Surface Of Human Platelets Research Articles

Hsp47 Inhibitor Col003 Attenuates Collagen-Induced Platelet Activation and Cerebral Ischemic-Reperfusion Injury in Rats.

Ischemic stroke is a major type of stroke worldwide currently without effective treatment, although antiplatelet therapy is an existing option for it. In previous studies, heat shock protein 47 (Hsp47) was found to be expressed on the surface of human and mice platelets and to strengthen the interaction between platelets and collagen. In recent years, Col003 was discovered to inhibit the interaction of Hsp47 with collagen. We evaluated whether the Hsp47 inhibitor Col003 is a promising therapeutic agent for ischemic stroke. Here, we first verified that Hsp47 is also expressed on the surface of rat platelets, and its inhibitor Col003 significantly inhibited thrombus formation in the FeCl3-induced rat carotid arterial thrombus model. Both Col003 and clopidogrel did not alter the bleeding time or coagulation parameters, while aspirin increased the tail-bleeding time (p < 0.05). The low cytotoxicity level of Col003 to rat platelets and human liver cells was similar to those of aspirin and clopidogrel. Col003 inhibited collagen-induced platelet aggregation, adhesion, [Ca2+]i mobilization, P-selectin expression, reactive oxygen species production and the downstream signal pathway of collagen receptors. The results of the middle cerebral artery occlusion model indicated that Col003 has a protective effect against cerebral ischemic–reperfusion injury in rats. The Hsp47 inhibitor Col003 exerted antiplatelet effect and protective effect against brain damage induced by ischemic stroke through the inhibition of glycoprotein VI (GPVI)and mitogen-activated protein kinase (MAPK) signaling events, which might yield a new antiplatelet agent and strategy to treat ischemic stroke.

An alternate covalent form of platelet αIIbβ3 integrin that resides in focal adhesions and has altered function

AbstractThe αIIbβ3 integrin receptor coordinates platelet adhesion, activation, and mechanosensing in thrombosis and hemostasis. Using differential cysteine alkylation and mass spectrometry, we have identified a disulfide bond in the αIIb subunit linking cysteines 490 and 545 that is missing in ∼1 in 3 integrin molecules on the resting and activated human platelet surface. This alternate covalent form of αIIbβ3 is predetermined as it is also produced by human megakaryoblasts and baby hamster kidney fibroblasts transfected with recombinant integrin. From coimmunoprecipitation experiments, the alternate form selectively partitions into focal adhesions on the activated platelet surface. Its function was evaluated in baby hamster kidney fibroblast cells expressing a mutant integrin with an ablated C490-C545 disulfide bond. The disulfide mutant integrin has functional outside-in signaling but extended residency time in focal adhesions due to a reduced rate of clathrin-mediated integrin internalization and recycling, which is associated with enhanced affinity of the αIIb subunit for clathrin adaptor protein 2. Molecular dynamics simulations indicate that the alternate covalent form of αIIb requires higher forces to transition from bent to open conformational states that is in accordance with reduced affinity for fibrinogen and activation by manganese ions. These findings indicate that the αIIbβ3 integrin receptor is produced in various covalent forms that have different cell surface distribution and function. The C490, C545 cysteine pair is conserved across all 18 integrin α subunits, and the disulfide bond in the αV and α2 subunits in cultured cells is similarly missing, suggesting that the alternate integrin form and function are also conserved.

Juglone prevents human platelet aggregation through inhibiting Akt and protein disulfide isomerase

Background/PurposeJuglone, a natural compound widely found in Juglandaceae plants, has been suggested as a potential drug candidate for treating cancer, inflammation, and diabetic vascular complications. In the present study, the antiplatelet effect and underlying mechanisms of juglone were investigated for the first time. Study design/methodsHuman platelet aggregation and activation were measured by turbidimetric aggregometry, flow cytometry, and Western blotting. In vitro antithrombotic activity of juglone was assessed using collagen-coated flow chambers under whole-blood flow conditions. The effect of juglone on protein disulfide isomerase (PDI) activity was determined by the dieosin glutathione disulfide assay. ResultsJuglone (1 – 5 μM) inhibited platelet aggregation and glycoprotein (GP) IIb/IIIa activation caused by various agonists. In a whole blood flow chamber system, juglone reduced thrombus formation on collagen-coated surfaces under arterial shear rates. Juglone abolished intracellular Ca2+ elevation and protein kinase C activation caused by collagen, but had no significant effect on that induced by G protein-coupled receptor agonists. In contrast, Akt activation caused by various agonists were inhibited in juglone-treated platelets. Additionally, juglone showed inhibitory effects on both recombinant human PDI and platelet surface PDI at concentrations similar to those needed to prevent platelet aggregation. ConclusionJuglone exhibits potent in vitro antiplatelet and antithrombotic effects that are associated with inhibition of Akt activation and platelet surface PDI activity.

Platelet Carcinoembryonic Antigen Cell Adhesion Molecule 5 (CEACAM5) as a Possible Novel Diagnostic Tool for Evaluation of Acute Coronary Syndrome.

BackgroundAcute coronary syndrome (ACS) occurs approximately every 40 seconds, and was an underlying cause of death in 1 out of every 7 deaths. More accurate indicators are needed to distinguish patients with ACS from patients manifesting negative changes in electrocardiogram (ECG) and myocardial enzymes. This study aimed to investigate whether the expression of platelet carcinoembryonic antigen cell adhesion molecule-5 (CEACAM5/CEA/CD66e) could help predict ACS.Material/MethodsWe enrolled 82 participants (mean age 60 years, 33 females and 49 males). The expression of CEA on washed human platelets was assessed using two-color flow cytometry. The CEA levels on platelets and in serum of these 82 consecutive patients were detected using two-color whole-blood flow cytometry analysis and a custom-made Luminex multiplex assay, respectively.ResultsCEA was expressed on the surface of human platelets. The expression of platelet CEA (P<0.01), but not serum CEA (P=0.30), was significantly higher in patients with ACS compared to patients with normal coronary artery. Increased platelet CEA levels could serve as a new independent indicator for ACS (P=0.0003). Platelet CEA testing (P=0.000002), as well as cardiac troponin I (cTnI) (P=0.0005), can diagnose ACS with high sensitivity and specificity, and, combined with cTnI (P<0.0001), can improve the diagnostic value.ConclusionsPlatelet CEA expression was higher in individuals presenting with ACS. Hence, platelet CEA might be a novel and reliable biomarker for ACS. Large-scale studies are needed to confirm this hypothesis.

Autoantibody-mediated desialylation impairs human thrombopoiesis and platelet lifespan.

Immune thrombocytopenia is a common bleeding disease caused by autoantibody-mediated accelerated platelet clearance and impaired thrombopoiesis. Accumulating evidence suggests that desialylation affects platelet lifespan in immune thrombocytopenia. Herein, we report on novel effector functions of autoantibodies from patients with immune thrombocytopenia, which might interfere with the clinical picture of the disease. Data from our study show that a subgroup of autoantibodies is able to induce cleavage of sialic acid residues from the surface of human platelets and megakaryocytes. Moreover, autoantibody-mediated desialylation interferes with the interaction between cells and extracellular matrix proteins leading to impaired platelet adhesion and megakaryocyte differentiation. Using a combination of an ex vivo model of thrombopoiesis, a humanized animal model, and a clinical cohort study, we demonstrate that cleavage of sialic acid induces significant impairment of the production, survival as well as function of human platelets. These data may indicate that prevention of desialylation should be investigated in the future in clinical studies as a potential therapeutic approach to treat bleeding in immune thrombocytopenia.

A Uniform Shear Assay for Human Platelet and Cell Surface Receptors via Cone-plate Viscometry.

Many biological cells/tissues sense the mechanical properties of their local environments via mechanoreceptors, proteins that can respond to forces like pressure or mechanical perturbations. Mechanoreceptors detect their stimuli and transmit signals via a great diversity of mechanisms. Some of the most common roles for mechanoreceptors are in neuronal responses, like touch and pain, or hair cells which function in balance and hearing. Mechanosensation is also important for cell types which are regularly exposed to shear stress such as endothelial cells, which line blood vessels, or blood cells which experience shear in normal circulation. Viscometers are devices that detect the viscosity of fluids. Rotational viscometers may also be used to apply a known shear force to fluids. The ability of these instruments to introduce uniform shear to fluids has been exploited to study many biological fluids including blood and plasma. Viscometry may also be used to apply shear to the cells in a solution, and to test the effects of shear on specific ligand-receptor pairs. Here, we utilize cone-plate viscometry to test the effects of endogenous levels of shear stress on platelets treated with antibodies against the platelet mechanosensory receptor complex GPIb-IX.

A Flow Cytometry‐Based Assay for Procoagulant Platelet Polyphosphate

Platelet polyphosphate is an inorganic procoagulant polymer of orthophosphate units that is stored in dense granules and is released upon platelet activation. Here, we describe an assay to measure polyphosphate on the surface of procoagulant human platelets. Recombinant Escherichia coli-expressed exopolyphosphatase deletion mutant PPX_Δ12 labeled with fluorescent Alexa488 dye was used as a polyphosphate probe in flow cytometry. PPX_Δ12-Alexa488-signal dose-dependently increased with long-chain polyphosphate binding to platelets. In contrast, short-chain polyphosphate that is found in the supernatant of activated platelets, did not bind to the platelet surface. Both exopolyphosphatase treatment and polyphosphate pre-incubation abolished PPX_Δ12-Alexa488 binding to polyphosphate on platelets. Stimulation of platelets with thrombin receptor agonist Trap6, and P2Y12 receptor activator ADP increased polyphosphate accumulation on platelet surfaces and PPX_Δ12-Alexa488 signal in a dose-dependent manner. This study indicates that long-chain polyphosphate binds to platelet plasma membranes and presents a promising diagnostic assay to measure this interaction on human platelets in platelet-rich plasma. Future investigations will aim to determine if polyphosphate can be used as a novel biomarker of thrombosis. © 2016 International Clinical Cytometry Society.

Evidence of an interaction between TGF-β1 and the SDF-1/CXCR4/CXCR7 axis in human platelets

BackgroundTGF-β1, SDF-1 and its cognate receptors CXCR4 and CXCR7 are expressed on the surface of human platelets and their expression levels are differently regulated in symptomatic coronary artery disease (CAD). All these proteins and receptors influence outcome in patients with symptomatic CAD. There might be a crosstalk between TGF-β1 and the SDF-1/CXCR4/CXCR7 axis. Interrelations in CAD, especially in the context of platelets, are poorly understood. Therefore, we aimed to provide clinical and experimental evidence of interactions between TGF-β1 and the SDF-1/CXCR4/CXCR7 axis in human platelets. Methods and resultsBlood samples of the complete cohort (n=284) were analysed for platelet surface expression levels of TGF-β1, SDF-1, CXCR4 and CXCR7 by flow cytometry. For stimulation assays platelet rich plasma was treated with TGF-β1 or SDF-1 and then analysed by flow cytometry. Multiple regression analyses were run to show independent associations of TGF-β1 with SDF-1, CXCR4, CXCR7 and clinical cofactors. Both, CXCR4 and CXCR7 significantly predicted TGF-β1 (p<0.001 and p<0.001, respectively). After stimulation with SDF-1, surface expression of TGF-β1 increased significantly when compared to resting platelets [mean TGF-β1 MFI 19.01 vs. mean TGF-β1 MFI 14.01, p<0.001]. Upon receptor blocking with either anti-CXCR4 or anti-CXCR7 monoclonal antibodies the enhancing effect of SDF-1 on TGF-β1 surface expression was significantly blunted. Stimulation with TGF-β1 did not alter SDF-1, CXCR4 or CXCR7 expression significantly. ConclusionsWe provide first clinical and experimental data suggesting a cross-talk between TGF-β and the SDF-1/CXCR4/CXCR7 axis in platelets which does not involve transcriptional modulation as shown previously for other cellular systems.

Homozygous loss of mouse tetraspanin CD82 enhances integrin αIIbβ3 expression and clot retraction in platelets

Integrin αIIbβ3 is critical for platelet-mediated blood clotting. Tetraspanins are well-established regulators of integrins and genetic loss of tetraspanin CD151 or TSSC6 in mice leads to increased bleeding due to inadequate integrin αIIbβ3 outside-in signaling. Conversely, mild but enhanced integrin αIIbβ3 activation and hyperaggregation is observed in CD9 and CD63 null mice respectively. CD82 is reportedly expressed in platelets; however its function is unknown. Using genetically engineered CD82 null mice, we investigated the role of the tetraspanin CD82 in platelet activation. Loss of CD82 resulted in reduced bleed times in vivo. CD82 was present on the surface of both human and mouse platelets, and its levels did not change upon platelet activation or degranulation. No differences in platelet activation, degranulation, or aggregation in response to ADP or collagen were detected in CD82 null mice. However, the kinetics of clot retraction was enhanced, which was intrinsic to the CD82-null platelets. Integrin αIIbβ3 surface expression was elevated on the platelets from CD82 null mice and they displayed enhanced adhesion and tyrosine kinase signaling on fibrinogen. This is the first report on CD82 function in platelets; which we found intrinsically modulates clot retraction, integrin αIIbβ3 expression, cell adhesion, and tyrosine signaling.

Regulation of Pannexin-1 channel activity.

Pannexin-1 (Panx1) forms anion-selective channels with a permeability up to 1kDa and represents a pathway for the release of cytosolic ATP. Several structurally similar connexin (Cx) proteins have been identified in platelets and shown to play roles in haemostasis and thrombosis. More recently, functional Panx1 channels have been demonstrated on the surface of human platelets [Taylor et al. (2014) J. Thromb. Haemost. 12, 987-998]. Since their identification in the year 2000, several mechanisms have been reported to activate Panx1 channels, including mechanical stimulation, oxygen-glucose deprivation, a rise of [Ca2+]i, caspase cleavage and phosphorylation. Within this review, the regulation of Panx1 channels is discussed, with a focus on how they may contribute to platelet function.

F11R Expression upon Hypoxia Is Regulated by RNA Editing

F11R is a cell adhesion molecule found on the surface of human platelets. It plays a role in platelet aggregation, cell migration and cell proliferation. F11R is subjected to RNA editing, a post-transcriptional modification which affects RNA structure, stability, localization, translation and splicing. RNA editing in the 3'UTR of F11R and RNA levels are increased upon hypoxia. We therefore set to examine if RNA editing plays a role in the increase of F11R RNA seen upon hypoxic conditions. We show that ADAR1, but not ADAR2, takes part in the editing of F11R however editing alone is not sufficient for obtaining an elevation in RNA levels. In addition we show that hyper-edited mature mRNAs are retained in the nucleus and are associated with p54nrb. We therefore conclude that hypoxia-induced edited RNAs of F11R are preferentially stabilized and accumulate in the nucleus preventing their export to the cytoplasm for translation. This mechanism may be used by additional proteins in the cell as part of the cell's effort to reduce metabolism upon hypoxic stress.

Human Platelets Express Functional Thymic Stromal Lymphopoietin Receptors: a Potential Role in Platelet Activation in Acute Coronary Syndrome

Background: Thymic stromal lymphopoietin (TSLP) has been shown to be expressed in various inflammatory tissues, such as human atherosclerotic plaques. Many types of myeloid cells involved in atherosclerosis, including mast cells, lymphocytes, dendritic cells and monocytes/macrophages, present TSLP receptors (TSLPR). However, it is unknown whether platelets, which also play important roles in atherothrombosis, express TSLPR. Methods and Results: We applied flow cytometry and western blotting to show that TSLPR was expressed on the surface of human platelets. Following the addition of TSLP to platelets, the expression of CD62P, CD63, PAC-1 and p-Akt as well as aggregation and ATP release were increased significantly. A TSLPR antibody and a PI3K (phosphatidylinositol 3-kinase) enzyme inhibitor (LY294002) significantly inhibited the platelet activation induced by TSLP. The expression of TSLPR, CD62P and CD63 and the increment of the expression of CD62P and CD63 induced by TSLP in the acute coronary syndrome (ACS) group were markedly higher than those in the control group and the stable angina pectoris (SAP) group. The expression and the increment of the expression of CD62P and CD63 induced by TSLP were positively correlated with the expression of TSLPR. Conclusion: Human platelets express functional TSLPR, which can be activated by TSLP to promote platelet activation. TSLP/TSLPR functions via activating the PI3K/AKT pathway, and this signalling pathway may be one of the mechanisms involved in thrombosis in ACS. In coronary disease patients, the determination of TSLPR in platelets may help to identify the risk of ACS.

The SLAM family member CD84 is regulated by ADAM10 and calpain in platelets

Ectodomain shedding is a major mechanism to modulate platelet receptor signaling and to downregulate platelet reactivity. Proteins of the a disintegrin and metalloproteinase (ADAM) family are implicated in the shedding of various platelet receptors. The signaling lymphocyte activation molecule (SLAM) family receptor CD84 is highly expressed in platelets and immune cells, but its role in platelet physiology is not well explored. Because of its ability to form homodimers, CD84 has been suggested to mediate contact-dependent signaling and contribute to thrombus stability. However, nothing is known about the cellular regulation of CD84. We studied the regulation of CD84 in murine platelets by biochemical approaches and use of three different genetically modified mouse lines. Regulation of CD84 in human platelets was studied using inhibitors and biochemical approaches. We show that CD84 is cleaved from the surface of human and murine platelets in response to different shedding inducing agents and platelet receptor agonists. CD84 downregulation occurs through ectodomain-shedding and intracellular cleavage. Studies in transgenic mice identified ADAM10 as the principal sheddase responsible for CD84 cleavage, whereas ADAM17 was dispensable. Western blot analyses revealed calpain-mediated intracellular cleavage of the CD84 C-terminus, occurring simultaneously with, but independently of, ectodomain shedding. Furthermore, analysis of plasma and serum samples from transgenic mice demonstrated that CD84 is constitutively shed from the platelet surface by ADAM10 in vivo. These results reveal a dual regulation mechanism for platelet CD84 by simultaneous extra- and intracellular cleavage that may modulate platelet-platelet and platelet-immune cell interactions.

Simvastatin and atorvastatin attenuate VCAM-1 and uPAR expression on human endothelial cells and platelet surface expression of CD40 ligand

In addition to their cholesterol lowering ability, statins have proven pleiotropic effects in the cardiovascular system. Chronic inflammation with interactions between platelets and endothelial cells leads to an upregulation of activity markers of atherosclerosis. The purpose of this study was to investigate the effects of simvastatin and atorvastatin on platelets and endothelial cells in an in vitro endothelial cell model. After a 24 h incubation period with either simvastatin or atorvastatin (1 μmol/L), human umbilical vein endothelial cells were stimulated for 1 h with lipopolysaccharide (LPS), and were then incubated in direct contact with activated platelets. Platelet surface expression of CD40L and CD62P and expression of ICAM-1, VCAM-1, uPAR and MT1-MMP on endothelial cells were measured by flow cytometry. Supernatants were analyzed by ELISA for soluble MMP-1. The increased expression of VCAM-1 and uPAR on endothelial cells by stimulation with LPS and by direct contact with activated platelets was significantly reduced to a similar extent through pre-incubation with both atorvastatin and simvastatin (p < 0.05). Platelets without endothelial cell contact, but in direct contact with either statin, showed similar significant reductions in surface expression of CD40L (p < 0.005). These effects may explain the ability of statins to reduce the progression of atherosclerosis in addition to their cholesterol-lowering properties.

Platelet-Mediated Thrombosis and Drug-Eluting Stents

Stent thrombosis is an example of device-induced, platelet-mediated arterial thrombosis. Rates of stent thrombosis can vary from 10% depending on the patient population, genetic predisposition, device type, pharmacological choices, and duration of antiplatelet pharmacotherapy. The Platelet Colloquium is an annual academic–industry–governmental think-tank meeting devoted to identifying research challenges in platelet biology and clinical applications. The latest meeting was held in Washington, DC, on January 25 to 26, 2011, and this review summarizes the discussions of biocompatible stent design, platelet function assessment, and prevention of thrombosis via short- and long-term P2Y12 platelet receptor antagonism. ### Stent Design and Surface-Mediated Platelet Activation The vascular injury induced by percutaneous coronary intervention (PCI) produces dynamic changes on the surface of human platelets.1 Activated platelets are among the first cells to arrive at the site of injury. Stent thrombosis results from the interaction of several procedural, anatomic, and genetically determined factors.2 Early cellular and inflammatory events are influenced by the properties of the stent or its coating. First-generation drug-eluting stents (DES) used relatively thick struts and durable polymers.3 Research efforts focused on development of nonerodable biocompatible materials that could control the release of antiproliferative medications over several weeks.4 In vitro models showed that these devices appeared to be associated with increased platelet activation and adhesion compared with identical bare metal stents (BMS).5 The continuous presence of a durable polymer and drug has been posited to be partly responsible for delayed arterial healing and enhanced stent thrombogenicity.6 Second-generation DES modified some of these components by reducing strut thickness and polymeric drug load.3 In vitro data suggest that the lower polymeric drug load used in current everolimus-eluting stents may have a more favorable thrombogenic profile than BMS controls.7 Recent data also suggest that these devices might favorably affect inflammation and vascular healing after …

Identification of a Region within the Cytoplasmic Domain of Sema4D That Binds Calmodulin and Regulates Shedding of the Sema4D Exodomain in Platelets,

Abstract 3249We have previously shown that the 150 kDa semaphorin family member, Sema4D (or CD100), is expressed on the surface of human and mouse platelets, where it is able to selectively reinforce collagen-initiated platelet activation by engaging receptors in trans on adjoining platelets in a contact-dependent manner. Key to this effect is the Sema4D extracellular domain, which in addition to being a ligand for Sema4D receptors, is a substrate for the metalloprotease, ADAM17 in platelets. Our previous studies suggest that ADAM17 cleaves Sema4D and other platelet surface proteins close to the platelet plasma membrane gradually producing, in the case of Sema4D, a single large (≈120 kDa) exodomain fragment and a smaller (≈28 kDa) fragment that includes the transmembrane domain and the cytoplasmic domain and remains associated with the platelet (Zhu, et al., PNAS 2007). Exodomain shedding in platelets can be triggered by the phorbol ester, PMA, and by physiologic agonists such as thrombin that raise the cytoplasmic Ca++ concentration, but the mechanisms that regulate the shedding of Sema4D have not been defined. Here we have studied the potential role of an interaction between calmodulin and the Sema4D cytoplasmic domain. Using a public resource (http://calcium.uhnres.utoronto.ca/ctdb/ctdb/home.html), we identified a potential calmodulin-binding sequence (GYLPRQCLKFRSALLIGKKKPKS-COOH, Gly758–Ser780) within the membrane-proximal region of the Sema4D cytoplasmic domain. To test whether this region binds calmodulin, a 23 amino acid peptide corresponding to the predicted Sema4D calmodulin binding sequence (SCBP) was synthesized, as was a scrambled control peptide (RLIKACRQPKPKYKLLGFGSSKL or scrambled SCBP), which is not predicted to bind calmodulin. The results show that SCBP, but not scrambled SCBP, was able to bind to calmodulin-agarose and retrieve calmodulin from platelet lysates. As constitutive association of calmodulin with glycoprotein (GP) Ib has been shown prevent ADAM17-dependent GPIb alpha shedding in platelets, we incubated human platelets with the calmodulin inhibitor, W7. The inhibitor induced gradual Sema4D shedding that was detectable after 5 min and reached a maximum at 60 min, kinetics that are similar to those we have observed with platelet agonists. However, in contrast to platelet agonists, W7-induced Sema4D shedding generated a smaller retained fragment (≈24 kDa Vs. 28 kDa) suggesting that there is either a second or different site of cleavage. Despite their polybasic sequences, flow cytometry and confocal microscopy showed that FITC-conjugated SCBP and scrambled-SCBP are able to cross the plasma membrane. Addition of SCBP, but not scrambled-SCBP, to platelet caused cleavage of Sema4D, producing the same 28 kDa fragment observed with thrombin and PMA. In all cases cleavage of Sema4D was blocked by the metalloprotease inhibitor, TAPI-2. Combined with our earlier observations, these results suggest that 1) Sema4D is a calmodulin binding protein with a site of interaction in the membrane-proximal cytoplasmic domain and a site of cleavage by ADAM17 in the membrane-proximal exodomain, 2) the detachment of calmodulin from Sema4D may be the trigger for Sema4D cleavage in response to platelet agonists, and 3) in contrast to W7, decoying calmodulin from binding sites on Sema4D and other metalloprotease substrates on the platelet surface, as we have done here with a Sema4D cytoplasmic domain peptide, may trigger the same events seen in activated platelets and provide a tool to understand the underlying mechanisms. Disclosures:No relevant conflicts of interest to declare.

Identification of Interacting Hot Spots in the β3 Integrin Stalk Using Comprehensive Interface Design

Protein-protein interfaces are usually large and complementary surfaces, but specific side chains, representing energetic "hot spots," often contribute disproportionately to binding free energy. We used a computational method, comprehensive interface design, to identify hot spots in the interface between the stalk regions of the β3 and the complementary αIIb and αv integrin subunits. Using the Rosetta alanine-scanning and design algorithms to predict destabilizing, stabilizing, and neutral mutations in the β3 region extending from residues Lys(532) through Gly(690), we predicted eight alanine mutations that would destabilize the αIIbβ3 interface as well as nine predicted to destabilize the αvβ3 interface, by at least 0.3 kcal/mol. The mutations were widely and unevenly distributed, with four between residues 552 and 563 and five between 590 and 610, but none between 565 and 589, and 611 and 655. Further, mutations destabilizing the αvβ3 and αIIbβ3 interfaces were not identical. The predictions were then tested by introducing selected mutations into the full-length integrins expressed in Chinese hamster ovary cells. Five mutations predicted to destabilize αIIb and β3 caused fibrinogen binding to αIIbβ3, whereas three of four predicted to be neutral or stabilizing did not. Conversely, a mutation predicted to destabilize αvβ3, but not αIIbβ3 (D552A), caused osteopontin binding to αvβ3, but not fibrinogen binding to αIIbβ3. These results indicate that stability of the distal stalk interface is involved in constraining integrins in stable, inactive conformations. Further, they demonstrate the ability of comprehensive interface design to identify functionally significant integrin mutations.

Heat Shock Protein 47: A New Platelet Collagen Receptor

AbstractAbstract 158We recently reported that Heat shock protein 47 (Hsp47), a well established collagen binding protein, was present on the surface of human platelets. In the current study we demonstrate that anti-Hsp47 antibodies and a small molecule inhibitor of Hsp47 selectively prevent aggregation of platelets induced with collagen fibrils. Inhibition of Hsp47 was also found to reduce the size of thrombi produced upon perfusion of blood under arterial flow conditions over collagen in vitro. Inclusion of Integrilin, to prevent platelet-platelet interactions via fibrinogen, indicated that the reduced size of thrombi was due to fewer direct platelet-collagen interactions. Collagen-induced tyrosine phosphorylation of Syk, an early marker of signalling from the collagen receptor GPVI was unaffected by the Hsp47 inhibitor, suggesting that any contribution to platelet function by Hsp47 may be independent of GPVI-proximal signalling. Surface plasmon resonance analysis of recombinant his-tagged human Hsp47 revealed its ability to interact directly with collagen fibrils, an interaction that was disrupted by the small molecule inhibitor of Hsp47.Mouse platelets were also found to contain Hsp47, and aggregation induced with collagen was reduced by inhibitors of Hsp47. Infusion of Hsp47 inhibitor into mice increased tail bleeding times and volumes of blood lost, even in the absence of anticoagulant. This effect was likely due to inhibition of platelet function, since the thrombi formed in cremaster muscle arterioles using an intravital laser-injury model of thrombosis were smaller following administration of the Hsp47 inhibitor.Together these data indicate Hsp47 to function on the platelet surface as a collagen receptor that potentially represents a new target for antithrombotic therapy. Disclosures:No relevant conflicts of interest to declare.

Small Molecules Can Selectively Inhibit Ephrin Binding to the EphA4 and EphA2 Receptors

The erythropoietin-producing hepatocellular (Eph) family of receptor tyrosine kinases regulates a multitude of physiological and pathological processes. Despite the numerous possible research and therapeutic applications of agents capable of modulating Eph receptor function, no small molecule inhibitors targeting the extracellular domain of these receptors have been identified. We have performed a high throughput screen to search for small molecules that inhibit ligand binding to the extracellular domain of the EphA4 receptor. This yielded a 2,5-dimethylpyrrolyl benzoic acid derivative able to inhibit the interaction of EphA4 with a peptide ligand as well as the natural ephrin ligands. Evaluation of a series of analogs identified an isomer with similar inhibitory properties and other less potent compounds. The two isomeric compounds act as competitive inhibitors, suggesting that they target the high affinity ligand-binding pocket of EphA4 and inhibit ephrin-A5 binding to EphA4 with K(i) values of 7 and 9 mum in enzyme-linked immunosorbent assays. Interestingly, despite the ability of each ephrin ligand to promiscuously bind many Eph receptors, the two compounds selectively target EphA4 and the closely related EphA2 receptor. The compounds also inhibit ephrin-induced phosphorylation of EphA4 and EphA2 in cells, without affecting cell viability or the phosphorylation of other receptor tyrosine kinases. Furthermore, the compounds inhibit EphA4-mediated growth cone collapse in retinal explants and EphA2-dependent retraction of the cell periphery in prostate cancer cells. These data demonstrate that the Eph receptor-ephrin interface can be targeted by inhibitory small molecules and suggest that the two compounds identified will be useful to discriminate the activities of EphA4 and EphA2 from those of other co-expressed Eph receptors that are activated by the same ephrin ligands. Furthermore, the newly identified inhibitors represent possible leads for the development of therapies to treat pathologies in which EphA4 and EphA2 are involved, including nerve injuries and cancer.

Crystal structure of rhodocytin, a ligand for the platelet‐activating receptor CLEC‐2

Binding of the snake venom protein rhodocytin to CLEC-2, a receptor on the surface of human platelets, initiates a signaling cascade leading to platelet activation and aggregation. We have previously solved the structure of CLEC-2. The 2.4 A resolution crystal structure of rhodocytin presented here demonstrates that it is the first snake venom or other C-type lectin-like protein to assemble as a non-disulfide linked (alphabeta)(2) tetramer. Rhodocytin is highly adapted for interaction with CLEC-2 and displays a concave binding surface, which is highly complementary to the experimentally determined binding interface on CLEC-2. Using computational dynamic methods, surface electrostatic charge and hydrophobicity analyses, and protein-protein docking predictions, we propose that the (alphabeta)(2) rhodocytin tetramer induces clustering of CLEC-2 receptors on the platelet surface, which will trigger major signaling events resulting in platelet activation and aggregation.