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Cell Adhesion & Migration Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kcam20
Manipulating Integrin Signaling for Anti-thrombotic Benefits a
Kushal U Naik & Ulhas P Naik
a
a
Delaware Cardiovascular Research Center, Department of Biological Sciences, Department of Chemistry and Biochemistry, Delaware Biotechnology Institute, University of Delaware, Newark, DE. USA Accepted author version posted online: 31 Oct 2014.Published online: 28 Sep 2014.
Click for updates To cite this article: Kushal U Naik & Ulhas P Naik (2014): Manipulating Integrin Signaling for Anti-thrombotic Benefits, Cell Adhesion & Migration, DOI: 10.4161/19336918.2014.968497 To link to this article: http://dx.doi.org/10.4161/19336918.2014.968497
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Manipulating Integrin Signaling for Anti-thrombotic Benefits
Kushal U. Naik and Ulhas P. Naik
ip t
Delaware Cardiovascular Research Center, Department of Biological Sciences,
us
cr
University of Delaware, Newark, DE. USA
an
Address correspondence to Ulhas P. Naik, email, [email protected]
ce
pt
ed
anti-thrombotic drug.
M
Keywords: Integrin IIb3, platelet, G13, talin, outside-in signaling, inside-out signaling,
Ac
Downloaded by [University of Nebraska, Lincoln] at 18:28 08 April 2015
Department of Chemistry and Biochemistry, Delaware Biotechnology Institute,
1
Blood platelets play an important role during physiological hemostasis and pathological thrombosis1. On unactivated platelets, integrin IIb3, the platelet fibrinogen (Fg) receptor, is in a low-affinity state, unable to bind soluble Fg. When platelet activation by physiological agonists such as collagen, thrombin, or ADP, occurs, a cascade of signaling
ip t
events induces a conformational change in the extracellular domains of IIb3. This
cr
inside-out signaling2. Once a ligand binds to an integrin receptor, a signal is once again
us
transmitted, this time from the extracellular domain of IIb3 through the transmembrane domain to the cytoplasmic domain, a phenomenon known as outside-in signaling2. The
an
outside-in signaling controls the process of platelet spreading and clot retraction, which
through integrin IIb33.
M
requires a significant contractile force between the platelet cytoskeleton and fibrin
ed
Most platelet agonists such as thrombin, ADP, epinephrine, and thromboxane A2 induce inside-out signaling through guanine nucleotide-binding protein (G-protein)
pt
coupled receptors (GPCRs), which activate heterotrimetric G-proteins consisting of G,
ce
G and G subunits4. Platelets express nine G subunits; among them Gi, Gq, G12, G13, and Gs are known to be essential mediators of hemostasis and thrombosis5. Activation of platelets through these GPCRs leads to activation of platelet integrin IIb3, an essential
Ac
Downloaded by [University of Nebraska, Lincoln] at 18:28 08 April 2015
converts it into a high-affinity state capable of binding Fg, through a process known as
common step in platelet aggregation leading to hemostasis and thrombosis. Antagonists of platelet integrin IIb3 are potent anti-thrombotic drugs, but they can cause bleeding in patients, which can be life threatening6,7. Recently it has been shown that G13 is essential for outside-in signaling through integrin IIb38,9. Shen et al, in a series of outstanding
2
experiments, now describe the discovery of a novel anti-thrombotic drug that does not cause bleeding10. Building on their previous finding that G13 is an essential regulator of integrin outside-in signaling9, Shen et al found that G13 binds to a conserved EXE motif of the
ip t
subunit of major platelet integrins, as indicated by the ability of G13 to bind integrin 1,
cr
are important for G13-binding as indicated by testing with various mutants and wild-type
us
3. Since the EXE motif is located in a talin-binding region of 3, using overexpression of the talin head domain (THD), which is sufficient to bind integrin, or G13, and co-
an
immunoprecipitation with 3, it was shown that they are mutually exclusive in 3 binding.
M
Additionally, involvement of a distinct temporal factor to the binding of talin and G13 to integrin signaling was unveiled. It was determined that the transition from the talin-bound
ed
to G13-bound state in IIb3 is initiated by macromolecular ligand binding to the integrin. These studies suggested that talin and G13 selectively mediate inside-out and outside-in
pt
signaling respectively, due to their opposing waves of binding to 3. This was confirmed
ce
using talin knockout platelets, which demonstrate defective aggregation to ADP, but could be corrected with manganese or integrin-activating antibody LIBS6, both of which independently activate integrins in the absence of talin-dependent inside-out signaling.
Ac
Downloaded by [University of Nebraska, Lincoln] at 18:28 08 April 2015
2, and 3, but not 8, which lacks this motif10. The first and third Glu in the EXE motif
When Fg changes conformation, whether due to immobilization or converting to fibrin, it is currently believed that it can interact with integrin independently of inside-out signaling. This is due to ligand-induced integrin activation caused by the contact of the integrin recognition sequence RGD of the ligand with the integrin. Interestingly, it was noted that there was defective adhesion of resting talin-knockout or -knockdown platelets 3
to immobilized Fg, which was rescued fully by adding manganese or LIBS6. This indicates that the importance of talin in resting platelet adhesion to fibrinogen is due to its role in integrin activation induced by ligands. Since platelet spreading occurs in the absence of talin after artificial activation of integrin, it indicates that talin is not required
cr
Shen et al next evaluated whether G13 binding to the EXE motif of 3 selectively
us
mediates outside-in signaling without perturbing talin-dependent integrin activation function10. Wild-type and AAA mutant 3-transfected 3 knockout mouse bone marrow
an
stem cells were transplanted into irradiated mice from the same background. Recipient mouse platelets expressed similar levels of the wild-type or AAA mutant. The 3
M
interaction with G13 was inhibited by the AAA mutation, but the interaction with talin was unaffected, as was the agonist-induced soluble Fg binding. This shows that the EXE
ed
is not necessary for talin-dependent inside-out signaling. On the other hand, 3-AAA mutant platelets showed defects when spreading on immobilized Fg. The results suggest
pt
that G13-binding deficiency in 3 causes a selective defect in platelet spreading and
ce
integrin outside-in signaling. Similarly, G13 and fibrinogen binding defects were also noted in 3-AAA, -DED, and -QSE mutants expressed in CHO cells. However, there was
Ac
Downloaded by [University of Nebraska, Lincoln] at 18:28 08 April 2015
ip t
during outside-in signaling once its role in integrin activation is no longer a factor.
no observed negative effect on THD binding. Additionally, cells expressing 3-AAA
mutant demonstrated defects in integrin-dependent activation of c-Src as seen by Y416 phosphorylation, as well as transient inhibition of Rho-A during cell spreading, both of which are important elements of outside-in signaling. This data in conjunction with previous studies identifying 3 sequences that mediate talin binding, suggest that G13
4
and talin serve as a molecular switch temporally controlling the direction of integrin signaling by interacting with distinct recognition sequences in the cytoplasmic domain of 3 . The particular role that the EXE motif seems to play in outside-in signaling led to
3
peptides:
mP5,
mP6,
and
mP1310.
The
inhibition
of
co-
cr
immunoprecipitation of G13 and 3 indicated that only the minimal EEERA sequence is
us
necessary to bind G13, whereas mP13 inhibited both talin and G13 binding to the
an
integrin 3. mP6 had no observed effect on agonist-induced Fg binding to platelets or adhesion of resting platelets to immobilized Fg. Platelet-dependent clot retraction,
M
however, was accelerated by mP6. It can be inferred from these data that mP6, which is an EXE-based inhibitor, inhibits the early phase of outside-in signaling selectively
ed
without any effect on integrin activation or clot retraction, which are both associated with talin. On the other hand, mP13 showed inhibition of both outside-in and inside-out
pt
signaling, as well as platelet adhesion, clot retraction, and Fg binding. Thus, while mP6
ce
interferes selectively with the early phase of outside-in signaling, mP13 affects all integrin signaling phases. Interestingly, the second wave of thrombin-induced platelet aggregation in vitro was inhibited by mP6. It would have been interesting to see the effect
Ac
Downloaded by [University of Nebraska, Lincoln] at 18:28 08 April 2015
containing
ip t
a designing of selective inhibitors of such, including several myristoylated EXE motif-
of mP13 on platelet aggregation induced by thrombin. Would it actually block platelet aggregation completely as envisioned? When injected into mice, mP6 proved to be an effective inhibitor of thrombosis as assessed by laser-induced cremaster arteriole injury as well as FeCl3-induced carotid artery injury, two well-established assays for in vivo thrombosis. Interestingly, mP6 had no effect on tail-bleeding time, a measure of 5
hemostatic functions. When compared to integrilin, a commonly-used integrin antagonist, it appears that mP6 is a potent anti-thrombotic agent with fewer adverse effects10. In summation, the authors unveil a novel molecular switch affecting integrin signaling. It is mediated by talin and G13 binding in opposing waves to nearby
ip t
sequences in the cytoplasmic domain of 3. The authors noted that it is possible to
cr
made use of this observation to design a potent integrin antagonist that can prevent
us
thrombosis without the bleeding complications present in current anti-thrombotics (Figure 1). There exists a potential for this knowledge to be used in clinical settings in the
an
realm of anti-integrin and -thrombotic therapies.
ce
pt
ed
M
References
Ac
Downloaded by [University of Nebraska, Lincoln] at 18:28 08 April 2015
selectively inhibit outside-in signaling without disrupting integrin ligand binding. They
6
1. Packham MA. Role of platelets in thrombosis and hemostasis. Can J Physiol Pharmacol. 1994;72(3):278‐284. 2. Shattil SJ. Signaling through platelet integrin alpha IIb beta 3: inside‐out, outside‐ in, and sideways. Thromb Haemost. 1999;82(2):318‐325.
ip t
3. Li Z, Delaney MK, O'Brien KA, Du X. Signaling during platelet adhesion and
cr
4. Amen S, Braun OO, Bengtsson A, Erlinge D. Gene expression profiling for the
us
identification of G‐protein coupled receptors in human platelets. Thromb Res. 2008;122(1):47‐57.
an
5. Offermanns S. Activation of platelet function through G protein‐coupled
M
receptors. Circ Res. 2006;99(12):1293‐1304.
6. Coller BS. Anti‐GPIIb/IIIa drugs: current strategies and future directions. Thromb
ed
Haemost. 2001;86(1):427‐443.
7. Serebruany VL, Malinin AI, Eisert RM, Sane DC. Risk of bleeding complications
pt
with antiplatelet agents: meta‐analysis of 338,191 patients enrolled in 50
ce
randomized controlled trials. Am J Hematol. 2004;75(1):40‐47. 8. Chari‐Turaga R, Naik UP. Integrin alphaIIbbeta3: a novel effector of Galpha13. Cell Adh Migr. 2011;5(1):4‐5.
Ac
Downloaded by [University of Nebraska, Lincoln] at 18:28 08 April 2015
activation. Arterioscler Thromb Vasc Biol. 2010;30(12):2341‐2349.
9. Gong H, Shen B, Flevaris P, et al. G protein subunit Galpha13 binds to integrin alphaIIbbeta3
and
mediates
integrin
"outside‐in"
signaling.
2010;327(5963):340‐343. 10. Shen B, Zhao X, O'Brien KA, et al. A directional switch of integrin signist alling and a new anti‐thrombotic strategy. Nature. 2013;503(7474):131‐135. 7
Science.
ip t cr us an
signaaling leading g to integrin
M
platellets adhere to the expo osed subend dothelial maatrix proteinns and initiaate inside-ouut IIb
3
activ vation. Acti vated integrrin binds solluble bivalennt
ed
Fg to o form an aggregate, whiich is stabiliized by outsiide-in signalling, thus forming a largge throm mbus capablle of vessell occlusion. Inhibition of integrin activation aas in currennt
pt
integrrin antagoniists, such ass integrilin, blocks plateelet adhesionn as well ass aggregatioon
ce
poten ntially causiing bleeding g. On the other o hand, mP6 throuugh virtue oof selectivelly inhibiting outsidee-in signalin ng, allows in nitial platelett adhesion aand activatioon, but blockks throm mbus growth h, thus allowiing hemostaasis to occur,, but protectiing from throombosis.
Ac
Downloaded by [University of Nebraska, Lincoln] at 18:28 08 April 2015
Figurre 1. Schemaatic represen ntation of thrrombus grow wth after vaascular injuryy. Circulatinng
8
Cell Adhesion & Migration Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kcam20
Manipulating Integrin Signaling for Anti-thrombotic Benefits a
Kushal U Naik & Ulhas P Naik
a
a
Delaware Cardiovascular Research Center, Department of Biological Sciences, Department of Chemistry and Biochemistry, Delaware Biotechnology Institute, University of Delaware, Newark, DE. USA Accepted author version posted online: 31 Oct 2014.Published online: 28 Sep 2014.
Click for updates To cite this article: Kushal U Naik & Ulhas P Naik (2014): Manipulating Integrin Signaling for Anti-thrombotic Benefits, Cell Adhesion & Migration, DOI: 10.4161/19336918.2014.968497 To link to this article: http://dx.doi.org/10.4161/19336918.2014.968497
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Manipulating Integrin Signaling for Anti-thrombotic Benefits
Kushal U. Naik and Ulhas P. Naik
ip t
Delaware Cardiovascular Research Center, Department of Biological Sciences,
us
cr
University of Delaware, Newark, DE. USA
an
Address correspondence to Ulhas P. Naik, email, [email protected]
ce
pt
ed
anti-thrombotic drug.
M
Keywords: Integrin IIb3, platelet, G13, talin, outside-in signaling, inside-out signaling,
Ac
Downloaded by [University of Nebraska, Lincoln] at 18:28 08 April 2015
Department of Chemistry and Biochemistry, Delaware Biotechnology Institute,
1
Blood platelets play an important role during physiological hemostasis and pathological thrombosis1. On unactivated platelets, integrin IIb3, the platelet fibrinogen (Fg) receptor, is in a low-affinity state, unable to bind soluble Fg. When platelet activation by physiological agonists such as collagen, thrombin, or ADP, occurs, a cascade of signaling
ip t
events induces a conformational change in the extracellular domains of IIb3. This
cr
inside-out signaling2. Once a ligand binds to an integrin receptor, a signal is once again
us
transmitted, this time from the extracellular domain of IIb3 through the transmembrane domain to the cytoplasmic domain, a phenomenon known as outside-in signaling2. The
an
outside-in signaling controls the process of platelet spreading and clot retraction, which
through integrin IIb33.
M
requires a significant contractile force between the platelet cytoskeleton and fibrin
ed
Most platelet agonists such as thrombin, ADP, epinephrine, and thromboxane A2 induce inside-out signaling through guanine nucleotide-binding protein (G-protein)
pt
coupled receptors (GPCRs), which activate heterotrimetric G-proteins consisting of G,
ce
G and G subunits4. Platelets express nine G subunits; among them Gi, Gq, G12, G13, and Gs are known to be essential mediators of hemostasis and thrombosis5. Activation of platelets through these GPCRs leads to activation of platelet integrin IIb3, an essential
Ac
Downloaded by [University of Nebraska, Lincoln] at 18:28 08 April 2015
converts it into a high-affinity state capable of binding Fg, through a process known as
common step in platelet aggregation leading to hemostasis and thrombosis. Antagonists of platelet integrin IIb3 are potent anti-thrombotic drugs, but they can cause bleeding in patients, which can be life threatening6,7. Recently it has been shown that G13 is essential for outside-in signaling through integrin IIb38,9. Shen et al, in a series of outstanding
2
experiments, now describe the discovery of a novel anti-thrombotic drug that does not cause bleeding10. Building on their previous finding that G13 is an essential regulator of integrin outside-in signaling9, Shen et al found that G13 binds to a conserved EXE motif of the
ip t
subunit of major platelet integrins, as indicated by the ability of G13 to bind integrin 1,
cr
are important for G13-binding as indicated by testing with various mutants and wild-type
us
3. Since the EXE motif is located in a talin-binding region of 3, using overexpression of the talin head domain (THD), which is sufficient to bind integrin, or G13, and co-
an
immunoprecipitation with 3, it was shown that they are mutually exclusive in 3 binding.
M
Additionally, involvement of a distinct temporal factor to the binding of talin and G13 to integrin signaling was unveiled. It was determined that the transition from the talin-bound
ed
to G13-bound state in IIb3 is initiated by macromolecular ligand binding to the integrin. These studies suggested that talin and G13 selectively mediate inside-out and outside-in
pt
signaling respectively, due to their opposing waves of binding to 3. This was confirmed
ce
using talin knockout platelets, which demonstrate defective aggregation to ADP, but could be corrected with manganese or integrin-activating antibody LIBS6, both of which independently activate integrins in the absence of talin-dependent inside-out signaling.
Ac
Downloaded by [University of Nebraska, Lincoln] at 18:28 08 April 2015
2, and 3, but not 8, which lacks this motif10. The first and third Glu in the EXE motif
When Fg changes conformation, whether due to immobilization or converting to fibrin, it is currently believed that it can interact with integrin independently of inside-out signaling. This is due to ligand-induced integrin activation caused by the contact of the integrin recognition sequence RGD of the ligand with the integrin. Interestingly, it was noted that there was defective adhesion of resting talin-knockout or -knockdown platelets 3
to immobilized Fg, which was rescued fully by adding manganese or LIBS6. This indicates that the importance of talin in resting platelet adhesion to fibrinogen is due to its role in integrin activation induced by ligands. Since platelet spreading occurs in the absence of talin after artificial activation of integrin, it indicates that talin is not required
cr
Shen et al next evaluated whether G13 binding to the EXE motif of 3 selectively
us
mediates outside-in signaling without perturbing talin-dependent integrin activation function10. Wild-type and AAA mutant 3-transfected 3 knockout mouse bone marrow
an
stem cells were transplanted into irradiated mice from the same background. Recipient mouse platelets expressed similar levels of the wild-type or AAA mutant. The 3
M
interaction with G13 was inhibited by the AAA mutation, but the interaction with talin was unaffected, as was the agonist-induced soluble Fg binding. This shows that the EXE
ed
is not necessary for talin-dependent inside-out signaling. On the other hand, 3-AAA mutant platelets showed defects when spreading on immobilized Fg. The results suggest
pt
that G13-binding deficiency in 3 causes a selective defect in platelet spreading and
ce
integrin outside-in signaling. Similarly, G13 and fibrinogen binding defects were also noted in 3-AAA, -DED, and -QSE mutants expressed in CHO cells. However, there was
Ac
Downloaded by [University of Nebraska, Lincoln] at 18:28 08 April 2015
ip t
during outside-in signaling once its role in integrin activation is no longer a factor.
no observed negative effect on THD binding. Additionally, cells expressing 3-AAA
mutant demonstrated defects in integrin-dependent activation of c-Src as seen by Y416 phosphorylation, as well as transient inhibition of Rho-A during cell spreading, both of which are important elements of outside-in signaling. This data in conjunction with previous studies identifying 3 sequences that mediate talin binding, suggest that G13
4
and talin serve as a molecular switch temporally controlling the direction of integrin signaling by interacting with distinct recognition sequences in the cytoplasmic domain of 3 . The particular role that the EXE motif seems to play in outside-in signaling led to
3
peptides:
mP5,
mP6,
and
mP1310.
The
inhibition
of
co-
cr
immunoprecipitation of G13 and 3 indicated that only the minimal EEERA sequence is
us
necessary to bind G13, whereas mP13 inhibited both talin and G13 binding to the
an
integrin 3. mP6 had no observed effect on agonist-induced Fg binding to platelets or adhesion of resting platelets to immobilized Fg. Platelet-dependent clot retraction,
M
however, was accelerated by mP6. It can be inferred from these data that mP6, which is an EXE-based inhibitor, inhibits the early phase of outside-in signaling selectively
ed
without any effect on integrin activation or clot retraction, which are both associated with talin. On the other hand, mP13 showed inhibition of both outside-in and inside-out
pt
signaling, as well as platelet adhesion, clot retraction, and Fg binding. Thus, while mP6
ce
interferes selectively with the early phase of outside-in signaling, mP13 affects all integrin signaling phases. Interestingly, the second wave of thrombin-induced platelet aggregation in vitro was inhibited by mP6. It would have been interesting to see the effect
Ac
Downloaded by [University of Nebraska, Lincoln] at 18:28 08 April 2015
containing
ip t
a designing of selective inhibitors of such, including several myristoylated EXE motif-
of mP13 on platelet aggregation induced by thrombin. Would it actually block platelet aggregation completely as envisioned? When injected into mice, mP6 proved to be an effective inhibitor of thrombosis as assessed by laser-induced cremaster arteriole injury as well as FeCl3-induced carotid artery injury, two well-established assays for in vivo thrombosis. Interestingly, mP6 had no effect on tail-bleeding time, a measure of 5
hemostatic functions. When compared to integrilin, a commonly-used integrin antagonist, it appears that mP6 is a potent anti-thrombotic agent with fewer adverse effects10. In summation, the authors unveil a novel molecular switch affecting integrin signaling. It is mediated by talin and G13 binding in opposing waves to nearby
ip t
sequences in the cytoplasmic domain of 3. The authors noted that it is possible to
cr
made use of this observation to design a potent integrin antagonist that can prevent
us
thrombosis without the bleeding complications present in current anti-thrombotics (Figure 1). There exists a potential for this knowledge to be used in clinical settings in the
an
realm of anti-integrin and -thrombotic therapies.
ce
pt
ed
M
References
Ac
Downloaded by [University of Nebraska, Lincoln] at 18:28 08 April 2015
selectively inhibit outside-in signaling without disrupting integrin ligand binding. They
6
1. Packham MA. Role of platelets in thrombosis and hemostasis. Can J Physiol Pharmacol. 1994;72(3):278‐284. 2. Shattil SJ. Signaling through platelet integrin alpha IIb beta 3: inside‐out, outside‐ in, and sideways. Thromb Haemost. 1999;82(2):318‐325.
ip t
3. Li Z, Delaney MK, O'Brien KA, Du X. Signaling during platelet adhesion and
cr
4. Amen S, Braun OO, Bengtsson A, Erlinge D. Gene expression profiling for the
us
identification of G‐protein coupled receptors in human platelets. Thromb Res. 2008;122(1):47‐57.
an
5. Offermanns S. Activation of platelet function through G protein‐coupled
M
receptors. Circ Res. 2006;99(12):1293‐1304.
6. Coller BS. Anti‐GPIIb/IIIa drugs: current strategies and future directions. Thromb
ed
Haemost. 2001;86(1):427‐443.
7. Serebruany VL, Malinin AI, Eisert RM, Sane DC. Risk of bleeding complications
pt
with antiplatelet agents: meta‐analysis of 338,191 patients enrolled in 50
ce
randomized controlled trials. Am J Hematol. 2004;75(1):40‐47. 8. Chari‐Turaga R, Naik UP. Integrin alphaIIbbeta3: a novel effector of Galpha13. Cell Adh Migr. 2011;5(1):4‐5.
Ac
Downloaded by [University of Nebraska, Lincoln] at 18:28 08 April 2015
activation. Arterioscler Thromb Vasc Biol. 2010;30(12):2341‐2349.
9. Gong H, Shen B, Flevaris P, et al. G protein subunit Galpha13 binds to integrin alphaIIbbeta3
and
mediates
integrin
"outside‐in"
signaling.
2010;327(5963):340‐343. 10. Shen B, Zhao X, O'Brien KA, et al. A directional switch of integrin signist alling and a new anti‐thrombotic strategy. Nature. 2013;503(7474):131‐135. 7
Science.
ip t cr us an
signaaling leading g to integrin
M
platellets adhere to the expo osed subend dothelial maatrix proteinns and initiaate inside-ouut IIb
3
activ vation. Acti vated integrrin binds solluble bivalennt
ed
Fg to o form an aggregate, whiich is stabiliized by outsiide-in signalling, thus forming a largge throm mbus capablle of vessell occlusion. Inhibition of integrin activation aas in currennt
pt
integrrin antagoniists, such ass integrilin, blocks plateelet adhesionn as well ass aggregatioon
ce
poten ntially causiing bleeding g. On the other o hand, mP6 throuugh virtue oof selectivelly inhibiting outsidee-in signalin ng, allows in nitial platelett adhesion aand activatioon, but blockks throm mbus growth h, thus allowiing hemostaasis to occur,, but protectiing from throombosis.
Ac
Downloaded by [University of Nebraska, Lincoln] at 18:28 08 April 2015
Figurre 1. Schemaatic represen ntation of thrrombus grow wth after vaascular injuryy. Circulatinng
8
