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Efficient in silico approaches are needed to identify strong integrin αIIbβ3 inhibitors through a small number of measurements. To address the challenge, we investigated the effect of learning dataset on the classification performance of machine learning models focusing on weak and inactive compounds. The structure and activity information of the compounds were obtained from ChEMBL, and pCHEMBL values were used to classify them as active, inactive, or weak. Datasets with various imbalance levels from active:inactive=1 : 1 to 1 : 1000 were used for the machine learning. The prediction scores of the weak samples were found to lie between the predictive values of active and inactive compounds. In addition, another dataset that consists of 149 actives and 6.9 million inactives was screened; the results indicated that the number of positive predictions decreased for models trained with a higher number of inactives. Although there is a trade‐off between false positives and false negatives, for determination of compounds with strong activity using a reduced number of measurements, it is better to use a large number of inactives for learning and identifying compounds that score higher than the weak samples. [ABSTRACT FROM AUTHOR]

Background: A nonpedigreed male cat presented with epistaxis, severe bladder hemorrhage, and secondary urethral obstruction after cystocentesis. Objectives: To characterize the phenotype of a cat with bleeding diathesis and use a precision medicine approach to identify the molecular genetic defect by whole genome sequencing. Methods: Adenosine diphosphate (ADP) and arachidonic acid (AA)‐induced whole blood platelet aggregometry was performed in the affected cat and a healthy cat. Platelet activation, measured by P‐selectin expression, and surface integrin subunit β3 expression were evaluated by flow cytometry in the affected cat and healthy control. Total integrin subunit αIIb expression was assessed by western blot. Whole genome sequencing at 30× coverage was used to identify genetic variants that segregated in the affected cat compared to 194 cats from the 99 Lives Sequencing Consortium. Results: Platelet aggregometry identified significant impairment in platelet aggregation in response to ADP and AA compared to the control cat. Targeted protein expression analyses by flow cytometry and immunoblot analysis determined that the surface expression and total expression of the integrin, αIIbβ3, was absent. Whole genome sequencing identified a homozygous c.1986delC frameshift variant in the integrin subunit αIIb (ITGA2B) gene that was not detected in the control population. The p.Pro662fs (ITGA2B P662X) variant terminates translation of the protein at the extracellular domain of the integrin prematurely, which is predicted to affect expression of the β3 unit. Conclusions and Clinical Importance: This novel ITGA2B variant and the associated phenotype closely resemble Glanzmann's thrombasthenia, which has never been reported in cats. [ABSTRACT FROM AUTHOR]

Abstract: During platelet activation, fibrinogen binds to its specific platelet receptor, integrin αIIbβ3, thus completing the final common pathway for platelet aggregation. Norcantharidin (NCTD) is a promising anticancer agent in China from medicinal insect blister beetle. In this study, we provided the evidence to demonstrate NCTD (0.1–1.0 μM) possesses very powerful antiplatelet activity in human platelets; nevertheless, it had no effects on surface P‐selectin expression and only slight inhibition on ATP‐release reaction in activated platelets. Moreover, NCTD markedly hindered integrin αIIbβ3 activation by interfering with the binding of FITC‐labelled PAC‐1. It also markedly reduced the number of adherent platelets and the single platelet spreading area on immobilized fibrinogen as well as clot retraction. Additionally, NCTD attenuated phosphorylation of proteins such as integrin β3, Src and FAK in platelets spreading on immobilized fibrinogen. These results indicate that NCTD restricts integrin αIIbβ3‐mediated outside‐in signalling in human platelets. Besides, NCTD substantially prolonged the closure time in human whole blood and increased the occlusion time of thrombotic platelet plug formation and prolonged the bleeding time in mice. In conclusion, NCTD has dual activities, it can be a chemotherapeutic agent for cancer treatment, and the other side it possesses powerful antiplatelet activity for treating thromboembolic disorders. [ABSTRACT FROM AUTHOR]

Cardiovascular disease, which is caused by unregulated platelet aggregation, is one of the main causes of deaths worldwide. Many studies have focused on natural products with antiplatelet effects as a safe alternative therapy to prevent the disease. In this context, an in-house chemical library was screened to find natural products capable of inhibiting the interaction between platelet integrin αIIbβ3 and fibrinogen, which is an essential step in platelet aggregation. On the basis of the screening results, indothiazinone, an alkaloid found in microbial cultures, was identified as a potential antiplatelet agent. Specifically, indothiazinone treatment significantly inhibited the binding of fibrinogen to Chinese hamster ovary cells expressing integrin αIIbβ3. It also restricted thrombin- and adenosine diphosphate-dependent spreading of human platelets on a fibrinogen matrix. More importantly, surface plasmon resonance and molecular dynamics studies suggested that indothiazinone suppressed talin-induced activation of integrin αIIbβ3 presumably by inhibiting talin-integrin interaction. In conclusion, these results suggest that indothiazinone can be used as a lead compound for the development of antiplatelet drugs with a novel mode of action. [ABSTRACT FROM AUTHOR]

There is mounting evidence that tumor angiogenesis can be regulated by platelets (Plts), which serve as major sources and delivery vehicles of many proangiogenic cytokines including transforming growth factor-β and vascular endothelial growth factor. Although considerable progress has been made in understanding the role for Plt secretion in tumor angiogenesis, very little is known about the precise mechanisms underlying cancer cell induction of Plt granule release. Here, we demonstrated that nonsmall cell lung cancer (NSCLC) cells directly induced Plt secretion of several angiogenic regulatory cytokines that promoted angiogenesis in concert. Moreover, we discovered that these Plt-derived angiogenesis modulators were regulated by different molecular pathways and could be largely inhibited by combination of multiple signaling inhibitors. Our present studies indicated that manipulation of Plt secretion of angiogenic cytokines without compromising hemostatic functions could provide a novel option for management of tumor angiogenesis and metastasis in NSCLC patients with thrombocytosis. © 2015 IUBMB Life, 67(8):626-633, 2015 [ABSTRACT FROM AUTHOR]

A panel of platelets expressing various human platelet antigens (HPAs) for a platelet antibody screening assay is difficult to prepare because some antigens are rarely expressed. Therefore, an alternative method without using platelets would be helpful in detecting HPA antibodies. This study describes the establishment of cell lines that stably express specific HPAs and their application for detecting specific antibodies. Wild-type β3, HPA-1b, -6b, -7b and -7 variant cDNA as well as wild-type αIIb and HPA-3b cDNA were individually co-transduced with wild-type αIIb and β3 cDNA in the K562 cell line. We performed an immunobead monoclonal antibody immobilisation of platelet antigens (MAIPA) assay to evaluate this cell line panel for antibody detection using identified sera containing HPA antibodies, whose specificities had been determined by the mixed passive haemagglutination test. Of the 12 sera containing HPA-1a ( n = 2), HPA-3a ( n = 6), HPA-6b ( n = 3) or HPA-7 variant ( n = 1) antibodies, all antibodies were detected and determined by our new method, except for two HPA-3a antibodies. One of the two antibodies was also negative for conventional platelet MAIPA, suggesting that the cell line panel might be used as an alternative source of platelet antigens in the MAIPA assay. [ABSTRACT FROM AUTHOR]

Abstract: We examined the influence of S-nitrosoglutathione (GSNO) on αIIbβ3 integrin-mediated platelet adhesion to immobilised fibrinogen. GSNO induced a time- and concentration-dependent inhibition of platelet adhesion. Inhibition was cGMP-independent and associated with both reduced platelet spreading and protein tyrosine phosphorylation. To investigate the cGMP-independent effects of NO we evaluated integrin β3 phosphorylation. Adhesion to fibrinogen induced rapid phosphorylation of β3 on tyrosines 773 and 785, which was reduced by GSNO in a cGMP independent manner. Similar results were observed in suspended platelets indicating that NO-induced effects were independent of spreading-induced signalling. This is the first demonstration that NO directly regulates integrin β3 phosphorylation. [Copyright &y& Elsevier]

Abstract: Platelet activation by potent, Ca2+-mobilizing agonists results in shedding of microparticles that are active in coagulation. Here we show that platelets under storage produce procoagulant microparticles in the absence of agonist. Microparticle formation by resting platelets results from αIIbβ3 signaling to destabilization of the actin cytoskeleton in the absence of calpain activation. Integrin-mediated spreading of platelets over fibrinogen similarly results in microparticle formation. After transfusion of stored platelet preparations to thrombocytopenic patients, the microparticles contribute to coagulant activity in vivo. [Copyright &y& Elsevier]

Calcium- and integrin-binding protein 1 (CIB1) is involved in the process of platelet aggregation by binding the cytoplasmic tail of the αIIb subunit of the platelet-specific integrin αIibβ3. Although poorly understood, it is widely believed that CIB1 acts as a global signaling regulator because it is expressed in many tissues that do not express integrin αIibβ3. We report the structure of human CIB1 to a resolution of 2.3 Å, crystallized as a dimer. The dimer interface includes an extensive hydrophobic patch in a crystal form with 80% solvent content. Although the dimer form of CIB1 may not be physiologically relevant, this intersub-unit surface is likely to be linked to αIIb binding and to the binding of other signaling partner proteins. The C-terminal domain of CIB1 is structurally similar to other EF-hand proteins such as calmodulin and calcineurin B. Despite structural homology to the C-terminal domain, the N-terminal domain of CIB1 lacks calcium-binding sites. The structure of CIB1 revealed a complex with a molecule of glutathione in the reduced state bond to the N-terminal domain of one of the two subunits poised to interact with the free thiol of C35. Glutathione bound in this fashion suggests CIB1 may be redox regulated. Next to the bound GSH, the orientation of residues C35, H31, and S48 is suggestive of a cysteine-type protein phosphatase active site. The potential enzymatic activity of CIB1 is discussed and suggests a mechanism by which it regulates a wide variety of proteins in cells in addition to platelets. [ABSTRACT FROM AUTHOR]

Summary. A Glanzmann thrombasthenia variant with a β3 Ser752 →Pro cytoplasmic domain substitution has platelets that fail to aggregate or bind soluble fibrinogen (Fg) after activation. Despite this, Fg is normally present in the α-granules. We have used immunoelectron microscopy to examine the reactivity of Fg with the different pools of αIIbβ3 in the patient's platelets. Immunogold labelling was performed on cryosections using an anti-ligand-induced binding site (LIBS) monoclonal antibody (mAb), which binds to αIIbβ3 only when Fg is bound, or a mixture of two anti-receptor-induced binding site (RIBS) mAbs that specifically recognize receptor-bound Fg. Labelling of the α-granule membrane and channels of the surface-connected canalicular system in unstimulated platelets confirmed that the mutated αIIbβ3 retains the capacity to transport Fg. When the patient's platelets were stimulated with ADP in the presence of Fg, as expected there was a much-decreased activation of surface-exposed αIIbβ3. However, thrombin-induced activation was associated with both secretion and a rapid increase in the labelling of internal membrane systems by anti-RIBS and anti-LIBS mAbs, with mobilization of theinternal Fg pool. Yet labelling on the surface of the patient's platelets was transient. Our studies implied that αIIbβ3 in platelets may bind fibrinogen in different activation states and that this patient specifically lacked high-affinity binding. [ABSTRACT FROM AUTHOR]

In spite of new therapeutics and diagnostics, thrombosis-related diseases including heart failure and stroke remain the leading cause of death globally and a major healthcare burden. Pharmacogenomic studies may reduce the burden of thrombotic diseases by aiding treatment individualization with the most appropriate anti-coagulant and anti-platelet therapeutics. Discovering and validating biomarkers for improved personalized treatment of thrombotic diseases will require massive investment. A concerted dedicated effort built upon a public-private partnership is warranted for addressing this foremost public health concern. [ABSTRACT FROM AUTHOR]

During platelet activation, fibrinogen binds to its specific platelet receptor, integrin α II b β 3 , thus completing the final common pathway for platelet aggregation. Norcantharidin (NCTD) is a promising anticancer agent in China from medicinal insect blister beetle. In this study, we provided the evidence to demonstrate NCTD (0.1-1.0 μM) possesses very powerful antiplatelet activity in human platelets; nevertheless, it had no effects on surface P-selectin expression and only slight inhibition on ATP-release reaction in activated platelets. Moreover, NCTD markedly hindered integrin α II b β 3 activation by interfering with the binding of FITC-labelled PAC-1. It also markedly reduced the number of adherent platelets and the single platelet spreading area on immobilized fibrinogen as well as clot retraction. Additionally, NCTD attenuated phosphorylation of proteins such as integrin β 3 , Src and FAK in platelets spreading on immobilized fibrinogen. These results indicate that NCTD restricts integrin α II b β 3 -mediated outside-in signalling in human platelets. Besides, NCTD substantially prolonged the closure time in human whole blood and increased the occlusion time of thrombotic platelet plug formation and prolonged the bleeding time in mice. In conclusion, NCTD has dual activities, it can be a chemotherapeutic agent for cancer treatment, and the other side it possesses powerful antiplatelet activity for treating thromboembolic disorders., (© 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)