scholarly journals PTP-1B is an essential positive regulator of platelet integrin signaling

2005 ◽  
Vol 170 (5) ◽  
pp. 837-845 ◽  
Author(s):  
Elena Garcia Arias-Salgado ◽  
Fawaz Haj ◽  
Christophe Dubois ◽  
Barry Moran ◽  
Ana Kasirer-Friede ◽  
...  
Keyword(s):  
Thrombus Formation ◽  
Tyrosine Phosphatase ◽  
Clot Retraction ◽  
Positive Regulator ◽  
Normal Platelet ◽  
Fibrinogen Binding ◽  
Src Activation ◽  
Ptp 1B

Outside-in integrin αIIbβ3 signaling is required for normal platelet thrombus formation and is triggered by c-Src activation through an unknown mechanism. In this study, we demonstrate an essential role for protein–tyrosine phosphatase (PTP)–1B in this process. In resting platelets, c-Src forms a complex with αIIbβ3 and Csk, which phosphorylates c-Src tyrosine 529 to maintain c-Src autoinhibition. Fibrinogen binding to αIIbβ3 triggers PTP-1B recruitment to the αIIbβ3–c-Src–Csk complex in a manner that is dependent on c-Src and specific tyrosine (tyrosine 152 and 153) and proline (proline 309 and 310) residues in PTP-1B. Studies of PTP-1B–deficient mouse platelets indicate that PTP-1B is required for fibrinogen-dependent Csk dissociation from αIIbβ3, dephosphorylation of c-Src tyrosine 529, and c-Src activation. Furthermore, PTP-1B–deficient platelets are defective in outside-in αIIbβ3 signaling in vitro as manifested by poor spreading on fibrinogen and decreased clot retraction, and they exhibit ineffective Ca2+ signaling and thrombus formation in vivo. Thus, PTP-1B is an essential positive regulator of the initiation of outside-in αIIbβ3 signaling in platelets.

Akt2 Supports aIIbb3-Dependent Reorganization of the Actin Cytoskeleton in Platelets.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3565-3565
Author(s):  
Shelley August ◽  
Donna S. Woulfe
Keyword(s):  
Platelet Rich Plasma ◽  
Thrombus Formation ◽  
Active Form ◽  
Dense Granule ◽  
Clot Retraction ◽  
Akt Activation ◽  
Cytoskeletal Remodeling ◽  
Fibrinogen Binding

Abstract Akt is a serine-threonine kinase with well-described roles in growth and metabolism. Previous studies from our laboratory have shown that Akt also plays important roles in platelet function in vitro and in thrombus formation in vivo. Two isoforms are present in mouse platelets, Akt1 and Akt2, with Akt2 being the dominant isoform. Our previous studies have shown that platelets from Akt2−/− mice have marked defects in aggregation, a- and dense granule secretion, and fibrinogen binding. Each of these platelet functions depends in part on the function of the major platelet integrin, aIIbb3. However, whether Akt might regulate the function of aIIbb3 is still unknown. To determine whether Akt regulates aIIbb3-dependent signaling, first the rate of thrombin-initiated clot retraction was compared in platelet-rich plasma (PRP) from wild-type versus Akt2−/− mice. Akt2−/− platelets have a delay in the aIIbb3-dependent retraction of the fibrin clot compared to their wildtype counterparts. Akt2−/− platelets also have a delay in aIIbb3-dependent spreading on immobilized fibrinogen. However, adhesion to immobilized fibrinogen was normal in these platelets, suggesting that the spreading defect is due to a defect in outside-in signaling by the integrin, rather than in fibrinogen binding. Furthermore, unstimulated platelets expressing a constitutively active form of Akt spread more rapidly on fibrinogen-coated slides and generate more filopodial extensions than wildtype platelets, suggesting that Akt activation may be sufficient to induce outside-in signaling and /or cytoskeletal remodeling. Interestingly, integrin activation was not sufficient to induce Akt phoshorylation, suggesting that integrin activity is downstream rather than upstream of Akt activation. Taken together, these results suggest that integrin aIIbb3 is not necessary for Akt activation; however, Akt promotes outside-in signaling and cytoskeletal remodeling by aIIbb3.

Abstract 13598: The G-protein BetaGamma Subunits Regulate Platelet Function

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Ahmed Alarabi ◽  
Zubair Karim ◽  
Victoria Hinojos ◽  
Patricia A Lozano ◽  
Keziah Hernandez ◽  
...  
Keyword(s):  
G Protein ◽  
Platelet Function ◽  
Thrombus Formation ◽  
Human Platelets ◽  
Inhibitory Effects ◽  
Clot Retraction ◽  
Betagamma Subunits

Platelet activation involves tightly regulated processes to ensure a proper hemostasis response, but when unbalanced, can lead to pathological consequences such as thrombus formation. G-protein coupled receptors (GPCRs) regulate platelet function by interacting with and mediating the response to various physiological agonists. To this end, an essential mediator of GPCR signaling is the G protein Gαβγ heterotrimers, in which the βγ subunits are central players in downstream signaling pathways. While much is known regarding the role of the Gα subunit in platelet function, that of the βγ remains poorly understood. Therefore, we investigated the role of Gβγ subunits in platelet function using a Gβγ (small molecule) inhibitor, namely gallein. We observed that gallein inhibits platelet aggregation and secretion in response to agonist stimulation, in both mouse and human platelets. Furthermore, gallein also exerted inhibitory effects on integrin αIIbβ3 activation and clot retraction. Finally, gallein’s inhibitory effects manifested in vivo , as documented by its ability to modulate physiological hemostasis and delay thrombus formation. Taken together, our findings demonstrate, for the first time, that Gβγ directly regulates GPCR-dependent platelet function, in vitro and in vivo . Moreover, these data highlight Gβγ as a novel therapeutic target for managing thrombotic disorders.

scholarly journals Characteristics of Food Protein-Derived Antidiabetic Bioactive Peptides: A Literature Update

2021 ◽  
Vol 22 (17) ◽  
pp. 9508
Author(s):  
Nhung Thi Phuong Nong ◽  
Jue-Liang Hsu
Keyword(s):  
Bioactive Peptides ◽  
Protein Hydrolysate ◽  
Tyrosine Phosphatase ◽  
Food Protein ◽  
Dpp Iv ◽  
Glucosidase Inhibitors ◽  
Commercial Applications ◽  
Ptp 1B

Diabetes, a glucose metabolic disorder, is considered one of the biggest challenges associated with a complex complication of health crises in the modern lifestyle. Inhibition or reduction of the dipeptidyl peptidase IV (DPP-IV), alpha-glucosidase, and protein-tyrosine phosphatase 1B (PTP-1B) enzyme activities or expressions are notably considered as the promising therapeutic strategies for the management of type 2 diabetes (T2D). Various food protein-derived antidiabetic bioactive peptides have been isolated and verified. This review provides an overview of the DPP-IV, PTP-1B, and α-glucosidase inhibitors, and updates on the methods for the discovery of DPP-IV inhibitory peptides released from food-protein hydrolysate. The finding of novel bioactive peptides involves studies about the strategy of separation fractionation, the identification of peptide sequences, and the evaluation of peptide characteristics in vitro, in silico, in situ, and in vivo. The potential of bioactive peptides suggests useful applications in the prevention and management of diabetes. Furthermore, evidence of clinical studies is necessary for the validation of these peptides’ efficiencies before commercial applications.

scholarly journals The secreted tyrosine kinase VLK is essential for normal platelet activation and thrombus formation

2021 ◽  
Author(s):  
Leila Revollo ◽  
Glenn Merrill-Skoloff ◽  
Karen De Ceunynck ◽  
James R. Dilks ◽  
Mattia Bordoli ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Platelet Activation ◽  
Platelet Function ◽  
Secretory Pathway ◽  
Thrombus Formation ◽  
Extracellular Proteins ◽  
Normal Platelet

AbstractTyrosine phosphorylation of extracellular proteins is observed in cell cultures and in vivo, but little is known about the functional roles of tyrosine phosphorylation of extracellular proteins. Vertebrate Lonesome Kinase (VLK) is a broadly expressed secretory pathway tyrosine kinase present in platelet ɑ-granules. It is released from platelets upon activation and phosphorylates substrates extracellularly. Its role in platelet function, however, has not been previously studied. In human platelets, we identified phosphorylated tyrosines mapped to luminal or extracellular domains of transmembrane and secreted proteins implicated in the regulation of platelet activation. To determine the role of VLK in extracellular tyrosine phosphorylation and platelet function, we generated mice with a megakaryocyte/platelet-specific deficiency of VLK. Platelets from these mice are normal in abundance and morphology, but have dramatic changes in function both in vitro and in vivo. Resting and thrombin-stimulated VLK-deficient platelets demonstrate a significant decrease of several tyrosine phosphobands. Functional testing of VLK-deficient platelets shows decreased PAR4- and collagen-mediated platelet aggregation, but normal responses to ADP. Dense granule and α-granule release are reduced in these platelets. Furthermore, VLK-deficient platelets exhibit decreased PAR4-mediated Akt (S473) and Erk1/2(T202/Y204) phosphorylation, indicating altered proximal signaling. In vivo, mice lacking VLK in megakaryocytes/platelets demonstrate strongly reduced platelet accumulation and fibrin formation following laser-injury of cremaster arterioles compared to controls. These studies demonstrate that the secretory pathway tyrosine kinase VLK is critical for stimulus-dependent platelet activation and thrombus formation, providing the first evidence that a secreted protein kinase is required for normal platelet function.

scholarly journals The secreted tyrosine kinase VLK is essential for normal platelet activation and thrombus formation

Blood ◽  
2021 ◽  
Author(s):  
Leila Denise Revollo ◽  
Glenn Merrill-Skoloff ◽  
Karen De Ceunynck ◽  
James R Dilks ◽  
Shihui Guo ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Platelet Activation ◽  
Platelet Function ◽  
Secretory Pathway ◽  
Thrombus Formation ◽  
Extracellular Proteins ◽  
Normal Platelet

Tyrosine phosphorylation of extracellular proteins is observed in cell cultures and in vivo, but little is known about the functional roles of tyrosine phosphorylation of extracellular proteins. Vertebrate Lonesome Kinase (VLK) is a broadly expressed secretory pathway tyrosine kinase present in platelet ɑ-granules. It is released from platelets upon activation and phosphorylates substrates extracellularly. Its role in platelet function, however, has not been previously studied. In human platelets, we identified phosphorylated tyrosines mapped to luminal or extracellular domains of transmembrane and secreted proteins implicated in the regulation of platelet activation. To determine the role of VLK in extracellular tyrosine phosphorylation and platelet function, we generated mice with a megakaryocyte/platelet-specific deficiency of VLK. Platelets from these mice are normal in abundance and morphology, but have significant changes in function both in vitro and in vivo. Resting and thrombin-stimulated VLK-deficient platelets demonstrate a significant decrease of several tyrosine phosphobands. Functional testing of VLK-deficient platelets shows decreased PAR4- and collagen-mediated platelet aggregation, but normal responses to ADP. Dense granule and a-granule release are reduced in these platelets. Furthermore, VLK-deficient platelets exhibit decreased PAR4-mediated Akt (S473) and Erk1/2 (T202/Y204) phosphorylation, indicating altered proximal signaling. In vivo, mice lacking VLK in megakaryocytes/platelets demonstrate strongly reduced platelet accumulation and fibrin formation following laser-injury of cremaster arterioles compared to controls, but normal bleeding times. These studies demonstrate that the secretory pathway tyrosine kinase VLK is critical for stimulus-dependent platelet activation and thrombus formation, providing the first evidence that a secreted protein kinase is required for normal platelet function.

Functional Studies of Young Versus Old Platelets in a Patient With Chronic Thrombocytopenia

Blood ◽  
1971 ◽  
Vol 37 (2) ◽  
pp. 163-171 ◽  
Author(s):  
CHRISTINE A. JOHNSON ◽  
CHARLES F. ABILDGAARD ◽  
IRVING SCHULMAN
Keyword(s):  
Electron Microscopy ◽  
Platelet Adhesiveness ◽  
Plasma Transfusion ◽  
Clot Retraction ◽  
Platelet Factor ◽  
Functional Studies ◽  
Normal Platelet ◽  
Test Cells

Abstract Results of a comparative study of the functional capacity of young versus old platelets are presented. A girl with thrombopoietin deficiency, who predictably produced platelets in response to plasma transfusion, was used as platelet donor. Her platelets obtained 4 days after infusion were used as young test cells. Platelets obtained at 18 or 21 days after infusion were used as old cells. Young platelets were found to be associated with normal bleeding times, normal clot retraction, normal or increased platelet adhesiveness, normal aggregation to ADP and collagen, and normal platelet factor 3 availability. Old platelets were found to be associated with long bleeding times, decreased platelet adhesiveness in vivo and in vitro, and deficient platelet factor 3 availability. The clot retraction and aggregation to ADP and collagen of old platelets, however, was normal. No differences between young and old platelets were observed by electron microscopy.

scholarly journals Thymosin β4 is essential for thrombus formation by controlling the G-actin/F-actin equilibrium in platelets

Haematologica ◽  
2021 ◽  
Author(s):  
Inga Scheller ◽  
Sarah Beck ◽  
Vanessa Göb ◽  
Carina Gross ◽  
Raluca A. I. Neagoe ◽  
...  
Keyword(s):  
Thrombus Formation ◽  
Critical Role ◽  
Actin Dynamics ◽  
Thymosin Β4 ◽  
Clot Retraction ◽  
And Function ◽  
External Signals

Coordinated rearrangements of the actin cytoskeleton are pivotal for platelet biogenesis from megakaryocytes (MKs) but also orchestrate key functions of peripheral platelets in hemostasis and thrombosis, such as granule release, the formation of filopodia and lamellipodia, or clot retraction. Along with profilin (Pfn) 1, thymosin β4 (encoded by Tmsb4x) is one of the two main G-actin sequestering proteins within cells of higher eukaryotes, and its intracellular concentration is particularly high in cells that rapidly respond to external signals by increased motility, such as platelets. Here, we analyzed constitutive Tmsb4x knockout (KO) mice to investigate the functional role of the protein in platelet production and function. Thymosin β4 deficiency resulted in a macrothrombocytopenia with only mildly increased platelet volume and an unaltered platelet life span. MK numbers in the bone marrow (BM) and spleen were unaltered, however, Tmsb4x KO MKs showed defective proplatelet formation in vitro and in vivo. Thymosin β4 deficient platelets displayed markedly decreased G-actin levels and concomitantly increased F-actin levels resulting in accelerated spreading on fibrinogen and clot retraction. Moreover, Tmsb4x KO platelets showed activation defects and an impaired immunoreceptor tyrosine-based activation motif (ITAM) signaling downstream of the activating collagen receptor glycoprotein (GP) VI. These defects translated into impaired aggregate formation under flow, protection from occlusive arterial thrombus formation in vivo and increased tail bleeding times. In summary, these findings point to a critical role of thymosin β4 for actin dynamics during platelet biogenesis, platelet activation downstream of GPVI and thrombus stability.

Agonist-Induced Binding of the Regulatory Scaffold Protein Spinophilin to Protein Phosphatase-1 In Platelets

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2029-2029
Author(s):  
Andrew Sinnamon ◽  
Peisong Ma ◽  
Lawrence F. Brass
Keyword(s):  
Platelet Activation ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Tyrosine Phosphatase ◽  
Critical Role ◽  
Scaffold Protein ◽  
Western Blots ◽  
Co Precipitation

Abstract Abstract 2029 Platelet regulation plays a critical role in hemostasis. Underactivation can result in failure to stop bleeding, whereas inappropriate platelet activation can cause thrombus formation. The 130-kDa scaffold protein spinophilin (SPL) has recently been shown to play a role in preventing platelet overactivation by forming a complex with the proteins RGS10, RGS18, and the tyrosine phosphatase SHP-1. This complex dissociates when platelet are activated by thrombin or thromboxane A2 and evidence from spinophilin knockout mice suggests that this regulates platelet activation in vitro and in vivo. Spinophilin was originally isolated as a binding partner for the serine/threonine phosphatase, PP-1, in neurons. Here we asked whether PP-1 forms a complex with spinophilin in human platelets and, if so, whether the complex is affected by platelet activation. The approaches that we used to answer this question included Western blotting with antibodies to PP-1 and spinophilin, and co-precipitation studies looking for an association between spinophilin and PP-1. The results of the Western blots confirm the presence of PP-1 in platelets. The initial co-precipitation studies show that little, if any, PP-1 is associated with spinophilin in resting platelets, but there is a time-dependent increase in the SPL/PP-1 complex when platelets are activated with the PAR1 (thrombin receptor) activating peptide, SFLLRN. Thus it appears that within approximately the same time frame that the SPL/RGS/SHP-1 complex is decaying in activated platelets, the SPL/PP-1 complex is forming. Targets for PP1 have not been fully identified in platelets, but it is known that spinophilin localizes to the plasma membrane upon platelet activation. Since spinophilin is thought to direct PP1 targeting in neurons, it is reasonable to propose that it may be directing PP1 to targets in platelets in a similar manner. The studies described in this abstract were supported in part by a 2010 ASH Trainee Research Award to Andrew Sinnamon, who is a first year medical student at the University of Pennsylvania. Disclosures: No relevant conflicts of interest to declare.

scholarly journals An Antithrombotic Strategy by Targeting Phospholipase D in Human Platelets

2018 ◽  
Vol 7 (11) ◽  
pp. 440 ◽  
Author(s):  
Wan Lu ◽  
Chi Chung ◽  
Ray Chen ◽  
Li Huang ◽  
Li Lien ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Phospholipase D ◽  
Thrombus Formation ◽  
Human Platelets ◽  
Platelet Activity ◽  
Clot Retraction ◽  
First Time

Phospholipase D (PLD) is involved in many biological processes. PLD1 plays a crucial role in regulating the platelet activity of mice; however, the role of PLD in the platelet activation of humans remains unclear. Therefore, we investigated whether PLD is involved in the platelet activation of humans. Our data revealed that inhibition of PLD1 or PLD2 using pharmacological inhibitors effectively inhibits platelet aggregation in humans. However, previous studies have showed that PLD1 or PLD2 deletion did not affect mouse platelet aggregation in vitro, whereas only PLD1 deletion inhibited thrombus formation in vivo. Intriguingly, our data also showed that the pharmacological inhibition of PLD1 or PLD2 does not affect mouse platelet aggregation in vitro, whereas the inhibition of only PLD1 delayed thrombus formation in vivo. These findings indicate that PLD may play differential roles in humans and mice. In humans, PLD inhibition attenuates platelet activation, adhesion, spreading, and clot retraction. For the first time, we demonstrated that PLD1 and PLD2 are essential for platelet activation in humans, and PLD plays different roles in platelet function in humans and mice. Our findings also indicate that targeting PLD may provide a safe and alternative therapeutic approach for preventing thromboembolic disorders.

scholarly journals Ras-induced activation of Raf-1 is dependent on tyrosine phosphorylation.

1996 ◽  
Vol 16 (3) ◽  
pp. 1027-1034 ◽  
Author(s):  
T Jelinek ◽  
P Dent ◽  
T W Sturgill ◽  
M J Weber
Keyword(s):  
Tyrosine Phosphorylation ◽  
Plasma Membranes ◽  
Tyrosine Phosphatase ◽  
3T3 Cells ◽  
Level Of Activity ◽  
Active Pool ◽  
Ptp 1B ◽  
Nih 3T3

Although Rafs play a central role in signal transduction, the mechanism(s) by which they become activated is poorly understood. Raf-1 activation is dependent on the protein's ability to bind Ras, but Ras binding is insufficient to activate Raf-1 tyrosine phosphorylation to this Ras-induced activation, in the absence of an over-expressed tyrosine kinase. We demonstrate that Raf-1 purified form Sf9 cells coinfected with baculovirus Ras but not Src could be inactivated by protein tyrosine phosphatase PTP-1B. 14-3-3 and Hsp90 proteins blocked both the tyrosine dephosphorylation and inactivation of Raf-1, suggesting that Raf-1 activity is phosphotyrosine dependent. In Ras-transformed NIH 3T3 cells, a minority of Raf-1 protein was membrane associated, but essentially all Raf-1 activity and Raf-1 phosphotyrosine fractionated with plasma membranes. Thus, the tyrosine-phosphorylated and active pool of Raf-1 constitute a membrane-localized subfraction which could also be inactivated with PTP-1B. By contrast, B-Raf has aspartic acid residues at positions homologous to those of the phosphorylated tyrosines (at 340 and 341) of Raf-1 and displays a high basal level of activity. B-Raf was not detectably tyrosine phosphorylated, membrane localized, or further activated upon Ras transformation, even though B-Raf has been shown to bind to Ras in vitro. We conclude that tyrosine phosphorylation is an essential component of the mechanism by which Ras activates Raf-1 kinase activity and that steady-state activated Ras is insufficient to activate B-Raf in vivo.

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