Integrin β3E726 Regulates the Switch Between Platelet Spreading and Clot Retraction by Interfering Gα13/RhoA Pathway

Abstract

Background

Platelet spreading and clot retraction, albeit both mediated by integrin outside-in signaling, lead to platelet shape changes in two opposite directions. The mechanisms by which these processes are regulated are not fully understood. Our previous study found that E726Q mutation in β3 integrin caused impaired spreading in Chinese hamster ovary (CHO) cells on immobilized fibrinogen.

Material and Methods

The current study further utilized knock-in mice bearing the β3^E726Q mutation to explore the underlying mechanisms whereby the E⁷²⁶ residue differentially influences platelet spreading and clot retraction.

Results

Compared to wild type (WT) platelets, β3^E726Q platelets displayed similar level of β3 expression but partially impaired fibrinogen binding associated with attenuated responses in platelet aggregation and P-selectin exposure. Notably, β3^E726Q mutation resulted in defective platelet spreading but accelerated clot retraction concomitant with increased clot density. Functionally, β3^E726Q mice displayed prolonged bleeding time and defective thrombogenesis in vitro and in vivo. Further mechanistic study showed that in β3^E726Q platelets the activities of RhoA and Rac1 were significantly enhanced following thrombin stimulation, possibly due to reduced binding of Gα13 to the β3 cytoplasmic tail.

Conclusion

Taken together, the β3E⁷²⁶ is a potential novel regulatory site that influences the direct interaction of β3 cytoplasmic tail with Gα13 and therefore the activity of downstream RhoA, a molecular switch that shifts platelet spreading into clot retraction.

Keywords

integrin β3 - platelet spreading - clot retraction - Gα13 - RhoA

Publication History

Received: 11 December 2024

Accepted: 23 July 2025

Accepted Manuscript online:
24 July 2025

Article published online:
05 August 2025

• References

• 1 Hynes RO. Integrins: bidirectional, allosteric signaling machines. Cell 2002; 110 (06) 673-687
• 2 Shattil SJ, Newman PJ. Integrins: dynamic scaffolds for adhesion and signaling in platelets. Blood 2004; 104 (06) 1606-1615
• 3 Durrant TN, van den Bosch MT, Hers I. Integrin α_IIbβ₃ outside-in signaling. Blood 2017; 130 (14) 1607-1619
• 4 Shattil SJ. Signaling through platelet integrin alpha IIb beta 3: inside-out, outside-in, and sideways. Thromb Haemost 1999; 82 (02) 318-325
• 5 Takagi J, Petre BM, Walz T, Springer TA. Global conformational rearrangements in integrin extracellular domains in outside-in and inside-out signaling. Cell 2002; 110 (05) 599-511
• 6 Nieswandt B, Varga-Szabo D, Elvers M. Integrins in platelet activation. J Thromb Haemost 2009; 7 (Suppl. 01) 206-209
• 7 Xiao B, Mao J, Sun B. et al. Integrin β3 deficiency results in hypertriglyceridemia via disrupting LPL (lipoprotein lipase) secretion. Arterioscler Thromb Vasc Biol 2020; 40 (05) 1296-1310
• 8 Vinogradova O, Velyvis A, Velyviene A. et al. A structural mechanism of integrin alpha(IIb)beta(3) “inside-out” activation as regulated by its cytoplasmic face. Cell 2002; 110 (05) 587-597
• 9 Shen B, Zhao X, O'Brien KA. et al. A directional switch of integrin signalling and a new anti-thrombotic strategy. Nature 2013; 503 (7474) 131-135
• 10 Su X, Mi J, Yan J. et al. RGT, a synthetic peptide corresponding to the integrin beta 3 cytoplasmic C-terminal sequence, selectively inhibits outside-in signaling in human platelets by disrupting the interaction of integrin alpha IIb beta 3 with Src kinase. Blood 2008; 112 (03) 592-602
• 11 Huang JS, Shi XF, Xi WD, Liu P, Xi XD. CRGT peptide in cytoplasm regulates thrombus formation via dissociating c-Src-beta 3 interaction. Blood 2013 122. 21
• 12 Chen YP, O'Toole TE, Ylänne J, Rosa JP, Ginsberg MH. A point mutation in the integrin beta 3 cytoplasmic domain (S752–>P) impairs bidirectional signaling through alpha IIb beta 3 (platelet glycoprotein IIb-IIIa). Blood 1994; 84 (06) 1857-1865
• 13 Wang R, Shattil SJ, Ambruso DR, Newman PJ. Truncation of the cytoplasmic domain of beta3 in a variant form of Glanzmann thrombasthenia abrogates signaling through the integrin alpha(IIb)beta3 complex. J Clin Invest 1997; 100 (09) 2393-2403
• 14 Karaman K, Yürektürk E, Geylan H. et al. Identification of three novel pathogenic ITGA2B and one novel pathogenic ITGB3 mutations in patients with hereditary Glanzmann's thrombasthenia living in Eastern Turkey. Platelets 2021; 32 (02) 238-242
• 15 Zhang Y, Zhao X, Shen B. et al. Integrin β₃ directly inhibits the Gα₁₃-p115RhoGEF interaction to regulate G protein signaling and platelet exocytosis. Nat Commun 2023; 14 (01) 4966
• 16 Flevaris P, Stojanovic A, Gong H, Chishti A, Welch E, Du X. A molecular switch that controls cell spreading and retraction. J Cell Biol 2007; 179 (03) 553-565
• 17 Kong YK, Zhang Y, Lin MH, Xi XD. [A single E726Q mutation in the membrane proximal α-helix of integrin β3 subunit induces membrane blebbing by disrupting the membrane-actin cortex interaction] [in Chinese]. Zhongguo Shi Yan Xue Ye Xue Za Zhi 2011; 19 (06) 1450-1455
• 18 Shi X, Yang J, Cui X. et al. Functional effect of the mutations similar to the cleavage during platelet activation at integrin β3 cytoplasmic tail when expressed in mouse platelets. PLoS One 2016; 11 (11) e0166136
• 19 Li D, Peng J, Li T, Liu Y, Chen M, Shi X. Itgb3-integrin-deficient mice may not be a sufficient model for patients with Glanzmann thrombasthenia. Mol Med Rep 2021; 23 (06) 449
• 20 Kunishima S, Kashiwagi H, Otsu M. et al. Heterozygous ITGA2B R995W mutation inducing constitutive activation of the αIIbβ3 receptor affects proplatelet formation and causes congenital macrothrombocytopenia. Blood 2011; 117 (20) 5479-5484
• 21 Hughes PE, Diaz-Gonzalez F, Leong L. et al. Breaking the integrin hinge. A defined structural constraint regulates integrin signaling. J Biol Chem 1996; 271 (12) 6571-6574
• 22 Peyruchaud O, Nurden AT, Milet S. et al. R to Q amino acid substitution in the GFFKR sequence of the cytoplasmic domain of the integrin IIb subunit in a patient with a Glanzmann's thrombasthenia-like syndrome. Blood 1998; 92 (11) 4178-4187
• 23 Ghevaert C, Salsmann A, Watkins NA. et al. A nonsynonymous SNP in the ITGB3 gene disrupts the conserved membrane-proximal cytoplasmic salt bridge in the alphaIIbbeta3 integrin and cosegregates dominantly with abnormal proplatelet formation and macrothrombocytopenia. Blood 2008; 111 (07) 3407-3414
• 24 Nurden AT, Pillois X, Fiore M, Heilig R, Nurden P. Glanzmann thrombasthenia-like syndromes associated with Macrothrombocytopenias and mutations in the genes encoding the αIIbβ3 integrin. Semin Thromb Hemost 2011; 37 (06) 698-706
• 25 Morais S, Oliveira J, Lau C. et al. αIIbβ3 variants in ten families with autosomal dominant macrothrombocytopenia: expanding the mutational and clinical spectrum. PLoS One 2020; 15 (12) e0235136
• 26 Lee J, Lee JM, Kim HS. et al. Twins with an identical novel mutation in ITGB3: a case report of Glanzmann thrombasthenia-like syndrome. Ann Lab Med 2024; 44 (03) 299-302
• 27 Ablooglu AJ, Kang J, Petrich BG, Ginsberg MH, Shattil SJ. Antithrombotic effects of targeting alphaIIbbeta3 signaling in platelets. Blood 2009; 113 (15) 3585-3592
• 28 Petrich BG, Fogelstrand P, Partridge AW. et al. The antithrombotic potential of selective blockade of talin-dependent integrin alpha IIb beta 3 (platelet GPIIb-IIIa) activation. J Clin Invest 2007; 117 (08) 2250-2259
• 29 Tomaiuolo M, Matzko CN, Poventud-Fuentes I, Weisel JW, Brass LF, Stalker TJ. Interrelationships between structure and function during the hemostatic response to injury. Proc Natl Acad Sci U S A 2019; 116 (06) 2243-2252
• 30 Ono A, Westein E, Hsiao S. et al. Identification of a fibrin-independent platelet contractile mechanism regulating primary hemostasis and thrombus growth. Blood 2008; 112 (01) 90-99
• 31 Nguyen HTT, Xu Z, Shi X. et al. Paxillin binding to the PH domain of kindlin-3 in platelets is required to support integrin αIIbβ3 outside-in signaling. J Thromb Haemost 2021; 19 (12) 3126-3138
• 32 Ridley AJ, Hall A. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 1992; 70 (03) 389-399
• 33 Ridley AJ, Paterson HF, Johnston CL, Diekmann D, Hall A. The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell 1992; 70 (03) 401-410
• 34 Nobes CD, Hall A. Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell 1995; 81 (01) 53-62
• 35 Waterman-Storer CM, Worthylake RA, Liu BP, Burridge K, Salmon ED. Microtubule growth activates Rac1 to promote lamellipodial protrusion in fibroblasts. Nat Cell Biol 1999; 1 (01) 45-50
• 36 Lamarche N, Hall A. GAPs for rho-related GTPases. Trends Genet 1994; 10 (12) 436-440
• 37 Zhou K, Wang Y, Gorski JL, Nomura N, Collard J, Bokoch GM. Guanine nucleotide exchange factors regulate specificity of downstream signaling from Rac and Cdc42. J Biol Chem 1998; 273 (27) 16782-16786
• 38 Metcalf DG, Moore DT, Wu Y. et al. NMR analysis of the alphaIIb beta3 cytoplasmic interaction suggests a mechanism for integrin regulation. Proc Natl Acad Sci U S A 2010; 107 (52) 22481-22486
• 39 Aslan JE, McCarty OJ. Rho GTPases in platelet function. J Thromb Haemost 2013; 11 (01) 35-46
• 40 Pleines I, Hagedorn I, Gupta S. et al. Megakaryocyte-specific RhoA deficiency causes macrothrombocytopenia and defective platelet activation in hemostasis and thrombosis. Blood 2012; 119 (04) 1054-1063
• 41 Flevaris P, Li Z, Zhang G, Zheng Y, Liu J, Du X. Two distinct roles of mitogen-activated protein kinases in platelets and a novel Rac1-MAPK-dependent integrin outside-in retractile signaling pathway. Blood 2009; 113 (04) 893-901
• 42 Gong H, Shen B, Flevaris P. et al. G protein subunit Galpha13 binds to integrin alphaIIbbeta3 and mediates integrin “outside-in” signaling. Science 2010; 327 (5963) 340-343