Platelet integrin αIIbß3 plays a key role in a venous thrombogenesis mouse model.

Venous thrombosis (VT) is a common vascular disease associated with reduced survival and a high recurrence rate. VT is initiated by the accumulation of platelets and neutrophils at sites of endothelial cell activation. A role for platelet αIIbß3 in VT is not established, a task complicated by the increased bleeding risk caused by partial agonists such as tirofiban. Here, we show that m-tirofiban, a modified version of tirofiban, does not agonize αIIbß3 based on lack of neoepitope expression and the cryo-EM structure of m-tirofiban/full-length αIIbß3 complex. m-tirofiban abolishes agonist-induced platelet aggregation while preserving clot retraction ex vivo and, unlike tirofiban, it suppresses venous thrombogenesis in a mouse model without increasing bleeding. These findings establish a key role for αIIbß3 in VT initiation and suggest that m-tirofiban and compounds with a similar structurally-defined mechanism of action merit consideration as potential thromboprophylaxis agents in patients at high risk for VT and hemorrhage.

[Platelet-specific Rictor knockout inhibits platelet production and activation and reduces thrombosis in mice].

OBJECTIVE: To investigate the effects of platelet-specific Rictor knockout on platelet activation and thrombus formation in mice. METHODS: PF4-Cre and Rictorfl/fl transgenic mice were crossed to obtain platelet-specific Rictor knockout (Rictor-KO) mice and wild-type mice (n=65), whose expression levels of Rictor, protein kinase B (AKT) and p-AKT were detected using Western blotting. Platelet counts of the mice were determined using routine blood tests, and hemostatic function was assessed by tail vein hemorrhage test. Venous thrombosis models were established in the mice to evaluate the effect of Rictor knockout on thrombosis. Platelet aggregation induced by ADP and thrombin was observed in Rictor-KO and wild-type mice, and flow cytometry was used to analyze the expression levels of integrin αIIbß3 and CD62P in resting and activated platelets. Plasma PF4 levels were determined with ELISA. Megakaryocytes from Rictor-KO and wild-type mice were incubated by vWF immunohistochemical antibody and APC-CD41 antibody to detect the number and ploidy of megakaryocytes, respectively. Platelet elongation on collagen surface was observed with scanning electron microscopy. RESULTS: Compared with the wild-type mice, Rictor-KO mice showed significantly decreased AKT phosphorylation, decreased platelet production, reduced thrombosis, and decreased platelet activation in response to ADP and thrombin stimulation. The Rictor-KO mice also showed lowered expression level of P-selectin protein and activation of integrin αIIbß3 with suppression of platelet extension, reduced plasma PF4 level and decreased number of megakaryocytes in the bone marrow. The ploidy of megakaryocytes and the mean area of proplatelets were both significantly decreased in Rictor-KO mice. CONCLUSION: Platelet-specific Rictor knockout inhibits platelet generation and activation to result in decreased thrombus formation in mice, suggesting the potential of mTORC2 activity inhibition as an efficient antithrombotic strategy.

USP15-Mediated Deubiquitination of FKBP 5 and Activation of the αIIbß3 Signaling Pathway Regulate Thrombosis in Mice.

BACKGROUND: Platelets have the hemostatic function, and their aberrant activation is associated with occlusive thrombus formation. Plasma exosomes are rich in platelets containing ubiquitin-specific peptidase 15 (USP15). Herein, we aim to explore the effect of USP15 on thrombosis, as well as expounding whether USP15 acts as an upstream target of FK506 binding protein 5 (FKBP5) to regulate occlusive thrombus formation. METHODS: Washed human platelets were treated with thrombin for measurement of USP15 and FKBP5 expressions. USP15 loss/gain-of-function variant in HEK293 cells was performed by cell transfection, and the interaction between USP15 and FKBP5 was examined using immunoprecipitation and ubiquitination assays. Mice with USP15-knockout platelets (Plt USP15-/-) were modeled, and subjected to calculation of bleeding time, artery thrombosis imaging and clot retraction assay. FKBP5 expression and the inhibitor of nuclear factor kappa B kinase subunit epsilon (IKBKE)/phosphatidylinositol 3-kinase (PI3K)/Rap1 pathway in wild-type and Plt USP15-/- mice-derived platelets were detected using Western blot. The activation of αIIbß3 in washed platelets was analyzed using flow cytometry. RESULTS: USP15 and FKBP5 expressions were upregulated in platelets after thrombin treatment. Following transfection of USP15 knockdown and USP15 overexpression plasmids into HEK293 cells, FKBP5 protein expression was downregulated by USP15 knockdown while being upregulated by USP15 overexpression. USP15 bound to FKBP5 and protected FKBP5 against ubiquitination. Knockdown of platelet USP15 prolonged bleeding time, inhibited arterial thrombosis and delayed clot retraction in mice. Knockdown of platelet USP15 also decreased protein expressions of FKBP5, IKBKE and Rap1, p-PI3K/PI3K ratio, and activation of αIIbß3 in mice. CONCLUSION: USP15 knockdown in platelets affects thrombosis in mice by promoting the instability of FKBP5 to repress the activation of IKBKE/PI3K/Rap1 pathway-mediated αIIbß3.

Differential Effect of Omega-3 Fatty Acids on Platelet Inhibition by Antiplatelet Drugs In Vitro.

The omega-3 polyunsaturated fatty acids (PUFAs) Docosahexaenoic acid (DHA) and Eicosapentaenoic acid (EPA) exert multiple cardioprotective effects, influencing inflammation, platelet activation, endothelial function and lipid metabolism, besides their well-established triglyceride lowering properties. It is not uncommon for omega-3 PUFAs to be prescribed for hypertriglyceridemia, alongside antiplatelet therapy in cardiovascular disease (CVD) patients. In this regard, we studied the effect of EPA and DHA, in combination with antiplatelet drugs, in platelet aggregation and P-selectin and αIIbß3 membrane expression. The antiplatelet drugs aspirin and triflusal, inhibitors of cyclooxygenase-1 (COX-1); ticagrelor, an inhibitor of the receptor P2Y12; vorapaxar, an inhibitor of the PAR-1 receptor, were combined with DHA or EPA and evaluated against in vitro platelet aggregation induced by agonists arachidonic acid (AA), adenosine diphosphate (ADP) and TRAP-6. We further investigated procaspase-activating compound 1 (PAC-1) binding and P-selectin membrane expression in platelets stimulated with ADP and TRAP-6. Both DHA and EPA displayed a dose-dependent inhibitory effect on platelet aggregation induced by AA, ADP and TRAP-6. In platelet aggregation induced by AA, DHA significantly improved acetylsalicylic acid (ASA) and triflusal's inhibitory activity, while EPA enhanced the inhibitory effect of ASA. In combination with EPA, ASA and ticagrelor expressed an increased inhibitory effect towards ADP-induced platelet activation. Both fatty acids could not improve the inhibitory effect of vorapaxar on AA- and ADP-induced platelet aggregation. In the presence of EPA, all antiplatelet drugs displayed a stronger inhibitory effect towards TRAP-6-induced platelet activation. Both omega-3 PUFAs inhibited the membrane expression of αIIbß3, though they had no effect on P-selectin expression induced by ADP or TRAP-6. The antiplatelet drugs exhibited heterogeneity regarding their effect on P-selectin and αIIbß3 membrane expression, while both omega-3 PUFAs inhibited the membrane expression of αIIbß3, though had no effect on P-selectin expression induced by ADP or TRAP-6. The combinatory effect of DHA and EPA with the antiplatelet drugs did not result in enhanced inhibitory activity compared to the sum of the individual effects of each component.

Biomedical application of TiO2NPs can cause arterial thrombotic risks through triggering procoagulant activity, activation and aggregation of platelets.

BACKGROUND: Titanium dioxide nanoparticles (TiO2NPs) are widely used in medical application. However, the relevant health risk has not been completely assessed, the potential of inducing arterial thrombosis (AT) in particular. METHODS: Alterations in platelet function and susceptibility to arterial thrombosis induced by TiO2NPs were examined using peripheral blood samples from healthy adult males and an in vivo mouse model, respectively. RESULTS: Here, using human platelets (hPLTs) freshly isolated from health volunteers, we demonstrated TiO2NP treatment triggered the procoagulant activity of hPLTs through phosphatidylserine exposure and microvesicles generation. In addition, TiO2NP treatment increased the levels of glycoprotein IIb/IIIa and P-selectin leading to aggregation and activation of hPLTs, which were exacerbated by providing physiology-mimicking conditions, including introduction of thrombin, collagen, and high shear stress. Interestingly, intracellular calcium levels in hPLTs were increased upon TiO2NP treatment, which were crucial in TiO2NP-induced hPLT procoagulant activity, activation and aggregation. Moreover, using mice in vivo models, we further confirmed that TiO2NP treatment a reduction in mouse platelet (mPLT) counts, disrupted blood flow, and exacerbated carotid arterial thrombosis with enhanced deposition of mPLT. CONCLUSIONS: Together, our study provides evidence for an ignored health risk caused by TiO2NPs, specifically TiO2NP treatment augments procoagulant activity, activation and aggregation of PLTs via calcium-dependent mechanism and thus increases the risk of AT.

Western Diet Modifies Platelet Activation Profiles in Male Mice.

The correlation between obesity and cardiovascular disease has long been understood, yet scant investigations endeavored to determine the impact of an obesogenic diet on platelet activation or function. As platelets drive clot formation, the terminus of cardiovascular events, we aimed to elucidate the longitudinal effect of an obesogenic diet on platelet phenotype by assessing markers of platelet activation using flow cytometry. Male, weanling mice were fed either a Western diet (30% kcal sucrose, 40% kcal fat, 8.0% sodium) or Control diet (7% kcal sucrose, 10% kcal fat, 0.24% sodium). At 12, 16 and 20 weeks on diets, platelets were collected and stained to visualize glycoprotein Ibα (GPIbα), P-selectin and the conformationally active state of αIIbß3 (a platelet specific integrin) after collagen stimulation. At all time points, a Western diet reduced GPIbα and αIIbß3 expression in platelets broadly while P-selectin levels were unaffected. However, P-selectin was diminished by a Western diet in the GPIbα- subpopulation. Thus, a Western diet persistently primed platelets towards a blunted activation response as indicated by reduced active αIIbß3 and P-selectin surface expression. This study provides a first look at the influence of diet on platelet activation and revealed that platelet activation is susceptible to dietary intervention.

An αIIbß3 monoclonal antibody traps a semiextended conformation and allosterically inhibits large ligand binding.

ABSTRACT: Monoclonal antibodies (mAbs) have provided valuable information regarding the structure and function of platelet αIIbß3. Protein disulfide isomerase (PDI) has been implicated in αIIbß3 activation and binds to thrombin-activated αIIbß3. Using human platelets as the immunogen, we identified a new mAb (R21D10) that inhibits the binding of PDI to platelets activated with thrombin receptor-activating peptide (T6). R21D10 also partially inhibited T6-induced fibrinogen and PAC-1 binding to platelets, as well as T6- and adenosine 5'-diphosphate-induced platelet aggregation. Mutual competition experiments showed that R21D10 does not inhibit the binding of mAbs 10E5 (anti-αIIb cap domain) or 7E3 (anti-ß3 ß-I domain), and immunoblot studies indicated that R21D10 binds to ß3. The dissociation of αIIbß3 by EDTA had a minimal effect on R21D10 binding. Cryogenic electron microscopy of the αIIbß3-R21D10 Fab complex revealed that R21D10 binds to the ß3 integrin-epidermal growth factor 1 (I-EGF1) domain and traps an intermediate conformation of αIIbß3 with semiextended leg domains. The binding of R21D10 produces a major structural change in the ß3 I-EGF2 domain associated with a new interaction between the ß3 I-EGF2 and αIIb thigh domains, which may prevent the swing-out motion of the ß3 hybrid domain required for high-affinity ligand binding and protect αIIbß3 from EDTA-induced dissociation. R21D10 partially reversed the ligand binding priming effect of eptifibatide, suggesting that it could convert the swung-out conformation into a semiextended conformation. We concluded that R21D10 inhibits ligand binding to αIIbß3 via a unique allosteric mechanism, which may or may not be related to its inhibition of PDI binding.

Prospective, international, multisite comparison of platelet isolation techniques for genome-wide transcriptomics: communication from the SSC of the ISTH.

Genome-wide platelet transcriptomics is increasingly used to uncover new aspects of platelet biology and as a diagnostic and prognostic tool. Nevertheless, platelet isolation methods for transcriptomic studies are not standardized, introducing challenges for cross-study comparisons, data integration, and replication. In this prospective multicenter study, called "Standardizing Platelet Transcriptomics for Discovery, Diagnostics, and Therapeutics in the Thrombosis and Hemostasis Community (STRIDE)" by the International Society on Thrombosis and Haemostasis Scientific and Standardization Committees, we assessed how 3 of the most commonly used platelet isolation protocols influence metrics from next-generation bulk RNA sequencing and functional assays. Compared with washing alone, more stringent removal of leukocytes by anti-CD45 beads or PALL filters resulted in a sufficient quantity of RNA for next-generation sequencing and similar quality of RNA sequencing metrics. Importantly, stringent removal of leukocytes resulted in the lower relative expression of known leukocyte-specific genes and the higher relative expression of known platelet-specific genes. The results were consistent across enrolling sites, suggesting that the techniques are transferrable and reproducible. Moreover, all 3 isolation techniques did not influence basal platelet reactivity, but agonist-induced integrin αIIbß3 activation is reduced by anti-CD45 bead isolation compared with washing alone. In conclusion, the isolation technique chosen influences genome-wide transcriptional and functional assays in platelets. These results should help the research community make informed choices about platelet isolation techniques in their own platelet studies.

Iron deficiency anemia and platelet dysfunction: A comprehensive analysis of the underlying mechanisms.

AIMS: This research aimed to study the changes in platelet function and their underlying mechanisms in iron deficiency anemia. MAIN METHODS: Initially, we evaluated platelet function in an IDA mice model. Due to the inability to accurately reduce intracellular Fe2+ concentrations, we investigated the impact of Fe2+ on platelet function by introducing varying concentrations of Fe2+. To probe the underlying mechanism, we simultaneously examined the dynamics of calcium in the cytosol, and integrin αIIbß3 activation in Fe2+-treated platelets. Ferroptosis inhibitors Lip-1 and Fer-1 were applied to determine whether ferroptosis was involved in this process. KEY FINDINGS: Our study revealed that platelet function was suppressed in IDA mice. Fe2+ concentration-dependently facilitated platelet activation and function in vitro. Mechanistically, Fe2+ promoted calcium mobilization, integrin αIIbß3 activation, and its downstream outside-in signaling. Additionally, we also demonstrated that ferroptosis might play a role in this process. SIGNIFICANCE: Our data suggest an association between iron and platelet activation, with iron deficiency resulting in impaired platelet function, while high concentrations of Fe2+ contribute to platelet activation and function by promoting calcium mobilization, αIIbß3 activation, and ferroptosis.

Application of Funnel Metadynamics to the Platelet Integrin αIIbß3 in Complex with an RGD Peptide.

Integrin αIIbß3 mediates platelet aggregation by binding the Arginyl-Glycyl-Aspartic acid (RGD) sequence of fibrinogen. RGD binding occurs at a site topographically proximal to the αIIb and ß3 subunits, promoting the conformational activation of the receptor from bent to extended states. While several experimental approaches have characterized RGD binding to αIIbß3 integrin, applying computational methods has been significantly more challenging due to limited sampling and the need for a priori information regarding the interactions between the RGD peptide and integrin. In this study, we employed all-atom simulations using funnel metadynamics (FM) to evaluate the interactions of an RGD peptide with the αIIb and ß3 subunits of integrin. FM incorporates an external history-dependent potential on selected degrees of freedom while applying a funnel-shaped restraint potential to limit RGD exploration of the unbound state. Furthermore, it does not require a priori information about the interactions, enhancing the sampling at a low computational cost. Our FM simulations reveal significant molecular changes in the ß3 subunit of integrin upon RGD binding and provide a free-energy landscape with a low-energy binding mode surrounded by higher-energy prebinding states. The strong agreement between previous experimental and computational data and our results highlights the reliability of FM as a method for studying dynamic interactions of complex systems such as integrin.

Essential role of glycoprotein Ibα in platelet activation.

ABSTRACT: Glycoprotein Ibα (GPIbα), the ligand-binding subunit of platelet GPIb-IX complex, interacts with von Willebrand factor (VWF) exposed at the injured vessel wall, initiating platelet adhesion, activation, hemostasis, and thrombus formation. The cytoplasmic tail of GPIbα interacts with 14-3-3ζ, regulating the VWF-GPIbα-elicited signal transduction and VWF binding function of GPIbα. However, we unexpectedly found that the GPIbα-14-3-3ζ association, beyond VWF-dependent function, is essential for general platelet activation. We found that the myristoylated peptide of GPIbα C-terminus MPαC, a potential GPIbα inhibitor, by itself induced platelet aggregation, integrin αIIbß3 activation, granule secretion, and phosphatidylserine (PS) exposure. Conversely, the deletion of the cytoplasmic tail of GPIbα in mouse platelets (10aa-/-) decreased platelet aggregation, integrin αIIbß3 activation, granule secretion, and PS exposure induced by various physiological agonists. Phosphoproteome-based kinase activity profiling revealed significantly upregulated protein kinase C (PKC) activity in MPαC-treated platelets. MPαC-induced platelet activation was abolished by the pan-PKC inhibitor and PKCα deletion. Decreased PKC activity was observed in both resting and agonist-stimulated 10aa-/- platelets. GPIbα regulates PKCα activity by sequestering 14-3-3ζ from PKCα. In vivo, the deletion of the GPIbα cytoplasmic tail impaired mouse hemostasis and thrombus formation and protected against platelet-dependent pulmonary thromboembolism. Therefore, our findings demonstrate an essential role for the GPIbα cytoplasmic tail in regulating platelet general activation and thrombus formation beyond the VWF-GPIbα axis.

Impact of Protein Corona Formation and Polystyrene Nanoparticle Functionalisation on the Interaction with Dynamic Biomimetic Membranes Comprising of Integrin.

Plastics, omnipresent in the environment, have become a global concern due to their durability and limited biodegradability, especially in the form of microparticles and nanoparticles. Polystyrene (PS), a key plastic type, is susceptible to fragmentation and surface alterations induced by environmental factors or industrial processes. With widespread human exposure through pollution and diverse industrial applications, understanding the physiological impact of PS, particularly in nanoparticle form (PS-NPs), is crucial. This study focuses on the interaction of PS-NPs with model blood proteins, emphasising the formation of a protein corona, and explores the subsequent contact with platelet membrane mimetics using experimental and theoretical approaches. The investigation involves αIIbß3-expressing cells and biomimetic membranes, enabling real-time and label-free nanoscale precision. By employing quartz-crystal microbalance with dissipation monitoring studies, the concentration-dependent cytotoxic effects of differently functionalised ~210 nm PS-NPs on HEK293 cells overexpressing αIIbß3 are evaluated in detail. The study unveils insights into the molecular details of PS-NP interaction with supported lipid bilayers, demonstrating that a protein corona formed in the presence of exemplary blood proteins offers protection against membrane damage, mitigating PS-NP cytotoxicity.

Regulation of platelet activation and thrombus formation in acute non-ST segment elevation myocardial infarction: Role of Beclin1.

This study aims to investigate the mechanism of platelet activation-induced thrombosis in patients with acute non-ST segment elevation myocardial infarction (NSTEMI) by detecting the expression of autophagy-associated proteins in platelets of patients with NSTEMI. A prospective study was conducted on 121 patients with NSTEMI who underwent emergency coronary angiography and optical coherence tomography. The participants were divided into two groups: the ST segment un-offset group (n = 64) and the ST segment depression group (n = 57). We selected a control group of 60 patients without AMI during the same period. The levels of autophagy-associated proteins and the expression of autophagy-associated proteins in platelets were measured using immunofluorescence staining and Western blot. In NSTEMI, the prevalence of red thrombus was higher in the ST segment un-offset myocardial infarction (STUMI) group, whereas white thrombus was more common in the ST segment depression myocardial infarction (STDMI) group. Furthermore, the platelet aggregation rate was significantly higher in the white thrombus group compared with the red thrombus group. Compared with the control group, the autophagy-related protein expression decreased, and the expression of αIIbß3 increased in NSTEMI. The overexpression of Beclin1 could activate platelet autophagy and inhibit the expression of αIIbß3. The results suggested that the increase in platelet aggregation rate in patients with NSTEMI may be potentially related to the change in autophagy. And the overexpression of Beclin1 could reduce the platelet aggregation rate by activating platelet autophagy. Our findings demonstrated that Beclin1 could be a potential therapeutic target for inhibiting platelet aggregation in NSTEMI.

1,25-Dihydroxyvitamin D3 attenuates platelet aggregation potentiated by SARS-CoV-2 spike protein via inhibiting integrin αIIbß3 outside-in signaling.

Platelet hyperreactivity contributes to the pathogenesis of COVID-19, which is associated with a hypercoagulability state and thrombosis disorder. It has been demonstrated that Vitamin D deficiency is associated with the severity of COVID-19 infection. Vitamin D supplement is widely used as a dietary supplement due to its safety and health benefits. In this study, we investigated the direct effects and underlying mechanisms of 1,25(OH)2D3 on platelet hyperreactivity induced by SRAS-CoV-2 spike protein via Western blot and platelet functional studies in vitro. Firstly, we found that 1,25(OH)2D3 attenuated platelet aggregation and Src-mediated signaling. We further observed that 1,25(OH)2D3 attenuated spike protein-potentiated platelet aggregation in vitro. Mechanistically, 1,25(OH)2D3 attenuated spike protein upregulated-integrin αIIbß3 outside-in signaling such as platelet spreading and the phosphorylation of ß3, c-Src and Syk. Moreover, using PP2, the Src family kinase inhibitor to abolish spike protein-stimulated platelet aggregation and integrin αIIbß3 outside-in signaling, the combination of PP2 and 1,25(OH)2D3 did not show additive inhibitory effects on spike protein-potentiated platelet aggregation and the phosphorylation of ß3, c-Src and Syk. Thus, our data suggest that 1,25(OH)2D3 attenuates platelet aggregation potentiated by spike protein via downregulating integrin αIIbß3 outside-in signaling.

Expression, Purification and Characterization of Recombinant Disintegrin from Gloydius Brevicaudus Venom in Escherichia Coli.

Disintegrins, a family of snake venom protein, which are capable of modulating the activity of integrins that play a fundamental role in the regulation of many physiological and pathological processes. The main purpose of this study is to obtain the recombinant disintegrin (r-DI) and evaluate its biological activity. In this study, we explored a high-level expression prokaryotic system and purification strategy for r-DI. Then, r-DI was treated to assay effects on cell growth, migration, and invasion. The affinity for the interactions of r-DI with integrin was determined using Surface plasmon resonance (SPR) analyses. The r-DI can be expressed in Escherichia coli and purified by one-step chromatography. The r-DI can inhibit B16F10 cells proliferation, migration, and invasion. Also, we found that r-DI could interact with the integrin αIIbß3 (GPIIb/IIIa). The r-DI can be expressed, purified, characterized through functional assays, and can also maintain strong biological activities. Thus, this study showed potential therapeutic effects of r-DI for further functional and structural studies.

In vitro study on device-induced damage to blood cellular components and degradation of von Willebrand factor in a CentriMag pump-assisted circulation.

BACKGROUND: High mechanical shear stress (HMSS) generated by blood pumps during mechanical circulatory support induces blood damage (or function alteration) not only of blood cell components but also of plasma proteins. METHODS: In the present study, fresh, healthy human blood was used to prime a blood circuit assisted by a CentriMag centrifugal pump at a flow rate of 4.5 L/min under three pump pressure heads (75, 150, and 350 mm Hg) for 4 h. Blood samples were collected for analyses of plasma-free hemoglobin (PFH), von Willebrand factor (VWF) degradation and platelet glycoprotein (GP) IIb/IIIa receptor shedding. RESULTS: The extent of all investigated aspects of blood damage increased with increasing cross-pump pressure and duration. Loss of high-molecular-weight multimers (HMWM)-VWF in Loop 2 and Loop 3 significantly increased after 2 h. PFH, loss of HMWM-VWF, and platelet GPIIb/IIIa receptor shedding showed a good linear correlation with mean shear stress corresponding to the three pump pressure heads. CONCLUSIONS: HMSS could damage red blood cells, cause pathological VWF degradation, and induce platelet activation and platelet receptor shedding. Different blood components can be damaged to different degrees by HMSS; VWF and VWF-enhanced platelet activation may be more susceptible to HMSS.

Full-length αIIbß3 cryo-EM structure reveals intact integrin initiate-activation intrinsic architecture.

Integrin αIIbß3 is the key receptor regulating platelet retraction and accumulation and a proven drug-target for antithrombotic therapies. Here we resolve the cryo-EM structures of the full-length αIIbß3, which covers three distinct states along the activation pathway. Firstly, we obtain the αIIbß3 structure at 3 Å resolution in the inactive state, revealing the overall topology of the heterodimer with the transmembrane (TM) helices and the ligand-binding domain tucked in a specific angle proximity to the TM region. After the addition of a Mn2+ agonist, we resolve two coexisting structures representing two new states between inactive and active state. Our structures show conformational changes of the αIIbß3 activating trajectory and a unique twisting of the integrin legs, which is required for platelets accumulation. Our structure provides direct structural evidence for how the lower legs are involved in full-length integrin activation mechanisms and offers a new strategy to target the αIIbß3 lower leg.

Contributions of the individual domains of αIIbß3 integrin to its extension: Insights from multiscale modeling.

The platelet integrin αIIbß3 undergoes long-range conformational transitions between bent and extended conformations to regulate platelet aggregation during hemostasis and thrombosis. However, how exactly αIIbß3 transitions between conformations remains largely elusive. Here, we studied how transitions across bent and extended-closed conformations of αIIbß3 integrin are regulated by effective interactions between its functional domains. We first carried out µs-long equilibrium molecular dynamics (MD) simulations of full-length αIIbß3 integrins in bent and intermediate conformations, the latter characterized by an extended headpiece and closed legs. Then, we built heterogeneous elastic network models, perturbed inter-domain interactions, and evaluated their relative contributions to the energy barriers between conformations. Results showed that integrin extension emerges from: (i) changes in interfaces between functional domains; (ii) allosteric coupling of the head and upper leg domains with flexible lower leg domains. Collectively, these results provide new insights into integrin conformational activation based on short- and long-range interactions between its functional domains and highlight the importance of the lower legs in the regulation of integrin allostery.

Recent advances in vascular thiol isomerases and redox systems in platelet function and thrombosis.

There have been substantial advances in vascular protein disulfide isomerases (PDIs) in platelet function and thrombosis in recent years. There are 4 known prothrombotic thiol isomerases; PDI, endoplasmic reticulum protein (ERp)57, ERp72, and ERp46, and 1 antithrombotic PDI; transmembrane protein 1. A sixth PDI, ERp5, may exhibit either prothrombotic or antithrombotic properties in platelets. Studies on ERp46 in platelet function and thrombosis provide insight into the mechanisms by which these enzymes function. ERp46-catalyzed disulfide cleavage in the αIIbß3 platelet integrin occurs prior to PDI-catalyzed events to maximally support platelet aggregation. The transmembrane PDI transmembrane protein 1 counterbalances the effect of ERp46 by inhibiting activation of αIIbß3. Recent work on the prototypic PDI found that oxidized PDI supports platelet aggregation. The a' domain of PDI is constitutively oxidized, possibly by endoplasmic reticulum oxidoreductase-1α. However, the a domain is normally reduced but becomes oxidized under conditions of oxidative stress. In contrast to the role of oxidized PDI in platelet function, reduced PDI downregulates activation of the neutrophil integrin αMß2. Intracellular platelet PDI cooperates with Nox1 and contributes to thromboxane A2 production to support platelet function. Finally, αIIb and von Willebrand factor contain free thiols, which alter the functions of these proteins, although the extent to which the PDIs regulate these functions is unclear. We are beginning to understand the substrates and functions of vascular thiol isomerases and the redox network they form that supports hemostasis and thrombosis. Moreover, the disulfide bonds these enzymes target are being defined. The clinical implications of the knowledge gained are wide-ranging.

Potential Mechanism of Platelet GPIIb/IIIa and Fibrinogen on Retinal Vein Occlusion.

PURPOSE: To explore the role of coagulation and fibrinolytic factors, and the potential mechanism of platelet aggregation in the pathogenesis of retinal vein occlusion. METHODS: Coagulation and fibrinolytic parameters in patients with retinal vein occlusion were determined using hemagglutinin and HISCL-5000. Relationships between these elevated parameters and factors representing typical clinical manifestations of retinal vein occlusion were examined, and these parameters were analyzed using a STRING database to indicate the potential role of platelet aggregation. Platelet glycoprotein IIb/IIIa (GPIIb/IIIa) levels were evaluated by flow cytometry after antiplatelet treatment in patients and mouse models. Furthermore, the GPIIb/IIIa ligand fibrinogen in peripheral blood and retina of mouse models was assessed by the turbidimetric method and real-time PCR, respectively. RESULTS: In patients, significant increases in peripheral blood fibrinogen and GPIIb/IIIa levels were observed (p = 0.0040, p < 0.0001, respectively). A positive correlation was observed between macular thickness (MT) and both fibrinogen and GPIIb/IIIa (r = 0.4528, p = 0.0063; r = 0.3789, p = 0.0427, respectively). After intravitreal injections of anti-vascular endothelial growth factor drugs, a significant reduction in fibrinogen levels was observed (p = 0.0072). In addition, the use of antiplatelet drugs resulted in a significant decrease in GPIIb/IIIa (p < 0.0001). In a mouse model, antiplatelet therapy significantly reduced both peripheral blood and retina fibrinogen levels and the overall rate of vein occlusion 3 days after occlusion (p < 0.0005). In addition, the reduction in GPIIb/IIIa levels after antiplatelet therapy was remarkable. CONCLUSION: Fibrinogen and GPIIb/IIIa may be involved in retinal vein occlusion and blocking platelet aggregation may be a new therapeutic approach for retinal vein occlusion.