Outside-in Signaling In Platelets Research Articles

Characterization of Integrin αIIbβ3-Mediated Outside-in Signaling by Protein Kinase Cδ in Platelets.

Engagement of integrin αIIbβ3 promotes platelet–platelet interaction and stimulates outside-in signaling that amplifies activation. Protein kinase Cδ (PKCδ) is known to play an important role in platelet activation, but its role in outside-in signaling has not been established. In the present study, we determined the role of PKCδ and its signaling pathways in integrin αIIbβ3-mediated outside-in signaling in platelets using PKCδ-deficient platelets. Platelet spreading to immobilized fibrinogen resulted in PKCδ phosphorylation, suggesting that αIIbβ3 activation caused PKCδ activation. αIIbβ3-mediated phosphorylation of Akt was significantly inhibited in PKCδ -/- platelets, indicating a role of PKCδ in outside-in signaling. αIIbβ3-mediated PKCδ phosphorylation was inhibited by proline-rich tyrosine kinase 2 (Pyk2) selective inhibitor, suggesting that Pyk2 contributes to the regulation of PKCδ phosphorylation in outside-in signaling. Additionally, Src-family kinase inhibitor PP2 inhibited integrin-mediated Pyk2 and PKCδ phosphorylation. Lastly, platelet spreading was inhibited in PKCδ -/- platelets compared to the wild-type (WT) platelets, and clot retraction from PKCδ -/- platelets was markedly delayed, indicating that PKCδ is involved in the regulation of αIIbβ3-dependent interactivities with cytoskeleton elements. Together, these results provide evidence that PKCδ plays an important role in outside-in signaling, which is regulated by Pyk2 in platelets.

Reelin Amplifies Glycoprotein VI Activation and AlphaIIb Beta3 Integrin Outside-In Signaling via PLC Gamma 2 and Rho GTPases.

Reelin, a secreted glycoprotein, was originally identified in the central nervous system, where it plays an important role in brain development and maintenance. In the cardiovascular system, reelin plays a role in atherosclerosis by enhancing vascular inflammation and in arterial thrombosis by promoting platelet adhesion, activation, and thrombus formation via APP (amyloid precursor protein) and GP (glycoprotein) Ib. However, the role of reelin in hemostasis and arterial thrombosis is not fully understood to date. Approach and Results: In the present study, we analyzed the importance of reelin for cytoskeletal reorganization of platelets and thrombus formation in more detail. Platelets release reelin to amplify alphaIIb beta3 integrin outside-in signaling by promoting platelet adhesion, cytoskeletal reorganization, and clot retraction via activation of Rho GTPases RAC1 (Ras-related C3 botulinum toxin substrate) and RhoA (Ras homolog family member A). Reelin interacts with the collagen receptor GP (glycoprotein) VI with subnanomolar affinity, induces tyrosine phosphorylation in a GPVI-dependent manner, and supports platelet binding to collagen and GPVI-dependent RAC1 activation, PLC gamma 2 (1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase gamma-2) phosphorylation, platelet activation, and aggregation. When GPVI was deleted from the platelet surface by antibody treatment in reelin-deficient mice, thrombus formation was completely abolished after injury of the carotid artery while being only reduced in either GPVI-depleted or reelin-deficient mice. Our study identified a novel signaling pathway that involves reelin-induced GPVI activation and alphaIIb beta3 integrin outside-in signaling in platelets. Loss of both, GPVI and reelin, completely prevents stable arterial thrombus formation in vivo suggesting that inhibiting reelin-platelet-interaction might represent a novel strategy to avoid arterial thrombosis in cardiovascular disease.

The Tyrosine Kinase c-Src Specifically Binds to the Active Integrin αIIbβ3 to Initiate Outside-in Signaling in Platelets

It is currently believed that inactive tyrosine kinase c-Src in platelets binds to the cytoplasmic tail of the β3 integrin subunit via its SH3 domain. Although a recent NMR study supports this contention, it is likely that such binding would be precluded in inactive c-Src because an auto-inhibitory linker physically occludes the β3 tail binding site. Accordingly, we have re-examined c-Src binding to β3 by immunoprecipitation as well as NMR spectroscopy. In unstimulated platelets, we detected little to no interaction between c-Src and β3. Following platelet activation, however, c-Src was co-immunoprecipitated with β3 in a time-dependent manner and underwent progressive activation as well. We then measured chemical shift perturbations in the (15)N-labeled SH3 domain induced by the C-terminal β3 tail peptide NITYRGT and found that the peptide interacted with the SH3 domain RT-loop and surrounding residues. A control peptide whose last three residues where replaced with those of the β1 cytoplasmic tail induced only small chemical shift perturbations on the opposite face of the SH3 domain. Next, to mimic inactive c-Src, we found that the canonical polyproline peptide RPLPPLP prevented binding of the β3 peptide to the RT- loop. Under these conditions, the β3 peptide induced chemical shift perturbations similar to the negative control. We conclude that the primary interaction of c-Src with the β3 tail occurs in its activated state and at a site that overlaps with PPII binding site in its SH3 domain. Interactions of inactive c-Src with β3 are weak and insensitive to β3 tail mutations.

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.

Evaluation of targeting c-Src by the RGT-containing peptide as a novel antithrombotic strategy

BackgroundInteraction of integrin β3 with c-Src plays critical roles in cellular signaling which is heavily implicated in platelet adhesion and aggregation, as well as in tumor cell proliferation and metastasis or in osteoclastic bone resorption. Selectively blocking integrin αIIbβ3 outside-in signaling in platelets has been a focus of attention because of its effective antithrombotic potential together with a sufficient hemostatic capacity. The myristoylated RGT peptide has been shown to achieve this blockade by targeting the association of c-Src with the integrin β3 tail, but the lack of key information regarding the mechanisms of action prevents this strategy from being further developed into practical antithrombotics. Therefore, in-depth knowledge of the precise mechanisms for RGT peptide in regulating platelet function is needed to establish the basis for a potential antithrombotic therapy by targeting c-Src.MethodsThe reduction-sensitive peptides were applied to rule out the membrane anchorage after cytoplasmic delivery. The c-Src activity was assayed at living cell or at protein levels to assess the direct effect of RGT targeting on c-Src. Thrombus formation under flow in the presence of cytoplasmic RGT peptide was observed by perfusing whole blood through the collagen-coated micro-chamber.ResultsThe RGT peptide did not depend on the membrane anchorage to inhibit outside-in signaling in platelets. The myr-AC ~ CRGT peptide readily blocked agonist-induced c-Src activation by disrupting the Src/β3 association and inhibited the RhoA activation and collagen-induced platelet aggregation in addition to the typical outside-in signaling events. The myr-AC ~ CRGT had no direct effect on the kinase activity of c-Src in living cells as evidenced by its inability to dissociate Csk from c-Src or to alter the phosphorylation level of c-Src Y416 and Y527, consistent results were also from in vitro kinase assays. Under flow conditions, the myr-AC ~ CRGT peptide caused an inhibition of platelet thrombus formation predominantly at high shear rates.ConclusionsThese findings provide novel insights into the molecular mechanisms by which the RGT peptide regulates integrin signaling and platelet function and reinforce the potential of the RGT peptide-induced disruption of Src/β3 association as a druggable target that would finally enable in vivo and clinical studies using the structure-based small molecular mimetics.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-015-0159-8) contains supplementary material, which is available to authorized users.

EMILIN2 regulates platelet activation, thrombus formation, and clot retraction.

Thrombosis, like other cardiovascular diseases, has a strong genetic component, with largely unknown determinants. EMILIN2, Elastin Microfibril Interface Located Protein2, was identified as a candidate gene for thrombosis in mouse and human quantitative trait loci studies. EMILIN2 is expressed during cardiovascular development, on cardiac stem cells, and in heart tissue in animal models of heart disease. In humans, the EMILIN2 gene is located on the short arm of Chromosome 18, and patients with partial and complete deletion of this chromosome region have cardiac malformations. To understand the basis for the thrombotic risk associated with EMILIN2, EMILIN2 deficient mice were generated. The findings of this study indicate that EMILIN2 influences platelet aggregation induced by adenosine diphosphate, collagen, and thrombin with both EMILIN2-deficient platelets and EMILIN2-deficient plasma contributing to the impaired aggregation response. Purified EMILIN2 added to platelets accelerated platelet aggregation and reduced clotting time when added to EMILIN2-deficient mouse and human plasma. Carotid occlusion time was 2-fold longer in mice with platelet-specific EMILIN2 deficiency, but stability of the clot was reduced in mice with both global EMILIN2 deficiency and with platelet-specific EMILIN2 deficiency. In vitro clot retraction was markedly decreased in EMILIN2 deficient mice, indicating that platelet outside-in signaling was dependent on EMILIN2. EMILIN1 deficient mice and EMILIN2:EMILIN1 double deficient mice had suppressed platelet aggregation and delayed clot retraction similar to EMILIN2 mice, but EMILIN2 and EMILIN1 had opposing affects on clot retraction, suggesting that EMILIN1 may attenuate the effects of EMILIN2 on platelet aggregation and thrombosis. In conclusion, these studies identify multiple influences of EMILIN2 in pathophysiology and suggest that its role as a prothrombotic risk factor may arise from its effects on platelet aggregation and platelet mediated clot retraction.

Integrin αIIbβ3-Mediated c-Src Activation: Differential Binding to Inactive and Active c-Src

Activation of the tyrosine kinase c-Src is a fundamental element of integrin αIIbβ3-mediated outside-in signaling in platelets. Presently, it is thought that c-Src is bound via its SH3 domain to the C-terminal Arg-Gly-Thr (RGT) sequence of the β3 cytoplasmic tail (CT). Although a recent NMR study supports this contention, it is likely that such binding would be precluded in the inactive conformation of c-Src because the linker connecting the SH2 and kinase domains of c-Src physically occludes the putative β3 CT binding site in the SH3 domain. Accordingly, we have re-examined the interaction between c-Src and β3 by immunoprecipitating β3 bound c-Src from resting and agonist-stimulated platelets and then applied the biophysical techniques of surface plasmon resonance (SPR) and NMR spectroscopy to directly characterize the interaction of the SH3 domain with the β3 CT. In unstimulated platelets, there was little or no association between c-Src and β3. However, following platelet stimulation with 1 unit/ml thrombin, c-Src binding to β3 is detectable within 10 sec of stimulation and binding persists for at least the next 90 sec. Phosphorylation of c-Src residue Tyr419 located in its activation loop is an indicator of c-Src activation. We detected the phosphorylation of β3-bound c-Src on Tyr419 at 20 sec following thrombin stimulation. Tyr419 phosphorylation then persisted for the next 40 sec after which it rapidly declined. Thus, platelet stimulation induces the rapid interaction of c-Src with the β3 subunit of αIIbβ3 and this interaction is accompanied by the induction of c-Src kinase activity. Since it has been reported that c-Src binds to β3 via its SH3 domain, we used SPR spectroscopy to measure the strength of this interaction. In initial experiments, we measured binding of the C-terminal β3 heptapeptide NITYRGT to the immobilized SH3 domain and detected NITYRGT binding with a dissociation constant (Kd) of approximately 700 mM. We then repeated the measurements by flowing the SH3 domain over the entire 43 residue β3 CT appended to the SPR chip. Under these conditions, we detected a Kd for SH3 domain binding to the β3 CT of 7.21 ± 2.42 µM. Previously, we reported similar behavior for talin-1 FERM domain binding to the β3 CT. Thus, these experiments suggest that like binding of the talin-1 FERM domain to the β3 CT, the interaction of c-Src with β3 is a two-dimensional ternary interaction in which the membrane surface makes an important contribution. Next, to identify the sites in the SH3 domain that interact with β3, we used NMR spectroscopy. In these experiments, we measured the interaction of the β3 peptide NITYRGT with the 15N-labeled SH3 domain. Because NITYRGT binding to the SH3 domain is weak, we used chemical shift perturbations (CSP) to identify the SH3 domain residues interacting with NITYRGT. We found that NITYRGT interacted with the SH3 domain RT-loop and surrounding residues. A control peptide whose last three residues where replaced with those of the β1 CT (NITYEGK) induced only small CSP on the opposite face of the SH3 domain. Next, to mimic inactive c-Src, we found that the canonical polyproline peptide RPLPPLP prevented binding of the β3 peptide to the RT-loop. Under these conditions, the β3 peptide induced CSP similar to the negative control. Thus, these studies indicate that the primary interaction of c-Src with the β3 CT occurs in its activated state at a site that overlaps with the polyproline binding site in its SH3 domain and suggest that protein-membrane interactions make an important contribution to the strength of binding. By contrast, interactions of inactive c-Src with β3 are weak and insensitive to β3 CT mutations. DisclosuresNo relevant conflicts of interest to declare.

CRGT Peptide In Cytoplasm Regulates Thrombus Formation Via Dissociating c-Src-β3 Interaction

The RGT sequences of the integrin β3 tail directly and constitutively bind the inactive c-Src, regulating integrin αIIbβ3 signaling and platelet function. Previous work has shown that disrupting the interaction of c-Src with β3 via myristoylated RGT peptide or deletion of the RGT sequences in β3 selectively inhibits integrin αIIbβ3 outside-in signaling in platelets. However, the precise molecular mechanisms by which the Src-β3 association regulates integrin αIIbβ3 signaling need to be clarified. We found that active c-Src phosphoylated the Y747 and Y759 residues of β3 directly at the in vitro protein/protein level or in CHO cell models bearing Tac-β3 chimeras, which were devoid of the intact β3 signal transduction. Furthermore, data from mass spectrometry, [γ-32P] ATP incorporation assays and CHO cell/Tac-β3 chimeras demonstrated that the direct phosphorylation of Y747 and Y759 by active c-Src did not depend on the binding of c-Src to the RGT sequences of the β3 tail. To further investigate the biological functions of Src-β3 association in signal transduction we employed a cell-permeable and reduction-sensitive peptide (myr-AC∼CRGT), which disrupted the Src-β3 association in platelets independent of membrane-anchorage, and found that when platelets were stimulated by thrombin the c-Src activation and the phosphorylation of the tyrosine residues of the β3 tail were substantially inhibited by the presence of the peptide. These results suggest that one of the crucial biological functions of Src-β3 association is to serve as a “bridge” linking integrin signaling with the c-Src full activation and phosphorylation of the tyrosines of the β3 tail. To answer whether the RGT peptide binding to Src is able to alter the enzymatic activity of c-Src, we examined the Src-Csk association, the phosphorylation status of Y416 and Y527 of c-Src and the c-Src kinase catalytic activity. Results showed that myr-AC∼CRGT did not dissociate Csk from c-Src in resting platelets and the phosphorylation level of Y416 and Y527 of c-Src remained unaltered. Consistent data were also obtained from in vitro analysis of the c-Src kinase catalytic activity in the presence of CRGT peptide. These results suggest that myr-AC∼CRGT peptide per se does not fully activate c-Src. Myr-AC∼CRGT was also found to inhibit integrin αIIbβ3 outside-in signaling in human platelets. To examine the effect of the myr-AC∼CRGT on platelet adhesion and aggregation under flow conditions, we measured the platelet thrombus formation under different shear rates. Myr-AC∼CRGT did not affect the platelet adhesion at a wall shear rate of 125 s-1. The inability of myr-AC∼CRGT to affect platelet adhesion and aggregation remained at 500 s-1 shear rates. At 1,500 s-1, or 5,000 s-1 rates, myr-AC∼CRGT partially inhibited platelet adhesion and aggregation. These observations indicate that the Src-regulated outside-in signaling plays a pivotal role in the stable thrombus formation and the thrombus growth under flow conditions. The present study reveals novel insights into the molecular mechanisms by which c-Src regulates integrin αIIbβ3 signaling, particularly the phorsphorylation of the β3 cytoplasmic tyrosines, and provides first evidence in human platelets that the RGT peptide or derivatives regulate thrombus formation through dissociating the Src-β3 interaction. The data of this work allow us to anticipate that intracellular delivery of the RGT peptide or its analogues may have potential in the development of a new antithrombotic strategy where only the Src-β3 interaction is specifically interrupted so as to provide an effective inhibition on thrombosis together with a decent hemostasis. Disclosures:No relevant conflicts of interest to declare.

2,3,5,4’-tetrahydroxystilbene-2-O-β-D-glucoside (THSG) attenuates human platelet aggregation, secretion and spreading in vitro

2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside(THSG) is a water-soluble component of the rhizome extract from the traditional Chinese herb Polygonum multiflorum. Recent studies have demonstrated that THSG has potent anti-oxidative and anti-inflammatory effects. In this study, we investigated the anti-platelet aggregation, secretion and spreading of THSG with different methods. The purpose was to explore the anti-platelet effect of THSG and the underlying mechanism. We investigated the anti-platelet activity of THSG on platelet aggregation induced by collagen (2 μg/mL), thrombin(0.04U/mL), U46619 (3 μM) and ADP (2 μM). ATP secretion induced by collagen (2 μg/mL) was also investigated. P-selectin expression and PAC-1 binding were measured by flow cytometry. In addition, human platelet spreading on immobilized fibrinogen and immunoblotting were also tested. THSG dose-dependently inhibited platelet aggregation and ATP secretion induced by collagen. It inhibited platelet P-selectin expression and PAC-1 binding induced by thrombin(0.1U/mL). THSG also inhibited human platelet spreading on immobilized fibrinogen, a process mediated by platelet outside-in signaling. Western blot analysis showed that THSG could inhibit platelet Fc γ RIIa, Akt(Ser473)and GSK3β(Ser9) phosphorylation. Our study indicates that THSG has potent anti-platelet activity to collagen induced aggregation. THSG is likely to exert protective effects in platelet-associated thromboembolic disorders by modulating human platelet.

Evaluation of CK2 inhibitor (E)-3-(2,3,4,5-tetrabromophenyl)acrylic acid (TBCA) in regulation of platelet function

Casein Kinase II (CK2) is a serine/threonine kinase which is expressed in platelets. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a substrate of CK2 and antagonizes PI 3-kinase-mediated pathways by dephosphorylating phosphatidylinositol 3,4,5-triphosphate (PIP3). Since the role of CK2 and its signaling mechanism in platelet activation is not understood, we have examined whether CK2 plays an important role in agonist-induced platelet functional responses through the regulation of PI 3-kinase pathways by using a new class of highly selective CK2 inhibitor TBCA [(E)-3-(2,3,4,5-tetrabromophenyl)acrylic acid]. TBCA dose-dependently inhibited platelet aggregation and secretion induced by various agonists including 2-MeSADP, AYPGKF, SFLLRN, and CRP. Extent of platelet response inhibited by TBCA was similar to the extent of inhibition induced by PI 3-kinase inhibitors. CK2 regulated phosphorylation of PTEN as the inhibition of CK2 resulted in the inhibition of AYPGKF-induced PTEN phosphorylation. Agonist-induced thromboxane A2 (TxA2) generation and ERK phosphorylation were significantly inhibited by TBCA. TBCA also inhibited phosphorylation of PDK1, Akt, and GSK3β induced by AYPGKF. However, CK2 inhibition had no effect on AYPGKF-induced phosphorylation of PKC substrate plekstrin, demonstrating the selective action of TBCA through Gi-mediated PI 3-kinase pathways. Finally, platelet spreading on immobilized fibrinogen surface and clot retraction mediated by integrin αIIbβ3 signaling were significantly inhibited in the presence of TBCA. We conclude that CK2 plays a key role in platelet aggregation, secretion, TxA2 generation, and Akt and ERK phosphorylation, through the regulation of PI 3-kinase pathways. Moreover, CK2 is involved in αIIbβ3-mediated outside-in signaling in platelets.

The Bleeding Defect Exhibited by Aryl Hydrocarbon Receptor-Null Mice Is Due to Defective Collagen-Dependent Outside-in Signaling

Abstract 3294We previously identified AHR as a novel regulator of megakaryocytic (Mk) polyploidization and differentiation (Lindsey et al. Brit J of Haem, 2011). Best known as a mediator of toxicological signals, we propose that AHR influences multiple aspects of normal hematopoietic differentiation (Lindsey et al. Stem Cell Rev, 2012), including unpublished data suggesting a role for AHR in mediating platelet function. AHR-null mice had 9% fewer platelets and 10.4% fewer reticulated, young RNA-containing platelets than WT mice. Abnormal Mk maturation played a role in this phenotype, as AhR-null mice had ca. 25% fewer high ploidy (= 32n) Mks residing within the murine bone marrow niche compared to WT mice. While investigating if AHR influenced platelet function, we found that AHR-null mice bleed 5.3 times longer (8 minutes for AHR-null mice compared to 1.5 minutes for WT mice) and lose 3 times as much blood as WT mice during bleeding time assays. Although significant, we felt that the decreased Mk polyploidization and resulting reduced platelet counts were not enough to explain the drastic bleeding phenotype in AHR-null mice. In agreement with our hypothesis that AHR impacts platelet function, others have suggested AHR is critical for blood clotting during Oryzias latipes embryogenesis (Kawamura et al. Zoolog Sci, 2002). We previously showed that treatment of bone marrow-derived progenitor cells with AHR ligands such as TCDD (dioxin, a prototypic AHR ligand and activator) during ex vivo expansion could produce polyploid CD41-expressing cells in the absence of any cytokines. Here, we show that ex vivo expansion of murine progenitor cells with 10 mM of the AHR inhibitor 6’,2’,4’-trimethoxyflavone (TMF) resulted in 37% fewer highly polyploid (≥32n) Mks by day 7 (n=3, p=0.017), effectively blocking the effects of TPO on Mk differentiation and suggesting that AHR activation is downstream of TPO signaling.To examine the dramatic bleeding phenotype present in AHR-null mice, we next turned our attention toward platelet function, mediated by both outside-in and inside-out signaling. Defects in either or both of these signaling cascades could result in the bleeding defect present in AHR-null mice. In our initial experiments, we found that platelets from AHR-null mice bind fibrinogen equivalently to WT platelets (n=3), suggesting that AHR is not involved in inside-out platelet signaling. As we investigated other measures of platelet activation, we found that although platelets from AHR-null mice efficiently aggregated in response to ADP and the PAR-4 agonist AYPGKF (n=3 p=0.897 and 0.914, respectively), only 20 percent of AHR-null murine platelets (compared to 60 percent for WT platelets) aggregated in response to collagen (n=3, p=0.013). Spreading assays further demonstrated defective collagen-dependent outside-in signaling in AHR-null mice. We found that 5 times as many AHR-null platelets remained round (lacking filipodia or lamellipodia) as WT platelets after resting on collagen-coated slides (100 ug/mL) for 5 minutes. Over 60 percent of WT platelets were fully spread after a 20 minute incubation on collagen, compared to only 35 percent of AHR-null platelets. Additionally, roughly 20 percent of AHR-null platelets failed to respond and maintained a round morphology, representing 8 times as many unresponsive platelets as WT mice. The extent of spreading also appeared altered in AHR-null platelets, as the surface area of AHR-null platelets spreading on collagen was reduced by 42% after 5 minutes and 39% after 20 minutes compared to WT (n=3; p<0.001 for both experiments). Similar responses were seen when AHR-null platelets were allowed to spread on fibrinogen (100 ug/mL) after activation by 1 ug/mL collagen, with a 39% and 28% reduction in the surface area of AHR-null platelets after 5 and 20 minutes, respectively (n=3; p<0.001 for both experiments). Based on these findings, we are now investigating the molecular mechanisms of the collagen signaling defects present in AHR-null platelets, beginning with known interactions between AHR and vav genes, critical mediators of collagen-dependant platelet outside-in signaling. Our work is significant in that it builds upon our previously reported data and provides evidence that AHR is a critical component of the physiologic response platelets undergo in response to collagen. This information may provide novel treatment options for patients with bleeding disorders. Disclosures:No relevant conflicts of interest to declare.

Integrin αIIb-Mediated PI3K/Akt Activation in Platelets

Integrin αIIbβ3 mediated bidirectional signaling plays a critical role in thrombosis and haemostasis. Signaling mediated by the β3 subunit has been extensively studied, but αIIb mediated signaling has not been characterized. Previously, we reported that platelet granule secretion and TxA2 production induced by αIIb mediated outside-in signaling is negatively regulated by the β3 cytoplasmic domain residues R724KEFAKFEEER734. In this study, we identified part of the signaling pathway utilized by αIIb mediated outside-in signaling. Platelets from humans and gene deficient mice, and genetically modified CHO cells as well as a variety of kinase inhibitors were used for this work. We found that aggregation of TxA2 production and granule secretion by β3Δ724 human platelets initiated by αIIb mediated outside-in signaling was inhibited by the Src family kinase inhibitor PP2 and the PI3K inhibitor wortmannin, respectively, but not by the MAPK inhibitor U0126. Also, PP2 and wortmannin, and the palmitoylated β3 peptide R724KEFAKFEEER734, each inhibited the phosphorylation of Akt residue Ser473 and prevented TxA2 production and storage granule secretion. Similarly, Akt phosphorylation in mouse platelets stimulated by the PAR4 agonist peptide AYPGKF was αIIbβ3-dependent, and blocked by PP2, wortmannin and the palmitoylated peptide p-RKEFAKFEEER. Akt was also phosphorylated in response to mAb D3 plus Fg treatment of CHO cells in suspension expressing αIIbβ3-Δ724 or αIIbβ3E724AERKFERKFE734, but not in cells expressing wild type αIIbβ3. In summary, SFK(s) and PI3K/Akt signaling is utilized by αIIb-mediated outside-in signaling to activate platelets even in the absence of all but 8 membrane proximal residues of the β3 cytoplasmic domain. Our results provide new insight into the signaling pathway used by αIIb-mediated outside-in signaling in platelets.

JAM-A protects from thrombosis by suppressing integrin αIIbβ3-dependent outside-in signaling in platelets

AbstractMounting evidence suggests that agonist-initiated signaling in platelets is closely regulated to avoid excessive responses to injury. A variety of physiologic agonists induce a cascade of signaling events termed as inside-out signaling that culminate in exposure of high-affinity binding sites on integrin αIIbβ3. Once platelet activation has occurred, integrin αIIbβ3 stabilizes thrombus formation by providing agonist-independent “outside-in” signals mediated in part by contractile signaling. Junctional adhesion molecule A (JAM-A), a member of the cortical thymocyte marker of the Xenopus (CTX) family, was initially identified as a receptor for a platelet stimulatory mAb. Here we show that JAM-A in resting platelets functions as an endogenous inhibitor of platelet function. Genetic ablation of Jam-A in mice enhances thrombotic function of platelets in vivo. The absence of Jam-A results in increase in platelet aggregation ex vivo. This gain of function is not because of enhanced inside-out signaling because granular secretion, Thromboxane A2 (TxA2) generation, as well as fibrinogen receptor activation, are normal in the absence of Jam-A. Interestingly, integrin outside-in signaling such as platelet spreading and clot retraction is augmented in Jam-A–deficient platelets. We conclude that JAM-A normally limits platelet accumulation by inhibiting integrin outside-in signaling thus preventing premature platelet activation.

Peptide LSARLAF induces integrin β3 dependent outside-in signaling in platelets

IntroductionPeptide LSARLAF (LSA) can bind and activate integrin αIIbβ3 in the absence of ‘inside-out’ signal. The active αIIbβ3 mediates ‘outside-in’ signaling that elicits platelet aggregation, granule secretion and TxA2 production. Here we identify the membrane glycoproteins which mediate LSA-induced platelet activation other than αIIbβ3, and determine the roles of Src, PLCγ2, FcRγ-chain, and SLP-76 in LSA-induced platelet activation. MethodLigand-receptor binding assay was performed to study the effect of peptide LSA or its control peptide FRALASL (FRA) on integrins binding to their ligands. Spreading of CHO cells expressing αIIbβ3 or αVβ3 on immobilized fibrinogen was measured in the presence of LSA or FRA. Washed β3, Src, FcRγ-chain, LAT and SLP-76 deficient platelets aggregation and secretion were tested in response to LSA. ResultsLigand-receptor binding assay indicated that LSA promoted the binding of multiple ligands to αIIbβ3 or αVβ3. LSA also enhanced CHO cells with αIIbβ3 or αVβ3 expression spreading on immobilized fibrinogen. β3 deficient platelets failed to aggregate and secrete in response to LSA. The phosphorylation of PLCγ2 and Syk was also β3 dependent. Src, FcRγ-chain, LAT and SLP-76 deficient platelets did not aggregate, secrete ATP or produce TxA2 in response to LSA. ConclusionLSA-induced platelet activation is β3 dependent, and signaling molecules Src, FcRγ-chain, SLP-76 and LAT play crucial roles in LSA-induced β3 mediated signaling.

The PLATELET INTEGRIN αIIbβ3 CHANGES FROM A LOWER- to A Higher-AFFINITY STATE DURING INTERACTION with FIBRINOGEN

Abstract 1130The integrin αIIbβ3 (GPIIb-IIIa) plays an essential role in platelet adhesion and cohesion by binding to a number of adhesive ligands including fibrinogen, von Willebrand factor, fibronectin, and vitronectin. Following ligand binding, αIIbβ3-mediated intracellular signaling is thought to stabilize platelet adhesive interactions, cause platelet spreading, and initiate clot retraction. Here we investigated the time course of interactions between purified αIIbβ3 and its major ligand fibrinogen when both were firmly attached to apposed surfaces, mimicking αIIbβ3-mediated platelet adhesive interactions. To measure the mechanical resistance of individual αIIbβ3-fibrinogen complexes under a constant tensile force, we used a previously described optical trap force clamp system (Litvinov et al., Biophys. J., 2011, 100, 165). Briefly, we trapped a fibrinogen-coated microscopic bead in a focused laser beam and intermittently brought it into contact with an αIIbβ3-coated silica pedestal. Repeated touching and separation of the bead and pedestal occurred with a compressive force of 20 pN and a constant pulling force of 50 pN. When the duration of contact between interacting molecules on the bead and pedestal was varied from 0.1s to 2s, the probability of αIIbβ3-fibrinogen interaction was time-dependent. This enabled us to extract two-dimensional kinetic parameters, i.e., how fast the αIIbβ3-fibrinogen complex forms and dissociates at the interface without the application of an external tensile force (zero-force kinetics). To convert the kinetic parameters to absolute values, we determined the density of αIIbβ3 molecules on the pedestal surface capable of binding 125I-fibrinogen to be ≈3,000 molecules per μm2. We found that the reactive αIIbβ3 exists in at least two states that differ significantly in their on-rates (kon1 =8×10−5 and kon2 =5.6×10−4 μm2/s), off-rates (koff1 =1.56 and koff2 =1.70 1/s) and affinities (Kd1 =2×104 and Kd2 =3×103 1/μm2) for fibrinogen. In the absence of αIIbβ3 activators, the interacting molecules reached a stable equilibrium in which 85% of the complexes were in the lower affinity form. In the presence of the activator Mn2+, the proportion of lower affinity forms decreased to 60%. Mn2+ also changed the on-rates (kon1 =3.38×10−5 and kon2 =9.9×10−4 μm2/s), off-rates (koff1 =2.99 and koff2 =4.82 1/s) and affinities (Kd1 =9×104 and Kd2 =5×103 1/μm2). We then tested whether the ratio between the two integrin activation states and the mechanical stability of the αIIbβ3-fibrinogen complex depends on the duration of contact between the interacting surfaces. As the contact duration was prolonged from 0.1s to 2s, the αIIbβ3-fibrinogen bond lifetime increased from shorter lifetimes (<2s) corresponding to the low-affinity state to longer lifetimes (up to 50s) corresponding to the high-affinity state. Moreover, the rate of transition from low- to high-affinity increased ∼3-fold, whereas the reverse rate decreased 1.5–2-fold, resulting in an overall 6-fold increase in the equilibrium rate constant. This means that reversible conformational transitions of αIIbβ3 from low- to high affinity states occur during its interaction with fibrinogen with the equilibrium gradually shifting toward the higher affinity state as contact duration increases. Thus, the strength of αIIbβ3-fibrinogen interactions is time-dependent due to a progressive increase in the affinity of the αIIbβ3-fibrinogen complex during the course of the interaction. These results provide kinetic and thermodynamic characteristics of the adhesive interaction between αIIbβ3 and fibrinogen at the single-molecule level and provide evidence for a direct fibrinogen-induced change in αIIbβ3 conformation as a potential mechanism for the initiation of αIIbβ3-mediated outside-in signaling in platelets. Disclosures:No relevant conflicts of interest to declare.

The Aryl Hydrocarbon Receptor Influences Multiple Stages of Megakaryocyte Differentiation

Abstract 1298 Previous microarray analyses (n=3) uncovered evidence that aryl hydrocarbon receptor (AHR) mRNA increased 4–6 fold during megakaryocytic (Mk) differentiation as compared to isogenic granulocytic cultures (Lindsey et al. Blood, 2010) and identified AHR as a novel regulator of Mk polyploidization and differentiation (Lindsey et al. Brit J of Haem, 2011). Best known as a mediator of toxicological signals, we now provide unpublished data suggesting AHR impacts several additional aspects of Mk differentiation, including the initial “decision” of hematopoietic stem cells (HSCs) to differentiate toward the Mk lineage, as well as platelet function. In our current work, we first investigated if the absence of AHR signaling within the bone marrow results in Mk polyploidization defects. We found that after 7 days of ex vivo expansion, AHR-null mice had 59.5% fewer Mks ≥128n compared to WT littermates (n=3; p=0.011). In separate experiments, treatment of murine progenitor cells (n=4) with 10 nM TCDD (Dioxin, a prototypic AHR ligand) generated polyploid CD41-expressing cells within 1 day of ex vivo expansion, indicating that AHR ligands can stimulate ex vivo expansion toward the Mk lineage in the absence of cytokines such as thrombopoietin (TPO). As one might expect, TCDD is not as effective as TPO; 18.5% of the Mks treated with TCDD were polyploid by day 7, as compared to 31.5% of the Mks treated with TPO (n=3, p=0.004). Adding TCDD (n=3) did not significantly enhance Mk differentiation in response to TPO (35.4% vs 31.5%; p=0.377). Ex vivo expanding murine progenitor cells with 10 mM of the AHR inhibitor 6',2',4'-trimethoxyflavone (TMF) resulted in 37% fewer highly polyploid (≥32n) Mks by day 7 (n=3, p=0.017), effectively blocking the effects of TPO on Mk differentiation and suggesting that AHR activation is downstream of TPO signaling. Further underscoring a role for AHR during the initial differentiation “decisions” of HSCs, AHR antagonists promote HSC expansion (Boitano et al. Science, 2010) and treatments with AHR agonists deplete the HSC pool within the bone marrow (Singh et al. Carcinogenesis, 2009). HSCs reside in hypoxic niches within the bone marrow and move toward areas of increasing oxygen tension as the differentiate (Laluppa et al. Exp Hematol, 1998), suggesting to many that HSCs prefer areas of hypoxia and that HIF-1α may play a role in this process. Preliminary data shows a 3.1-fold decrease in AHR protein level under hypoxic conditions at a time when HIF-1α expression increases by 2.1-fold. AHR and HIF-1α expression is mediated by the same nuclear chaperone, HIF-1β, suggesting AHR and HIF-1α competition for HIF-1β may serve as a molecular switch by which hematopoietic cells respond to differences in oxygen levels. AHR-null mice bleed 5.3 times longer and lose three times as much blood volume than WT mice in bleeding time assays. While AHR-null mice had 9% fewer platelets and 10.4% fewer reticulated, young RNA-containing platelets than WT mice, we felt that this was not enough to explain the drastic bleeding phenotype in AHR-null mice. In agreement with our hypothesis that AHR impacts platelet function, others have suggested AHR is critical for blood clotting during Oryzias latipes embryogenesis (Kawamura et al. Zoolog Sci, 2002). Platelet function is mediated by both outside-in and inside-out signaling; defects in one or both of these signaling cascades result in bleeding disorders. In the current study, platelets from AHR-null mice bind fibrinogen as well or better than WT platelets (n=3), suggesting that AHR is not involved in inside-out platelet signaling. Based on these findings and work that shows AHR regulates vav2, a critical mediator of platelet outside-in signaling (Pearce et al. JBC, 2004), we pursued the possibility that AHR mediates platelet outside-in signaling. In agreement with a role in outside-in signaling, platelets from AHR-null mice demonstrate defective aggregation in response to collagen as compared to WT platelets. Current work seeks to further investigate the role of AHR in outside-in signaling using spreading assays. Ideally, new therapeutic approaches for Mk/platelet diseases should target specific biological events such as the initial “decisions” leading to Mk expansion from HSCs, Mk polyploidization, or platelet function. By impacting all of these processes, AHR is quickly becoming a very interesting therapeutic target and definitely warrants further investigation. Disclosures: No relevant conflicts of interest to declare.

Tyrosine phosphorylated c-Cbl regulates platelet functional responses mediated by outside-in signaling

Abstractc-Cbl protein functions as an E3 ligase and scaffolding protein, where 3 residues, Y700, Y731, and Y774, upon phosphorylation, have been shown to initiate several signaling cascades. In this study, we investigated the role of these phospho-tyrosine residues in the platelet functional responses after integrin engagement. We observed that c-Cbl Y700, Y731 and Y774 undergo phosphorylation upon platelet adhesion to immobilized fibrinogen, which was inhibited in the presence of PP2, a pan-src family kinase (SFK) inhibitor, suggesting that c-Cbl is phosphorylated downstream of SFKs. However, OXSI-2, a Syk inhibitor, significantly reduced c-Cbl phosphorylation at residues Y774 and Y700, without affecting Y731 phosphorylation. Interestingly, PP2 inhibited both platelet-spreading on fibrinogen as well as clot retraction, whereas OXSI-2 blocked only platelet-spreading, suggesting a differential role of these tyrosine residues. The physiologic role of c-Cbl and Y731 was studied using platelets from c-Cbl KO and c-CblYF/YF knock-in mice. c-Cbl KO and c-CblYF/YF platelets had a significantly reduced spreading over immobilized fibrinogen. Furthermore, clot retraction with c-Cbl KO and c-CblYF/YF platelets was drastically delayed. These results indicate that c-Cbl and particularly its phosphorylated residue Y731 plays an important role in platelet outside-in signaling contributing to platelet-spreading and clot retraction.

C-Cbl-PI3K Interaction Is Important for Platelet Outside-In Signaling

AbstractAbstract 2014Platelet activation by outside-in signaling is initiated by the binding of fibrinogen to alphaIIbbeta3, an integrin only expressed in platelets and megakaryocytes. Signals transduced by alphaIIbbeta3 regulate actin cytoskeleton resulting in filopodia and lamellipodia formation, cell spreading and retraction.c-Cbl protein is abundantly expressed in platelets and functions as E3 ubiquitin ligase and scaffolding protein to mediate protein-protein interactions. Importantly, c-Cbl tyrosine 731 has been shown to interact with p85 subunit of phosphotidylinositol 3-kinase (PI3K) modulating the actin cytoskeleton. Although previous reports showed c-Cbl activation downstream of alphaIIbbeta3, the mechanisms and implications of this activation or the downstream targets remain to be elucidated. We have studied the role of c-Cbl in platelet outside-in signaling: Using human platelets we have demonstrated that c-Cbl Y700, Y731 and Y774 residues undergoes tyrosine phosphorylation upon platelet adhesion to immobilized fibrinogen. These phosphorylation events are completely inhibited in the presence of the pan Src Family Kinases (SFKs) inhibitor (PP2) suggesting that c-Cbl is phosphorylated downstream of SFKs. Spleen tyrosine kinase (Syk) is also involved in this signaling pathway since its inhibition significantly reduce c-Cbl phosphorylation at residues Y774 and Y700; interestingly, tyrosine 731 phosphorylation, which allows the interaction with the p85-subunit of PI3K, is not affected by Syk inhibition. The physiological role of c-Cbl in platelet outside-in signaling was studied using c-Cbl knock-out mice. We found that in contrast to WT platelets, c-Cbl KO platelets had a significantly reduced spreading over a fibrinogen-coated surface. Furthermore, clot retraction analysis demonstrated that c-Cbl KO platelets retraction time was delayed when compared to WT platelets, suggesting a retraction defect. To further elucidate the physiological role of c-Cbl-PI3K interaction we used a knock-in mouse in which the c-Cbl residue Y 731 was substituted with phenylalanine (Y731F) thereby abolishing the PI3K binding site on c-Cbl. Importantly, platelets from Y731F mice showed spreading and clot retraction defect that were comparable with the c-Cbl KO. These result indicates that in large part, the role of c-Cbl in platelets outside-in signaling is determined by its interaction with PI3K.In conclusion, we have demonstrated that c-Cbl plays an important role in platelet outside-in signaling, and its interaction with PI3K through tyrosine 731 is of pivotal importance in platelet spreading and retraction. Disclosures:No relevant conflicts of interest to declare.

Junctional Adhesion Molecule-A Is a Novel Csk-Binding Protein Which Recruits Csk to the Integrin aIIbb3 and Suppresses Outside-In Signaling In Platelets

AbstractAbstract 2120Platelet activation is regulated by both positive and negative regulators present within the platelets so that unwanted activation is suppressed, but, when needed, occurs rapidly. A significant amount of effort has been devoted towards understanding the positive regulators of platelet function. However, very little is known about the negative regulators. Dysregulation of the endogenous negative regulators may aid the thrombotic complications seen in various diseases. Upon ligand binding to integrin aIIbb3, a cascade of signaling known as outside-in signaling is induced through the integrin that regulates platelet aggregation and clot retraction. How endogenous negative regulators suppress these events is not well understood. We have previously identified junctional adhesion molecule A (JAM-A), on the platelet surface. We found that Jam-a knockout mice show a prothrombotic phenotype as assessed by significantly (P<0.00001) shortened tail bleeding time, decreased carotid vessel occlusion time and increased pulmonary thromboembolism. Platelet functional studies revealed that Jam-a null platelets were hyperactive to physiological agonists. Surprisingly, inside-out signaling events were not affected in Jam-a null platelets. It is therefore possible that observed hyper-reactivity of Jam-a null platelets could be due to enhanced outside-in signaling. To test this, we performed a clot retraction assay. The clot retraction in wild type (Wt) occurred normally, which began after 1 h and about 50% was completed by 2 h. On the contrary, in Jam-a null platelets, the process of clot retraction was significantly enhanced (P<0.0001). It was initiated before 1h and was completed within 2 h. When analyzed for outside-in signaling events such as b3 tyrosine phosphorylation and c-Src phosphorylation, we found both significantly enhanced in Jam-a null platelets. To assess the mechanism by which JAM-A suppresses outside-in signaling, we analyzed the phosphorylation status of JAM-A. Interestingly, JAM-A was found to be phosphorylated on Y280 in unactivated platelets and rapidly dephosphorylated upon initiation of outside-in signaling. On the other hand, in resting platelets, a minimally phosphorylated S284 residue of JAM-A is rapidly phosphorylated, suggesting that there is a dephosphorylation/phosphorylation switch that may be involved in regulating outside-in signaling. Furthermore, we found that JAM-A associates with integrin aIIbb3 in unactivated human platelets, but this association was disrupted during outside-in signaling as determined by co-immunoprecipitation. We also found Csk, a C-terminal Src kinase, coimmunoprecipitating (IP) with JAM-A from resting, but not activated, platelet lysates, suggesting that JAM-A may be recruiting Csk to unactivated integrins and thus suppressing signaling. To test this, we analyzed association of Csk with integrin in Jam-a null platelets and found that in Wt platelets Csk was abundantly present in the integrin IP, but was completely absent in the integrin IP of the Jam-a null platelet lysates. These results clearly suggest that JAM-A recruits Csk to the integrin and thus suppresses outside-in signaling. Disclosures:No relevant conflicts of interest to declare.

Origin of the nonadhesive properties of fibrinogen matrices probed by force spectroscopy.

The deposition of a multilayered fibrinogen matrix on various surfaces results in a dramatic reduction of integrin-mediated cell adhesion and outside-in signaling in platelets and leukocytes. The conversion of a highly adhesive, low-density fibrinogen substrate to the nonadhesive high-density fibrinogen matrix occurs within a very narrow range of fibrinogen coating concentrations. The molecular events responsible for this transition are not well understood. Herein, single-cell and molecular force spectroscopy were used to determine the early steps in the formation of nonadhesive fibrinogen substrates. We show that the adsorption of fibrinogen in the form of a molecular bilayer coincides with a several-fold reduction in the adhesion forces generated between the AFM tip and the substrate as well as between a cell and the substrate. The subsequent deposition of new layers at higher coating concentrations of fibrinogen results in a small additional decrease in adhesion forces. The poorly adhesive fibrinogen bilayer is more extensible under an applied tensile force than is the surface-bound fibrinogen monolayer. Following chemical cross-linking, the stabilized bilayer displays the mechanical and adhesive properties characteristic of a more adhesive fibrinogen monolayer. We propose that a greater compliance of the bi- and multilayer fibrinogen matrices has its origin in the interaction between the molecules forming the adjacent layers. Understanding the mechanical properties of nonadhesive fibrinogen matrices should be of importance in the therapeutic control of pathological thrombosis and in biomaterials science.