Soluble Fibrinogen Binding Research Articles

Opposing Roles for the α Isoform of the Catalytic Subunit of Protein Phosphatase 1 in Inside-Out and Outside-In Integrin Signaling in Murine Platelets.

Platelet activation during hemostasis and thrombosis is facilitated by agonist-induced inside-out and integrin αIIbβ3-initiated outside-in signaling via protein kinases and phosphatases. Pharmacological inhibitor studies suggest that the serine/threonine protein phosphatase 1 (PP1) promotes platelet activation. However, since phosphatase inhibitors block all the isoforms of the catalytic subunit of PP1 (PP1c), the role of specific PP1c isoform in platelet signaling remains unclear. Here, we employed a platelet-specific PP1cα-/- mice to explore the contribution of a major PP1 isoform in platelet functions. Loss of PP1cα moderately decreased activation of integrin αIIbβ3, binding of soluble fibrinogen, and aggregation to low-dose thrombin, ADP, and collagen. In contrast, PP1cα-/- platelets displayed increased adhesion to immobilized fibrinogen, fibrin clot retraction, and thrombus formation on immobilized collagen. Mechanistically, post-fibrinogen engagement potentiated p38 mitogen-activated protein kinase (MAPK) activation in PP1cα-/- platelets and the p38 inhibitor blocked the increased integrin-mediated outside-in signaling function. Tail bleeding time and light-dye injury-induced microvascular thrombosis in the cremaster venules and arterioles were not altered in PP1cα-/- mice. Thus, PP1cα displays pleiotropic signaling in platelets as it amplifies agonist-induced signaling and attenuates integrin-mediated signaling with no impact on hemostasis and thrombosis.

Platelet SHARPIN regulates platelet adhesion and inflammatory responses through associations with αIIbβ3 and LUBAC

AbstractPlatelets form hemostatic plugs to prevent blood loss, and they modulate immunity and inflammation in several ways. A key event during hemostasis is activation of integrin αIIbβ3 through direct interactions of the β3 cytoplasmic tail with talin and kindlin-3. Recently, we showed that human platelets express the adapter molecule Shank-associated RH domain interacting protein (SHARPIN), which can associate directly with the αIIb cytoplasmic tail and separately promote NF-κB pathway activation as a member of the Met-1 linear ubiquitination activation complex (LUBAC). Here we investigated the role of SHARPIN in platelets after crossing Sharpin flox/flox (fl/fl) mice with PF4-Cre or GPIbα-Cre mice to selectively delete SHARPIN in platelets. SHARPIN-null platelets adhered to immobilized fibrinogen through αIIbβ3, and they spread more extensively than littermate control platelets in a manner dependent on feedback stimulation by platelet adenosine diphosphate (ADP) (P < .01). SHARPIN-null platelets showed increased colocalization of αIIbβ3 with talin as assessed by super-resolution microscopy and increased binding of soluble fibrinogen in response to submaximal concentrations of ADP (P < .05). However, mice with SHARPIN-null platelets showed compromised thrombus growth on collagen and slightly prolonged tail bleeding times. Platelets lacking SHARPIN also showed reduced NF-κB activation and linear ubiquitination of protein substrates upon challenge with classic platelet agonists. Furthermore, the loss of platelet SHARPIN resulted in significant reduction in inflammation in murine models of colitis and peritonitis (P < .01). Thus, SHARPIN plays differential and context-dependent roles in platelets to regulate important inflammatory and integrin adhesive functions of these anucleate cells.

Paxillin binding to the PH domain of kindlin‐3 in platelets is required to support integrin αIIbβ3 outside‐in signaling

BackgroundKindlin‐3 is essential for supporting the bidirectional signaling of integrin αIIbβ3 in platelets by bridging the crosstalk between integrin αIIbβ3 and the cytoplasmic signaling adaptors. ObjectiveIn this study, we identified a previously unrecognized paxillin binding site in the pleckstrin homology (PH) domain of kindlin‐3 and verified its functional significance. MethodsStructure‐based approaches were employed to identify the paxillin binding site in the PH domain of kindlin‐3. In addition, the bidirectional signaling of integrin αIIbβ3 were evaluated in both human and mouse platelets. ResultsIn brief, we found that a β1‐β2 loop in the PH domain of kindlin‐3, an important part of the canonical membrane phospholipid binding pocket, was also involved in mediating paxillin interaction. Interestingly, the binding sites of paxillin and membrane phospholipids in the PH domain of kindlin‐3 were mutually exclusive. Specific disruption of paxillin binding to the PH domain by point mutations inhibited platelet spreading on immobilized fibrinogen while having no inhibition on soluble fibrinogen binding to stimulated platelets. In addition, a membrane‐permeable peptide derived from the β1‐β2 loop in the PH domain of kindlin‐3 was capable of inhibiting platelet spreading and clot retraction, but it had no effect on soluble fibrinogen binding to platelets and platelet aggregation. Treatment with this peptide significantly reduced thrombus formation in mice. ConclusionTaken together, these findings suggest that interaction between paxillin and the PH domain of kindlin‐3 plays an important role in supporting integrin αIIbβ3 outside‐in signaling in platelets, thus providing a novel antithrombotic target.

Agonist-Induced Signaling and Integrin-Mediated Function in Murine Platelets Is Distinctly Regulated By the α Isoform of the Catalytic Subunit of Protein Phosphatase 1

Platelet activation at the site of injury is promoted by agonist-induced inside-out and integrin αIIbβ3-initiated outside-in signaling. Our understanding of the agonist and integrin mediated signaling pathways has largely been driven by studies that have examined the role of protein kinases. As models of signal transduction have been refined to include protein phosphorylation and protein dephosphorylation events, the contribution of the protein phosphatases to platelet signaling and function have garnered fresh attention. This is particularly evident for the catalytic subunit of serine/threonine protein phosphatase 1 (PP1c), wherein, its role in platelet biology has been previously assessed with pharmacological agents like calyculin A and okadaic acid. Although such chemical inhibitors represent a useful tool, it lacks specificity to discriminate the different isoforms of PP1c. PP1cα represents a major platelet expressed isoform and its contribution to platelet function is not known. To explore the potential role of PP1cα isoform in platelet signaling, we generated a platelet specific PP1cα -/- mice by crossing the PP1cα floxed mice with a platelet factor 4 (PF4) cre mice. Immunoblotting studies from PP1cα -/- mice confirmed that PP1cα was lost in platelets but not in lymphocytes. A compensatory upregulation of the PP1cβ but not PP1cγ1 isoform was noticed in PP1cα -/- platelets. Loss of PP1cα decreased platelet aggregation and soluble fibrinogen binding to low dose thrombin, thromboxane analog and convulxin, suggesting that PP1cα promotes agonist-induced inside-out signaling. In contrast, PP1cα -/- platelets displayed enhanced adhesiveness to immobilized fibrinogen and increased fibrin clot retraction, processes that are dependent on integrin-mediated outside-in signaling. In a microfluidic perfusion system, whole blood from PP1cα -/- mice displayed increased thrombus formation on collagen. p38 mitogen activated protein kinase (MAPK) was activated in PP1cα -/- platelets, and p38 inhibitor blocked the increased integrin-mediated PP1cα -/- platelet functions. These observations suggest that PP1cα suppress integrin-mediated function in part by decreasing the activation of p38 MAPK. Thus, unlike the conclusion gathered from generic phosphatase inhibitors that PP1c promotes platelet function, our studies using a genetic approach indicate that PP1cα contributes to platelet signaling in a fashion that is specific to the mode of signaling. Specifically, PP1cα promotes platelet agonist-induced inside-out signaling and suppress integrin-mediated outside-in signaling. This observation suggests that chemical activators of PP1cα isoform could serve as a potential antithrombotic agent. DisclosuresNo relevant conflicts of interest to declare.

Conformation-Specific Blockade of αIIbβ3 by a Non-RGD Peptide to Inhibit Platelet Activation without Causing Significant Bleeding and Thrombocytopenia.

Bleeding and thrombocytopenia to readministration are the most serious side effects of clinical integrin αIIbβ3 antagonists such as RGD-containing peptides. Here we show that a non-RGD peptide ZDPI, identified from skin secretions of Amolops loloensis, inhibited platelet aggregation induced by agonists, such as adenosine diphosphate, collagen, arachidonic acid, PAR1AP, and integrin αIIbβ3 allosteric activator, and reduces soluble fibrinogen binding to activated platelets without perturbing adhesion numbers on immobilized fibrinogen. Further study showed that ZDPI preferred to bind to the active conformation of integrin αIIbβ3, and thus inhibited c-Src-mediated integrin signaling transduction. In contrast to currently used clinical blockers of integrin αIIbβ3, which are all conformation-unspecific blockers, ZDPI conformation specifically binds to activated integrin αIIbβ3, subsequently suppressing platelet spreading. In vivo study revealed that ZDPI inhibited carotid arterial thrombosis with limited bleeding and thrombocytopenia. A non-RGD peptide which targets the active conformation of integrin αIIbβ3, such as ZDPI, might be an excellent candidate or template to develop antithrombotics without significant bleeding and thrombocytopenia side effects.

Tannic Acid Inhibits Protein Disulfide Isomerase, Platelet Activation and Thrombus Formation

Tannic acid (TA) was a polyphenol that harbors anti-oxidant capacity. A recent report implied that surface coating with TA might blunt thrombosis via altering the structure of fibrinogen. However, the effect of TA on platelet function and in vivo thrombus formation has not been reported. In this study, we showed that TA inhibits PDI activity and attenuates platelet activation.To explore the effects of TA on platelet aggregation, gel-filtered human platelets from healthy human donors were pretreated with TA (10/30/50 μM) or vehicle (0.9% sodium chloride) before being stimulated by various agonists. Turbidity analyses on a Chronolog aggregometer showed that TA dose-dependently inhibited platelet aggregation induced by thrombin, SFLLRN, GYQGQV, collagen, CRP, U46619, and ristocetin. Next, we employed flow cytometry (FACS) to determine the role of TA in platelet activation, including α-granule secretion and integrin activation. Pretreatment of platelets with TA led to significant reductions in surface P-selectin expression and soluble fibrinogen binding, supporting the inhibition of diverse platelet activation pathways. Supportively, platelet spreading on immobilized fibrinogen was significantly suppressed by TA treatment. In addition, cell viability assay with Almar blue agent showed no detrimental impact of TA on the survival of platelets.To ask whether the antiplatelet role of TA might be translated into an antithrombotic efficacy, we tested the effect of TA in both ex vivo and in vivo thrombosis models. Calcein-labeled human whole blood was perfused through microfluidic channels coated with collagen, and adherent platelets were visualized under a fluorescent microscopy. However, treatment with TA suppressed the number of adherent platelets under flow conditions. Moreover, in laser-induced mouse cremaster muscle arteries, administration of TA (5mg/kg) significantly reduced the size of forming thrombi compared with the vehicle. Verification of bleeding risk using tail truncation assay indicated no prolongation of bleeding time in mice receiving TA. Thus, TA shows an antiplatelet effect and may also attenuate thrombus formation.To gain a mechanistic insight to the role of TA in platelet function, we performed a molecular docking screen of the structure of TA and platelet surface proteins using the Autodock Vina software, which displayed the binding of TA with protein disulfide isomerase at the enzymatic active center. We then measured the impact of TA on PDI reductase activity with the dieosin glutathione disulfide assay in vitro (di-GSSG), showing that TA significantly inhibited PDI activity in a concentration-dependent manner. The results were verified in platelets using the 3-(N-Maleimidylpropionyl) biocytin (MPB) labeling, which showed that TA abrogated thrombin-stimulated free thiol formation on platelet surface. Supportively, FACS demonstrated that TA significantly suppressed the binding of fluorescent-labeled PDI to Mn2+-activated platelet integrin β3. Taken together, our findings demonstrated that TA inhibits PDI activity and may become a novel antithrombotic agent. DisclosuresNo relevant conflicts of interest to declare.

The heterotrimeric G protein Gβ1 interacts with the catalytic subunit of protein phosphatase 1 and modulates G protein–coupled receptor signaling in platelets

Thrombosis is caused by the activation of platelets at the site of ruptured atherosclerotic plaques. This activation involves engagement of G protein-coupled receptors (GPCR) on platelets that promote their aggregation. Although it is known that protein kinases and phosphatases modulate GPCR signaling, how serine/threonine phosphatases integrate with G protein signaling pathways is less understood. Because the subcellular localization and substrate specificity of the catalytic subunit of protein phosphatase 1 (PP1c) is dictated by PP1c-interacting proteins, here we sought to identify new PP1c interactors. GPCRs signal via the canonical heterotrimeric Gα and Gβγ subunits. Using a yeast two-hybrid screen, we discovered an interaction between PP1cα and the heterotrimeric G protein Gβ1 subunit. Co-immunoprecipitation studies with epitope-tagged PP1c and Gβ1 revealed that Gβ1 interacts with the PP1c α, β, and γ1 isoforms. Purified PP1c bound to recombinant Gβ1-GST protein, and PP1c co-immunoprecipitated with Gβ1 in unstimulated platelets. Thrombin stimulation of platelets induced the dissociation of the PP1c-Gβ1 complex, which correlated with an association of PP1c with phospholipase C β3 (PLCβ3), along with a concomitant dephosphorylation of the inhibitory Ser1105 residue in PLCβ3. siRNA-mediated depletion of GNB1 (encoding Gβ1) in murine megakaryocytes reduced protease-activated receptor 4, activating peptide-induced soluble fibrinogen binding. Thrombin-induced aggregation was decreased in PP1cα-/- murine platelets and in human platelets treated with a small-molecule inhibitor of Gβγ. Finally, disruption of PP1c-Gβ1 complexes with myristoylated Gβ1 peptides containing the PP1c binding site moderately decreased thrombin-induced human platelet aggregation. These findings suggest that Gβ1 protein enlists PP1c to modulate GPCR signaling in platelets.

Mechanistic Basis for the Binding of RGD- and AGDV-Peptides to the Platelet Integrin αIIbβ3.

Binding of soluble fibrinogen to the activated conformation of the integrin αIIbβ3 is required for platelet aggregation and is mediated exclusively by the C-terminal AGDV-containing dodecapeptide (γC-12) sequence of the fibrinogen γ chain. However, peptides containing the Arg-Gly-Asp (RGD) sequences located in two places in the fibrinogen Aα chain inhibit soluble fibrinogen binding to αIIbβ3 and make substantial contributions to αIIbβ3 binding when fibrinogen is immobilized and when it is converted to fibrin. Here, we employed optical trap-based nanomechanical measurements and computational molecular modeling to determine the kinetics, energetics, and structural details of cyclic RGDFK (cRGDFK) and γC-12 binding to αIIbβ3. Docking analysis revealed that NMR-determined solution structures of cRGDFK and γC-12 bind to both the open and closed αIIbβ3 conformers at the interface between the αIIb β-propeller domain and the β3 βI domain. The nanomechanical measurements revealed that cRGDFK binds to αIIbβ3 at least as tightly as γC-12. A subsequent analysis of molecular force profiles and the number of peptide-αIIbβ3 binding contacts revealed that both peptides form stable bimolecular complexes with αIIbβ3 that dissociate in the 60-120 pN range. The Gibbs free energy profiles of the αIIbβ3-peptide complexes revealed that the overall stability of the αIIbβ3-cRGDFK complex was comparable with that of the αIIbβ3-γC-12 complex. Thus, these results provide a mechanistic explanation for previous observations that RGD- and AGDV-containing peptides are both potent inhibitors of the αIIbβ3-fibrinogen interactions and are consistent with the observation that RGD motifs, in addition to AGDV, support interaction of αIIbβ3 with immobilized fibrinogen and fibrin.

Characterization of the Interactions of Arg-Gly-Asp- and Ala-Gly-Asp-Val-Containing Peptides with the Platelet Integrin αIIbβ3

Binding of soluble fibrinogen to the activated open conformation of the integrin αIIbβ3 is required for platelet aggregation and is mediated exclusively by the C-terminal AGDV-containing dodecapeptide (γC-12) sequence of the fibrinogen γ chain. Paradoxically, however, peptides containing the Arg-Gly-Asp (RGD) sequences located in two places in the fibrinogen Aα chain inhibit soluble fibrinogen binding to αIIbβ3. Moreover, the Aα chain RGD motifs make substantial contributions to αIIbβ3 binding when fibrinogen is immobilized and when it is converted to fibrin. The interaction of αIIbβ3 with a variety of RGD- and γC-12 peptides has been studied extensively, but the characteristics of their binding to the active open and inactive closed conformations of αIIbβ3 are largely unknown. Here, we have used both experimental and computational approaches to compare the two-dimensional kinetics, thermodynamics and structural details of cyclic RGDFK (cRGDFK) and γC-12 binding to αIIbβ3. First, we employed optical trap-based single-molecule nanomechanical measurements to determine the probability of peptide binding to αIIbβ3 as a function of the time the peptides interact with αIIbβ3. In the optical trap-based experimental system, a microscopic bead coated with either cRGDFK or γC-12 is trapped in a fluid chamber by a focused laser beam and moved in an oscillatory manner to touch a stationary pedestal coated with αIIbβ3. When the peptide on the bead interacts with αIIbβ3 on the pedestal, tension is generated when the bead is displaced from the laser focus until the αIIbβ3-peptide bond ruptures. The percentage of binding/unbinding events at a particular time of contact duration enabled us to plot the force-free binding probability as a function of contact duration. From these curves, we extracted first-order binding rates: 0.61×10-14 cm2/s for γC-12 and 0.71×10-14 cm2/s for cRGDFK and unbinding rates: 2.05/s and 1.53/s for the γC-12 and cRGDFK, respectively. Corresponding binding affinity constants were 0.3×10-14 cm2 and 0.46×10-14 cm2 for the γC-12 and cRGDFK, respectively, indicating that cRGDFK binds to αIIbβ3 somewhat tighter than γC-12. These measurements were then followed by computational modeling using NMR-determined 3D solution structures of cRGDFK and γC-12, as well as the crystallographically resolved αIIbβ3 conformers of the αIIbβ3 headpiece (PDB entries: 3ZDX and 3ZE2). Docking analysis confirmed that both cRGDFK and γC-12 bound to the αIIbβ3 conformers at a site located in the interface between the α subunit β-propeller domain and the β subunit βI domain. Estimated docking energies revealed that the closed αIIbβ3 conformation is almost as reactive towards the peptides as the open conformation. Next, we probed the strength of the αIIbβ3-peptide interactions and determined their comparative binding energies and the dynamics of interatomic contacts by performing dynamic force-ramp unbinding in silico using all-atom Molecular Dynamics simulations in implicit solvent on Graphics Processing Units. Force was increased linearly at a pulling rate of 2×104 μm/s and applied to specified Cα-atom of the peptides. Analysis of the molecular force profiles and the number of ligand-receptor binding contacts revealed that both peptides interact strongly with αIIbβ3, forming stable bimolecular complexes that dissociate in the 60-120 pN range through several competing unbinding pathways that display multi-step kinetics and distinct bond lifetimes. We profiled the Gibbs free energy (ΔG) of the αIIbβ3-peptide complexes as a function of the separation distance between the open form of αIIbβ3 headpiece and the peptide using the Umbrella Sampling technique. The results showed that the energies of ligand binding to the αIIbβ3 headpiece are ΔG~16 kcal/mol for cRGDFK and ΔG~13 kcal/mol for γC-12, meaning that the overall thermodynamic stability of the αIIbβ3-peptide complex is higher for cRGDFK than for γC-12. Thus, these results provide a kinetic and thermodynamic explanation for previous observations that the affinity of RGD for αIIbβ3 is greater than AGDV and support our hypothesis that the RGD motifs preferentially support the interaction of αIIbβ3 with immobilized fibrinogen and fibrin. DisclosuresWeisel:Bayer: Research Funding.

Functional Effect of the Mutations Similar to the Cleavage during Platelet Activation at Integrin β3 Cytoplasmic Tail when Expressed in Mouse Platelets.

Previous studies in Chinese hamster ovary cells showed that truncational mutations of β3 at sites of F754 and Y759 mimicking calpain cleavage regulate integrin signaling. The roles of the sequence from F754 to C-terminus and the conservative N756ITY759 motif in platelet function have yet to be elaborated. Mice expressing β3 with F754 and Y759 truncations, or NITY deletion (β3-ΔTNITYRGT, β3-ΔRGT, or β3-ΔNITY) were established through transplanting the homozygous β3-deficient mouse bone marrow cells infected by the GFP tagged MSCV MigR1 retroviral vector encoding different β3 mutants into lethally radiated wild-type mice. The platelets were harvested for soluble fibrinogen binding and platelet spreading on immobilized fibrinogen. Platelet adhesion on fibrinogen- and collagen-coated surface under flow was also tested to assess the ability of the platelets to resist hydrodynamic drag forces. Data showed a drastic inhibition of the β3-ΔTNITYRGT platelets to bind soluble fibrinogen and spread on immobilized fibrinogen in contrast to a partially impaired fibrinogen binding and an almost unaffected spreading exhibited in the β3-ΔNITY platelets. Behaviors of the β3-ΔRGT platelets were consistent with the previous observations in the β3-ΔRGT knock-in platelets. The adhesion impairment of platelets with the β3 mutants under flow was in different orders of magnitude shown as: β3-ΔTNITYRGT>β3-ΔRGT>β3-ΔNITY to fibrinogen-coated surface, and β3-ΔTNITYRGT>β3-ΔNITY>β3-ΔRGT to collagen-coated surface. To evaluate the interaction of the β3 mutants with signaling molecules, GST pull-down and immunofluorescent assays were performed. Results showed that β3-ΔRGT interacted with kindlin but not c-Src, β3-ΔNITY interacted with c-Src but not kindlin, while β3-ΔTNITYRGT did not interact with both proteins. This study provided evidence in platelets at both static and flow conditions that the calpain cleavage-related sequences of integrin β3, i.e. T755NITYRGT762, R760GT762, and N756ITY759 participate in bidirectional, outside-in, and inside-out signaling, respectively and the association of c-Src or kindlin with β3 integrin may regulate these processes.

Roles of integrin β3 cytoplasmic tail in bidirectional signal transduction in a trans-dominant inhibition model

We evaluated the roles of calpain cleavage-related mutations of the integrin β3 cytoplasmic tail in integrin αIIbβ3 bidirectional signaling using a trans-dominant inhibition model. Chimeric Tac-β3 proteins (i.e., Tac-β3, Tac-β3Δ741, Tac-β3Δ747, Tac-β3Δ754, Tac-β3Δ759, and Tac-β3ΔNITY) consisting of the extracellular and transmembrane domains of human IL-2 receptor (Tac) and the human integrin β3 cytoplasmic domain were stably expressed in the 123 CHO cells harboring human glycoprotein Ib-IX and wild-type integrin αIIbβ3. The different cells were assayed for stable adhesion and spreading on immobilized fibrinogen, and for binding soluble fibrinogen representing outside-in and inside-out signaling events, respectively. The chimeric protein Tac-β3 inhibited, and Tac-β3ΔNITY partially attenuated stable adhesion and spreading. Tac-β3, Tac-β3Δ759, Tac-β3ΔNITY, and Tac-β3Δ754, but not Tac-β3Δ747 or Tac-β3Δ741, impaired the soluble fibrinogen binding. Results indicated that the bidirectional signaling was significantly inhibited by Tac-β3 and Tac-β3ΔNITY, albeit to a much lesser extent. Moreover, only inside-out signaling was impaired in the 123/Tac-β3Δ759 and 123/Tac-β3Δ754 cells in contrast to an intact bidirectional signaling in the 123/Tac-β3Δ747 and 123/Tac-β3Δ741 cells. In conclusion, the calpain cleavage of integrin β3 resulted in the regulatory effects on signaling by interrupting its interaction with cytoplasmic proteins rather than altering its conformation, and may thus regulate platelet function.

Dok-1 negatively regulates platelet integrin αIIbβ3 outside-in signalling and inhibits thrombosis in mice.

Adaptor proteins play a critical role in the assembly of signalling complexes after engagement of platelet receptors by agonists such as collagen, ADP and thrombin. Recently, using proteomics, the Dok (downstream of tyrosine kinase) adapter proteins were identified in human and mouse platelets. In vitro studies suggest that Dok-1 binds to platelet integrin β3, but the underlying effects of Dok-1 on αIIbβ3 signalling, platelet activation and thrombosis remain to be elucidated. In the present study, using Dok-1-deficient (Dok-1-/-) mice, we determined the phenotypic role of Dok-1 in αIIbβ3 signalling. We found that platelets from Dok-1-/- mice displayed normal aggregation, activation of αIIbβ3 (assessed by binding of JON/A), P-selectin surface expression (assessed by anti-CD62P), and soluble fibrinogen binding. These findings indicate that Dok-1 does not affect "inside-out" platelet signalling. Compared with platelets from wild-type (WT) mice, platelets from Dok-1-/- mice exhibited increased clot retraction (p < 0.05 vs WT), increased PLCγ2 phosphorylation, and enhanced spreading on fibrinogen after thrombin stimulation (p < 0.01 vs WT), demonstrating that Dok-1 negatively regulates αIIbβ3 "outside-in" signalling. Finally, we found that Dok-1-/- mice exhibited significantly shortened bleeding times and accelerated carotid artery thrombosis in response to photochemical injury (p < 0.05 vs WT mice). We conclude that Dok-1 modulates thrombosis and haemostasis by negatively regulating αIIbβ3 outside-in signalling.

Myr-RKEFAK Peptide Selectively Regulates Outside-in Signaling Transduction-related Functions in Human Platelets

To study the effect of interaction of the talin rod domain integrin binding site 2 with integrin β3 on platelet signal transduction. A peptide that mimics the membrane proximal α helix 6 residues R724 KEFAK729 of the integrin β3 cytoplasmic tails was designed and synthesized, to which the myristoylation was covalently linked to the N-terminal of the peptide enabling membrane penetration. The effects of myr-RKEFAK peptide on the typical platelet outside-in signaling ovent (stable adhesion and spreading on immobilized fibrinogen, aggregation, fibrin clot retraction) and inside-out signaling events (soluble fibrinogen binding) were tested. myr-RKEFAK peptide dose-dependently inhibited platelet stable adhesion and spreading on immobilized fibrinogen, irreversible aggregation, as well as fibrin clot retraction, but not soluble fibrinogen binding and reversible phase of platelet aggregation. The cell-penetrating peptide myr-RKEFAK causes an inhibitory effect on integrin β3 outside-in signaling-regulated platelets functions, but did not affect inside-out signaling-regulated platelets functions.

The Interaction of Integrin αIIbβ3 with Fibrin Occurs through Multiple Binding Sites in the αIIb β-Propeller Domain

The currently available antithrombotic agents target the interaction of platelet integrin αIIbβ3 (GPIIb-IIIa) with fibrinogen during platelet aggregation. Platelets also bind fibrin formed early during thrombus growth. It was proposed that inhibition of platelet-fibrin interactions may be a necessary and important property of αIIbβ3 antagonists; however, the mechanisms by which αIIbβ3 binds fibrin are uncertain. We have previously identified the γ370-381 sequence (P3) in the γC domain of fibrinogen as the fibrin-specific binding site for αIIbβ3 involved in platelet adhesion and platelet-mediated fibrin clot retraction. In the present study, we have demonstrated that P3 can bind to several discontinuous segments within the αIIb β-propeller domain of αIIbβ3 enriched with negatively charged and aromatic residues. By screening peptide libraries spanning the sequence of the αIIb β-propeller, several sequences were identified as candidate contact sites for P3. Synthetic peptides duplicating these segments inhibited platelet adhesion and clot retraction but not platelet aggregation, supporting the role of these regions in fibrin recognition. Mutant αIIbβ3 receptors in which residues identified as critical for P3 binding were substituted for homologous residues in the I-less integrin αMβ2 exhibited reduced cell adhesion and clot retraction. These residues are different from those that are involved in the coordination of the fibrinogen γ404-411 sequence and from auxiliary sites implicated in binding of soluble fibrinogen. These results map the binding of fibrin to multiple sites in the αIIb β-propeller and further indicate that recognition specificity of αIIbβ3 for fibrin differs from that for soluble fibrinogen.

Agonist-Induced Kindlin-3 Phsphorylation Regulates αIIbβ3 Integrin Activation In HEL Cells and Platelets

The contributions of integrins to platelet responses depend upon the dynamic regulation of their activation status, which in turn depends on engagement of binding partners by their cytoplasmic tails. It is well-established that not only talin but also kindlin family members are essential for integrin activation, and both must present for optimal integrin function. Recent studies in humans have specifically emphasized the vital role of kindlin-3 in integrin functions in hematopoietic cells, including platelets, where kindlin-3 deficiency can lead to episodic bleeding, frequent infections and osteopetrosis, consequences of an inability to activate β1, β2 and β3 integrins. Despite this evidence, little is known about kindlin-3 structure-function relationship. Here, we used human platelets and human erythroleukemic HEL cell line that expresses integrin αIIbβ3 to investigate whether posttranslational modification(s) of kindlin-3 occurs and can influence its integrin activity. Non-stimulated HEL cells are suspension cells, and they do not adhere to fibrinogen or bind soluble fibrinogen and PAC-1 antibody (specific for activated αIIbβ3) readily. Thrombopoietin or PMA stimulation activated αIIbβ3 such that the cells adhered and spread on fibrinogen and increased their binding of PAC-1 and soluble fibrinogen. β3 integrin and kindlin-3 colocalized in focal adhesions in the adherent cells, and there was enhanced β3 integrin-kindlin-3 association as detected by coimmunoprecipitation. Kindlin-3 knockdown impaired agonist-stimulated adhesion and spreading on fibrinogen. Since, as we have shown previously, β3 integrin phosphorylation regulates kindlin and integrin interaction, we sought to determine whether kindlin-3 is also phosphorylated. Human platelets were stimulated with thrombin and HEL cells with PMA, and kindlin-3 was immunoprecipitated from lysates of control and stimulated cells. A kindlin-3 peptide showing significant increase in phosphorylation upon agonist stimulation was identified in both platelets and HEL cells by mass spectrometry. T482 or S484 were identified as phosphorylation sites in sequence that resides in the kindlin-3 variable region, which is not present either in kindlin-1 or kindlin-2 but is conserved across all species in which kindlin-3 has been sequenced. When expressed in HEL cells, TS/AA kindlin-3 mutant displayed decreased soluble fibrinogen binding and cell spreading on immobilized fibrinogen when compared to wild-type kindlin-3. Membrane-permeable, poly-arginine tagged kindlin-3 peptide containing the candidate phosphorylation sites kindlin-3 was introduced into HEL cells and platelets. HEL cell adhesion and spreading was blunted by the kindlin-3 peptide when compared to a scramble poly-arginine control peptide. Moreover, thrombin-induced platelet aggregation was inhibited by kindlin-3 peptide but not by the scramble peptide. Thus, our data emphasizes a role of previously unknown, agonist-induced kindlin-3 phosphorylation, in integrin αIIbβ3 activation in HEL cells and platelets and provides a basis for functional differences between kindlin-3 and its other two paralogs, kindlin-1 and kindlin-2. Disclosures:No relevant conflicts of interest to declare.

Gβ1 a Component Of The Heterotrimeric G Protein Is a New Protein Phosphatase 1c Interacting Protein That Regulates Platelet Activation

Platelet activation at the site of injury is tied to signal transduction events that are mediated by protein kinases and phosphatases. Reversible tyrosine, serine/threonine (Ser/Thr) phosphorylation-dependent assembly and/or disassembly of effector (cytoskeletal, signaling and adaptor) protein complexes propagate signaling downstream of G protein coupled receptors (GPCRs). Compared to kinases, the contribution of Ser/Thr phosphatases and its effectors in GPCR signaling studies is not well explored. Our previous studies had revealed that the catalytic subunit of protein phosphatase 1γ (PP1cγ) support GPCR signaling and thrombus formation. Since cell signaling networks are dependent on protein-protein interactions, we sought to identify the potential effectors of PP1cγ. We employed yeast two-hybrid interaction studies with the full length PP1cγ fused to GAL4 activating domain as bait and screened human bone marrow library. A novel interaction of PP1cγ with a protein called Gβ1 (GNB1) was identified. Gβ1 is a component of the heterotrimeric G proteins like the Gα and couple to GPCR. However, unlike Gα subunits, Gβ1 is unexplored in platelets. Co-immunoprecipitation (co-IP) studies validated PP1cγ-Gβ1 interaction in 293 cells expressing PP1cγ-HA and Gβ1-FLAG. Importantly, Gβ1 interacted with all the PP1c isoforms, suggesting that Gβ1 could target all PP1c isoforms to the GPCR complex. Purified PP1c bound to recombinant Gβ1-GST protein but not to GST protein, indicating that the in vitro interaction of PP1c with Gβ1 was direct and independent of Gα and Gγ subunits. A small molecule inhibitor of G protein βγ, gallein decreased thrombin-induced human platelet aggregation and adhesion to immobilized fibrinogen. There is a paucity of Gβ1-/- platelets because Gβ1-/- mice die within 2 days of birth due to microencephaly. siRNA mediated depletion of Gβ1 in murine megakaryocytes reduced PAR4-activating peptide induced soluble fibrinogen binding to αIIbβ3. These studies suggest a functional role for Gβ1 in GPCR signaling. PP1c co-immunoprecipitated with Gβ1 in resting platelets and agonist (thrombin and ADP) treatment under non-stirring conditions induced dissociation of PP1c from Gβ1. These studies indicate that Gβ1-PP1c complex in platelets is responsive to agonist. Furthermore, PP1c and Gβ1 associated with P2Y12 receptor in resting but not agonist activated platelets in a co-IP assay, suggesting a role for this complex in G protein signaling. Finally, agonist induced dissociation of PP1c from Gβ1 correlated with the association of PP1c with the downstream GPCR effector phospholipase C β3 (PLCβ3) with a concomitant dephosphorylation of PLCβ3 at Ser1105. Since previous studies have revealed that PLCβ3 activity is inhibited by Ser1105 phosphorylation, our observation suggest that agonist-induced association of PP1c with PLCβ3 facilitates dephosphorylation and activation of PLCβ3. These studies highlight a coupling of GPCR signaling with the phosphatase driven signal transduction during platelet activation. Disclosures:No relevant conflicts of interest to declare.

Abstract 27: Dok-1 Negatively Regulates Integrin aIIbß3 Outside-in Signaling and Inhibits Thrombosis

Background Platelet agonists activate integrin αIIbβ3 to allow the binding of soluble fibrinogen (a process known as inside-out signaling). Subsequent platelet aggregation leads to αIIbβ3 outside-in signaling, which results in tyrosine phosphorylation of β3 and other proteins, cytoskeletal reorganization, and platelet spreading. It has been reported that the adapter protein Dok-1 binds to the cytoplasmic tail of β3 and inhibits αIIbβ3 activation, but the specific role of Dok-1 in regulating inside-out or outside-in platelet signaling remains undefined. Methods We assessed the effects of Dok-1 on platelet signaling and thrombosis in Dok-1 null (Dok-1-/-) mice. Inside-out signaling was assessed by measuring αIIbβ3 activation (using the JON/A antibody) and fibrinogen binding by flow cytometry after stimulation of platelets with thrombin, ADP, and/or the thromboxane A2 receptor agonist U46619. Outside-in signaling was examined by measuring platelet spreading and clot retraction. Tail-transection bleeding time and susceptibility to thrombotic occlusion of the carotid artery in response to photochemical injury (rose bengal) were also measured. Results No significant differences in JON/A or fibrinogen binding were detected between wild-type (Dok-1+/+) and Dok-1-/- platelets, suggesting that Dok-1 does not regulate inside-out signaling. In contrast, Dok-1-/- platelets exhibited increased clot retraction and enhanced spreading on fibrinogen upon thrombin stimulation compared to Dok-1+/+ platelets (P&lt;0.05), suggesting that Dok-1 negatively regulates outside-in signaling. Compared with Dok-1+/+ mice, Dok-1-/- mice had shorter bleeding times (181±168 vs. 379±193 seconds; P&lt;0.001) and Dok-1-/- mice had shorter times to stable occlusion times of the carotid artery after photochemical injury compared with Dok-1+/+ mice (16.4±6.1 vs. 25.0±8.1 minutes; P&lt;0.05). Conclusions The adaptor protein Dok-1 functions to negatively regulate integrin αIIbβ3 outside-in signaling. Deficiency of Dok-1 results in a prothrombotic phenotype, with shorten bleeding times and enhanced arterial thrombosis.

Unique Disulfide Bonds in Epidermal Growth Factor (EGF) Domains of β3 Affect Structure and Function of αIIbβ3 and αvβ3 Integrins in Different Manner

The β3 subunit of αIIbβ3 and αvβ3 integrins contains four epidermal growth factor (EGF)-like domains. Each domain harbors four disulfide bonds of which one is unique for integrins. We previously discerned a regulatory role of the EGF-4 Cys-560-Cys-583 unique bond for αIIbβ3 activation. In this study we further investigated the role of all four integrin unique bonds in both αIIbβ3 and αvβ3. We created β3 mutants harboring serine substitutions of each or both cysteines that disrupt the four unique bonds (Cys-437-Cys-457 in EGF-1, Cys-473-Cys-503 in EGF-2, Cys-523-Cys-544 in EGF-3, and Cys-560-Cys-583 in EGF-4) and transfected them into baby hamster kidney cells together with normal αv or αIIb. Flow cytometry was used to measure surface expression of αIIbβ3 and αvβ3 and their activity state by soluble fibrinogen binding. Most cysteine substitutions caused similarly reduced surface expression of both receptors. Disrupting all four unique disulfide bonds by single cysteine substitutions resulted in variable constitutive activation of αIIbβ3 and αvβ3. In contrast, whereas double C437S/C457S and C473S/C503S mutations yielded constitutively active αIIbβ3 and αvβ3, the C560S/C583S mutation did not, and the C523S/C544S mutation only yielded constitutively active αIIbβ3. Activation of C523S/C544S αvβ3 mutant by activating antibody and dithiothreitol was also impaired. Molecular dynamics of C523S/C544S β3 in αIIbβ3 but not in αvβ3 displayed an altered stable conformation. Our findings indicate that unique disulfide bonds in β3 differently affect the function of αIIbβ3 and αvβ3 and suggest a free sulfhydryl-dependent regulatory role for Cys-560-Cys-583 in both αIIbβ3 and αvβ3 and for Cys-523-Cys-544 only in αvβ3.

A mutation in the β3 cytoplasmic tail causes variant Glanzmann thrombasthenia by abrogating transition of αIIbβ3 to an active state

The cytoplasmic tails of α(IIb) and β(3) regulate essential α(IIb) β(3) functions. We previously described a variant Glanzmann thrombasthenia mutation in the β(3) cytoplasmic tail, IVS14: -3C>G, which causes a frameshift with an extension of β(3) by 40 residues. The aim of this study was to characterize the mechanism by which the mutation abrogates transition of α(IIb) β(3) from a resting state to an active state. We expressed the natural mutation, termed 742ins, and three artificial mutations in baby hamster kidney (BHK) cells along with wild-type (WT) α(IIb) as follows: β(3) -742stop, a truncated mutant to evaluate the effect of deleted residues; β(3) -749stop, a truncated mutant that preserves the NPLY conserved sequence; and β(3) -749ins, in which the aberrant tail begins after the conserved sequence. Flow cytometry was used to determine ligand binding to BHK cells. Surface expression of α(IIb) β(3) of all four mutants was at least 60% of WT expression, but there was almost no binding of soluble fibrinogen following activation with activating antibodies (anti-ligand-induced-binding-site 6 [antiLIBS6] or PT25-2). Activation of the α(IIb) β(3) mutants was only achieved when both PT25-2 and antiLIBS6 were used together or following treatment with dithiothreitol. These data suggest that the ectodomain of the four mutants is tightly locked in a resting conformation but can be forced to become active by strong stimuli. These data and those of others indicate that the middle part of the β(3) tail is important for maintaining α(IIb) β(3) in a resting conformation.

β-arrestin-1 participates in thrombosis and regulates integrin αIIbβ3 signalling without affecting P2Y receptors desensitisation and function

β-arrestin-1 (β-arr1) and β-arrestin-2 (β-arr2) are cytosolic proteins well-known to participate in G protein-coupled receptor desensitisation and signalling. We used genetically-inactivated mice to evaluate the role of β-arr1 or β-arr2 in platelet function, P2Y receptor desensitisation, haemostasis and thrombosis. Platelet aggregation, soluble fibrinogen binding and P-selectin exposure induced by various agonists were near normal in β-arr1-/- and β-arr2-/- platelets. In addition, deficiency in β-arr1 or β-arr2 was not critical for P2Y receptors desensitisation. A functional redundancy between β-arr1 and β-arr2 may explain these unchanged platelet responses. Interestingly, β-arr1-/- but not β-arr2-/- mice were protected against laser- and FeCl3-induced thrombosis. The tail bleeding times, number of rebleeds and volume of blood loss were unchanged in β-arr1-/- and β-arr2-/- mice, suggesting no defect in haemostasis. β-arr1-/- platelet activation upon adhesion to immobilised fibrinogen was inhibited, as attested by a 37 ± 5% (n = 3, p<0.0001) decrease in filopodia extension, suggesting defective signalling through integrin αIIbβ3. β-arr1 appeared to be located downstream of Src family kinases and to regulate αIIbβ3 signalling by increasing Akt phosphorylation. Overall, this study supports a role for β-arr1 in promoting thrombus formation, in part through its participation in αIIbβ3 signalling, and no role of β-arr1 and β-arr2 in agonist-induced platelet activation and P2Y receptors desensitisation.