Bruton’s Tyrosine Kinase Is Essential for Botrocetin/vWf-Induced Signaling and GPIb-Dependent Thrombus Formation In Vivo.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3903-3903
Author(s):  
Junling Liu ◽  
Malinda Fitzgerald ◽  
Michael C. Berndt ◽  
Carl W. Jackson ◽  
T. Kent Gartner
Keyword(s):  
Tyrosine Kinase ◽  
Ferric Chloride ◽  
Thrombus Formation ◽  
Bruton’S Tyrosine Kinase ◽  
Dependent Manner ◽  
Bruton's Tyrosine Kinase ◽  
Akt Phosphorylation ◽  
Arterial Thrombus ◽  
Txa2 Receptor

Abstract Botrocetin (bt) facilitated binding of von Willebrand factor (vWf) to the platelet membrane glycoprotein (GP) Ib-IX-V complex on platelets in suspension initiates a signaling cascade that causes αIIbβ3 activation and platelet aggregation. Previous work has demonstrated that bt/vWf-mediated agglutination activates αIIbβ3 and elicits ATP secretion in a thromboxane A2 (TxA2)-dependent manner. The signaling that results in TxA2 production is initiated by Lyn, enhanced by Src and propagated through Syk, SLP-76, PI3K, PLCγ2 and PKC. Here, we demonstrate that the signaling elicited by GPIb-mediated agglutination that results in TxA2 production is dependent on Bruton’s tyrosine kinase (Btk). The results demonstrate that Btk is downstream of Lyn, Syk, SLP-76 and PI3K, upstream of ERK1/2, PLCγ2 and PKC, and greatly enhances Akt phosphorylation. The relationship(s), if any between ERK1/2, PLCγ2 and PKC were not elucidated. The requirement for Btk and TxA2 receptor function in GPIb-dependent arterial thrombosis was confirmed in vivo by characterizing blood flow in ferric chloride treated mouse carotid arteries. These results demonstrate that the Btk family kinase, Tec, cannot provide the function(s) missing because of the absence of normal Btk function, and that Btk is essential for both bt/vWf-mediated agglutination-induced TxA2 production and GPIb-dependent stable arterial thrombus formation in vivo. Finally, consistent with the TxA2 receptor requirement for stable arterial thrombus formation in vivo, aspirin (at the appropriate concentration) prevented GPIb-dependent stable thrombus formation in vivo on ferric chloride damaged arterial endothelium.

scholarly journals Bruton tyrosine kinase is essential for botrocetin/VWF-induced signaling and GPIb-dependent thrombus formation in vivo

Blood ◽  
2006 ◽  
Vol 108 (8) ◽  
pp. 2596-2603 ◽  
Author(s):  
Junling Liu ◽  
Malinda E. Fitzgerald ◽  
Michael C. Berndt ◽  
Carl W. Jackson ◽  
T. Kent Gartner
Keyword(s):  
Tyrosine Kinase ◽  
Thrombus Formation ◽  
Dependent Manner ◽  
Akt Phosphorylation ◽  
Atp Secretion ◽  
Arterial Thrombus ◽  
Bruton Tyrosine Kinase ◽  
Von Willebrand ◽  
Willebrand Factor

AbstractBotrocetin (bt)-facilitated binding of von Willebrand factor (VWF) to the platelet membrane glycoprotein (GP) Ib-IX-V complex on platelets in suspension initiates a signaling cascade that causes αIIbβ3 activation and platelet aggregation. Previous work has demonstrated that bt/VWF-mediated agglutination activates αIIbβ3 and elicits ATP secretion in a thromboxane A2 (TxA2)-dependent manner. The signaling that results in TxA2 production was shown to be initiated by Lyn, enhanced by Src, and propagated through Syk, SLP-76, PI3K, PLCγ2, and PKC. Here, we demonstrate that the signaling elicited by GPIb-mediated agglutination that results in TxA2 production is dependent on Bruton tyrosine kinase (Btk). The results demonstrate that Btk is downstream of Lyn, Syk, SLP-76, and PI3K; upstream of ERK1/2, PLCγ2, and PKC; and greatly enhances Akt phosphorylation. The relationship(s), if any, between ERK1/2, PLCγ2, and PKC were not elucidated. The requirement for Btk and TxA2 receptor function in GPIb-dependent arterial thrombosis was confirmed in vivo by characterizing blood flow in ferric chloride-treated mouse carotid arteries. These results demonstrate that the Btk family kinase, Tec, cannot provide the function(s) missing because of the absence of Btk and that Btk is essential for both bt/VWF-mediated agglutination-induced TxA2 production and GPIb-dependent stable arterial thrombus formation in vivo.

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.

scholarly journals Hirudin interruption of heparin-resistant arterial thrombus formation in baboons

Blood ◽  
1991 ◽  
Vol 77 (5) ◽  
pp. 1006-1012 ◽  
Author(s):  
AB Kelly ◽  
UM Marzec ◽  
W Krupski ◽  
A Bass ◽  
Y Cadroy ◽  
...  
Keyword(s):  
Bleeding Time ◽  
Thrombus Formation ◽  
Dependent Manner ◽  
Fibrin Deposition ◽  
Recombinant Hirudin ◽  
Arterial Thrombus ◽  
Dependent Inhibition ◽  
Antithrombotic Effects ◽  
Dose Dependent

Abstract To determine the role of thrombin in high blood flow, platelet- dependent thrombotic and hemostatic processes we measured the relative antithrombotic and antihemostatic effects in baboons of hirudin, a highly potent and specific antithrombin, and compared the effects of heparin, an antithrombin III-dependent inhibitor of thrombin. Thrombus formation was determined in vivo using three relevant models (homologous endarterectomized aorta, collagen-coated tubing, and Dacron vascular graft) by measuring: (1) platelet deposition, using gamma camera imaging of 111In-platelets; (2) fibrin deposition, as assessed by the incorporation of circulating 125I-fibrinogen; and (3) occlusion. The continuous intravenous infusion of 1, 5, and 20 nmol/kg per minute of recombinant hirudin (desulfatohirudin) maintained constant plasma levels of 0.16 +/- 0.03, 0.79 +/- 0.44, and 3.3 +/- 0.77 mumol/mL, respectively. Hirudin interrupted platelet and fibrin deposition in a dose-dependent manner that was profound at the highest dose for all three thrombogenic surfaces and significant at the lowest dose for thrombus formation on endarterectomized aorta. Thrombotic occlusion was prevented by all doses studied. In contrast, heparin did not inhibit either platelet or fibrin deposition when administered at a dose that maximally prolonged clotting times (100 U/kg) (P greater than .1), and only intermediate effects were produced at 10-fold that dose (1,000 U/kg). Moreover, heparin did not prevent occlusion of the test segments. Hirudin inhibited platelet hemostatic function in concert with its antithrombotic effects (bleeding times were prolonged by the intermediate and higher doses). By comparison, intravenous heparin failed to affect the bleeding time at the 100 U/kg dose (P greater than .5), and only minimally prolonged the bleeding time at the 1,000 U/kg dose (P less than .05). We conclude that platelet-dependent thrombotic and hemostatic processes are thrombin-mediated and that the biologic antithrombin hirudin produces a potent, dose-dependent inhibition of arterial thrombus formation that greatly exceeds the minimal antithrombotic effects produced by heparin.

scholarly journals Discovery of Tricyclic Pyranochromenone as Novel Bruton’s Tyrosine Kinase Inhibitors with In Vivo Antirheumatic Activity

2020 ◽  
Vol 21 (21) ◽  
pp. 7919
Author(s):  
Hyewon Cho ◽  
Eun Lee ◽  
Hye Ah Kwon ◽  
Lee Seul ◽  
Hui-Jeon Jeon ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Kinase Inhibitors ◽  
Docking Study ◽  
Bruton’S Tyrosine Kinase ◽  
Collagen Induced Arthritis ◽  
Bruton's Tyrosine Kinase ◽  
Ic50 Values ◽  
Novel Scaffold ◽  
Btk Inhibitors

Bruton’s tyrosine kinase (BTK) is an attractive target for treating patients with B cell malignancies and autoimmune diseases. Many BTK inhibitors have been identified; however, like other kinase inhibitors, they lack diversity in their core structures. Therefore, it is important to secure a novel scaffold that occupies the adenine-binding site of BTK. We screened an in-house library of natural products and their analogs via a biochemical assay to identify a novel scaffold for targeting BTK. A pyranochromenone scaffold, derived from a natural active component decursin, was found to be effective at targeting BTK and was selected for further optimization. A series of pyranochromenone analogs was synthesized through the modification of pyranochromenone at the C7 position. Pyranochromenone compounds with an electrophilic warhead exhibited promising BTK inhibitory activity, with IC50 values in the range of 0.5–0.9 µM. A docking study of the representative compound 8 provided a reasonable explanation for compound activity. Compound 8 demonstrated good selectivity over other associated kinases and decreased the production of proinflammatory cytokines in THP cells. Moreover, compound 8 presented significant in vivo efficacy in a murine model of collagen-induced arthritis.

scholarly journals Discovery of 5-Phenoxy-2-aminopyridine Derivatives as Potent and Selective Irreversible Inhibitors of Bruton’s Tyrosine Kinase

2020 ◽  
Vol 21 (21) ◽  
pp. 8006
Author(s):  
Eun Lee ◽  
Hyewon Cho ◽  
Da Kyung Lee ◽  
JuHyun Ha ◽  
Byeong Jo Choi ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Unmet Need ◽  
Cell Receptor ◽  
Bruton’S Tyrosine Kinase ◽  
Type Ii ◽  
Irreversible Inhibitor ◽  
Bruton's Tyrosine Kinase ◽  
Irreversible Inhibitors ◽  
Btk Inhibitors

As a member of the tyrosine protein kinase Tec (TEC) family, Bruton’s tyrosine kinase (BTK) is considered a promising therapeutic target due to its crucial roles in the B cell receptor (BCR) signaling pathway. Although many types of BTK inhibitors have been reported, there is an unmet need to achieve selective BTK inhibitors to reduce side effects. To obtain BTK selectivity and efficacy, we designed a novel series of type II BTK inhibitors which can occupy the allosteric pocket induced by the DFG-out conformation and introduced an electrophilic warhead for targeting Cys481. In this article, we have described the structure–activity relationships (SARs) leading to a novel series of potent and selective piperazine and tetrahydroisoquinoline linked 5-phenoxy-2-aminopyridine irreversible inhibitors of BTK. Compound 18g showed good potency and selectivity, and its biological activity was evaluated in hematological tumor cell lines. The in vivo efficacy of 18g was also tested in a Raji xenograft mouse model, and it significantly reduced tumor size, with 46.8% inhibition compared with vehicle. Therefore, we have presented the novel, potent, and selective irreversible inhibitor 18g as a type II BTK inhibitor.

scholarly journals Silencing Bruton's tyrosine kinase in alveolar neutrophils protects mice from LPS/immune complex-induced acute lung injury

2014 ◽  
Vol 307 (6) ◽  
pp. L435-L448 ◽  
Author(s):  
Agnieszka Krupa ◽  
Marek Fol ◽  
Moshiur Rahman ◽  
Karen Y. Stokes ◽  
Jon M. Florence ◽  
...  
Keyword(s):  
Acute Lung Injury ◽  
Lung Injury ◽  
Tyrosine Kinase ◽  
Immune Complex ◽  
Distress Syndrome ◽  
Bruton’S Tyrosine Kinase ◽  
Bruton's Tyrosine Kinase ◽  
Neutrophil Survival

Previous observations made by our laboratory indicate that Bruton's tyrosine kinase (Btk) may play an important role in the pathophysiology of local inflammation in acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). We have shown that there is cross talk between FcγRIIa and TLR4 in alveolar neutrophils from patients with ALI/ARDS and that Btk mediates the molecular cooperation between these two receptors. To study the function of Btk in vivo we have developed a unique two-hit model of ALI: LPS/immune complex (IC)-induced ALI. Furthermore, we conjugated F(ab)2 fragments of anti-neutrophil antibodies (Ly6G1A8) with specific siRNA for Btk to silence Btk specifically in alveolar neutrophils. It should be stressed that we are the first group to perform noninvasive transfections of neutrophils, both in vitro and in vivo. Importantly, our present findings indicate that silencing Btk in alveolar neutrophils has a dramatic protective effect in mice with LPS/IC-induced ALI, and that Btk regulates neutrophil survival and clearance of apoptotic neutrophils in this model. In conclusion, we put forward a hypothesis that Btk-targeted neutrophil specific therapy is a valid goal of research geared toward restoring homeostasis in lungs of patients with ALI/ARDS.

Phosphoinositide3-Kinase Independent Regulation of Bruton’s Tyrosine Kinase in Platelet Immunoreceptor Signaling.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3536-3536
Author(s):  
Naohide Watanabe ◽  
Hideaki Nakajima ◽  
Atsushi Oda ◽  
Yasuo Ikeda ◽  
Makoto Handa
Keyword(s):  
Tyrosine Kinase ◽  
Gene Knockout ◽  
Antigen Receptor ◽  
Cellular Responses ◽  
Bruton’S Tyrosine Kinase ◽  
Dependent Manner ◽  
Ph Domain ◽  
Bruton's Tyrosine Kinase ◽  
Cell Antigen Receptor ◽  
Cell Antigen

Abstract Phosphoinositide 3-kinase (PI3K)-dependent activation of Bruton’s tyrosine kinase (Btk) is an indispensable step of B cell antigen receptor (BCR)-mediated signaling leading to cell development and function. Btk is a cytosolic tyrosine kinase and its recruitment to the plasma membrane is a necessary step for its function. In the BCR pathway, class 1A PI3K is though to play a major role in Btk recruitment by generating the D3 phosphoinositide as a docking site for the pleckstrin homology (PH) domain of this effecter kinase. This widely accepted hypothesis has been tested in platelets from gene knockout or mutant mice, since the cells utilize sets of transducers in collagen-induced GP VI signaling similar to those involved in immunoreceptor tyrosine-based activation motif-mediated signaling cascades activated by BCR and T cell antigen receptor (TCR) ligation. GP VI stimulation by collagen or collagen related peptide induces cellular responses including aggregation, granular secretion and adhesion, and Btk/phospholipase C (PLC) γ2 activation. As compared with control mice, these cellular responses and PLCγ2 tyrosine phosphorylation of either Btk or PI3K p85α−/− platelets were readily impaired, but the defect was greater in Btk−/− than p85α−/− platelets. Most strikingly, platelets from double-deficiency mice showed a most severely compromised phenotype implying the existence of a PI3K-independent pathway for Btk activation. Moreover, unlike B cells, as compared with Btk−/− platelets, only subtle functional defect was observed in X-linked immunodeficiency (Xid) platelets in which PI3K-dependent Btk activation is selectively lacking due to a naturally occurring point mutation of the gene encoding the PH domain of the kinase. In the TCR pathway, an adaptor complex formed by LAT, Gads and SLP-76 proteins that is membrane-bound via LAT palmitoylation readily recruits Itk, which is a counterpart Btk/Tec family kinase specific for TCR. Indeed, Btk was found to be associated with LAT/Gads/SLP-76 complex in platelets in a GP VI-stimulation dependent manner, and this phenomenon was unaffected by either PI3K defect or PI3K inhibitor. These results indicate that in platelet immunoreceptor signaling, Btk function is under control, at least in part, by a mechanism independent of PI3K engagement.

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