Blood coagulation - GPCRs in platelet aggregation

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GPCRs in platelet aggregation

Platelets circulate in the blood and adhere at places of vascular injury to the vessel wall resulted in the formation of platelet plug that is responsible for primary hemostasis. Platelet activation is initiated by their interaction with adhesive macromolecules like Collagen and von Willebrand factor at the subendothelial surface inducing the recruitment of additional platelets via diffusible mediators [1].

These mediators include ADP, Thromboxane A2, ADP and Serotonin that are secreted or released from activated platelets as well as Thrombin produced on the surface of activated platelets. These platelet stimuli are commonly transduced via G protein-coupled receptors (GPCRs). Prostaglandin I2, in contrast to ADP, Thromboxane A2, Thrombin, ADP and Serotonin, inhibits platelet function [2].

Activation of G-protein alpha-13 by TBXA2R and Thrombin receptors (PAR1 and PAR4) stimulates activation of the small GTAase RhoA, likely through the activation of the Rho guanine nucleotide exchange factors ARHGEF1 (p115RhoGEF) and LARG [2], [3], [4]. RhoA activates ROCK that inhibits Myosin light chain phosphatase (MLCP) activity via phosphorylation of the MLCP (reg). Attenuation of dephosphorylation of the regulatory subunits of non-muscle Myosin II, MELC and MRLC, leads to formation of actomyosin fibrils [2], [5], [6].

ROCK kinases also phosphorylate LIMK1 and LIMK2, which in turn phosphorylate actin-modulating protein Cofilin followed by reorganization of Actin cytoskeletal [7], [8]. Cytoskeleton rearrangement results in platelet shape change that is necessary for proper platelet aggregation.

G-protein alpha-q is activated by ADP via P2Y1, Thromboxane A2 via TBXA2R, Serotonin via HTR2A, and Thrombin via PAR1 and PAR4 [1].

G-protein alpha-q activates PLC-beta2 and PLC-beta3, which stimulate the production of 1,2-Diacyglycerol and IP3, with the consequent release of Ca('2+) from the dense tubular system and stimulation of Protein kinase C, namely PKC-alpha [1], [9], [10].

PKC-alpha regulates platelet aggregation by mediating the secondary secretion of mediators (such as ADP, ADP and Serotonin, etc.) from dense granules, thereby promoting platelet stimulation and contributing to alpha-IIb/beta-3 integrin activation [10], [11], [12].

alpha-IIb/beta-3 integrin is the major platelet integrin. It is required for platelet interactions with plasma proteins (such as Fibrinogen (fibrin)) and is essential for platelet adhesion and aggregation. alpha-IIb/beta-3 integrin is a protein complex composed of ITGA2B and ITGB3 subunits. The process of transforming integrin into a competent receptor for Fibrinogen (fibrin) is the final step in platelet activation [12].

PKC-alpha has been shown to activate RAS-related protein RAP-1A that induces the formation of an "integrin activation complex" containing the RAP-1A effector, PREL1, and a cytoskeletal protein Talin. Talin binds to ITGB3 cytoplasmic domains and positively regulates alpha-IIb/beta-3 integrin affinity [13].

Calcium binding protein Calmyrin binds directly to ITGA2B and inhibits Talin binding to ITGB3 thereby limiting the extent of ligand binding to alpha-IIb/beta-3 integrin [14].

Another ITGB3 binding protein, NRIF3, increases the affinity state of alpha-IIb/beta-3 integrin [15].

G-protein alpha-i family, which is activated by PAR1, Purinergic receptor P2Y12 (another ADP receptor) and Alpha-2A adrenergic receptor (ADP receptor), inhibits the production of Cyclic AMP by Adenylate cyclase and stimulates Phosphoinositide-3-kinase (PI3K)-dependent pathways.

Stimulation of G-protein alpha-i family coupled receptors results in formation of free G-protein beta/gamma dimers that stimulate PI3K cat class IB (p110-gamma) and PI3K cat class IA (p110-beta) activity. Both these lipid-metabolizing enzymes have been shown to activate RAS-related protein RAP-1B that binds to alpha-IIb/beta-3 integrin and positively regulates its affinity [11], [16], [17], [18], [19], [20], [21]. Ca('2+) and 1,2-Diacyglycerol -regulated Rap1 exchange factor CALDAG-GEFI is also involved in the activation of RAP-1A and RAP-1B [22], [23].

Prostacyclin receptor, which inhibits platelet function, is coupled to G-protein alpha-s that stimulates Adenylate cyclase. High levels of cytosolic Cyclic AMP suppress platelet activation, in part by PKA-cat (cAMP-dependent)-mediated suppression of the IP3R1, which triggers Ca('2+) release from the dense tubular system [24].

Thereby, each of the agonists binds to GPCRs and regulates platelet functions. Activation of platelets by strong agonists (such as ADP, Thromboxane A2 and Thrombin) initiates rapid shape change, enhances secretion of platelet granule contents and triggers Fibrinogen (fibrin) binding to alpha-IIb/beta-3 integrin. Weak agonists (such as ADP and Serotonin) potentiate these signaling pathways. Fibrinogen (fibrin) can simultaneously bind two adjacent platelets. This binding initiates the process of platelet aggregation that can lead to formation of very large aggregates at sites of vascular injury. Platelets can also become activated under pathological conditions, e.g., in ruptured atherosclerotic plaques, leading to arterial thrombosis [9], [24], [25].



Objects list:

1,2-Diacyglycerol 1,2-Diacyglycerol Compound group
ADP Chemical IUPAC name [(2R,3S,4R,5R)-5-(6-aminopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphono hydrogen phosphate
ARHGEF1 (p115RhoGEF) Rho guanine nucleotide exchange factor 1
Actin cytoskeletal Actin cytoskeletal Protein group
Adenylate cyclase Adenylate cyclase Protein group
Alpha-2A adrenergic receptor Alpha-2A adrenergic receptor
CALDAG-GEFI RAS guanyl-releasing protein 2
Ca('2+) Chemical IUPAC name calcium(+2) cation
Calmyrin Calcium and integrin-binding protein 1
Cofilin Cofilin Protein group
Cyclic AMP Chemical IUPAC name (1S,6R,8R,9R)-8-(6-amino-8-bromopurin-9-yl)-3-hydroxy-3-oxo-2,4,7-trioxa-35-phosphabicyclo[4.3.0]nonan-9-ol
Fibrinogen (fibrin) Fibrinogen Complex
G-protein alpha-13 Guanine nucleotide-binding protein subunit alpha-13
G-protein alpha-i family G-protein alpha-i family Protein group
G-protein alpha-q Guanine nucleotide-binding protein G(q) subunit alpha
G-protein alpha-s Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
G-protein beta/gamma G-protein beta/gamma Group of complexes
HTR2A 5-hydroxytryptamine receptor 2A
IP3 Chemical IUPAC name [(1R,2S,3R,4R,5S,6R)-2,4,5-trihydroxy-3,6-diphosphonooxycyclohexyl] dihydrogen phosphate
IP3R1 Inositol 1,4,5-trisphosphate receptor type 1
ITGA2B Integrin alpha-IIb
ITGB3 Integrin beta-3
LARG Rho guanine nucleotide exchange factor 12
LIMK1 LIM domain kinase 1
LIMK2 LIM domain kinase 2
MELC MELC Protein group
MLCP (reg) Protein phosphatase 1 regulatory subunit 12A
MRLC MRLC Protein group
Myosin II Myosin II Group of complexes
NRIF3 Centromere protein R
P2Y1 P2Y purinoceptor 1
P2Y12 P2Y purinoceptor 12
PAR1 Proteinase-activated receptor 1
PAR4 Proteinase-activated receptor 4
PI3K cat class IA (p110-beta) Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit beta isoform
PI3K cat class IB (p110-gamma) Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gamma isoform
PKA-cat (cAMP-dependent) Protein kinase, cAMP-dependent, catalytic Protein group
PKC-alpha Protein kinase C alpha type
PLC-beta2 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase beta-2
PLC-beta3 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase beta-3
PREL1 Amyloid beta A4 precursor protein-binding family B member 1-interacting protein
Prostacyclin receptor Prostacyclin receptor
Prostaglandin I2 Chemical IUPAC name 5-[(3aR,4R,5R,6aS)-5-Hydroxy-4-[(E,3S)-3-hydroxyoct-1-enyl]-3,3a,4,5,6, 6a-hexahydrocyclopenta[d]furan-2-ylidene]pentanoic acid
RAP-1A Ras-related protein Rap-1A
RAP-1B Ras-related protein Rap-1b
ROCK Rho-kinase Protein group
RhoA Transforming protein RhoA
Serotonin Chemical IUPAC name 3-(2-Amino-ethyl)-1H-indol-5-ol
TBXA2R Thromboxane A2 receptor
Talin Talin Protein group
Thrombin Prothrombin
Thromboxane A2 Chemical IUPAC name (Z)-7-[(1S,2S,3R,5S)-3-[(E,3S)-3-Hydroxyoct-1-enyl]-4, 7-dioxabicyclo[3.1.1]heptan-2-yl]hept-5-enoic acid
alpha-IIb/beta-3 integrin alpha-IIb/beta-3 integrin Complex

References:

  1. Wettschureck N, Offermanns S
    Mammalian G proteins and their cell type specific functions. Physiological reviews 2005 Oct;85(4):1159-204
  2. Smyth SS, Jennings JL
    PPARgamma agonists: a new strategy for antithrombotic therapy. Journal of thrombosis and haemostasis : JTH 2005 Oct;3(10):2147-8
  3. Kim SD, Sung HJ, Park SK, Kim TW, Park SC, Kim SK, Cho JY, Rhee MH
    The expression patterns of RGS transcripts in platelets. Platelets 2006 Nov;17(7):493-7
  4. Huang JS, Dong L, Kozasa T, Le Breton GC
    Signaling through G(alpha)13 switch region I is essential for protease-activated receptor 1-mediated human platelet shape change, aggregation, and secretion. The Journal of biological chemistry 2007 Apr 6;282(14):10210-22
  5. Somlyo AP, Somlyo AV
    Signal transduction by G-proteins, rho-kinase and protein phosphatase to smooth muscle and non-muscle myosin II. The Journal of physiology 2000 Jan 15;522 Pt 2:177-85
  6. Somlyo AP, Somlyo AV
    Ca2+ sensitivity of smooth muscle and nonmuscle myosin II: modulated by G proteins, kinases, and myosin phosphatase. Physiological reviews 2003 Oct;83(4):1325-58
  7. Amano T, Tanabe K, Eto T, Narumiya S, Mizuno K
    LIM-kinase 2 induces formation of stress fibres, focal adhesions and membrane blebs, dependent on its activation by Rho-associated kinase-catalysed phosphorylation at threonine-505. The Biochemical journal 2001 Feb 15;354(Pt 1):149-59
  8. Riento K, Ridley AJ
    Rocks: multifunctional kinases in cell behaviour. Nature reviews. Molecular cell biology 2003 Jun;4(6):446-56
  9. Abrams CS
    Intracellular signaling in platelets. Current opinion in hematology 2005 Sep;12(5):401-5
  10. Strehl A, Munnix IC, Kuijpers MJ, van der Meijden PE, Cosemans JM, Feijge MA, Nieswandt B, Heemskerk JW
    Dual role of platelet protein kinase C in thrombus formation: stimulation of pro-aggregatory and suppression of procoagulant activity in platelets. The Journal of biological chemistry 2007 Mar 9;282(10):7046-55
  11. Woulfe D, Jiang H, Mortensen R, Yang J, Brass LF
    Activation of Rap1B by G(i) family members in platelets. The Journal of biological chemistry 2002 Jun 28;277(26):23382-90
  12. Shattil SJ, Newman PJ
    Integrins: dynamic scaffolds for adhesion and signaling in platelets. Blood 2004 Sep 15;104(6):1606-15
  13. Han J, Lim CJ, Watanabe N, Soriani A, Ratnikov B, Calderwood DA, Puzon-McLaughlin W, Lafuente EM, Boussiotis VA, Shattil SJ, Ginsberg MH
    Reconstructing and deconstructing agonist-induced activation of integrin alphaIIbbeta3. Current biology : CB 2006 Sep 19;16(18):1796-806
  14. Leisner TM, Yuan W, DeNofrio JC, Liu J, Parise LV
    Tickling the tails: cytoplasmic domain proteins that regulate integrin alphaIIbbeta3 activation. Current opinion in hematology 2007 May;14(3):255-61
  15. Kashiwagi H, Schwartz MA, Eigenthaler M, Davis KA, Ginsberg MH, Shattil SJ
    Affinity modulation of platelet integrin alphaIIbbeta3 by beta3-endonexin, a selective binding partner of the beta3 integrin cytoplasmic tail. The Journal of cell biology 1997 Jun 16;137(6):1433-43
  16. Lova P, Paganini S, Sinigaglia F, Balduini C, Torti M
    A Gi-dependent pathway is required for activation of the small GTPase Rap1B in human platelets. The Journal of biological chemistry 2002 Apr 5;277(14):12009-15
  17. Bertoni A, Tadokoro S, Eto K, Pampori N, Parise LV, White GC, Shattil SJ
    Relationships between Rap1b, affinity modulation of integrin alpha IIbbeta 3, and the actin cytoskeleton. The Journal of biological chemistry 2002 Jul 12;277(28):25715-21
  18. Lova P, Paganini S, Hirsch E, Barberis L, Wymann M, Sinigaglia F, Balduini C, Torti M
    A selective role for phosphatidylinositol 3,4,5-trisphosphate in the Gi-dependent activation of platelet Rap1B. The Journal of biological chemistry 2003 Jan 3;278(1):131-8
  19. Jackson SP, Schoenwaelder SM, Goncalves I, Nesbitt WS, Yap CL, Wright CE, Kenche V, Anderson KE, Dopheide SM, Yuan Y, Sturgeon SA, Prabaharan H, Thompson PE, Smith GD, Shepherd PR, Daniele N, Kulkarni S, Abbott B, Saylik D, Jones C, Lu L, Giuliano S, Hughan SC, Angus JA, Robertson AD, Salem HH
    PI 3-kinase p110beta: a new target for antithrombotic therapy. Nature medicine 2005 May;11(5):507-14
  20. Sun DS, Lo SJ, Tsai WJ, Lin CH, Yu MS, Chen YF, Chang HH
    PI3-kinase is essential for ADP-stimulated integrin alpha(IIb)beta3-mediated platelet calcium oscillation, implications for P2Y receptor pathways in integrin alpha(IIb)beta3-initiated signaling cross-talks. Journal of biomedical science 2005 Dec;12(6):937-48
  21. Voss B, McLaughlin JN, Holinstat M, Zent R, Hamm HE
    PAR1, but not PAR4, activates human platelets through a Gi/o/phosphoinositide-3 kinase signaling axis. Molecular pharmacology 2007 May;71(5):1399-406
  22. Clyde-Smith J, Silins G, Gartside M, Grimmond S, Etheridge M, Apolloni A, Hayward N, Hancock JF
    Characterization of RasGRP2, a plasma membrane-targeted, dual specificity Ras/Rap exchange factor. The Journal of biological chemistry 2000 Oct 13;275(41):32260-7
  23. Eto K, Murphy R, Kerrigan SW, Bertoni A, Stuhlmann H, Nakano T, Leavitt AD, Shattil SJ
    Megakaryocytes derived from embryonic stem cells implicate CalDAG-GEFI in integrin signaling. Proceedings of the National Academy of Sciences of the United States of America 2002 Oct 1;99(20):12819-24
  24. Woulfe DS
    Platelet G protein-coupled receptors in hemostasis and thrombosis. Journal of thrombosis and haemostasis : JTH 2005 Oct;3(10):2193-200
  25. Gawaz M, Langer H, May AE
    Platelets in inflammation and atherogenesis. The Journal of clinical investigation 2005 Dec;115(12):3378-84

  1. Wettschureck N, Offermanns S
    Mammalian G proteins and their cell type specific functions. Physiological reviews 2005 Oct;85(4):1159-204
  2. Smyth SS, Jennings JL
    PPARgamma agonists: a new strategy for antithrombotic therapy. Journal of thrombosis and haemostasis : JTH 2005 Oct;3(10):2147-8
  3. Kim SD, Sung HJ, Park SK, Kim TW, Park SC, Kim SK, Cho JY, Rhee MH
    The expression patterns of RGS transcripts in platelets. Platelets 2006 Nov;17(7):493-7
  4. Huang JS, Dong L, Kozasa T, Le Breton GC
    Signaling through G(alpha)13 switch region I is essential for protease-activated receptor 1-mediated human platelet shape change, aggregation, and secretion. The Journal of biological chemistry 2007 Apr 6;282(14):10210-22
  5. Somlyo AP, Somlyo AV
    Signal transduction by G-proteins, rho-kinase and protein phosphatase to smooth muscle and non-muscle myosin II. The Journal of physiology 2000 Jan 15;522 Pt 2:177-85
  6. Somlyo AP, Somlyo AV
    Ca2+ sensitivity of smooth muscle and nonmuscle myosin II: modulated by G proteins, kinases, and myosin phosphatase. Physiological reviews 2003 Oct;83(4):1325-58
  7. Amano T, Tanabe K, Eto T, Narumiya S, Mizuno K
    LIM-kinase 2 induces formation of stress fibres, focal adhesions and membrane blebs, dependent on its activation by Rho-associated kinase-catalysed phosphorylation at threonine-505. The Biochemical journal 2001 Feb 15;354(Pt 1):149-59
  8. Riento K, Ridley AJ
    Rocks: multifunctional kinases in cell behaviour. Nature reviews. Molecular cell biology 2003 Jun;4(6):446-56
  9. Abrams CS
    Intracellular signaling in platelets. Current opinion in hematology 2005 Sep;12(5):401-5
  10. Strehl A, Munnix IC, Kuijpers MJ, van der Meijden PE, Cosemans JM, Feijge MA, Nieswandt B, Heemskerk JW
    Dual role of platelet protein kinase C in thrombus formation: stimulation of pro-aggregatory and suppression of procoagulant activity in platelets. The Journal of biological chemistry 2007 Mar 9;282(10):7046-55
  11. Woulfe D, Jiang H, Mortensen R, Yang J, Brass LF
    Activation of Rap1B by G(i) family members in platelets. The Journal of biological chemistry 2002 Jun 28;277(26):23382-90
  12. Shattil SJ, Newman PJ
    Integrins: dynamic scaffolds for adhesion and signaling in platelets. Blood 2004 Sep 15;104(6):1606-15
  13. Han J, Lim CJ, Watanabe N, Soriani A, Ratnikov B, Calderwood DA, Puzon-McLaughlin W, Lafuente EM, Boussiotis VA, Shattil SJ, Ginsberg MH
    Reconstructing and deconstructing agonist-induced activation of integrin alphaIIbbeta3. Current biology : CB 2006 Sep 19;16(18):1796-806
  14. Leisner TM, Yuan W, DeNofrio JC, Liu J, Parise LV
    Tickling the tails: cytoplasmic domain proteins that regulate integrin alphaIIbbeta3 activation. Current opinion in hematology 2007 May;14(3):255-61
  15. Kashiwagi H, Schwartz MA, Eigenthaler M, Davis KA, Ginsberg MH, Shattil SJ
    Affinity modulation of platelet integrin alphaIIbbeta3 by beta3-endonexin, a selective binding partner of the beta3 integrin cytoplasmic tail. The Journal of cell biology 1997 Jun 16;137(6):1433-43
  16. Lova P, Paganini S, Sinigaglia F, Balduini C, Torti M
    A Gi-dependent pathway is required for activation of the small GTPase Rap1B in human platelets. The Journal of biological chemistry 2002 Apr 5;277(14):12009-15
  17. Bertoni A, Tadokoro S, Eto K, Pampori N, Parise LV, White GC, Shattil SJ
    Relationships between Rap1b, affinity modulation of integrin alpha IIbbeta 3, and the actin cytoskeleton. The Journal of biological chemistry 2002 Jul 12;277(28):25715-21
  18. Lova P, Paganini S, Hirsch E, Barberis L, Wymann M, Sinigaglia F, Balduini C, Torti M
    A selective role for phosphatidylinositol 3,4,5-trisphosphate in the Gi-dependent activation of platelet Rap1B. The Journal of biological chemistry 2003 Jan 3;278(1):131-8
  19. Jackson SP, Schoenwaelder SM, Goncalves I, Nesbitt WS, Yap CL, Wright CE, Kenche V, Anderson KE, Dopheide SM, Yuan Y, Sturgeon SA, Prabaharan H, Thompson PE, Smith GD, Shepherd PR, Daniele N, Kulkarni S, Abbott B, Saylik D, Jones C, Lu L, Giuliano S, Hughan SC, Angus JA, Robertson AD, Salem HH
    PI 3-kinase p110beta: a new target for antithrombotic therapy. Nature medicine 2005 May;11(5):507-14
  20. Sun DS, Lo SJ, Tsai WJ, Lin CH, Yu MS, Chen YF, Chang HH
    PI3-kinase is essential for ADP-stimulated integrin alpha(IIb)beta3-mediated platelet calcium oscillation, implications for P2Y receptor pathways in integrin alpha(IIb)beta3-initiated signaling cross-talks. Journal of biomedical science 2005 Dec;12(6):937-48
  21. Voss B, McLaughlin JN, Holinstat M, Zent R, Hamm HE
    PAR1, but not PAR4, activates human platelets through a Gi/o/phosphoinositide-3 kinase signaling axis. Molecular pharmacology 2007 May;71(5):1399-406
  22. Clyde-Smith J, Silins G, Gartside M, Grimmond S, Etheridge M, Apolloni A, Hayward N, Hancock JF
    Characterization of RasGRP2, a plasma membrane-targeted, dual specificity Ras/Rap exchange factor. The Journal of biological chemistry 2000 Oct 13;275(41):32260-7
  23. Eto K, Murphy R, Kerrigan SW, Bertoni A, Stuhlmann H, Nakano T, Leavitt AD, Shattil SJ
    Megakaryocytes derived from embryonic stem cells implicate CalDAG-GEFI in integrin signaling. Proceedings of the National Academy of Sciences of the United States of America 2002 Oct 1;99(20):12819-24
  24. Woulfe DS
    Platelet G protein-coupled receptors in hemostasis and thrombosis. Journal of thrombosis and haemostasis : JTH 2005 Oct;3(10):2193-200
  25. Gawaz M, Langer H, May AE
    Platelets in inflammation and atherogenesis. The Journal of clinical investigation 2005 Dec;115(12):3378-84

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