aggregation. Our data show that the multimeric pattern of each mutated rvWF is similar to that of WT-rvWF but the extent of spontaneous binding and the capacity to induce platelet activation and aggregation are more important for the R543Q and V553M mutations than for the L697V and A698V mutations. Both the binding of mutated rvWFs to platelets and platelet aggregation induced by type 2B rvWFs are inhibited by monoclonal anti-GPIb and anti-vWF antibodies, inhibitors of vWF binding to platelets in the presence of ristocetin, as well as by aurin tricarboxylic acid. On the other hand, EDTA and a monoclonal antibody directed against GPIIb/IIIa only inhibit platelet aggregation. Furthermore, the incubation of type 2B rvWFs with platelets, under stirring conditions, results in the decrease in high molecular weight vWF multimers in solution, the extent of which appears correlated with that of plasma vWF from type 2B vWD patients harboring the corresponding missense mutation. This study supports that the binding of different mutated type 2B vWFs onto platelet GPIb induces various degrees of platelet activation and aggregation and thus suggests that the phenotypic heterogeneity of type 2B vWD may be related to the nature and/or location of the causative point mutation.

Download Full-text

Reversibly Sampling Conformations and Binding Modes Using Molecular Darting

10.26434/chemrxiv.12670676.v2 ◽

2020 ◽

Author(s):

Samuel C. Gill ◽

David Mobley

Keyword(s):

Monte Carlo ◽

Ligand Binding ◽

Binding Sites ◽

Degrees Of Freedom ◽

Dynamics Simulation ◽

Hiv Integrase ◽

Binding Modes ◽

System A ◽

Multiple Binding ◽

Internal Degrees Of Freedom

<div>Sampling multiple binding modes of a ligand in a single molecular dynamics simulation is difficult. A given ligand may have many internal degrees of freedom, along with many different ways it might orient itself a binding site or across several binding sites, all of which might be separated by large energy barriers. We have developed a novel Monte Carlo move called Molecular Darting (MolDarting) to reversibly sample between predefined binding modes of a ligand. Here, we couple this with nonequilibrium candidate Monte Carlo (NCMC) to improve acceptance of moves.</div><div>We apply this technique to a simple dipeptide system, a ligand binding to T4 Lysozyme L99A, and ligand binding to HIV integrase in order to test this new method. We observe significant increases in acceptance compared to uniformly sampling the internal, and rotational/translational degrees of freedom in these systems.</div>

Download Full-text