Integrin αIIbβ3-mediated Outside-in Signaling Research Articles

Intraplatelet miRNA-126 regulates thrombosis and its reduction contributes to platelet inhibition.

MicroRNA-126 (miR-126), one of the most abundant microRNAs in platelets, is involved in the regulation of platelet activity and the circulating miR-126 is reduced during antiplatelet therapy. However, whether intraplatelet miR-126 plays a role in thrombosis and platelet inhibition remains unclear. Here, using tissue-specific knockout mice, we reported that the deficiency of miR-126 in platelets and vascular endothelial cells significantly prevented thrombosis and prolonged bleeding time. Using chimeric mice, we identified that the lack of intraplatelet miR-126 significantly prevented thrombosis. Ex vivo experiments further demonstrated that miR-126-deficient platelets displayed impaired platelet aggregation, spreading and secretory functions. Next, miR-126 was confirmed to target phosphoinositol-3 kinase regulatory subunit 2 (PIK3R2) in platelet, which encodes a negative regulator of the PI3 K/AKT pathway, enhancing platelet activation through activating the integrin αIIbβ3-mediated outside-in signaling. After undergoing myocardial infarction (MI), chimeric mice lacking intraplatelet miR-126 displayed reduced microvascular obstruction and prevented MI expansion in vivo. In contrast, overexpression of miR-126 by the administration of miR-126 agonist (agomiR-126) in wild-type mice aggravated microvascular obstruction and promoted MI expansion, which can be almost abolished by aspirin administration. In patients with cardiovascular diseases, antiplatelet therapies, either aspirin alone or combined with clopidogrel, decreased the level of intraplatelet miR-126. The reduction of intraplatelet miR-126 level was associated with the decrease of platelet activity. Our murine and human data reveal that (i) intraplatelet miR-126 contributes to platelet activity and promotes thrombus formation, and (ii) the reduction of intraplatelet miR-126 contributes to platelet inhibition during antiplatelet therapy.

Lipoxin A4 analogue, BML-111, reduces platelet activation and protects from thrombosis

Formyl peptide receptors (FPRs) are members of seven transmembrane G protein-coupled receptors superfamily that exhibit different responses based on the nature of stimulating ligand type. FPRs have been shown to be present in platelets and regulate their function. However, the effect of formyl peptide receptor 2 (FPR2/ALX) lipid ligands on platelets has not yet been addressed. Hence, we sought to study the role of FPR2/ALX ligand and lipoxin A4 lipid analogue, BML-111, in the modulation of platelet function and thrombus formation. Immunofluorescence microscopy showed subcellular distribution and peripheral mobilisation of FPR2/ALX in stimulated platelets. This variation in distribution was further confirmed using flow cytometry. BML-111 inhibited a range of platelet activities in a dose-dependent manner in response to several platelet agonists. This included aggregation, fibrinogen binding to integrin αIIbβ3, α-granule secretion, dense granule secretion, Ca2 + mobilisation and integrin αIIbβ3-mediated outside-in signaling. The selectivity of BML-111 for FPR2/ALX was confirmed using FPR2/ALX deficient mice in flow cytometry assays. In vitro thrombus formation was also inhibited by various concentrations of BML-111. Moreover, the levels of vasodilator stimulated phosphorylation (VASP-S157) increased significantly after BML-111 treatment in resting and stimulated platelets via protein kinase A (PKA) independently of cyclic adenosine monophosphate (cAMP) signaling. Together, our findings demonstrate the significance of BML-111 as a modulator of platelet function via FPR2/ALX and unravel the thrombo-protective potentials of BML-111 induced signaling against thrombo-inflammatory diseases.

Myricetin as a promising inhibitor of platelet fibrinogen receptor in humans

Platelets play a vital role in the formation of dangerous arterial thrombosis. Platelets are activated by adhesive proteins or soluble agonists through their specific receptors. The receptor-mediated signaling pathways lead to common signaling events, which result in shape changes and inside–out signaling, leading fibrinogen binding to glycoprotein IIb/IIIa complex (integrin αIIbβ3). This interaction initiates integrin αIIbβ3-mediated outside-in signaling, subsequently culminating in granule secretion and aggregation. Myricetin is a flavonoid that occurs in a variety of plants. Although myricetin has been demonstrated to have several bioactive properties, its role in platelet activation has not been extensively studied. The present study demonstrated the ability of myricetin to inhibit platelet aggregation stimulated by collagen, thrombin, and U46619. Myricetin reduced the ATP-release, cytosolic Ca2+ mobilization, and P-selectin expression and the activation of PLCγ2/PKC, PI3K/Akt/GSK3β, and MAPK. Myricetin exerted a direct inhibitory effect on the activation of integrin αIIbβ3 by disrupting the binding between FITC-PAC-1 and the integrin. Moreover, myricetin suppressed integrin αIIbβ3-mediated outside–in signaling, such as integrin β3, Src, and Syk phosphorylation on immobilized fibrinogen. In animal studies, myricetin significantly prolonged the occlusion time of thrombotic platelet plug formation in mesenteric microvessels without extending bleeding time. This study concludes that myricetin is a natural integrin αIIbβ3 inhibitor and a novel antithrombotic agent.

The Antithrombotic Agent Pterostilbene Interferes with Integrin αIIbβ3-Mediated Inside-Out and Outside-In Signals in Human Platelets.

Platelets play a crucial role in the physiology of primary hemostasis and pathological processes such as arterial thrombosis; thus, developing a therapeutic target that prevents platelet activation can reduce arterial thrombosis. Pterostilbene (PTE) has remarkable pharmacological activities, including anticancer and neuroprotection. Few studies have reported the effects of pterostilbene on platelet activation. Thus, we examined the inhibitory mechanisms of pterostilbene in human platelets and its role in vascular thrombosis prevention in mice. At low concentrations (2–8 μM), pterostilbene strongly inhibited collagen-induced platelet aggregation. Furthermore, pterostilbene markedly diminished Lyn, Fyn, and Syk phosphorylation and hydroxyl radical formation stimulated by collagen. Moreover, PTE directly hindered integrin αIIbβ3 activation through interfering with PAC-1 binding stimulated by collagen. In addition, pterostilbene affected integrin αIIbβ3-mediated outside-in signaling, such as integrin β3, Src, and FAK phosphorylation, and reduced the number of adherent platelets and the single platelet spreading area on immobilized fibrinogen as well as thrombin-stimulated fibrin clot retraction. Furthermore, pterostilbene substantially prolonged the occlusion time of thrombotic platelet plug formation in mice. This study demonstrated that pterostilbene exhibits a strong activity against platelet activation through the inhibition of integrin αIIbβ3-mediated inside-out and outside-in signaling, suggesting that pterostilbene can serve as a therapeutic agent for thromboembolic disorders.

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.

Migfilin supports hemostasis and thrombosis through regulating platelet αIIbβ3 outside-in signaling.

Elucidating the regulation mechanism of integrin aIIbβ3 is key to understanding platelet biology and thrombotic diseases. Previous in vitro studies have implicated a role of migfilin in the support of platelet aIIbβ3 activation, however, contribution of migfilin to thrombosis and hemostasis in vivo and a detailed mechanism of migfilin in platelets are not known. In this study, through migfilin knock-out (migfilin-/-) mice, we report that migfilin is a pivotal positive regulator of hemostasis and thrombosis. Migfilin-/- mice show a nearly doubled tail-bleeding time and a prolonged occlusion time in FeCl3-induced mesenteric arteriolar thrombosis. Migfilin deficiency impedes platelet thrombi formation on a collagen surface and impairs platelet aggregation and dense-granule secretion. Supported by characteristic functional readings and the phosphorylation status of distinctive signaling molecules in the bidirectional signaling processes of aIIbβ3, the functional defects of migfilin-/- platelets appear to be mechanistically associated with a compromised outside-in signaling, rather than inside-out signaling. A synthesized cell-permeable migfilin peptide harboring filamin A binding sequence rescued the defective function and phosphorylation of signaling molecules of migfilin-/- platelets. Finally, migfilin does not influence the binding of filamin A and β3 subunit of aIIbβ3 in resting platelets, but hampers the re-association of filamin A and β3 during the conduct of outside- in signaling, suggesting that migfilin functions through regulating the interaction dynamics of aIIbβ3 and filamin A in platelets. Our study enhances the current understanding of platelet integrin aIIbβ3-mediated outside-in signaling and proves that migfilin is an important regulator for platelet activation, hemostasis and thrombosis.

Suppression of Human Platelet Activation via Integrin αIIbβ3 Outside-In Independent Signal and Reduction of the Mortality in Pulmonary Thrombosis by Auraptene.

Auraptene is the most abundant coumarin derivative from plants. The pharmacological value of this compound has been well demonstrated, especially in the prevention of cancer and neurodegenerative diseases. Platelet activation is a major factor contributing to arterial thrombosis. Thus, this study evaluated the influence of auraptene in platelet aggregation and thrombotic formation. Auraptene inhibited platelet aggregation in human platelets stimulated with collagen only. However, auraptene was not effective in inhibiting platelet aggregation stimulated with thrombin, arachidonic acid, and U46619. Auraptene also repressed ATP release, [Ca2+]i mobilization, and P-selectin expression. Moreover, it markedly blocked PAC-1 binding to integrin αIIbβ3. However, it had no influence on properties related to integrin αIIbβ3-mediated outside-in signaling, such as the adhesion number, spreading area of platelets, and fibrin clot retraction. Auraptene inhibited the phosphorylation of Lyn-Fyn-Syk, phospholipase Cγ2 (PLCγ2), protein kinase C (PKC), Akt, and mitogen-activated protein kinases (MAPKs; extracellular-signal-regulated kinase (ERK1/2), and c-Jun N-terminal kinase (JNK1/2), but not p38 MAPK). Neither SQ22536, an adenylate cyclase inhibitor, nor ODQ, a guanylate cyclase inhibitor, reversed the auraptene-mediated inhibition of platelet aggregation. Auraptene reduced mortality caused by adenosine diphosphate (ADP)-induced pulmonary thromboembolism. In conclusion, this study provides definite evidence that auraptene signifies a potential therapeutic agent for preventing thromboembolic disorders.

Sin1 (Stress-Activated Protein Kinase-Interacting Protein) Regulates Ischemia-Induced Microthrombosis Through Integrin αIIbβ3-Mediated Outside-In Signaling and Hypoxia Responses in Platelets.

Objective- Microthrombosis as a serious consequence of myocardial infarction, impairs the microvascular environment and increases the occurrences of heart failure, arrhythmia, and death. Sin1 (stress-activated protein kinase-interacting protein) as an essential component of mTORC2 (mammalian target of rapamycin complex 2) is required for cell proliferation and metabolism in response to nutrients, stress, and reactive oxygen species and activates Akt and PKC (protein kinase C). However, the activation and function of Sin1/mTORC2 in ischemia-induced microthrombosis remain poorly understood. Approach and Results- The phosphorylation of the mTORC2 target Akt at S473 (serine 473) was significantly elevated in platelets from the distal end of left anterior descending obstructions from patients who underwent off-pump coronary artery bypass grafting compared with platelets from healthy subjects. Consistent with this finding, phosphorylation of T86 in Sin1 was also dramatically increased. Importantly, the augmented levels of phosphorylated Sin1 and Akt in platelets from 61 preoperative patients with ST-segment-elevation myocardial infarction correlated well with the no-reflow phenomena observed after revascularization. Platelet-specific Sin1 deficiency mice and Sin1 T86 phosphorylation deficiency mice were established to explore the underlying mechanisms in platelet activation. Mechanistically, Sin1 T86 phosphorylation amplifies mTORC2-mediated downstream signals; it is also required for αIIbβ3-mediated outside-in signaling and plays a role in generating hypoxia/reactive oxygen species through NAD+/Sirt3 (sirtuin 3)/SOD2 (superoxide dismutase 2) pathway. Importantly, Sin1 deletion in platelets protected mice from ischemia-induced microvascular embolization and subsequent heart dysfunction in a mouse model of myocardial infarction. Conclusions- Together, the results of our study reveal a novel role for Sin1 in platelet activation. Thus, Sin1 may be a valuable therapeutic target for interventions for ischemia-induced myocardial infarction deterioration.

A novel ruthenium (II)-derived organometallic compound, TQ-6, potently inhibits platelet aggregation: Ex vivo and in vivo studies

Arterial thrombosis plays a key role in cardiovascular diseases. Hence, developing more effective antithrombotic agents is necessary. We designed a ruthenium (II)-derived complex, [Ru(η6-cymene)2-(1H-benzoimidazol-2-yl)-quinoline Cl]BF4 (TQ-6), as a new antiplatelet drug. TQ-6 (0.3 µM) exhibited extremely strong inhibitory activity against platelet aggregation, Src, and Syk phosphorylation stimulated by agonists in human platelets. In collagen-activated platelets, TQ-6 also inhibited ATP-release, [Ca+2]i, P-selectin expression, FITC-PAC-1 binding, and hydroxyl radical formation, as well as the phosphorylation of phospholipase Cγ2, protein kinase C, mitogen-activated protein kinases, and Akt. Neither FITC-JAQ1 nor FITC-triflavin binding or integrin β3 phosphorylation stimulated by immobilized fibrinogen were diminished by TQ-6. Furthermore, TQ-6 had no effects in cyclic nucleotide formation. Moreover, TQ-6 substantially prolonged the closure time in whole blood, increased the occlusion time of thrombotic platelet plug formation and bleeding time in mice. In conclusion, TQ-6 has a novel role in inhibiting platelet activation through the inhibition of the agonist receptors-mediated inside-out signaling such as Src-Syk-PLCγ2 cascade and subsequent suppression of granule secretion, leading to disturb integrin αIIbβ3-mediated outside-in signaling, and ultimately inhibiting platelet aggregation. Therefore, TQ-6 has potential to develop as a therapeutic agent for preventing or treating thromboembolic disorders.

The Phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) Binder Rasa3 Regulates Phosphoinositide 3-kinase (PI3K)-dependent Integrin αIIbβ3 Outside-in Signaling

The class I PI3K family of lipid kinases plays an important role in integrin αIIbβ3 function, thereby supporting thrombus growth and consolidation. Here, we identify Ras/Rap1GAP Rasa3 (GAP1IP4BP) as a major phosphatidylinositol 3,4,5-trisphosphate-binding protein in human platelets and a key regulator of integrin αIIbβ3 outside-in signaling. We demonstrate that cytosolic Rasa3 translocates to the plasma membrane in a PI3K-dependent manner upon activation of human platelets. Expression of wild-type Rasa3 in integrin αIIbβ3-expressing CHO cells blocked Rap1 activity and integrin αIIbβ3-mediated spreading on fibrinogen. In contrast, Rap1GAP-deficient (P489V) and Ras/Rap1GAP-deficient (R371Q) Rasa3 had no effect. We furthermore show that two Rasa3 mutants (H794L and G125V), which are expressed in different mouse models of thrombocytopenia, lack both Ras and Rap1GAP activity and do not affect integrin αIIbβ3-mediated spreading of CHO cells on fibrinogen. Platelets from thrombocytopenic mice expressing GAP-deficient Rasa3 (H794L) show increased spreading on fibrinogen, which in contrast to wild-type platelets is insensitive to PI3K inhibitors. Together, these results support an important role for Rasa3 in PI3K-dependent integrin αIIbβ3-mediated outside-in signaling and cell spreading.

Integrin αIIbβ3-Mediated Outside-in Signaling: Brake or Amplifier of Platelet Activation?

αIIbβ3 is the most prominent integrin in platelets, and binding to its ligands, in addition to supporting platelet aggregation, also results in the transmission of so-called αIIbβ3-mediated outside-in signals into the cell interior. While it is well accepted that integrin-mediated outside-in signaling functions as an amplifier of platelet activation, accumulating evidence suggests that outside-in signaling can, under certain conditions, function as an inhibitor of platelet activation. In this regard, previous studies have shown that ligand binding and platelet aggregation activate the inositol phosphatase SHIP-1, a negative regulator of the PI3K/Akt signaling pathway, to shift activated integrins back to their resting state, leading to dissociation of platelet aggregates. Because the PI3K/Akt signaling pathway is also involved in platelet granule secretion, we examined whether ligand binding to αIIbβ3 might transmit inhibitory signals that suppress platelet granule secretion. Interestingly, we found that antagonists of integrin αIIbβ3 promote both platelet dense- and α-granule secretion stimulated by low dose agonists. In support of this finding, both mouse and human platelets lacking expression of αIIbβ3 exhibited increased granule secretion compared to their wild-type counterparts. Conversely, Mn++-induced fibrinogen binding to αIIbβ3 inhibited low-dose agonist-induced platelet granule secretion. Biochemical analysis revealed that blocking ligand binding to, or absence of, αIIbβ3, enhanced agonist-induced Akt phosphorylation, while at the same time prevented the activation of the inhibitory enzyme, SHIP-1. To further investigate the role of SHIP-1 in inhibitory signaling, we examined the effect on platelet secretion of 3AC, a specific inhibitor of SHIP-1. We found that 3AC not only restores ADP-induced platelet granule secretion, but also increases CRP- or TRAP-induced platelet granule secretion. Taken together, these data demonstrate that integrin αIIbβ3-mediated outside-in signaling act as a brake to restrict unnecessary platelet activation that occurs in the presence of low-dose agonist stimulation. DisclosuresNo relevant conflicts of interest to declare.

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.

Hyperreactivity of junctional adhesion molecule A-deficient platelets accelerates atherosclerosis in hyperlipidemic mice.

Besides their essential role in hemostasis, platelets also have functions in inflammation. In platelets, junctional adhesion molecule (JAM)-A was previously identified as an inhibitor of integrin αIIbβ3-mediated outside-in signaling and its genetic knockdown resulted in hyperreactivity. This gain-of-function was specifically exploited to investigate the role of platelet hyperreactivity in plaque development. JAM-A-deficient platelets showed increased aggregation and cellular and sarcoma tyrosine-protein kinase activation. On αIIbβ3 ligation, JAM-A was shown to be dephosphorylated, which could be prevented by protein tyrosine phosphatase nonreceptor type 1 inhibition. Mice with or without platelet-specific (tr)JAM-A-deficiency in an apolipoprotein e (apoe(-/-)) background were fed a high-fat diet. After ≤12 weeks of diet, trJAM-A(-/-)apoe-/- mice showed increased aortic plaque formation when compared with trJAM-A(+/+) apoe(-/-) controls, and these differences were most evident at early time points. At 2 weeks, the plaques of the trJAM-A(-/-) apoe(-/-) animals revealed increased macrophage, T cell, and smooth muscle cell content. Interestingly, plasma levels of chemokines CC chemokine ligand 5 and CXC-chemokine ligand 4 were increased in the trJAM-A(-/-) apoe(-/-)mice, and JAM-A-deficient platelets showed increased binding to monocytes and neutrophils. Whole-blood perfusion experiments and intravital microscopy revealed increased recruitment of platelets and monocytes to the inflamed endothelium in blood of trJAM-A(-/-) apoe(-/-)mice. Notably, these proinflammatory effects of JAM-A-deficient platelets could be abolished by the inhibition of αIIbβ3 signaling in vitro. Deletion of JAM-A causes a gain-of-function in platelets, with lower activation thresholds and increased inflammatory activities. This leads to an increase of plaque formation, particularly in early stages of the disease.

Disaggregation Signals Initiated by Ligand Binding and Platelet Aggregation

Abstract 2196Accumulating evidence suggests that activation of the major platelet integrin, αIIbβ3, and subsequent platelet aggregation are intrinsically dynamic and reversible processes, especially when platelets are activated by low-dose agonists like ADP. When aggregation is not robust enough to activate feed-forward autocrine amplification pathways, platelet integrins revert to their inactivated state and fibrinogen dissociates, resulting in platelet disaggregation. Failure to disaggregate following low-dose agonist stimulation has been known for many years to correlate with occlusive arterial disease and diabetes mellitus, however the mechanisms by which integrins become inactivated leading to dissociation of fibrinogen is not well understood. Because phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signaling is known to be required to maintain integrins in an activated state and to support the formation of stable platelet aggregates, we examined whether the PI3K/Akt signaling might be actively reversed when platelets aggregate in response to ADP. We found that Akt becomes completely dephosphorylated in an aggregation-dependent manner. SHIP-1, a negative regulator of PI3K signaling, also becomes phosphorylated and activated during the early stages of platelet aggregation. These results suggest that fibrinogen binding and platelet aggregation initiate negative feedback through the PI3K/Akt signaling pathway to control unnecessary platelet aggregation responses. To gain further insight into the mechanism of platelet disaggregation, we examined the dynamic interaction of αIIbβ3 with talin – a cytosolic protein whose association with the integrin β3 cytoplasmic domain is required for integrin activation. We found that talin translocates to αIIbβ3 upon ADP stimulation, but dissociates from it during the process of platelet disaggregation. Blocking ADP-induced platelet aggregation with RGDW inhibited dissociation of talin from the integrin, again supporting the notion that that platelet aggregation initiates negative, as well as positive, signals to regulate the extent of agonist-induced platelet aggregation. Finally, we found that platelets missing PLCγ2 or FcγRIIa, each of which plays a positive role downstream of αIIbβ3-mediated outside-in signaling, are less prone to disaggregate following low-dose agonist stimulation than are their wild-type counterparts, although neither of them plays a role in ADP-induced platelet aggregation. Taken together, these data demonstrate that platelet aggregation and integrin αIIbβ3-mediated outside-in signaling not only initiates signals that amplify integrin activation and platelet aggregation, but also guards against inadvertent activation by shifting activated integrin αIIbβ3 back to its resting state, triggering platelet contraction and leading to dissociation of platelet aggregates. Disclosures:No relevant conflicts of interest to declare.

Calcium oscillation and phosphatidylinositol 3-kinase positively regulate integrin αIIbβ3-mediated outside-in signaling

The frequency of calcium oscillation reveals the platelet activation status, however, the biological significance of the periodic calcium responses and methods of communication with other integrin-mediated signals are not clear. RGD-containing disintegrin rhodostomin coated substrates were employed to enhance platelet spreading and calcium oscillation through direct binding and clustering of the receptor integrin alpha(IIb)beta3. The results showed that the activation of phosphatidylinositol 3-kinase (PI3-K) and internal calcium pathways were crucial for alpha(IIb)beta3 outside-in signaling. PI3-K antagonists wortmannin and LY294002 inhibited disintegrin substrates and induced platelet spreading and calcium oscillation. At the same time, pretreatment of platelets with the microsomal calcium-ATPase inhibitor thapsigargin to deplete internal calcium stores severely impaired the calcium oscillation as well as PI3-K activation and spreading on disintegrin substrates. Because inhibition of one pathway could inhibit the other, our data indicates that PI3-K and calcium oscillation are synergistically operated and form a positive-feedback regulation in integrin alpha(IIb)beta3-mediated outside-in signaling.