Integrin α9β1

Integrins are transmembrane heterodimeric receptors responsible for transducing and modulating signals between the extracellular matrix and cytoskeleton, ultimately influencing cell functions such as adhesion and migration. Integrin α9β1 is classified within a two member sub-family of integrins highlighted in part by its specialized role in cell migration. The importance of this role is demonstrated by its regulation of numerous biological functions including lymphatic valve morphogenesis, lymphangiogenesis, angiogenesis and hematopoietic homeostasis. Compared to other integrins the signaling mechanisms that transduce α9β1-induced cell migration are not well described. We have recently shown that Src tyrosine kinase plays a key proximal role to control α9β1 signaling. Specifically it activates inducible nitric oxide synthase (iNOS) and in turn nitric oxide (NO) production as a means to transduce cell migration. Furthermore, we have also described a role for FAK, Erk and Rac1 in α9β1 signal transduction. Here we provide an over view of known integrin α9β1 signaling pathways and highlight its roles in diverse biological conditions.     

Cell biological roles of αB-crystallin

αB-crystallin, also called HspB5, is a molecular chaperone able to interact with unfolding proteins. By interacting, it inhibits further unfolding, thereby preventing protein aggregation and allowing ATP-dependent chaperones to refold the proteins. αB-crystallin belongs to the family of small heat-shock proteins (sHsps), which in humans consists of 10 different members. The protein forms large oligomeric complexes, containing up to 40 or more subunits, which in vivo consist of heterooligomeric complexes formed by a mixture of αB-crystallin and other sHsps. αB-crystallin is highly expressed in the lens and to a lesser extent in several other tissues, among which heart, skeletal muscle and brain. αB-crystallin plays a role in several cellular processes, such as signal transduction, protein degradation, stabilization of cytoskeletal structures and apoptosis. Mutations in the αB-crystallin gene can have detrimental effects, leading to pathologies such as cataract and cardiomyopathy. This review describes the biological roles of αB-crystallin, with a special focus on its function in the eye lens, heart muscle and brain. In addition its therapeutic potential is discussed.     

‘Satellite DNA transcripts have diverse biological roles in Drosophila'

Recent years have seen considerable progress in applying single nucleotide polymorphisms (SNPs) to population genetics studies. However, relatively few have attempted to use them to study the genetic differentiation of wild bird populations and none have examined possible differences of exonic and intronic SNPs in these studies. Here, using 144 SNPs, we examined population genetic differentiation in the saker falcon (Falco cherrug) across Eurasia. The position of each SNP was verified using the recently sequenced saker genome with 108 SNPs positioned within the introns of 10 fragments and 36 SNPs in the exons of six genes, comprising MHC, MC1R and four others. In contrast to intronic SNPs, both Bayesian clustering and principal component analyses using exonic SNPs consistently revealed two genetic clusters, within which the least admixed individuals were found in Europe/central Asia and Qinghai (China), respectively. Pairwise D analysis for exonic SNPs showed that the two populations were significantly differentiated and between the two clusters the frequencies of five SNP markers were inferred to be influenced by selection. Central Eurasian populations clustered in as intermediate between the two main groups, consistent with their geographic position. But the westernmost populations of central Europe showed evidence of demographic isolation. Our work highlights the importance of functional exonic SNPs for studying population genetic pattern in a widespread avian species.     

Human Parechovirus 1 Infection Occurs via αVβ1 Integrin

Human parechovirus 1 (HPeV-1) (family Picornaviridae) is a global cause of pediatric respiratory and CNS infections for which there is no treatment. Although biochemical and in vitro studies have suggested that HPeV-1 binds to αVβ1, αVβ3 and αVβ6 integrin receptor(s), the actual cellular receptors required for infectious entry of HPeV-1 remain unknown. In this paper we analyzed the expression profiles of αVβ1, αVβ3, αVβ6 and α5β1 in susceptible cell lines (A549, HeLa and SW480) to identify which integrin receptors support HPeV-1 internalization and/or replication cycle. We demonstrate by antibody blocking assay, immunofluorescence microscopy and RT-qPCR that HPeV-1 internalizes and replicates in cell lines that express αVβ1 integrin but not αVβ3 or αVβ6 integrins. To further study the role of β1 integrin, we used a mouse cell line, GE11-KO, which is deficient in β1 expression, and its derivate GE11-β1 in which human integrin β1 subunit is overexpressed. HPeV-1 (Harris strain) and three clinical HPeV-1 isolates did not internalize into GE11-KO whereas GE11-β1 supported the internalization process. An integrin β1-activating antibody, TS2/16, enhanced HPeV-1 infectivity, but infection occurred in the absence of visible receptor clustering. HPeV-1 also co-localized with β1 integrin on the cell surface, and HPeV-1 and β1 integrin co-endocytosed into the cells. In conclusion, our results demonstrate that in some cell lines the cellular entry of HPeV-1 is primarily mediated by the active form of αVβ1 integrin without visible receptor clustering.     

Characterization of the signaling pathways Regulating α2β1 integrin-mediated events by a pharmacological approach

In certain instances of developing and adult organism, epithelial cells can change morphology and transform into mesenchynial-like type in order to move through the extracellular matrix. However, because of the multiplicity and complexity of signaling pathways that contribute to these processes, their molecular dissection has remained difficult. By using a pharmacological approach on the rat bladder carcinoma cell line NBT-II dispersion system, we have identified distinct signaling events for adhesion and motility in response to collagen, both activities depending on α2β1 integrin. Treatment of cells with PKC inhibitors markedly impaired initial attachment on collagen without affecting the capacity of cells to move, suggesting that PKC activity is required for initial adhesion strength during cell translocation. Both adhesion and motility were diminished by tyrosine kinase inhibitors herbimycin and tyrphostin whereas tyrosine phosphatase inhibitors amplified cell scattering. The collagen-induced dispersion was insensitive to genistein which we previously showed to abrogate growth factor-induced scattering, thus demonstrating inducer specificity. Finally. Ras inhibitors and expression of a dominant negative form of Ras (N 17Ras) while affecting initial cell attachment, did not prevent cell migration, and instead favored the dissociated state on collagen. The specific signaling pathways identified for adhesion and motility should help to understand the sequential processes associated with cell migration.     

Physiological and Pathological Roles of α3β1 Integrin

α3β1 integrin has been considered to be a mysterious adhesion molecule due to the pleiotropy in its ligand-binding specificity. However, recent studies have identified laminin isoforms as high-affinity ligands for this integrin, and demonstrated that α3β1 integrin plays a number of essential roles in development and differentiation, mainly by mediating the establishment and maintenance of epithelial tissues. Furthermore, α3β1 integrin is also implicated in many other biological phenomena, including cell growth and apoptosis, angiogenesis and neural functions. This integrin receptor forms complexes with various other membrane proteins, such as the transmembrane-4 superfamily proteins (tetraspanins), cytoskeletal proteins and signaling molecules. Recently, lines of evidence have been reported showing that complex formation regulates integrin functions in cell adhesion and migration, signal transduction across cell membranes, and cytoskeletal organization. In addition to these roles in physiological processes, α3β1 integrin performs crucial functions in various pathological processes, especially in wound healing, tumor invasion and metastasis, and infection by pathogenic microorganisms.This revised version was published online in June 2005 with a corrected cover date.     

Integrin α1β1 participates in chondrocyte transduction of osmotic stress

Background/purpose

The goal of this study was to determine the role of the collagen binding receptor integrin α1β1 in regulating osmotically induced [Ca²⁺]_i transients in chondrocytes.

Method

The [Ca²⁺]_i transient response of chondrocytes to osmotic stress was measured using real-time confocal microscopy. Chondrocytes from wildtype and integrin α1-null mice were imaged ex vivo (in the cartilage of intact murine femora) and in vitro (isolated from the matrix, attached to glass coverslips). Immunocytochemistry was performed to detect the presence of the osmosensor, transient receptor potential vanilloid-4 (TRPV4), and the agonist GSK1016790A (GSK101) was used to test for its functionality on chondrocytes from wildtype and integrin α1-null mice.

Results/interpretation

Deletion of the integrin α1 subunit inhibited the ability of chondrocytes to respond to a hypo-osmotic stress with [Ca²⁺]_i transients ex vivo and in vitro. The percentage of chondrocytes responding ex vivo was smaller than in vitro and of the cells that responded, more single [Ca²⁺]_i transients were observed ex vivo compared to in vitro. Immunocytochemistry confirmed the presence of TRPV4 on wildtype and integrin α1-null chondrocytes, however application of GSK101 revealed that TRPV4 could be activated on wildtype but not integrin α1-null chondrocytes. Integrin α1β1 is a key participant in chondrocyte transduction of a hypo-osmotic stress. Furthermore, the mechanism by which integrin α1β1 influences osmotransduction is independent of matrix binding, but likely dependent on the chondrocyte osmosensor TRPV4.     

Enhancing Integrin α1 Inserted (I) Domain Affinity to Ligand Potentiates Integrin α1β1-mediated Down-regulation of Collagen Synthesis

Integrin α1β1 binding to collagen IV, which is mediated by the α1-inserted (I) domain, down-regulates collagen synthesis. When unligated, a salt bridge between Arg²⁸⁷ and Glu³¹⁷ is thought to keep this domain in a low affinity conformation. Ligand binding opens the salt bridge leading to a high-affinity conformation. How modulating integrin α1β1 affinity alters collagen homeostasis is unknown. To address this question, we utilized a thermolysin-derived product of the α1α2α1 network of collagen IV (α1α2α1(IV) truncated protomer) that selectively binds integrin α1β1. We show that an E317A substitution enhanced binding to the truncated protomer, consistent with a previous finding that this substitution eliminates the salt bridge. Surprisingly, we show that an R287A substitution did not alter binding, whereas R287E/E317R substitutions enhanced binding to the truncated protomer. NMR spectroscopy and molecular modeling suggested that eliminating the Glu³¹⁷ negative charge is sufficient to induce a conformational change toward the open state. Thus, the role played by Glu³¹⁷ is largely independent of the salt bridge. We further show that cells expressing E317A or R287E/E317R substitutions have enhanced down-regulation of collagen IV synthesis, which is mediated by the ERK/MAPK pathway. In conclusion, we have demonstrated that modulating the affinity of the extracellular α1 I domain to collagen IV enhances outside-in signaling by potentiating ERK activation and enhancing the down-regulation of collagen synthesis.     

Distinct roles for Rap1b protein in platelet secretion and integrin αIIbβ3 outside-in signaling

Rap1b is activated by platelet agonists and plays a critical role in integrin α(IIb)β(3) inside-out signaling and platelet aggregation. Here we show that agonist-induced Rap1b activation plays an important role in stimulating secretion of platelet granules. We also show that α(IIb)β(3) outside-in signaling can activate Rap1b, and integrin outside-in signaling-mediated Rap1b activation is important in facilitating platelet spreading on fibrinogen and clot retraction. Rap1b-deficient platelets had diminished ATP secretion and P-selectin expression induced by thrombin or collagen. Importantly, addition of low doses of ADP and/or fibrinogen restored aggregation of Rap1b-deficient platelets. Furthermore, we found that Rap1b was activated by platelet spreading on immobilized fibrinogen, a process that was not affected by P2Y(12) or TXA(2) receptor deficiency, but was inhibited by the selective Src inhibitor PP2, the PKC inhibitor Ro-31-8220, or the calcium chelator demethyl-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis. Clot retraction was abolished, and platelet spreading on fibrinogen was diminished in Rap1b-deficient platelets compared with wild-type controls. The defects in clot retraction and spreading on fibrinogen of Rap1b-deficient platelets were not rescued by addition of MnCl(2), which elicits α(IIb)β(3) outside-in signaling in the absence of inside-out signaling. Thus, our results reveal two different activation mechanisms of Rap1b as well as novel functions of Rap1b in platelet secretion and in integrin α(IIb)β(3) outside-in signaling.     

Glycosylation Modulates Melanoma Cell α2β1 and α3β1 Integrin Interactions with Type IV Collagen

Although type IV collagen is heavily glycosylated, the influence of this post-translational modification on integrin binding has not been investigated. In the present study, galactosylated and nongalactosylated triple-helical peptides have been constructed containing the α1(IV)382–393 and α1(IV)531–543 sequences, which are binding sites for the α2β1 and α3β1 integrins, respectively. All peptides had triple-helical stabilities of 37 °C or greater. The galactosylation of Hyl³⁹³ in α1(IV)382–393 and Hyl⁵⁴⁰ and Hyl⁵⁴³ in α1(IV)531–543 had a dose-dependent influence on melanoma cell adhesion that was much more pronounced in the case of α3β1 integrin binding. Molecular modeling indicated that galactosylation occurred on the periphery of α2β1 integrin interaction with α1(IV)382–393 but right in the middle of α3β1 integrin interaction with α1(IV)531–543. The possibility of extracellular deglycosylation of type IV collagen was investigated, but no β-galactosidase-like activity capable of collagen modification was found. Thus, glycosylation of collagen can modulate integrin binding, and levels of glycosylation could be altered by reduction in expression of glycosylation enzymes but most likely not by extracellular deglycosylation activity.     

Cell adhesion to anosmin via α5β1, α4β1, and α9β1 integrins

Anosmin is an extracellular matrix protein, and genetic defects in anosmin result in human Kallmann syndrome. It functions in neural crest formation, cell adhesion, and neuronal migration. Anosmin consists of multiple domains, and it has been reported to bind heparan sulfate, FGF receptor, and UPA. In this study, we establish cell adhesion/spreading assays for anosmin and use them for antibody inhibition analyses to search for an integrin adhesion receptor. We find that α5β1, α4β1, and α9β1 integrins are needed for effective adhesive receptor function in cell adhesion and cell spreading on anosmin; adhesion is inhibited by both RGD and α4β1 CS1-based peptides. This identification of anosmin-integrin adhesion receptors should facilitate studies of anosmin function in cell and developmental biology.     

Integrin α6β4 cooperates with LPA signaling to stimulate Rac through AKAP-Lbc-mediated RhoA activation

The α(6)β(4) integrin promotes carcinoma invasion through its ability to promote directed migration and polarization of carcinoma cells. In this study, we explore how the α(6)β(4) integrin cooperates with lysophosphatidic acid (LPA) to activate Rho and Rac small GTPases. Through the use of dominant negative Rho constructs, C3 exotransferase, and Rho kinase inhibitor, we find that Rho is critical for LPA-dependent chemotaxis and lamellae formation. However, utilization of specific Rho isoforms depends on integrin α(6)β(4) expression status. Integrin α(6)β(4)-negative MDA-MB-435 cells utilize only RhoC for motility, whereas integrin α(6)β(4)-expressing cells utilize RhoC but additionally activate and utilize RhoA for LPA-dependent cell motility and lamellae formation. Notably, the activation of RhoA by cooperative LPA and integrin α(6)β(4) signaling requires the Rho guanine nucleotide exchange factor AKAP-Lbc. We also determine that integrin α(6)β(4) cannot activate Rac1 directly but promotes LPA-mediated Rac1 activation that is dependent on RhoA activity and de novo β(1) integrin ligation. Finally, we find that the regulation of Rac1 and RhoA in response to LPA is differentially regulated by phosphodiesterases, PKA, and phosphatidylinositol 3-kinase, thus supporting their spatially distinct compartmentalization. In summary, signaling from integrin α(6)β(4) facilitates LPA-stimulated chemotaxis through preferential activation of RhoA, which, in turn, facilitates activation of Rac1.     

Integrin α9β1-mediated cell migration in glioblastoma via SSAT and Kir4.2 potassium channel pathway

The α9β1 integrin accelerates cell migration through binding of the α9 cytoplasmic domain to SSAT, which catalyzes the catabolism of higher order polyamines, spermidine and spermine, to the lower order polyamine, putrescine. SSAT levels were downregulated at both the mRNA and protein levels by shRNA-mediated simultaneous knockdown of MMP-9 and uPAR/cathepsin B. In addition, we noted a prominent reduction in the expression of SSAT with MMP-9 and uPAR/cathepsin B knockdown in the tumor regions of 5310 injected nude mice brains. Further, SSAT knockdown in glioma xenograft cells significantly reduced their migration potential. Interestingly, MMP-9, uPAR and cathepsin B overexpression in these xenograft cells significantly elevated SSAT mRNA and protein levels. The migratory potential of MMP-9/uPAR/cathepsin B-overexpressed 4910 and 5310 cells was not affected by either glybenclamide (Kir 6.x inhibitor) or tertiapin-Q (Kir 1.1 and 3.x inhibitor) but instead was significantly inhibited by either barium or Kir4.2 siRNA treatments. Co-localization of α9 integrin with Kir4.2 was observed in both 4910 and 5310 xenograft cells. However, MMP-9 and uPAR/cathepsin B knockdown in these cells prominently reduced the co-localization of α9 with Kir4.2. Taken together, our results clearly demonstrate that α9β1 integrin-mediated cell migration utilizes SSAT and the Kir4.2 potassium channel pathway, and inhibition of the migratory potential of these glioma xenograft cells by simultaneous knockdown of MMP-9 and uPAR/cathepsin B could be attributed to the reduced SSAT levels and co-localization of α9 integrin with Kir4.2 inward rectifier potassium channels.     

Integrin αEβ7+ T cells direct intestinal stem cell fate decisions via adhesion signaling

Intestinal stem cell (ISC) differentiation is regulated precisely by a niche in the crypt,where lymphocytes may interact with stem and transient amplifying (TA)...     

Discoidin Domain Receptors Promote α1β1- and α2β1-Integrin Mediated Cell Adhesion to Collagen by Enhancing Integrin Activation

The discoidin domain receptors, DDR1 and DDR2, are receptor tyrosine kinases that bind to and are activated by collagens. Similar to collagen-binding β1 integrins, the DDRs bind to specific motifs within the collagen triple helix. However, these two types of collagen receptors recognize distinct collagen sequences. While GVMGFO (O is hydroxyproline) functions as a major DDR binding motif in fibrillar collagens, integrins bind to sequences containing Gxx’GEx”. The DDRs are thought to regulate cell adhesion, but their roles have hitherto only been studied indirectly. In this study we used synthetic triple-helical collagen-derived peptides that incorporate either the DDR-selective GVMGFO motif or integrin-selective motifs, such as GxOGER and GLOGEN, in order to selectively target either type of receptor and resolve their contributions to cell adhesion. Our data using HEK293 cells show that while cell adhesion to collagen I was completely inhibited by anti-integrin blocking antibodies, the DDRs could mediate cell attachment to the GVMGFO motif in an integrin-independent manner. Cell binding to GVMGFO was independent of DDR receptor signalling and occurred with limited cell spreading, indicating that the DDRs do not mediate firm adhesion. However, blocking the interaction of DDR-expressing cells with collagen I via the GVMGFO site diminished cell adhesion, suggesting that the DDRs positively modulate integrin-mediated cell adhesion. Indeed, overexpression of the DDRs or activation of the DDRs by the GVMGFO ligand promoted α1β1 and α2β1 integrin-mediated cell adhesion to medium- and low-affinity integrin ligands without regulating the cell surface expression levels of α1β1 or α2β1. Our data thus demonstrate an adhesion-promoting role of the DDRs, whereby overexpression and/or activation of the DDRs leads to enhanced integrin-mediated cell adhesion as a result of higher integrin activation state.     

Integrin αIIbβ3 intermediates: From molecular dynamics to adhesion assembly

The platelet integrin α_IIbβ₃ undergoes long-range conformational transitions associated with its functional conversion from inactive (low-affinity) to active (high-affinity) during hemostasis. Although new conformations that are intermediate between the well-characterized bent and extended states have been identified, their molecular dynamic properties and functions in the assembly of adhesions remain largely unexplored. In this study, we evaluated the properties of intermediate conformations of integrin α_IIbβ₃ and characterized their effects on the assembly of adhesions by combining all-atom simulations, principal component analysis, and mesoscale modeling. Our results show that in the low-affinity, bent conformation, the integrin ectodomain tends to pivot around the legs; in intermediate conformations, the headpiece becomes partially extended, away from the lower legs. In the fully open, active state, α_IIbβ₃ is flexible, and the motions between headpiece and lower legs are accompanied by fluctuations of the transmembrane helices. At the mesoscale, bent integrins form only unstable adhesions, but intermediate or open conformations stabilize the adhesions. These studies reveal a mechanism by which small variations in ligand binding affinity and enhancement of the ligand-bound lifetime in the presence of actin retrograde flow stabilize α_IIbβ₃ integrin adhesions.     

Integrin αIIbβ3: A novel effector of Gα13

Under physiological conditions, circulating platelets are discoid in shape.¹ On these platelets, the fibrinogen receptor (integrin α_IIbβ₃) is in a low-affinity state, unable to bind soluble fibrinogen (Fg). Activation by agonists such as ADP and thrombin leads to a change in the conformation of the integrin α_IIbβ₃ through a process known as inside-out signaling. This enables the integrin to bind soluble Fg, which initiates a cascade of events referred to as outside-in signaling.² Outside-in signaling control processes, such as platelet spreading and clot retraction, by regulating small G-proteins such as RhoA, Rac and cdc42.Key words: platelets, integrin α_IIbβ₃, Galpha13, RhoA, clot retraction, thrombin, fibrinogenThe majority of the physiological platelet agonists (except collagen) induce inside-out signaling by binding to specific G-protein-coupled receptors (GPCRs). A G-protein plays a crucial role in translating the signal from GPCR to downstream effector molecules, ultimately leading to affinity modulation of integrin α_IIbβ₃. Platelets express nine Gα subunits; namely G_q, G_i1, G_i2, G_i3, G_z, G₁₂, G₁₃, G_s and G₁₆. Previous studies have shown that a small G-protein, RhoA, is activated by the G_12/13 family and plays a crucial role in calcium-independent platelet shape change.³ However, RhoA is also activated by α_IIbβ₃ and inhibits platelet spreading to trigger clot retraction.⁴ Recently, in a series of elegant experiments, Gong et al. have described the dynamic regulation of RhoA through a signaling crosstalk between Gα₁₃ and α_IIbβ₃.⁵By generating mice in which the platelets were depleted of Gα₁₃ using siRNA technology, Gong et al. investigated the role of Gα₁₃-mediated signaling on platelet spreading on immobilized Fg.⁵ The confocal images very clearly showed that, in the absence of Gα₁₃, platelets spread poorly on Fg, which was rescued by pretreatment with the Rho-kinase inhibitor Y27632, confirming previous findings that RhoA activated downstream of integrin α_IIbβ₃ inhibits platelet spreading. Interestingly, Gα₁₃-depleted platelets failed to activate c-Src but accelerated RhoA activation. From these observations, the authors infer that Gα₁₃ is important for integrin-mediated c-Src activation and RhoA inhibition, leading to increased cell spreading.⁵Since Gα₁₃ regulates integrin-mediated cell spreading and c-Src activation, Gong et al. examined the interaction of Gα₁₃ with α_IIbβ₃ using co-immunoprecipitation and GST pull-down assays.⁵ They found that the GTP-bound form of Gα₁₃ shows enhanced interaction with the integrin β₃ subunit. This interaction is required for the activation of c-Src and the inhibition of RhoA. However, they found that the inhibition of RhoA is transient. RhoA activation is suppressed for the first 15 min of platelet spreading, after which RhoA is activated. This initial suppression is rescued by blocking Gα₁₃ and β₃ cytoplasmic domain (β3-CD) interaction. Furthermore, they observed that RhoA activation parallels clot retraction.⁵ These findings indicate that Gα₁₃ is a key regulator of platelet spreading and clot retraction phenomena.According to Gong et al., thrombin-induced inside-out signaling through GPCR leads to GTP loading of Gα₁₃ (Fig. 1A). This GTP-bound Gα₁₃ interacts with integrin β3-CD of ligand-bound integrin, thus facilitating c-Src activation, which leads to platelet spreading. Blockade of the interaction between Gα₁₃ and β3-CD or cleavage of β3-CD by calpain results in clot retraction (Fig. 1B).Open in a separate windowFigure 1Schematic representation of the dynamic regulation of RhoA by Gα₁₃ during platelet activation. (A) Activation of platelets by thrombin receptors coupled to Gα₁₃ leads to the activation of RhoA, leading to platelet shape change. (B) The change in the conformation of integrin to a high-affinity form results in fibrinogen binding to α_IIbβ₃. Active Gα13 binds to the cytoplasmic domain of β₃ leading to the activation of c-Src, resulting in platelet spreading. The rise in intracellular calcium activates calpain, which cleaves the β₃ cytoplasmic domain, releasing c-Src, which, resulting in the activation of RhoA, leads to cell retraction. *Denotes GTP-bound active form of G-proteins.Perhaps the most significant and novel finding of the study is the identification of integrin α_IIbβ₃ as an effector of Gα₁₃. The study also convincingly shows that Gα₁₃ bound to integrin regulates RhoA via c-Src. Furthermore, achieving 80% knockdown of Gα₁₃ in an in vivo setting using siRNA represents a technological advancement. Since Gα₁₃ binds to integrin β3-CD in a 1:1 stoichiometry, it appears that only a small population of integrin is regulated by Gα₁₃, as there are far less Gα₁₃ molecules in a single platelet than the number of α_IIbβ₃ molecules. This will require further investigation. Gong et al. also finds that an appreciable amount of Gα₁₃ is associated with β₃ in resting platelets, which requires some explanation.⁵ It is also not clear if Gα₁₃ remains bound to β3-CD or dissociates from the integrin during clot retraction.Overall, this is a paradigm-shifting study that establishes the importance of the dynamic regulation of RhoA by Gα₁₃ in order to achieve efficient platelet spreading and clot retraction.