scholarly journals Integrin α2β1 mediates outside-in regulation of platelet spreading on collagen through activation of Src kinases and PLCγ2

2003 ◽  
Vol 160 (5) ◽  
pp. 769-780 ◽  
Author(s):  
Osamu Inoue ◽  
Katsue Suzuki-Inoue ◽  
William L. Dean ◽  
Jon Frampton ◽  
Steve P. Watson
Keyword(s):  
Kinase Inhibitor ◽  
Thrombus Formation ◽  
Critical Role ◽  
Chain Complex ◽  
Minor Effect ◽  
Specific Sequence ◽  
Glycoprotein Vi ◽  
Intracellular Signals ◽  
Vessel Injury ◽  
Platelet Spreading

Collagen plays a critical role in hemostasis by promoting adhesion and activation of platelets at sites of vessel injury. In the present model of platelet–collagen interaction, adhesion is mediated via the inside-out regulation of integrin α2β1 and activation through the glycoprotein VI (GPVI)–Fc receptor (FcR) γ-chain complex. The present study extends this model by demonstrating that engagement of α2β1 by an integrin-specific sequence from within collagen or by collagen itself generates tyrosine kinase–based intracellular signals that lead to formation of filopodia and lamellipodia in the absence of the GPVI–FcR γ-chain complex. The same events do not occur in platelet suspensions. α2β1 activation of adherent platelets stimulates tyrosine phosphorylation of many of the proteins in the GPVI–FcR γ-chain cascade, including Src, Syk, SLP-76, and PLCγ2 as well as plasma membrane calcium ATPase and focal adhesion kinase. α2β1-mediated spreading is dramatically inhibited in the presence of the Src kinase inhibitor PP2 and in PLCγ2-deficient platelets. Spreading is abolished by chelation of intracellular Ca2+. Demonstration that adhesion of platelets to collagen via α2β1 generates intracellular signals provides a new insight into the mechanisms that control thrombus formation and may explain the unstable nature of β1-deficient thrombi and why loss of the GPVI–FcR γ-chain complex has a relatively minor effect on bleeding.

Tyrosine Kinase Inhibitor Sunitinib Delays Platelet-Induced Coagulation: Additive Effects of Aspirin

2021 ◽  
Author(s):  
Delia I. Fernández ◽  
Alicia Veninga ◽  
Bibian M. E. Tullemans ◽  
Constance C. F. M. J. Baaten ◽  
Linsey J. F. Peters ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitor ◽  
Cancer Patients ◽  
Kinase Inhibitor ◽  
Synergistic Effects ◽  
Thrombus Formation ◽  
Type I ◽  
Glycoprotein Vi ◽  
Fibrin Formation ◽  
Increased Risk

Abstract Background Sunitinib is a multitarget tyrosine kinase inhibitor (TKI) used for cancer treatment. In platelets, sunitinib affects collagen-induced activation under noncoagulating conditions. We investigated (1) the effects of sunitinib on thrombus formation induced by other TK-dependent receptors, and (2) the effects under coagulating conditions. Cardiovascular disease is a comorbidity in cancer patients, resulting in possible aspirin treatment. Sunitinib and aspirin are associated with increased bleeding risk, and therefore we also investigated (3) the synergistic effects of these compounds on thrombus and fibrin formation. Methods Blood or isolated platelets from healthy volunteers or cancer patients were incubated with sunitinib and/or aspirin or vehicle. Platelet activation was determined by TK phosphorylation, flow cytometry, changes in [Ca2+]i, aggregometry, and whole blood perfusion over multiple surfaces, including collagen with(out) tissue factor (TF) was performed. Results Sunitinib reduced thrombus formation and phosphatidylserine (PS) exposure under flow on collagen type I and III. Also, sunitinib inhibited glycoprotein VI-induced TK phosphorylation and Ca2+ elevation. Upon TF-triggered coagulation, sunitinib decreased PS exposure and fibrin formation. In blood from cancer patients more pronounced effects of sunitinib were observed in lung and pancreatic as compared to neuroglioblastoma and other cancer types. Compared to sunitinib alone, sunitinib plus aspirin further reduced platelet aggregation, thrombus formation, and PS exposure on collagen under flow with(out) coagulation. Conclusion Sunitinib suppresses collagen-induced procoagulant activity and delays fibrin formation, which was aggravated by aspirin. Therefore, we urge for awareness of the combined antiplatelet effects of TKIs with aspirin, as this may result in increased risk of bleeding.

Local delivery of soluble platelet collagen receptor glycoprotein VI inhibits thrombus formation in vivo

2006 ◽  
Vol 95 (05) ◽  
pp. 763-766 ◽  
Author(s):  
Andreas Bültmann ◽  
Christian Herdeg ◽  
Zhongmin Li ◽  
Götz Münch ◽  
Christine Baumgartner ◽  
...  
Keyword(s):  
Carotid Artery ◽  
Vascular Injury ◽  
Carotid Arteries ◽  
Thrombus Formation ◽  
Critical Role ◽  
Local Delivery ◽  
Computer Assisted ◽  
Glycoprotein Vi ◽  
Collagen Receptor

SummaryPlatelet-mediated thrombus formation at the site of vascular injury isa major trigger for thrombo-ischemic complications after coronary interventions. The platelet collagen receptor glycoprotein VI (GPVI) plays a critical role in the initiation of arterial thrombus formation. Endothelial denudation of the right carotid artery in rabbits was induced through balloon injury. Subsequently, local delivery of soluble, dimeric fusion protein of GPVI (GPVI-Fc) (n=7) or control Fc (n=7) at the site of vascular injury was performed with a modified double-balloon drugdelivery catheter.Thrombus area within the injured carotid artery was quantified using a computer-assisted image analysis and was used as index of thrombus formation.The extent of thrombus formation was significantly reduced in GPVI-Fc- compared with control Fc-treated carotid arteries (relative thrombus area, GPVI-Fc vs. Fc: 9.3 ± 4.2 vs. 2.3 ± 1.7, p<0.001). Local delivery of soluble GPVI resulted in reduced thrombus formation after catheter-induced vascular injury.These data suggest a selective pharmacological modulation of GPVI-collagen interactions to be important for controlling onset and progression of pathological arterial thrombosis, predominantly or even exclusively at sites of injured carotid arteries in the absence of systemic platelet therapy.

scholarly journals The fibronectin synergy site re-enforces cell adhesion and mediates a crosstalk between integrin classes

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Maria Benito-Jardón ◽  
Sarah Klapproth ◽  
Irene Gimeno-LLuch ◽  
Tobias Petzold ◽  
Mitasha Bharadwaj ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Thrombus Formation ◽  
Critical Role ◽  
Initial Contact ◽  
Αvβ3 Integrin ◽  
Rgd Motif ◽  
Cell Coverage ◽  
Vessel Injury ◽  
Close Proximity ◽  
External Forces

Fibronectin (FN), a major extracellular matrix component, enables integrin-mediated cell adhesion via binding of α5β1, αIIbβ3 and αv-class integrins to an RGD-motif. An additional linkage for α5 and αIIb is the synergy site located in close proximity to the RGD motif. We report that mice with a dysfunctional FN-synergy motif (Fn1syn/syn) suffer from surprisingly mild platelet adhesion and bleeding defects due to delayed thrombus formation after vessel injury. Additional loss of β3 integrins dramatically aggravates the bleedings and severely compromises smooth muscle cell coverage of the vasculature leading to embryonic lethality. Cell-based studies revealed that the synergy site is dispensable for the initial contact of α5β1 with the RGD, but essential to re-enforce the binding of α5β1/αIIbβ3 to FN. Our findings demonstrate a critical role for the FN synergy site when external forces exceed a certain threshold or when αvβ3 integrin levels decrease below a critical level.

scholarly journals Critical role of Src-Syk-PLCγ2 signaling in megakaryocyte migration and thrombopoiesis

Blood ◽  
2010 ◽  
Vol 116 (5) ◽  
pp. 793-800 ◽  
Author(s):  
Alexandra Mazharian ◽  
Steve G. Thomas ◽  
Tarvinder S. Dhanjal ◽  
Christopher D. Buckley ◽  
Steve P. Watson
Keyword(s):  
Kinase Inhibitor ◽  
Tyrosine Phosphatase ◽  
Critical Role ◽  
Src Family Kinases ◽  
Surface Glycoprotein ◽  
Platelet Recovery ◽  
Glycoprotein Vi ◽  
Syk Kinase ◽  
And Migration

Migration of megakaryocytes (MKs) from the proliferative osteoblastic niche to the capillary-rich vascular niche is essential for proplatelet formation and platelet release. In this study, we explore the role of surface glycoprotein receptors and signaling proteins in regulating MK migration and platelet recovery after immune-induced thrombocytopenia. We show that spreading and migration of mouse primary bone marrow–derived MKs on a fibronectin matrix are abolished by the Src family kinases inhibitor PP1, the Syk kinase inhibitor R406 and the integrin αIIbβ3 antagonist lotrafiban. We also demonstrate that these responses are inhibited in primary phospholipase C γ2 (PLCγ2)–deficient MKs. Conversely, MK spreading and migration were unaltered in the absence of the collagen receptor, the glycoprotein VI–FcRγ-chain complex. We previously reported a correlation between a defect in MK migration and platelet recovery in the absence of platelet endothelial cell adhesion molecule-1 and the tyrosine phosphatase CD148. This correlation also holds for mice deficient in PLCγ2. This study identifies a model in which integrin signaling via Src family kinases and Syk kinase to PLCγ2 is required for MK spreading, migration, and platelet formation.

scholarly journals HDAC6 controls the kinetics of platelet activation

Blood ◽  
2012 ◽  
Vol 120 (20) ◽  
pp. 4215-4218 ◽  
Author(s):  
Karin Sadoul ◽  
Jin Wang ◽  
Boubou Diagouraga ◽  
Anne-Laure Vitte ◽  
Thierry Buchou ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Thrombus Formation ◽  
Shape Change ◽  
Lysine Acetylation ◽  
Vessel Injury ◽  
Marginal Band ◽  
Platelet Spreading ◽  
Kinetics Of ◽  
Fine Tune ◽  
Shape Changes

Abstract HDAC6, a major cytoplasmic deacetylase, is shown here to fine-tune the kinetics of platelet activation, a process that must be precisely regulated to ensure hemostasis after blood vessel injury while preventing pathologic thrombus formation. The discoid shape of resting platelets in the circulation is maintained by several highly acetylated microtubules organized in a marginal band. During platelet activation, microtubules undergo major reorganizations, which contribute to the shape change of activating platelets. We show that, during these activation-induced shape changes, a dramatic HDAC6-mediated tubulin deacetylation takes place, followed by microtubule reacetylation in spread platelets. In addition, although HDAC6-controlled tubulin deacetylation is not required for platelet activation, the capacity of HDAC6 to prevent tubulin hyperacetylation influences the speed of platelet spreading. These results are particularly important in view of HDAC6 inhibitors being currently used in clinical trials and represent the first example of cell signaling by lysine acetylation in platelet biology.

Rac, a small guanosine triphosphate–binding protein, and p21-activated kinase are activated during platelet spreading on collagen-coated surfaces: roles of integrin α2β1

Blood ◽  
2001 ◽  
Vol 98 (13) ◽  
pp. 3708-3716 ◽  
Author(s):  
Katsue Suzuki-Inoue ◽  
Yutaka Yatomi ◽  
Naoki Asazuma ◽  
Mie Kainoh ◽  
Toshiaki Tanaka ◽  
...  
Keyword(s):  
Kinase Inhibitor ◽  
Shape Change ◽  
Rho Kinase ◽  
Pi3 Kinase ◽  
Glycoprotein Vi ◽  
The Difference ◽  
P21 Activated Kinase ◽  
Coated Surfaces ◽  
Platelet Spreading

Abstract In this study, the receptors and signals involved in collagen-induced platelet spreading were examined. It was found that platelet spreading on collagen (presenting a polygon shape with a number of filopodialike projections) was inhibited by the anti–integrin α2 antibody, suggesting the involvement of integrin α2β1 in this process. Studies with a glutathione-S-transferase fusion protein that binds specifically to activated Rac and in vitro p21-activated kinase (PAK) kinase assays revealed that Rac and PAK were activated during this collagen-activated process. Platelet spreading on collagen-coated surfaces was inhibited strongly by PP1 (a Src family kinase inhibitor) or weakly by wortmannin (a phosphatidylinositol 3-kinase [PI3-kinase] inhibitor) but not at all by Y-27632 (a Rho kinase inhibitor). The surfaces coated with anti–integrin α2β1antibodies also induced platelet spreading (presenting an almost complete round shape) and activation of Rac and PAK, although more slowly than collagen-coated surfaces. The antibody-induced responses were strongly inhibited by PP1 or wortmannin but not by Y-27632. The same concentration of Y-27632 inhibited collagen-induced shape change of platelets in suspension. These findings suggest that Rac and/or PAK activation, but not Rho, may play certain roles in platelet spreading via integrin α2β1 and that Src family kinases and PI3-kinase participate in these processes. Furthermore, the difference between spreading on collagen and the anti-integrin antibody suggests the involvement of other receptor(s) (in addition to the integrin α2β1) for collagen-induced spreading, the most likely candidate being glycoprotein VI.

scholarly journals RhoG Protein Regulates Glycoprotein VI-Fc Receptor γ-Chain Complex-mediated Platelet Activation and Thrombus Formation

2013 ◽  
Vol 288 (47) ◽  
pp. 34230-34238 ◽  
Author(s):  
Soochong Kim ◽  
Carol Dangelmaier ◽  
Dheeraj Bhavanasi ◽  
Shu Meng ◽  
Hong Wang ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Thrombus Formation ◽  
Chain Complex ◽  
Fc Receptor ◽  
Glycoprotein Vi

Thrombin overcomes the thrombosis defect associated with platelet GPVI/FcRγ deficiency

Blood ◽  
2006 ◽  
Vol 107 (11) ◽  
pp. 4346-4353 ◽  
Author(s):  
Pierre Mangin ◽  
Cindy L. Yap ◽  
Christelle Nonne ◽  
Sharelle A. Sturgeon ◽  
Isaac Goncalves ◽  
...  
Keyword(s):  
Vascular Injury ◽  
Thrombus Formation ◽  
Chain Complex ◽  
Concurrent Administration ◽  
Glycoprotein Vi ◽  
Thrombin Inhibition ◽  
Arterial Thrombus ◽  
Major Defect

AbstractFibrillar collagens are among the most potent activators of platelets and play an important role in the initiation of thrombosis. The glycoprotein VI (GPVI)/FcRγ-chain complex is a central collagen receptor and inhibitors of GPVI produce a major defect in arterial thrombogenesis. In this study we have examined arterial thrombus formation in mice lacking the GPVI/FcRγ-chain complex (FcRγ–/–). Using 3 distinct arterial thrombosis models involving deep vascular injury, we demonstrate that deficiency of GPVI/FcRγ is not associated with a major defect in arterial thrombus formation. In contrast, with milder vascular injury deficiency of GPVI/FcRγ was associated with a 30% reduction in thrombus growth. Analysis of FcRγ–/– platelets in vitro, using thrombin-dependent and -independent thrombosis models, demonstrated a major role for thrombin in overcoming the thrombosis defect associated with GPVI/FcRγ deficiency. Inhibition of thrombin in vivo produced a much greater defect in thrombus formation in mice lacking GPVI/FcRγ compared with normal controls. Similarly, thrombin inhibition produced a marked prolongation in bleeding time in FcRγ–/– mice relative to wild-type mice. Our studies define an important role for thrombin in overcoming the hemostatic and thrombotic defect associated with GPVI/FcRγ deficiency. Moreover, they raise the interesting possibility that the full antithrombotic potential of GPVI receptor antagonists may only be realized through the concurrent administration of anticoagulant agents.

scholarly journals Antithrombotic effects of targeting αIIbβ3 signaling in platelets

Blood ◽  
2009 ◽  
Vol 113 (15) ◽  
pp. 3585-3592 ◽  
Author(s):  
Ararat J. Ablooglu ◽  
Jian Kang ◽  
Brian G. Petrich ◽  
Mark H. Ginsberg ◽  
Sanford J. Shattil
Keyword(s):  
Tyrosine Phosphorylation ◽  
Adenosine Diphosphate ◽  
Spontaneous Bleeding ◽  
Glycoprotein Vi ◽  
Fibrinogen Binding ◽  
Vessel Injury ◽  
Antithrombotic Effects ◽  
Platelet Spreading

Abstract αIIbβ3 interaction with fibrinogen promotes Src-dependent platelet spreading in vitro. To determine the consequences of this outside-in signaling pathway in vivo, a “β3(Δ760-762)” knockin mouse was generated that lacked the 3 C-terminal β3 residues (arginine-glycine-threonine [RGT]) necessary for αIIbβ3 interaction with c-Src, but retained β3 residues necessary for talin-dependent fibrinogen binding. β3(Δ760-762) mice were compared with wild-type β3+/+ littermates, β3+/− heterozygotes, and knockin mice where β3 RGT was replaced by β1 C-terminal cysteine-glycine-lysine (EGK) to potentially enable signaling by Src kinases other than c-Src. Whereas β3+/+, β3+/− and β3/β1(EGK) platelets spread and underwent tyrosine phosphorylation normally on fibrinogen, β3(Δ760-762) platelets spread poorly and exhibited reduced tyrosine phosphorylation of c-Src substrates, including β3 (Tyr747). Unlike control mice, β3(Δ760-762) mice were protected from carotid artery thrombosis after vessel injury with FeCl3. Some β3(Δ760-762) mice exhibited prolonged tail bleeding times; however, none demonstrated spontaneous bleeding, excess bleeding after surgery, fecal blood loss, or anemia. Fibrinogen binding to β3(Δ760-762) platelets was normal in response to saturating concentrations of protease-activated receptor 4 or glycoprotein VI agonists, but responses to adenosine diphosphate were impaired. Thus, deletion of β3 RGT disrupts c-Src–mediated αIIbβ3 signaling and confers protection from arterial thrombosis. Consequently, targeting αIIbβ3 signaling may represent a feasible antithrombotic strategy.

scholarly journals Physical and Functional Association of the Src Family Kinases Fyn and Lyn with the Collagen Receptor Glycoprotein VI-Fc Receptor γ Chain Complex on Human Platelets

1998 ◽  
Vol 188 (2) ◽  
pp. 267-276 ◽  
Author(s):  
Yasuharu Ezumi ◽  
Keisuke Shindoh ◽  
Masaaki Tsuji ◽  
Hiroshi Takayama
Keyword(s):  
Tyrosine Phosphorylation ◽  
Platelet Activation ◽  
Critical Role ◽  
Specific Inhibitor ◽  
Chain Complex ◽  
Human Platelets ◽  
Fc Receptor ◽  
Cross Linking ◽  
Glycoprotein Vi ◽  
Collagen Receptor

We have previously shown that uncharacterized glycoprotein VI (GPVI), which is constitutively associated and coexpressed with Fc receptor γ chain (FcRγ) in human platelets, is essential for collagen-stimulated tyrosine phosphorylation of FcRγ, Syk, and phospholipase Cγ2 (PLCγ2), leading to platelet activation. Here we investigated involvement of the Src family in the proximal signals through the GPVI–FcRγ complex, using the snake venom convulxin from Crotalus durissus terrificus, which specifically recognizes GPVI and activates platelets through cross-linking GPVI. Convulxin-coupled beads precipitated the GPVI–FcRγ complex from platelet lysates. Collagen and convulxin induced tyrosine phosphorylation of FcRγ, Syk, and PLCγ2 and recruited tyrosine-phosphorylated Syk to the GPVI–FcRγ complex. Using coprecipitation methods with convulxin-coupled beads and antibodies against FcRγ and the Src family, we showed that Fyn and Lyn, but not Yes, Src, Fgr, Hck, and Lck, were physically associated with the GPVI–FcRγ complex irrespective of stimulation. Furthermore, Fyn was rapidly activated by collagen or cross-linking GPVI. The Src family–specific inhibitor PP1 dose-dependently inhibited collagen- or convulxin-induced tyrosine phosphorylation of proteins including FcRγ, Syk, and PLCγ2, accompanied by a loss of aggregation and ATP release reaction. These results indicate that the Src family plays a critical role in platelet activation via the collagen receptor GPVI–FcRγ complex.

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