Integrin αIIb cytoplasmic Tail Is Essential for Integrin Outside-in Signaling and Regulation of in Vivo Thrombosis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4160-4160
Author(s):  
Meghna Ulhas Naik ◽  
Ulhas P Naik
Keyword(s):  
Platelet Aggregation ◽  
Conflicts Of Interest ◽  
Cytoplasmic Tail ◽  
Cytoplasmic Domain ◽  
P38 Map Kinase ◽  
Clot Retraction ◽  
Integrin Αiibβ3 ◽  
Signaling Events ◽  
Inside Out

Abstract Platelet aggregation plays an important role in physiological hemostasis and pathological thrombosis. Platelet agonists induce a series of events called inside-out signaling that lead to the activation of integrin αIIbβ3. Fibrinogen binding to the activated integrin relay signals termed as outside-in signaling that regulate thrombus growth and stability. Talin and kindlin bind to the integrin β3 cytoplasmic tail to induce inside-out signaling. Although, integrin αIIb cytoplasmic domain also bind to a number of proteins, its importance in hemostasis or thrombosis is not well understood. Previously, we have identified a calcium- and integrin-binding protein (CIB1) that specifically interacts with the integrin αIIb cytoplasmic tail. Using a novel technique to inhibit interaction of CIB1 with integrin αIIb in intact human platelets, we have shown that CIB1 regulates outside-in signaling through integrin αIIbβ3. Recently, using Cib1-/- mice, we showed that CIB1 is a key regulator of thrombosis in vivo. Interestingly, agonist-induced platelet aggregation and secretion was normal in Cib1-/- platelets. Furthermore, expression or activation of integrin αIIbβ3 was also not affected by Cib1 deficiency, suggesting that integrin inside-out signaling is not affected in Cib1-/- platelets. However, adhesion and spreading on immobilized fibrinogen (Fg) was severely affected in Cib1-/- platelets. When we analyzed the rate of clot retraction, we found that significantly (P<0.001) delayed clot retraction was observed in Cib1-/- platelets compared to WT littermates, suggesting that integrin outside-in signaling is impaired in the absence of Cib1. To delineate the molecular mechanism regulated by CIB1 during platelet spreading and clot retraction, we analyzed the known signaling events activated during outside-in signaling. We found that Fg-dependent activation of ERK1 and p38 MAP kinase was significantly reduced in Cib1-/- null platelets. Furthermore, phosphorylation of the myosin light chain was also blocked in Cib1-/- platelets adhered to immobilized Fg. Furthermore, outside-in signaling-dependent tyrosine phosphorylation of β3 was greatly reduced in Cib1-/- platelets. When analyzed for the candidate tyrosine kinase responsible for reduced β3 phosphorylation, both Src and FAK activation was significantly reduced in Cib1-/- platelets. Furthermore, downstream signaling events such as activation of PAK1, PI3K, PDK1, as well as Akt were significantly affected in Cib1-/- platelets adhered to immobilized Fg. To test if impaired inhibition of GSK3-β is the cause of defective outside in signaling in Cib1-/- platelets we treated Cib1-/- platelets with SB216763, a specific GSK3-β inhibitor. We found that inhibition of GSK3-β rescued defective platelet adhesion and clot retraction observed in Cib1-/- platelets. It also rescued activation of p38 and Erk2 activation as well as MLC phosphorylation. However, activation of FAK, Src, PAK1, PI3K, PDK1, and Akt was not rescued, suggesting that these are upstream of GSK3-β. Furthermore, we found that outside-in dependent recruitment of FAK to the integrin-c-Src complex is greatly reduced in the absence of Cib1 suggesting that integrin αIIb cytoplasmic domain serves as a docking site for CIB1 so that it can recruit FAK to the integrin-c-Src complex and propagate outside-in signaling leading to GSK3-β inhibition, which is crucial for thrombus growth and stability. These in vivo and in vitro results clearly show that CIB1 regulates thrombosis by regulating outside-in signaling without affecting inside-out signaling through integrin αIIbβ3. Our results highlight an essential function to integrin αIIb cytoplasmic tail in regulating integrin outside-in signaling and thus thrombus growth and stability. Disclosures No relevant conflicts of interest to declare.

CIB1 Regulates Thrombosis through Integrin αaIIbβ3 Outside-in Signaling, but Not Inside-out Signaling.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2992-2992
Author(s):  
Meghna Ulhas Naik ◽  
Ulhas Naik
Keyword(s):  
Platelet Aggregation ◽  
Tyrosine Phosphorylation ◽  
Conflicts Of Interest ◽  
P38 Map Kinase ◽  
In Vivo Model ◽  
Clot Retraction ◽  
Pulmonary Vessel ◽  
Signaling Events ◽  
Inside Out

Abstract Abstract 2992 Poster Board II-969 Platelet aggregation plays an important role in physiological hemostasis and pathological thrombosis. Platelet agonists induce a series of signaling events called inside-out that leads to the activation of integrin αaIIbβ3. Upon ligand binding to integrin αaIIbβ3, a cascade of signaling known as outside-in signaling is induced through the integrin that regulates platelet aggregation and clot retraction. The regulation of these signaling events is not well understood. Previously, we had identified a calcium- and integrin-binding protein (CIB1) that specifically interacts with integrin αaIIbβ3. Using a novel technique to inhibit interaction of CIB1 with integrin αaIIbβ3 in intact platelets, we have shown that CIB1 regulates outside-in signaling through integrin αaIIbβ3. Recently, using Cib1-/- mice, we confirmed that CIB1 is a key regulator of hemostasis. FeCl3-induced carotid artery thrombosis, a well recognized in vivo model of thrombosis showed a significantly extended time of occlusion in Cib1-/- mice compared to wild type (WT) mice. Since CIB1 is expressed both in the endothelium and in platelets, the observed defect could arise from the lack of CIB1 in either cell types. To identify the specific involvement of platelets, we performed a collagen-epinephrine-induced pulmonary thromboembolism assay. In this assay, pulmonary vessel occlusion occurs due to platelet thrombus formation without injury to the endothelium. We found no difference in the number of Cib1-/- mice and WT mice that died within two minutes (n=20). When analyzed for the extent of pulmonary vascular occlusion by Evans blue exclusion as well as histochemical analysis, no difference between Cib1-/- and WT mice was observed. Consistent with this finding, agonist-induced platelet aggregation and secretion was normal in Cib1-/- platelets. Furthermore, expression or activation of integrin αaIIbβ3 was also not affected by Cib1 deficiency. When we analyzed the rate of clot retraction, we found that incomplete and significantly (P<0.001) delayed clot retraction was observed in Cib1-/- platelets compared to WT littermates. To delineate the molecular mechanism regulated by CIB1, we analyzed the known signaling cascade involved in clot retraction. We found that outside-in signaling-induced activation of ERK1 and p38 MAP kinase was significantly reduced in Cib1-/- null platelets. Furthermore, phosphorylation of the myosin light chain during outside-in signaling was blocked in Cib1-/- platelets. Interestingly, agonist-induced tyrosine phosphorylation of integrin β3 chain was unaffected in Cib1-/- platelets, but Mn2+-induced outside-in signaling-dependent tyrosine phosphorylation of β3 was greatly reduced. When analyzed for the candidate tyrosine kinase responsible for β3 phosphorylation, both Src and FAK activation was significantly reduced in Cib1-/- platelets. These in vivo and in vitro results clearly show that CIB1 regulates thrombosis by regulating outside-in signaling without affecting inside-out signaling through integrin αaIIbβ3. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Coenzyme Q10 Attenuates Platelet Integrin αIIbβ3 Outside-in Signaling through Targeting cAMP/PKA Pathway and Inhibits Atherosclerosis

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2423-2423
Author(s):  
Yan Yang ◽  
Xiaohong Ruby Xu ◽  
Heyu Ni ◽  
Liping Ma ◽  
Wenhua Ling ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Clot Retraction ◽  
Integrin Αiibβ3 ◽  
Granule Secretion ◽  
Platelet Spreading ◽  
Pka Pathway ◽  
Inside Out

Abstract Introduction: Platelet integrin αIIbβ3 outside-in signaling is crucial for platelet adhesion and aggregation, and contributes to atherogenesis. Coenzyme Q10 (CoQ10) has been implicated as a protective factor against cardiovascular diseases (CVDs), particularly atherosclerosis. However, whether CoQ10 attenuates atherosclerosis through inhibiting platelet function and αIIbβ3 outside-in signaling is unknown. The aim of this study was to explore whether CoQ10 affects platelet function and αIIbβ3 outside-in signalling and thus inhibiting the progress of atherosclerosis in vivo and the underlying mechanisms in vitro. Methods: In vitro study, The murine platelet rich plasma (PRP) from C57BL/6J wild-type (WT) mice or human PRP and gel-filtered platelets were incubated with different concentrations (1, 10 or 100 μM) of CoQ10 or the vehicle control for 50 min. Platelet aggregation, spreading on fibrinogen (Fg) and clot retraction were determined. In addition, the effects of CoQ10 on platelet integrin αIIbβ3 inside-out signalling (e.g., talin-1 and kindlin-3 binding to integrin β3) were determined by immunoprecipitation, and outside-in signalling (e.g., phosphorylation of sarcoma tyrosine-protein kinase (c-Src), focal adhesion kinase (FAK), and β3 cytoplasmic tail, myosin light chain (MLC)) were determined by Western blotting. The levels of platelet ATP and cAMP were measured by ELISA assays. In vivo study, male homozygous apolipoprotein E-deficient (apoE-/-) mice (C57BL/6 genetic background) were fed either a standard normal AIN-93G diet (NC group), a Western-type diet (HFD group) or a Western-type diet supplemented with CoQ10 (1800 mg/kg diet) (CoQ10 group) for 12 weeks. Platelet aggregation, granule secretion, platelet spreading, clot retraction, integrin αIIbβ3 outside-in signalling, platelet-leukocyte interactions and carotid artery plaque area were also examined. In our randomized, double-blind, placebo-controlled trial, 101 hypercholesterolemic subjects were randomly administrated to 120 mg CoQ10 or placebo daily for 24 weeks. Platelet intracellular CoQ10 levels, platelet aggregation in PRP, platelet platelet factor 4 (PF-4) and C-C motif ligand 5 (CCL5) release, and platelet integrin αIIbβ3 outside-in signalling were also evaluated before and after 24 weeks of intervention. Results: We found that CoQ10 inhibited human and WT mouse platelet aggregation, platelet spreading, granule secretion, and clot retraction in vitro and apoE-/- mice on a high fat diet. CoQ10 also reduced atherosclerosis and platelet-monocyte aggregation in apoE-/- mice. The inhibitory effects of CoQ10 is mediated by attenuated αIIbβ3 outside-in signalling pathway (e.g., attenuation of phosphorylation of c-Src, FAK, and β3 cytoplasmic tail, and MLC in thrombin-activated platelets or platelets exposed to immobilized Fg), which requires up-regulation of the cAMP/PKA pathway, where CoQ10 inhibited phosphodiesterase 3A activity and activated the A2A adenosine receptor. However, CoQ10 did not affect platelet integrin αIIbβ3 inside-out signalling pathway, platelet cellular ATP, or platelet apoptosis (the mitochondrial membrane potential and phosphatidylserine exposure). Moreover, our clinical trial in dyslipidemic patients demonstrated that CoQ10 supplementation attenuated platelet aggregation, which was positively correlated with the increased platelet CoQ10 concentrations, inhibited αIIbβ3 outside-in signalling and decreased platelet PF-4 and CCL5 secretion. Conclusions: We present new data to suggest that CoQ10 plays a novel role in attenuating platelet function and integrin αIIbβ3 outside-in signalling though targeting cAMP/PKA signalling cascade and thus inhibiting the progress of atherosclerosis. CoQ10 is therefore a promising agent for the prevention and/or treatment for cardiovascular disease. Disclosures No relevant conflicts of interest to declare.

Platelet Junctional Adhesion Molecule-A Regulates Thrombosis by Negatively Regulating Outside-in Signaling through Integrin αIIbβ3.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 155-155
Author(s):  
Meghna Ulhas Naik ◽  
Timothy J. Stalker ◽  
Lawrence F Brass ◽  
Ulhas P Naik
Keyword(s):  
Platelet Aggregation ◽  
Adhesion Molecule ◽  
Wild Type ◽  
Clot Retraction ◽  
Vessel Occlusion ◽  
Platelet Surface ◽  
Pulmonary Vessel ◽  
Integrin Αiibβ3 ◽  
Signaling Events

Abstract Abstract 155 Platelet aggregation plays an important role in physiological hemostasis and pathological thrombosis. Platelet agonists induce a series of signaling events called inside-out signaling that leads to the activation of integrin αIIbβ3. Upon ligand binding to integrin αIIbβ3, a cascade of signaling known as outside-in signaling is induced through the integrin that regulates platelet aggregation and clot retraction. How these signaling events are regulated is not well understood. We have previously identified JAM-A, a junctional adhesion molecule, on the platelet surface. Genetic ablation of Jam-A significantly (P<0.00001) shortened tail bleeding times (mean 54 seconds) compared to wild-type littermates (mean 98 seconds), suggesting an enhanced clot formation. FeCl3-induced carotid artery thrombosis and laser-induced cremaster arteriole thrombosis, two well recognized in vivo thrombosis model, both showed greatly shortened time of vessel occlusion and increased thrombus formation compared to wild-type (WT). Since JAM-A is expressed both on the endothelium and on platelets, the observed defect could arise from the lack of JAM-A in either cell type. To identify the involvement of platelet derived JAM-A, we performed a collagen-epinephrine-induced pulmonary thromboembolism assay. In this assay, pulmonary vessel occlusion occurs primarily through platelet thrombus without injury to the endothelium. We found that a significantly increased number of Jam-A−/− mice died within two minutes compared to WT mice. When analyzed for the extent of pulmonary vascular occlusion by Evans blue exclusion as well as histochemical analysis, we found a significantly greater extent of thromoembolism in Jam-A−/− mice compared to WT mice. Consistent with this finding, agonist-induced platelet aggregation, but not secretion, was significantly enhanced in Jam-A−/− platelets. Interestingly, however, the expression or activation of integrin αIIbβ;3 was not affected by Jam-A deficiency. When we analyzed the rate of clot retraction, we found that Jam-A−/− platelets showed 85% clot retraction within 90 minutes compared to only a 20% clot retraction in WT platelets, indicating that the absence of Jam-A significantly increases the rate of clot retraction (P<0.0001). JAM-A was found to associate with integrin αIIbβ3 in unactivated human platelets, but this association was disrupted during outside-in signaling as determined by co-immunoprecipitation assay suggesting that JAM-A may suppress signaling through the integrin. To delineate the molecular mechanism influenced by JAM-A, we analyzed the known signaling cascade involved in clot retraction. We found that outside-in signaling-induced activation of ERK1 and p38 MAP kinase was significantly enhanced in Jam-A−/− platelets. However, activation of focal adhesion kinase was unaffected in Jam-A−/− platelets compared to WT. Furthermore, outside-in signaling-induced phosphorylation of the myosin light chain was increased in Jam-A−/− platelets. These in vivo and in vitro results clearly show that JAM-A negatively regulates outside-in signaling through integrin αIIbβ;3, thus protecting from thrombosis. Disclosures: No relevant conflicts of interest to declare.

Ga13 and Talin Coordinate integrin Inside-out and Outside-In Signaling by “time-sharing” of b3 cytoplasmic Domain

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 162-162
Author(s):  
Haixia Gong ◽  
Xiaoping Du
Keyword(s):  
Conflicts Of Interest ◽  
Cytoplasmic Domain ◽  
Time Sharing ◽  
Protein Subunit ◽  
Fibrinogen Binding ◽  
Bidirectional Signaling ◽  
And Migration ◽  
Inside Out

Abstract Abstract 162 The bidirectional signaling of integrins mediates cell adhesion, spreading, retraction and migration. The binding of talin and kindlins to the cytoplasmic domain of integrin b3 subunit transmits inside-out signals to induce integrin activation. Ligand-induced outside-in signaling requires the binding of a G protein subunit, Ga13, and a tyrosine kinase, c-Src, to the b3 cytoplasmic domain. It is unclear how the short cytoplasmic domain of b3 accommodates these molecules and allows coordinated bidirectional signaling. Here we show that Ga13 and talin are mutually exclusive in binding to b3 both in vivo and in vitro. Increasing expression level of talin head or full-length talin in CHO123 cell decreases Ga13-b3 association. Ga13 also competes with talin head for GST-b3 binding in purified binding system. More importantly, talin is associated with b3 only in inside-out signaling during platelet aggregation. Following integrin ligation, however, Ga13 binds to b3, replacing talin. The Ga13 binding site located between K729-T741 within the talin binding region. However, Ga13 binding and signaling require a distinct ExE733 motif (EEE in b3) conserved in most integrin b subunits that is not required for talin binding but flanked by talin binding sequences on both sides. Interference of Ga13 binding to integrin b3 cytoplasmic domain by myristorylated b3 peptide (Myr-EEERA735) or by point-mutating the EEE motif to AAA selectively inhibits outside-in signaling, thus inhibited cell spreading on fibrinogen, accelerated RhoA activation and inhibited c-Src activity. But they have no effect on talin-dependent inside-out signaling judged by fibrinogen binding assay. In conclusion, our data suggest that the timed share of binding sites in b3 between Ga13 and talin coordinates bidirectional integrin signaling. Disclosures: No relevant conflicts of interest to declare.

A Mechanism For Switch Of Integrin Signaling Direction and a New Anti-Thrombotic Strategy Through Selective Outside-In Signaling Inhibition

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2295-2295
Author(s):  
Bo Shen ◽  
Xiaojuan Zhao ◽  
Kelly A O'Brien ◽  
Aleksandra Stojanovic-Terpo ◽  
Michael Keegan Delaney ◽  
...  
Keyword(s):  
Side Effect ◽  
Early Phase ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Late Phase ◽  
Integrin Signaling ◽  
Clot Retraction ◽  
Integrin Antagonists ◽  
Inside Out

Abstract Antagonists of platelet integrin alphaIIbbeta3 are potent anti-thrombotics due to critical roles of integrins in thrombosis. However, integrins are also important in hemostasis, and thus integrin antagonists have potentially life-threatening bleeding side effect. It would be ideal if we can develop integrin antagonists without bleeding side effect. Integrins transmit signals bidirectionally. Intracellular signals activate integrin alphaIIbbeta3, leading to talin-dependent integrin ligation, which is required for platelet adhesion and initial hemostatic thrombus formation. Integrin ligation in turn mediates Galpha13/Src-dependent outside-in signaling that stabilizes and amplify thrombi, which is crucial for occlusive arterial thrombosis. Here we show that talin and Galpha13 bind to mutually exclusive but distinct sites in integrin beta3, and their bindings are dynamically regulated during integrin signaling. The first talin binding wave mediates inside-out signaling and also “ligand-induced integrin activation”, but is not required for early phase outside-in signaling. Integrin ligation induces talin dissociation and Galpha13 binding, which selectively mediates early phase outside-in signaling. The second talin binding wave is associated with late phase outside-in signaling and clot retraction. Based on these findings, we have designed a selective inhibitor of Galpha13-integrin interaction, which specifically abolished outside-in signaling without affecting inside-out signaling and integrin ligation. Strikingly, this inhibitor potently inhibits occlusive thrombosis in vivo, but has no effect on tail bleeding time, in contrast to the current integrin antagonists. Thus, we have discovered a mechanism for switching the direction of integrin signaling and a new anti-thrombotic that does not cause bleeding. Disclosures: No elevant conflicts of interest to declare.

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

Blood ◽  
2012 ◽  
Vol 119 (14) ◽  
pp. 3352-3360 ◽  
Author(s):  
Meghna U. Naik ◽  
Timothy J. Stalker ◽  
Lawrence F. Brass ◽  
Ulhas P. Naik
Keyword(s):  
Platelet Activation ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Receptor Activation ◽  
Clot Retraction ◽  
Integrin Αiibβ3 ◽  
Genetic Ablation ◽  
Fibrinogen Receptor ◽  
Inside Out

Abstract Mounting 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.

Platelets Derived From Patients with Myelodysplastic Syndromes (MDS) Show Defective Platelet Aggregation and Integrin aIIbβ3 Receptor (GPIIb/IIIa) Signaling Caused by a Reduced Expression of Proteins Required for Inside-out Signaling.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 598-598
Author(s):  
Akos G. Czibere ◽  
Julia Fröbel ◽  
Sonja Hartwig ◽  
Ron-Patrick Cadeddu ◽  
Matthias Wilk ◽  
...  
Keyword(s):  
Platelet Count ◽  
Platelet Aggregation ◽  
Conflicts Of Interest ◽  
Active Form ◽  
Clinical Problem ◽  
Receptor Activation ◽  
In Vivo Model ◽  
Inside Out ◽  
Platelet Transfusions

Abstract Abstract 598 Thrombocytopenia is prevalent in up to 65% of patients with myelodysplastic syndrome (MDS) at the time of diagnosis and thrombocytopenic hemorrhage is a significant clinical problem that is often complicated by platelet aggregation defects. Little is known about the pathophysiology of this insufficient platelet function. Here, we delineate a reduced expression of critical platelet aggregation-related proteins by analyzing the platelet proteome of 7 patients with MDS and 7 normal donors. Patients' median platelet count was 60 × 10E9/L (range 37–109 × 109/L) and none of the patients examined had received prior anticoagulant treatment, chemotherapy or platelet transfusions. Differential 2-dimensional in-gel electrophoresis coupled with a time-of-flight Ultraflex-Tof/Tof mass spectrometer enabled the discovery of 120 differential protein spots. Among these, we identified a total of 35 proteins including 26 proteins that are integral part of the integrin aIIbβ3 receptor (GPIIb/IIIa, Fibrinogen receptor) signaling such as Talin-1 and Vinculin. In resting platelets the integrin aIIbβ3 receptor exhibits a low-affinity (inactive) state which is shifted to a high-affinity (active) state following inside-out activation. Talin-1 expression has been shown to be essential for this inside-out activation of the integrin aIIbβ3 receptor and consecutive platelet aggregation in an in-vivo model. We hypothesized that the reduced expression of Talin-1 and its co-factor Vinculin may inhibit activation of the integrin aIIbβ3 receptor and thereby contribute to the platelet aggregation defect seen in patients with MDS. Therefore, we performed further functional studies on integrin aIIbβ3 receptor activation and platelet spreading/aggregation with platelets derived from an independent cohort of 7 patients with MDS and 7 normal donors. In this new cohort, patients' median platelet count was 94 × 109/L (range 60–120 × 109/L) and again all patients had never received prior platelet transfusions or anti-coagulant treatment. When we looked at the surface expression of the integrin aIIbβ3 receptor on resting platelets by means of flow-cytometry, we did not detect any differences between platelets from patients with MDS and normal donors. Then, we activated platelets from normal donors and patients with MDS with 0.01U/μl and 0.001U/μl thrombin and measured binding of PAC-1, which is highly specific for detection of the active form of the integrin aIIbβ3 receptor. Here, we found a significantly lower shift from the inactive to the active form in platelets derived from patients with MDS dropping from 92.15% and 91.46% in normal donors to 41.97% and 48.45% (p = 0.01 and p = 0.006), respectively. We confirmed this suggested lack of adhesion and aggregation capacities in MDS platelets by confocal microscopy and single platelet imaging of washed platelets stimulated with 0.01U/μl thrombin which were adhered to immobilized fibrinogen. Consecutive platelet aggregation assays also revealed an insufficient response to stimuli like Collagen and ADP. Our findings provide for the first-time insight into the molecular pathology of defective platelet aggregation in MDS and suggest a mechanism of defective inside-out signaling caused by a reduced expression of proteins required for integrin aIIbβ3 receptor activation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Adaptor Protein CIN85 Supports Platelet Integrin αIIbβ3 outside-in Signaling and Thrombus Formation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 713-713
Author(s):  
Tanvir Khatlani ◽  
Subhashree Pradhan ◽  
Qi Da ◽  
K. Vinod Vijayan
Keyword(s):  
Shear Rate ◽  
Platelet Adhesion ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Adaptor Protein ◽  
Human Platelets ◽  
Protein Loss ◽  
Clot Retraction ◽  
Integrin Αiibβ3 ◽  
Inside Out

Abstract Activation of platelets in the setting of an atherosclerotic rupture contributes to thrombosis. Platelet responses at these sites are dependent on agonist induced inside-out and integrin αIIbβ3 mediated outside-in signaling processes, which in turn are regulated by protein kinases and phosphatases. Reversible tyrosine and/or serine/threonine phosphorylation dependent assembly of effector proteins propagate signaling downstream of platelet receptors. Adaptor proteins are key effectors in signal transmission because they are endowed with multiple structural domains, which enable them to engage with a wide variety of proteins in a spatial and temporal fashion to fine tune signaling. Emerging studies in the field suggest a potential for targeting integrin-induced outside-in signaling processes to attenuate thrombotic responses. During the course of our studies to understand how the catalytic subunit of protein phosphatase 2A (PP2Ac) regulates platelet integrin outside-in signaling, we previously identified a new complex of PP2Ac with an adaptor protein CIN85 (Cbl-interacting protein of 85kDa) in human platelets. Disruption of an endogenous PP2Ac-CIN85 complex with a cell permeable myristylated P3 synthetic peptide decreased platelet integrin αIIbβ3-dependent signaling functions. However, since the adaptor protein CIN85 has not been characterized in platelets, the contribution of CIN85 in integrin signaling is unknown. To explore the potential role of CIN85 in integrin function, we generated a platelet specific CIN85-/- mice by crossing the CIN85 flox/flox mice with a PF4Cre mice. Immunoblotting studies confirmed that platelets but not the non-megakaryocytic tissues from CIN85-/- mice lost the ~85kDa CIN85 protein. Loss of CIN85 did not significantly alter agonist-induced primary aggregation response, suggesting comparable inside-out signaling response. In contrast, integrin αIIbβ3 outside-in signaling responses such as spreading on immobilized fibrinogen and fibrin-mediated clot retraction was decreased in CIN85-/- platelets. Activation of Src that promotes integrin outside-in signaling was decreased in fibrinogen engaged CIN85-/- platelets. Perfusion of whole blood from the CIN85-/- mice on collagen at a shear rate of 1000 s-1 revealed significantly decreased platelet adhesion and thrombus formation. PLCγ2 is activated downstream of platelet collagen receptor engagement, and CIN85-/- platelets showed decreased PLCγ2 Tyr 529 phosphorylation. These studies indicate that the adaptor protein CIN85 supports platelet integrin outside in signaling functions. To extend these findings in human platelets, we disrupted CIN85-PP2A complex with the myristylated P3 peptide. P3 peptide but not the scrambled peptide significantly decreased human platelet adhesion and thrombus formation on collagen at a shear rate of 1000 s-1. Thus, loss of CIN85 in murine platelets or disruption of CIN85-PP2A complex in human platelets attenuates integrin outside-in signaling and thrombus formation. These studies suggest that CIN85 may represent a new potential anti-thrombotic target. Disclosures No relevant conflicts of interest to declare.

scholarly journals Syndecan Transmembrane Domain Specifically Regulates Downstream Signaling Events of the Transmembrane Receptor Cytoplasmic Domain

2021 ◽  
Vol 22 (15) ◽  
pp. 7918
Author(s):  
Jisun Hwang ◽  
Bohee Jang ◽  
Ayoung Kim ◽  
Yejin Lee ◽  
Joonha Lee ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Growth Factor Receptor ◽  
Cytoplasmic Domain ◽  
Nih3t3 Cells ◽  
C Elegans ◽  
Downstream Signaling ◽  
Downstream Effectors ◽  
Signaling Events

Despite the known importance of the transmembrane domain (TMD) of syndecan receptors in cell adhesion and signaling, the molecular basis for syndecan TMD function remains unknown. Using in vivo invertebrate models, we found that mammalian syndecan-2 rescued both the guidance defects in C. elegans hermaphrodite-specific neurons and the impaired development of the midline axons of Drosophila caused by the loss of endogenous syndecan. These compensatory effects, however, were reduced significantly when syndecan-2 dimerization-defective TMD mutants were introduced. To further investigate the role of the TMD, we generated a chimera, 2eTPC, comprising the TMD of syndecan-2 linked to the cytoplasmic domain of platelet-derived growth factor receptor (PDGFR). This chimera exhibited SDS-resistant dimer formation that was lost in the corresponding dimerization-defective syndecan-2 TMD mutant, 2eT(GL)PC. Moreover, 2eTPC specifically enhanced Tyr 579 and Tyr 857 phosphorylation in the PDGFR cytoplasmic domain, while the TMD mutant failed to support such phosphorylation. Finally, 2eTPC, but not 2eT(GL)PC, induced phosphorylation of Src and PI3 kinase (known downstream effectors of Tyr 579 phosphorylation) and promoted Src-mediated migration of NIH3T3 cells. Taken together, these data suggest that the TMD of a syndecan-2 specifically regulates receptor cytoplasmic domain function and subsequent downstream signaling events controlling cell behavior.

FUNDC2 regulates platelet activation through AKT/GSK-3β/cGMP axis

2018 ◽  
Vol 115 (11) ◽  
pp. 1672-1679 ◽  
Author(s):  
Qi Ma ◽  
Weilin Zhang ◽  
Chongzhuo Zhu ◽  
Junling Liu ◽  
Quan Chen
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Platelet Rich Plasma ◽  
Thrombus Formation ◽  
Mitochondrial Protein ◽  
Dependent Manner ◽  
Clot Retraction ◽  
Akt Phosphorylation ◽  
Gsk 3Β

Abstract Aims AKT kinase is vital for regulating signal transduction in platelet aggregation. We previously found that mitochondrial protein FUNDC2 mediates phosphoinositide 3-kinase (PI3K)/phosphatidylinositol-3,4,5-trisphosphate (PIP3)-dependent AKT phosphorylation and regulates platelet apoptosis. The aim of this study was to evaluate the role of FUNDC2 in platelet activation and aggregation. Methods and results We demonstrated that FUNDC2 deficiency diminished platelet aggregation in response to a variety of agonists, including adenosine 5′-diphosphate (ADP), collagen, ristocetin/VWF, and thrombin. Consistently, in vivo assays of tail bleeding and thrombus formation showed that FUNDC2-knockout mice displayed deficiency in haemostasis and thrombosis. Mechanistically, FUNDC2 deficiency impairs the phosphorylation of AKT and downstream GSK-3β in a PI3K-dependent manner. Moreover, cGMP also plays an important role in FUNDC2/AKT-mediated platelet activation. This FUNDC2/AKT/GSK-3β/cGMP axis also regulates clot retraction of platelet-rich plasma. Conclusion FUNDC2 positively regulates platelet functions via AKT/GSK-3β/cGMP signalling pathways, which provides new insight for platelet-related diseases.

Sign in / Sign up

Export Citation Format

Share Document