Protein interactions with the platelet integrin αIIb regulatory motif

Integrins are transmembrane proteins regulating cellular shape, mobility and the cell cycle. A highly conserved signature motif in the cytoplasmic tail of the integrin α‐subunit, KXGFFKR, plays a critical role in regulating integrin function. To date, six proteins have been identified that target this motif of the platelet‐specific integrin α_IIbβ₃. We employ peptide‐affinity chromatography followed‐up with LC‐MS/MS analysis as well as protein chips to identify new potential regulators of integrin function in platelets and put them into their biological context using information from protein:protein interaction (PPI) databases. Totally, 44 platelet proteins bind with high affinity to an immobilized LAMWKVGFFKR‐peptide. Of these, seven have been reported in the PPI literature as interactors with integrin α‐subunits. 68 recombinant human proteins expressed on the protein chip specifically bind with high affinity to biotin‐tagged α‐integrin cytoplasmic peptides. Two of these proteins are also identified in the peptide‐affinity experiments, one is also found in the PPI databases and a further one is present in the data to all three approaches. Finally, novel short linear interaction motifs are common to a number of proteins identified.     

The effect of differentiation inducers on the integrin expression of K562 erythroleukemia cells.

We studied the effects of differentiation-inducers on the integrin profile and adhesive properties of K562 leukemia cells. The fibronectin (Fn) receptor integrin, α₅β₁, was the only integrin expressed in suspension cultured K562 cells. When the cells were exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA) immunoreactivity for the β₁ integrin subunit was slightly enhanced. TPA exposure also induced the appearance of the α₂, α₃, α_v and β₃ integrin subunits, but the platelet integrin subunit α_IIb was not detected. On the other hand, hemin chloride-induced erythroid differentiation of K562 cells diminished the expression of the α₅β₁ integrin on the surface of the cells. Adhesion experiments with TPA-exposed K562 cells indicated that although the adherence to the extracellular matrix (ECM) proteins as a rule was low a few cells spread on these proteins. The present results specify the effects of differentiation inducers on the integrin profile of K562 cells and excludes the comprehension that TPA would induce expression of the platelet integrin α_IIb on their surface. Our results also show, that an increased expression of a certain integrin does not necessarily lead to a comparable adhesion ability on its ligand in vitro.     

Relevance of N-terminal residues for amyloid-β binding to platelet integrin αIIbβ3, integrin outside-in signaling and amyloid-β fibril formation

A pathological hallmark of Alzheimer's disease (AD) is the aggregation of amyloid-β peptides (Aβ) into fibrils, leading to deposits in cerebral parenchyma and vessels known as cerebral amyloid angiopathy (CAA). Platelets are major players of hemostasis but are also implicated in AD. Recently we provided strong evidence for a direct contribution of platelets to AD pathology. We found that monomeric Aβ40 binds through its RHDS sequence to integrin α_IIbβ₃, and promotes the formation of fibrillar Aβ aggregates by the secretion of adenosine diphosphate (ADP) and the chaperone protein clusterin (CLU) from platelets. Here we investigated the molecular mechanisms of Aβ binding to integrin α_IIbβ₃ by using Aβ11 and Aβ16 peptides. These peptides include the RHDS binding motif important for integrin binding but lack the central hydrophobic core and the C-terminal sequence of Aβ. We observed platelet adhesion to truncated N-terminal Aβ11 and Aβ16 peptides that was not mediated by integrin α_IIbβ₃. Thus, no integrin outside-in signaling and reduced CLU release was detected. Accordingly, platelet mediated Aβ fibril formation was not observed. Taken together, the RHDS motif of Aβ is not sufficient for Aβ binding to platelet integrin α_IIbβ₃ and platelet mediated Aβ fibril formation but requires other recognition or binding motifs important for platelet mediated processes in CAA. Thus, increased understanding of the molecular mechanisms of Aβ binding to platelet integrin α_IIbβ₃ is important to understand the role of platelets in amyloid pathology.     

Regulation of adaptor protein GIT1 in platelets, leading to the interaction between GIT1 and integrin alpha(IIb)beta3

GIT1 is an adaptor protein, which links signaling proteins to focal adhesion, thereby regulating cytoskeletal reorganization. Platelets undergo dynamic cytoskeletal reorganization during platelet activation, for which a large number of adaptor proteins are required. However, there has been no report of GIT1 in platelets. We found that GIT1 was abundantly expressed in platelets and underwent tyrosine phosphorylation downstream of integrin α_IIbβ₃, which was inhibited by the Src kinase inhibitor PP2. Furthermore, GIT1 constitutively associated with βPIX, a guanine nucleotide exchange factor (GEF) for Rac. The GIT1/βPIX complex associated with α_IIbβ₃, concomitantly with GIT1 tyrosine phosphorylation. Moreover, both GIT1 and α_IIbβ₃ rapidly translocated to the cytoskeletal fraction during platelet aggregation, which was not observed in the absence of aggregation. These results suggest that tyrosine phosphorylation of GIT1 by Src kinases may regulate cytoskeletal reorganization downstream of α_IIbβ₃ by bringing the Rac GEF βPIX to the vicinity of the integrin.     

Differential binding of ICln in platelets to integrin-derived activating and inhibitory peptides

The capacity of platelets to form a thrombus is mediated by integrin α_IIbβ₃. The cytoplasmic tail of α_IIb contains a highly conserved motif, ⁹⁸⁹KVGFFKR⁹⁹⁵, which plays a critical role in regulating integrin activation and acts as a recognition site for various intracellular proteins, e.g. CIB1, PP1, ICln and RN181. Previously, we demonstrated that a cell-permeable integrin-derived activating (IDA) peptide, KVGFFKR, induces platelet activation, whereas an integrin-derived inhibitory (IDI) peptide, KVGAAKR, is antithrombotic. To elucidate the molecular mechanism underlying these opposite effects we investigate the affinity of known integrin α_IIb binding proteins for the two immobilized peptides in dependence on the activation state of platelets by means of peptide-affinity chromatography, blotting techniques and protein:peptide docking studies.Our results provide a model for the inhibition of ICln interaction with the integrin in activated platelets by the IDI-peptide. Thus, ICln:IDI-peptide interaction profiles can have a pivotal purpose in the search for consensus pharmacophores specifically inhibiting ICln function in platelets potentially leading to the development of integrin-derived antithrombotic drugs.     

Predominant inhibition of ganodermic acid S on the thromboxane A2-dependent pathway in human platelets response to collagen

Ganodermic acid S (GAS), a membrane acting agent, exerts multiple effects on human platelet function (C.N. Wang et al. (1991) Biochem. J. 277, 189–197). The study reported how GAS affected the response of human gel-filtered platelets (GFP) to collagen. The agent inhibited cell aggregation by prolonging lag and shape change periods and decreasing the initial cell aggregation rate. However, the inhibitory efficiency was less than its inhibition on GFP response to U46619, a thromboxane (TX) A₂ mimetic. In the agent-effect on biochemical events, GAS effectively inhibited Ca²⁺ mobilization, phosphorylation of myosin light chain, dense granule secretion and TXB₂ generation. The inhibitions might originate from blocking Ca²⁺ mobilization of the TXA₂-dependent pathway. GAS partially decreased the phosphorylation of most phosphotyrosine proteins from early activation to the integrin α_IIbβ₃-regulated steps. The agent did not affect the phosphorylation of three proteins at the steps regulated by integrin α_IIbβ₃. The results suggest that GAS inhibits the collagen response predominantly on the TXA₂-dependent signaling, and the tyrosine kinase-dependent pathway in collagen response plays a major role in aggregation.     

Structural basis of integrin transmembrane activation

Integrins are cell adhesion receptors that transmit bidirectional signals across plasma membrane and are crucial for many biological functions. Recent structural studies of integrin transmembrane (TM) and cytoplasmic domains have shed light on their conformational changes during integrin activation. A structure of the resting state was solved based on Rosetta computational modeling and experimental data using intact integrins on mammalian cell surface. In this structure, the α_IIb GXXXG motif and their β₃ counterparts of the TM domains associate with ridge‐in‐groove packing, and the α_IIb GFFKR motif and the β₃ Lys‐716 in the cytoplasmic segments play a critical role in the α/β association. Comparing this structure with the NMR structures of the monomeric α_IIb and β₃ (represented as active conformations), the α subunit helix remains similar after dissociation whereas β subunit helix is tilted by embedding additional 5–6 residues into the lipid bilayer. These conformational changes are critical for integrin activation and signaling across the plasma membrane. We thus propose a new model of integrin TM activation in which the recent NMR structure of the α_IIbβ₃ TM/cytoplasmic complex represents an intermediate or transient state, and the electrostatic interaction in the cytoplasmic region is important for priming the initial α/β association, but not absolutely necessary for the resting state. J. Cell. Biochem. 109: 447–452, 2010. © 2009 Wiley‐Liss, Inc.     

Serine/threonine phosphatases regulate platelet αIIbβ3 integrin receptor outside-in signaling mechanisms and clot retraction

Aims

We studied the role of serine/threonine phosphatases (PSTPs) on α_IIbβ₃ signaling and the potential selectivity of the level of PSTP inhibition with okadaic acid (OA) on α_IIbβ₃ signaling for regulation of platelet aggregation and clot retraction.

Main methods

We used washed platelets from normal donors and OA as inhibitor of PSTPs. Clot retraction was induced by 1 U/mL of thrombin. Reorganized cytoskeleton was isolated from Triton X-100 lysed platelets. The presence of proteins incorporated to the cytoskeleton was assayed by immunoblotting with specific antibodies.

Key findings

We found that both 100 and 500 nM OA blocked platelet mediated clot retraction. In contrast, only 500 nM OA inhibited thrombin-induced inside-out α_IIbβ₃ activation, platelet aggregation, and cytoskeletal reorganization. Among markers of α_IIbβ₃ outside-in signaling, 500 nM OA inhibited the incorporation to the cytoskeleton of syk, src, and FAK (Focal Adhesion Kinase) tyrosine kinases and the incorporation and phosphorylation at Tyr⁷⁵⁹ of the β₃ chain of α_IIbβ₃, while 100 nM OA only inhibited the FAK translocation and its tyrosine phosphorylation.

Significance

The level of inhibition of PSTPs by low or high OA concentration (33% and 73% inhibition, respectively) in intact whole cells differentially regulates platelet aggregation and integrin signaling, but have a common effect in blocking clot retraction. The latter may be associated with the presence of phosphorylated FAK in the cytoskeleton. This study reveals a novel target for anti-platelet treatment to block clot retraction without affecting the platelet hemostatic function by a partial inhibition of PSTPs.     

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.     

Cystine-knot peptides engineered with specificities for α(IIb)β(3) or α(IIb)β(3) and α(v)β(3) integrins are potent inhibitors of platelet aggregation

A truncated form of the Agouti‐related protein (AgRP), a member of the cystine‐knot family, has shown promise as a scaffold for engineering novel peptides with new molecular recognition properties. In this study, we replaced a constrained six amino acid loop in AgRP with a nine amino acid loop containing an Arg–Gly–Asp integrin recognition motif, and randomized the neighboring residues to create a library of ～20 million AgRP variants. We displayed the AgRP mutants as fusions on the surface of yeast and used high‐throughput fluorescence‐activated cell sorting (FACS) to isolate peptides that bound specifically to the platelet integrin α_IIbβ₃, a clinically important target for the prevention and treatment of thrombosis. These AgRP peptides had equilibrium dissociation (K_D) constants for α_IIbβ₃ integrin ranging from 60 to 90 nM, and did not bind to α_vβ₃, α_vβ₅, or α₅β₁ integrins. Using an alternate library screening strategy, we identified AgRP peptides that bound to both α_IIbβ₃ and α_vβ₃ integrins with K_D values ranging from 40 to 70 nM and 20 to 30 nM, respectively, and did not bind to α_vβ₅ or α₅β₁ integrins. Unique consensus sequences were identified within both series of AgRP peptides suggesting alternative molecular recognition events that dictate different integrin binding specificities. In addition, the engineered AgRP peptides prevented platelet aggregation as well as or slightly better than the FDA‐approved cyclic peptide eptifibatide. Collectively, these data demonstrate that cystine‐knot peptides can be generated with high affinity and specificity to closely‐related integrins, and provide insights into molecular interactions between small, structured peptide ligands and their receptors. Copyright © 2010 John Wiley & Sons, Ltd.     

Mitochondrially Mediated Integrin αIIbβ3 Protein Inactivation Limits Thrombus Growth

When platelets are strongly stimulated, a procoagulant platelet subpopulation is formed that is characterized by phosphatidylserine (PS) exposure and epitope modulation of integrin α_IIbβ₃ or a loss of binding of activation-dependent antibodies. Mitochondrial permeability transition pore (mPTP) formation, which is essential for the formation of procoagulant platelets, is impaired in the absence of cyclophilin D (CypD). Here we investigate the mechanisms responsible for these procoagulant platelet-associated changes in integrin α_IIbβ₃ and the physiologic role of procoagulant platelet formation in the regulation of platelet aggregation. Among strongly stimulated adherent platelets, integrin α_IIbβ₃ epitope changes, mPTP formation, PS exposure, and platelet rounding were closely associated. Furthermore, platelet mPTP formation resulted in a decreased ability to recruit additional platelets. In the absence of CypD, integrin α_IIbβ₃ function was accentuated in both static and flow conditions, and, in vivo, a prothrombotic phenotype occurred in mice with a platelet-specific deficiency of CypD. CypD-dependent proteolytic events, including cleavage of the integrin β₃ cytoplasmic domain, coincided closely with integrin α_IIbβ₃ inactivation. Calpain inhibition blocked integrin β₃ cleavage and inactivation but not mPTP formation or PS exposure, indicating that integrin inactivation and PS exposure are mediated by distinct pathways subsequent to mPTP formation. mPTP-dependent alkalinization occurred in procoagulant platelets, suggesting a possible alternative mechanism for enhancement of calpain activity in procoagulant platelets. Together, these results indicate that, in strongly stimulated platelets, mPTP formation initiates the calpain-dependent cleavage of integrin β₃ and associated regulatory proteins, resulting in integrin α_IIbβ₃ inactivation, and demonstrate a novel CypD-dependent negative feedback mechanism that limits platelet aggregation and thrombotic occlusion.     

Dok-2 Adaptor Protein Regulates the Shear-dependent Adhesive Function of Platelet Integrin αIIbβ3 in Mice

The Dok proteins are a family of adaptor molecules that have a well defined role in regulating cellular migration, immune responses, and tumor progression. Previous studies have demonstrated that Doks-1 to 3 are expressed in platelets and that Dok-2 is tyrosine-phosphorylated downstream of integrin α_IIbβ₃, raising the possibility that it participates in integrin α_IIbβ₃ outside-in signaling. We demonstrate that Dok-2 in platelets is primarily phosphorylated by Lyn kinase. Moreover, deficiency of Dok-2 leads to dysregulated integrin α_IIbβ₃-dependent cytosolic calcium flux and phosphatidylinositol(3,4)P₂ accumulation. Although agonist-induced integrin α_IIbβ₃ affinity regulation was unaltered in Dok-2^−/− platelets, Dok-2 deficiency was associated with a shear-dependent increase in integrin α_IIbβ₃ adhesive function, resulting in enhanced platelet-fibrinogen and platelet-platelet adhesive interactions under flow. This increase in adhesion was restricted to discoid platelets and involved the shear-dependent regulation of membrane tethers. Dok-2 deficiency was associated with an increased rate of platelet aggregate formation on thrombogenic surfaces, leading to accelerated thrombus growth in vivo. Overall, this study defines an important role for Dok-2 in regulating biomechanical adhesive function of discoid platelets. Moreover, they define a previously unrecognized prothrombotic mechanism that is not detected by conventional platelet function assays.     

RN181, a novel ubiquitin E3 ligase that interacts with the KVGFFKR motif of platelet integrin alpha(IIb)beta3

We previously identified proteins that bind with high affinity to a peptide corresponding to the cytoplasmic regulatory domain (KVGFFKR) of the platelet-specific integrin subunit α_IIb. These included a hypothetical protein termed HSPC238, recently renamed as RING finger protein, RN181. Here, we establish the presence of RN181 in human platelets by RT-PCR, Western blotting and mass spectrometry and confirm its affinity for the platelet integrin. We demonstrate that RN181 has ubiquitin E3 ligase activity and that all other components of the ubiquitination pathway are abundant in platelets, suggesting a novel link of integrin signal transduction pathways with ubiquitin-conjugation events.     

Phosphoinositide 3-Kinase p110�� Regulates Integrin ��IIb��3 Avidity and the Cellular Transmission of Contractile Forces

Phosphoinositide (PI) 3-kinase (PI3K) signaling processes play an important role in regulating the adhesive function of integrin α_IIbβ₃, necessary for platelet spreading and sustained platelet aggregation. PI3K inhibitors are effective at reducing platelet aggregation and thrombus formation in vivo and as a consequence are currently being evaluated as novel antithrombotic agents. PI3K regulation of integrin α_IIbβ₃ activation (affinity modulation) primarily occurs downstream of G_i-coupled and tyrosine kinase-linked receptors linked to the activation of Rap1b, AKT, and phospholipase C. In the present study, we demonstrate an important role for PI3Ks in regulating the avidity (strength of adhesion) of high affinity integrin α_IIbβ₃ bonds, necessary for the cellular transmission of contractile forces. Using knock-out mouse models and isoform-selective PI3K inhibitors, we demonstrate that the Type Ia p110β isoform plays a major role in regulating thrombin-stimulated fibrin clot retraction in vitro. Reduced clot retraction induced by PI3K inhibitors was not associated with defects in integrin α_IIbβ₃ activation, actin polymerization, or actomyosin contractility but was associated with a defect in integrin α_IIbβ₃ association with the contractile cytoskeleton. Analysis of integrin α_IIbβ₃ adhesion contacts using total internal reflection fluorescence microscopy revealed an important role for PI3Ks in regulating the stability of high affinity integrin α_IIbβ₃ bonds. These studies demonstrate an important role for PI3K p110β in regulating the avidity of high affinity integrin α_IIbβ₃ receptors, necessary for the cellular transmission of contractile forces. These findings may provide new insight into the potential antithrombotic properties of PI3K p110β inhibitors.     

Concept and progress in the development of RGD-containing peptide pharmaceuticals

The cell adhesion domain, arginine–glycine–aspartic acid (RGD), has been incorporated into synthetic peptides to perform either of two modes of drug action, antagonist or agonist. Short, conformationally constrained peptides have been developed as antagonists for the platelet membrane glycopivtein complex, the integrin α_IIbβ₃, using cell-based and integrin-based assays. In combination with a comparative molecular modeling study, these results have helped identify common conformalional elements in the pharmacophore of this class of molecules. Peptides are presented that are highly potent, integrin specific, and that possess reduced pharmacological side effects. Also presented is the development of a peptide that modifies, noncovalently, the surfaces of a wide variety of this molecule is evident from its ability to stimulate cell attachment on these surfaces. This is shown to translate into an in vivo activity of faster and more complete tissue integration, and a reduction in foreign body response. © 1994 John Wiley & Sons, Inc.     

Reorientation and Dimerization of the Membrane-Bound Antimicrobial Peptide PGLa from Microsecond All-Atom MD Simulations

Platelet spreading is critical for hemostatic plug formation and thrombosis. However, the detailed dynamics of platelet spreading as a function of receptor-ligand adhesive interactions has not been thoroughly investigated. Using reflection interference contrast microscopy, we found that both adhesive interactions and PAR4 activation affect the dynamics of platelet membrane contact formation during spreading. The initial growth of close contact area during spreading was controlled by the combination of different immobilized ligands or PAR4 activation on fibrinogen, whereas the growth of the total area of spreading was independent of adhesion type and PAR4 signaling. We found that filopodia extend to their maximal length and then contract over time; and that filopodial protrusion and expansion were affected by PAR4 signaling. Upon PAR4 activation, the integrin α_IIbβ₃ mediated close contact to fibrinogen substrata and led to the formation of ringlike patterns in the platelet contact zone. A systematic study of platelet spreading of GPVI-, α₂-, or β₃-deficient platelets on collagen or fibrinogen suggests the integrin α₂ is indispensable for spreading on collagen. The platelet collagen receptors GPVI and α₂ regulate integrin α_IIbβ₃-mediated platelet spreading on fibrinogen. This work elucidates quantitatively how receptor-ligand adhesion and biochemical signals synergistically control platelet spreading.     

The Interaction of Integrin αIIbβ3 with Fibrin Occurs through Multiple Binding Sites in the αIIb β-Propeller Domain

The currently available antithrombotic agents target the interaction of platelet integrin α_IIbβ₃ (GPIIb-IIIa) with fibrinogen during platelet aggregation. Platelets also bind fibrin formed early during thrombus growth. It was proposed that inhibition of platelet-fibrin interactions may be a necessary and important property of α_IIbβ₃ antagonists; however, the mechanisms by which α_IIbβ₃ binds fibrin are uncertain. We have previously identified the γ370–381 sequence (P3) in the γC domain of fibrinogen as the fibrin-specific binding site for α_IIbβ₃ involved in platelet adhesion and platelet-mediated fibrin clot retraction. In the present study, we have demonstrated that P3 can bind to several discontinuous segments within the α_IIb β-propeller domain of α_IIbβ₃ enriched with negatively charged and aromatic residues. By screening peptide libraries spanning the sequence of the α_IIb β-propeller, several sequences were identified as candidate contact sites for P3. Synthetic peptides duplicating these segments inhibited platelet adhesion and clot retraction but not platelet aggregation, supporting the role of these regions in fibrin recognition. Mutant α_IIbβ₃ receptors in which residues identified as critical for P3 binding were substituted for homologous residues in the I-less integrin α_Mβ₂ exhibited reduced cell adhesion and clot retraction. These residues are different from those that are involved in the coordination of the fibrinogen γ404–411 sequence and from auxiliary sites implicated in binding of soluble fibrinogen. These results map the binding of fibrin to multiple sites in the α_IIb β-propeller and further indicate that recognition specificity of α_IIbβ₃ for fibrin differs from that for soluble fibrinogen.     

Derivatives of tetrahydroisoquinoline: Synthesis and initial evaluation of novel non-peptide antagonists of the αIIbβ3-integrin

The novel RGDF mimetics were synthesized with the use of 4-(1,2,3,4-tetrahydroisoquinoline-7-yl)amino-4-oxobutyric or 5-(1,2,3,4-tetrahydroisoquinoline-7-yl)amino-5-oxopentanoic acids as a surrogate of Arg-Gly motif. The synthesized compounds have demonstrated a high potency to inhibit platelet aggregation in vitro and to block FITC-Fg binding to α_IIbβ₃ on washed human platelets.     

PI3-kinase is essential for ADP-stimulated integrin α<Subscript>IIb</Subscript>β<Subscript>3</Subscript>-mediated platelet calcium oscillation,implications for P2Y receptor pathways in integrin α<Subscript>IIb</Subscript>β<Subscript>3</Subscript>-initiated signaling cross-talks

Summary Phosphatidylinositol 3-kinase (PI3K) pathway is important for platelet activation. Recent studies showed that PI3K and oscillative calcium could cross talk to each other and positively regulate integrin α _IIbβ₃-mediated outside-in signaling. However, the mechanism of this feedback regulation remains to be further characterized. Here we found that treatments of both PI3K inhibitor wortmannin and P2Y1 inhibitor A3P5P could inhibit granular secretion in platelets. Additionally, when RGD-substrate adherent platelets were treated with the ADP scavenger apyrase to deplete the granular-released ADP, their attachments in engaging with substrates became looser and the frequency of calcium oscillation decreased. Since it is known that ADP stimulates the PI3K and calcium signal primarily through P2Y12 and P2Y1 receptors respectively, our data indicated that integrin α_IIbβ₃ downstream PI3K and calcium activation might be not completely coupled to integrin associated signaling complex, but in part through feedback stimulation by granular released ADP. Our data indicates the important roles of PI3K and granular released ADP in coordinating the feedback regulations in integrin α_IIbβ₃-mediated platelet activation.     

Structural determinants of the integrin transmembrane domain required for bidirectional signal transmission across the cell membrane

Studying the tight activity regulation of platelet-specific integrin α_IIbβ₃ is foundational and paramount to our understanding of integrin structure and activation. α_IIbβ₃ is essential for the aggregation and adhesion function of platelets in hemostasis and thrombosis. Structural and mutagenesis studies have previously revealed the critical role of α_IIbβ₃ transmembrane (TM) association in maintaining the inactive state. Gain-of-function TM mutations were identified and shown to destabilize the TM association leading to integrin activation. Studies using isolated TM peptides have suggested an altered membrane embedding of the β₃ TM α-helix coupled with α_IIbβ₃ activation. However, controversies remain as to whether and how the TM α-helices change their topologies in the context of full-length integrin in native cell membrane. In this study, we utilized proline scanning mutagenesis and cysteine scanning accessibility assays to analyze the structure and function correlation of the α_IIbβ₃ TM domain. Our identification of loss-of-function proline mutations in the TM domain suggests the requirement of a continuous TM α-helical structure in transmitting activation signals bidirectionally across the cell membrane, characterized by the inside-out activation for ligand binding and the outside-in signaling for cell spreading. Similar results were found for α_Lβ₂ and α₅β₁ TM domains, suggesting a generalizable mechanism. We also detected a topology change of β₃ TM α-helix within the cell membrane, but only under conditions of cell adhesion and the absence of α_IIb association. Our data demonstrate the importance of studying the structure and function of the integrin TM domain in the native cell membrane.