Platelet Integrin Research Articles

Abstract 132: The Mammalian Target of Rapamycin Regulates Platelet Integrin Activation, Aggregation, and Ischemic Stroke

Rationale: Platelets are critical cellular effectors in the development of ischemic stroke. Translational control pathways, such as the mammalian target of rapamycin (mTOR), are active in platelets, and can be targeted therapeutically, but have not been examined in stroke. Objective: To determine if activation of the mTOR pathway regulates platelet activation and the development of ischemic stroke following stroke in vivo . Methods: We generated mice lacking mTOR specifically in platelets (KO). We assessed mTOR activation, platelet aggregation, integrin activation, and clot retraction in KO and wild-type control mice (WT). In both KO and WT mice, we also performed a transient middle cerebral artery occlusion (tMCAO) stroke model (t=1h followed with reperfusion for 24hrs). Following tMACO, blood flow in the MCA and stroke infarct size were determined. We also examined mTOR activation in human platelets from individuals with ischemic stroke (n=5) and healthy controls (n=4). Results: We confirmed the absence of mTOR in KO platelets. In platelets from WT mice, the mTOR pathway is activatable, triggering platelet activation and clot retraction in vitro . In comparison, indices of mTOR activation, including pAKT and pS6K, were significantly (p<0.05) reduced in platelets from KO mice. Platelets from KO mice also had significant reductions in platelet aggregation (50% less), integrin αIIbβ3 activation (40% less), and clot retraction compared to WT mice. These differences were not due to alterations in platelet count. In the tMCAO model, KO mice had significantly smaller infarct sizes compared to WT mice (36.8±9.4mm 3 , n=7 vs. 80.3±8.1mm 3 , n=8; p<0.004). Smaller infarct sizes in KO mice were not due to differences in blood flow during the ischemia or reperfusion phase, which were similar to WT mice. In platelets from human ischemic stroke patients, the mTOR pathway was activated as p4EBP1, a downstream target of mTOR, was 1.72-fold higher compared to healthy controls. Conclusion: These data demonstrate that inhibition of the mTOR pathway in platelets significantly reduces platelet activation and protects from ischemic stroke . Therapeutic targeting of the mTOR pathway in platelets may provide clinical benefit in the setting of ischemic stroke.

Endoglin as an Adhesion Molecule in Mature and Progenitor Endothelial Cells: A Function Beyond TGF-β.

Endoglin (ENG) is a transmembrane glycoprotein expressed on endothelial cells that functions as a co-receptor for several ligands of the transforming growth factor beta (TGF-β) family. ENG is also a recognized marker of angiogenesis and mutations in the endoglin gene are responsible for Hereditary Hemorrhagic Telangiectasia (HHT) type 1, a vascular disease characterized by defective angiogenesis, arteriovenous malformations, telangiectasia, and epistaxis. In addition to its involvement in the TGF-β family signaling pathways, several lines of evidence suggest that the extracellular domain of ENG has a role in integrin-mediated cell adhesion via its RGD motif. Indeed, we have described a role for endothelial ENG in leukocyte trafficking and extravasation via its binding to leukocyte integrins. We have also found that ENG is involved in vasculogenic properties of endothelial progenitor cells known as endothelial colony forming cells (ECFCs). Moreover, the binding of endothelial ENG to platelet integrins regulate the resistance to shear during platelet-endothelium interactions under inflammatory conditions. Because of the need for more effective treatments in HHT and the involvement of ENG in angiogenesis, current studies are aimed at identifying novel biological functions of ENG which could serve as a therapeutic target. This review focuses on the interaction between ENG and integrins with the aim to better understand the role of this protein in blood vessel formation driven by progenitor and mature endothelial cells.

Computational and experimental analysis of bioactive peptide linear motifs in the integrin adhesome.

Therapeutic modulation of protein interactions is challenging, but short linear motifs (SLiMs) represent potential targets. Focal adhesions play a central role in adhesion by linking cells to the extracellular matrix. Integrins are central to this process, and many other intracellular proteins are components of the integrin adhesome. We applied a peptide network targeting approach to explore the intracellular modulation of integrin function in platelets. Firstly, we computed a platelet-relevant integrin adhesome, inferred via homology of known platelet proteins to adhesome components. We then computationally selected peptides from the set of platelet integrin adhesome cytoplasmic and membrane adjacent protein-protein interfaces. Motifs of interest in the intracellular component of the platelet integrin adhesome were identified using a predictor of SLiMs based on analysis of protein primary amino acid sequences (SLiMPred), a predictor of strongly conserved motifs within disordered protein regions (SLiMPrints), and information from the literature regarding protein interactions in the complex. We then synthesized peptides incorporating these motifs combined with cell penetrating factors (tat peptide and palmitylation for cytoplasmic and membrane proteins respectively). We tested for the platelet activating effects of the peptides, as well as their abilities to inhibit activation. Bioactivity testing revealed a number of peptides that modulated platelet function, including those derived from α-actinin (ACTN1) and syndecan (SDC4), binding to vinculin and syntenin respectively. Both chimeric peptide experiments and peptide combination experiments failed to identify strong effects, perhaps characterizing the adhesome as relatively robust against within-adhesome synergistic perturbation. We investigated in more detail peptides targeting vinculin. Combined experimental and computational evidence suggested a model in which the positively charged tat-derived cell penetrating part of the peptide contributes to bioactivity via stabilizing charge interactions with a region of the ACTN1 negatively charged surface. We conclude that some interactions in the integrin adhesome appear to be capable of modulation by short peptides, and may aid in the identification and characterization of target sites within the complex that may be useful for therapeutic modulation.

Structure and dynamics of the platelet integrin-binding C4 domain of von Willebrand factor

AbstractVon Willebrand factor (VWF) is a key player in the regulation of hemostasis by promoting recruitment of platelets to sites of vascular injury. An array of 6 C domains forms the dimeric C-terminal VWF stem. Upon shear force activation, the stem adopts an open conformation allowing the adhesion of VWF to platelets and the vessel wall. To understand the underlying molecular mechanism and associated functional perturbations in disease-related variants, knowledge of high-resolution structures and dynamics of C domains is of paramount interest. Here, we present the solution structure of the VWF C4 domain, which binds to the platelet integrin and is therefore crucial for the VWF function. In the structure, we observed 5 intra- and inter-subdomain disulfide bridges, of which 1 is unique in the C4 domain. The structure further revealed an unusually hinged 2-subdomain arrangement. The hinge is confined to a very short segment around V2547 connecting the 2 subdomains. Together with 2 nearby inter-subdomain disulfide bridges, this hinge induces slow conformational changes and positional alternations of both subdomains with respect to each other. Furthermore, the structure demonstrates that a clinical gain-of-function VWF variant (Y2561) is more likely to have an effect on the arrangement of the C4 domain with neighboring domains rather than impairing platelet integrin binding.

Activated platelets in the tumor microenvironment for targeting of antibody-drug conjugates to tumors and metastases.

Rationale: Platelets are increasingly recognized as mediators of tumor growth and metastasis. Hypothesizing that activated platelets in the tumor microenvironment provide a targeting epitope for tumor-directed chemotherapy, we developed an antibody-drug conjugate (ADC), comprised of a single-chain antibody (scFv) against the platelet integrin GPIIb/IIIa (scFvGPIIb/IIIa) linked to the potent chemotherapeutic microtubule inhibitor, monomethyl auristatin E (MMAE).Methods: We developed an ADC comprised of three components: 1) A scFv which specifically binds to the high affinity, activated integrin GPIIb/IIIa on activated platelets. 2) A highly potent microtubule inhibitor, monomethyl auristatin E. 3) A drug activation/release mechanism using a linker cleavable by cathepsin B, which we demonstrate to be abundant in the tumor microenvironment. The scFvGPIIb/IIIa-MMAE was first conjugated with Cyanine7 for in vivo imaging. The therapeutic efficacy of the scFvGPIIb/IIIa-MMAE was then tested in a mouse metastasis model of triple negative breast cancer.Results: In vitro studies confirmed that this ADC specifically binds to activated GPIIb/IIIa, and cathepsin B-mediated drug release/activation resulted in tumor cytotoxicity. In vivo fluorescence imaging demonstrated that the newly generated ADC localized to primary tumors and metastases in a mouse xenograft model of triple negative breast cancer, a difficult to treat tumor for which a selective tumor-targeting therapy remains to be clinically established. Importantly, we demonstrated that the scFvGPIIb/IIIa-MMAE displays marked efficacy as an anti-cancer agent, reducing tumor growth and preventing metastatic disease, without any discernible toxic effects.Conclusion: Here, we demonstrate the utility of a novel ADC that targets a potent cytotoxic drug to activated platelets and specifically releases the cytotoxic agent within the confines of the tumor. This unique targeting mechanism, specific to the tumor microenvironment, holds promise as a novel therapeutic approach for the treatment of a broad range of primary tumors and metastatic disease, particularly for tumors that lack specific molecular epitopes for drug targeting.

Management of Glanzmann's Thrombasthenia – Guidelines based on an expert panel consensus from gulf cooperation council countries

Glanzmann thrombasthenia is a rare disorder, due to quantitative and/or qualitative abnormalities of the platelet integrin αIIbβ3 and/or αIIbβ3. Although it is considered a rare disorder with a global incidence of 1/1,000,000 population, the case is different at the Gulf Cooperation Council countries, where prevalence rate as high as 1/40,000 in Madinah, Saudi Arabia. This makes it necessary to develop patient's management guidelines. Due to limited resources in the literature, experts' consensus was important to develop such guidelines. Experts panel elected to use recombinant activated factor VII (rFVIIa) as the first line of treatment of acute bleeds and reserve platelets transfusion for nonresponding patients or severe bleeds, rFVIIa at high dose (270 μg/kg body weight) may tried upfront. rFVIIa may be tried as prophylactic treatment in patients with frequents bleeds. Experts panel elected to allow girls with Glanzmann thrombasthenia to have menstruation and to adapt a special protocol for this purpose (Madinah protocol). Pregnancy should be managed carefully, where normal delivery encouraged under coverage of rFVIIa. Risk of bleeding should be expected in neonates. Minor surgeries and dental workup can be managed under coverage of rFVIIa. Glanzmann thrombasthenia patients with severe disease should be encouraged for hematopoietic stem cell transplant.

Fast Force Loading Disrupts Molecular Binding Stability in Human and Mouse Cell Adhesions.

Force plays critical roles in cell adhesion and mechano-signaling, partially by regulating the dissociation rate, i.e., off-rate, of receptor-ligand bonds. However, the mechanism of such regulation still remains elusive. As a controversial topic of the field, when measuring the "off-rate vs. force" relation of the same molecular system, different dynamic force spectroscopy (DFS) assays (namely, force-clamp and force-ramp assays) often yield contradictive results. Such discrepancies hurdled our further understanding of molecular binding, and casted doubt on the existing theoretical models. In this work, we used a live-cell DFS technique, biomembrane force probe, to measure the single-bond dissociation in three receptor-ligand systems which respectively have important functions in vascular and immune systems: human platelet GPIbα-VWF, mouse T cell receptor-OVA peptide:MHC, and mouse platelet integrin αIIbβ3-fibrinogen. Using force-clamp and force-ramp assays in parallel, we identified that the force loading disrupted the stability of molecular bonds in a rate-dependent manner. This disruptive effect was achieved by the transitioning of bonds between two dissociation states: faster force loading induces more bonds to adopt the fast-dissociating state (and less to adopt the slow-dissociating state). Based on this mechanism, a new biophysical model of bond dissociation was established which took into account the effects of both force magnitude and loading rate. Remarkably, this model reconciled the results from the two assays in all three molecular systems under study. Our discoveries provided a new paradigm for understanding how force regulates receptor-ligand interactions and a guideline for the proper use of DFS technologies. Furthermore, our work highlighted the opportunity of using different DFS assays to answer specific biological questions in the field of cell adhesion and mechano-signaling.

Effects of water-soluble tomato concentrate on platelet aggregation

Objective: To investigate the antiplatelet aggregation effect of water-soluble tomato concentrate (WSTC) and explore the underlying molecular mechanisms. Materials and Methods: Platelet aggregometry was used to quantify rat platelet aggregation with the maximum aggregation rate in vitro and ex vivo. Then, the fibrinogen (FIB) binding assay was employed to detect the effect of WSTC on the activation of platelet integrin αIIβ3 (GP IIb/IIIa). Furthermore, Western blot was performed to assess the platelet protein levels of phosphoinositide 3-kinase 110 β (PI3K110 β), protein disulfide isomerase (PDI), platelet endothelial cell adhesion molecule 1 (PECAM-1), and β1-Tubulin. Results: WSTC inhibited adenosine diphosphate (ADP) and collagen-induced platelet aggregation in a concentration-dependent manner in vitro, at IC50values of 3.05 g/L and 8.03 g/L, respectively. Significantly reduced ex vivo ADP induced platelet aggregation was observed after oral consumption of WSTC for 4 weeks in rats; average inhibition rates were 24.42%, 21.48%, and 20.87% for 25 mg/Kg, 75 mg/Kg, and 150 mg/Kg WSTC, respectively. It appeared that WSTC had no influence on coagulation function in rats. Incubation with WSTC decreased FIB binding to GP IIb/IIIa by 17.47% and 32.29% at the concentrations of 0.6 and 6 g/L, respectively. WSTC at 0.6 and 6 g/L markedly downregulated PI3K110 β, PDI, and PECAM-1 in platelets, and upregulated β1-Tubulin, in a concentration-dependent manner. Conclusion: WSTC inhibits platelet activation through modulation of platelet skeletal stability and suppresses GP IIb/IIIa receptor-mediated platelet aggregation, likely via the PI3K signaling pathway and PDI inhibition.

Development and Validation of Tumor-educated Blood Platelets Integrin Alpha 2b (ITGA2B) RNA for Diagnosis and Prognosis of Non-small-cell Lung Cancer through RNA-seq.

Background: Currently, there are no molecular biomarkers for the early detection of non-small-cell lung cancer (NSCLC). This study focused on identifying RNAs found on tumor-educated blood platelets (TEPs) for detecting stage I NSCLC.Methods: Platelet RNAs, isolated from the blood of 9 patients with NSCLC (stages I and II) and 8 healthy controls, were analyzed using RNA-seq. ITGA2B was selected as a candidate marker. Two different Polymerase Chain Reactions (PCR) were used to measure ITGA2B in platelet samples from healthy controls (n = 150), patients with NSCLC (n = 243), and patients with benign pulmonary nodules (n = 141) in two cohorts.Results: Platelet ITGA2B levels were significantly higher (p < 0.001) in patients with NSCLC than in all controls. The diagnostic accuracy of ITGA2B was area under the curve (AUC) of 0.922 [95% confidence interval (CI), 0.892-0.952], sensitivity of 92.8%, and specificity of 78.6% in the test cohort and 0.888, 91.2%, and 56.5% in the validation cohort for NSCLC by quantitative real time PCR (q-PCR). Furthermore, ITGA2B maintained diagnostic accuracy for patients with NSCLC using Droplet Digital PCR (ddPCR) and the other type of internal control, Ribosomal Protein L32 (RPL32) [ddPCR: 0.967 (0.929-1.000) and RPL32: 0.847(0.773-0.920)]. A nomogram incorporating ITGA2B, carcinoembryonic antigen (CEA) and stage could predict the overall survival (C-index = 0.756).Conclusions: TEP ITGA2B is a promising marker to improve identification of patients with stage I NSCLC and differentiate malignant from benign lung nodules.

Direct Rap1/Talin1 interaction regulates platelet and neutrophil integrin activity in mice

AbstractTargeting Talin1 to the plasma membrane is a crucial step in integrin activation, which in leukocytes is mediated by a Rap1/RIAM/Talin1 pathway, whereas in platelets, it is RIAM independent. Recent structural, biochemical, and cell biological studies have suggested direct Rap1/Talin1 interaction as an alternative mechanism to recruit Talin1 to the membrane and induce integrin activation. To test whether this pathway is of relevance in vivo, we generated Rap1 binding–deficient Talin1 knockin (Tln13mut) mice. Although Tln13mut mice showed no obvious abnormalities, their platelets exhibited reduced integrin activation, aggregation, adhesion, and spreading, resulting in prolonged tail-bleeding times and delayed thrombus formation and vessel occlusion in vivo. Surprisingly, neutrophil adhesion to different integrin ligands and β2 integrin–dependent phagocytosis were also significantly impaired, which caused profound leukocyte adhesion and extravasation defects in Tln13mut mice. In contrast, macrophages exhibited no defect in adhesion or spreading despite reduced integrin activation. Taken together, our findings suggest that direct Rap1/Talin1 interaction is of particular importance in regulating the activity of different integrin classes expressed on platelets and neutrophils, which both depend on fast and dynamic integrin-mediated responses.

Evidence that ITGB3 promoter variants increase serotonin blood levels by regulating platelet serotonin transporter trafficking.

Elevated serotonin (5-HT) blood levels, the first biomarker identified in autism research, has been consistently found in 20-30% of patients with Autism Spectrum Disorder (ASD). Hyperserotonemia is mainly due to greater 5-HT uptake into platelets, mediated by the 5-HT transporter (SERT) located at the platelet plasma membrane. The protein complex involved in platelet SERT trafficking and externalization includes integrin β3, the beta subunit of the platelet membrane adhesive GP IIb/IIIa. Integrin β3 is encoded by the ITGB3 gene, previously identified as a quantitative trait locus (QTL) for 5-HT blood levels in ASD at single nucleotide polymorphism (SNP) rs2317385. The present study aims to identify the functional ITGB3 gene variants contributing to hyperserotonemia. ITGB3 gene sequencing in 20 individuals selected on the basis of rs2317385 genotypes defined four haplotypes encompassing six SNPs located in the ITGB3 gene promoter region, all in linkage disequilibrium with rs2317385. Luciferase assays in two hematopoietic cell lines, K-562 and HEL 92.1.7, demonstrate that ITGB3 gene promoter activity is enhanced by the presence of the C allele at rs55827077 specifically during differentiation into megakaryocytes (P<0.01), with modulatory effects by flanking SNPs. This same allele is strongly associated with (a) higher 5-HT blood levels in 176 autistic individuals (P<0.001), (b) greater platelet integrin β3 protein expression (P<0.05) and (c) enhanced SERT trafficking from the cytosol toward the platelet plasma membrane (P=4.05×10-11). Our results support rs55827077 as the functional ITGB3 gene promoter variant contributing to elevated 5-HT blood levels in ASD and define a mechanistic chain of events linking ITGB3 to hyperserotonemia.

Coenzyme Q10 Attenuates Platelet Integrin αIIbβ3 Outside-in Signaling through Targeting cAMP/PKA Pathway and Inhibits Atherosclerosis

Introduction: Platelet integrin αIIbβ3 outside-in signaling is crucial for platelet adhesion and aggregation, and contributes to atherogenesis. Coenzyme Q10 (CoQ10) has been implicated as a protective factor against cardiovascular diseases (CVDs), particularly atherosclerosis. However, whether CoQ10 attenuates atherosclerosis through inhibiting platelet function and αIIbβ3 outside-in signaling is unknown. The aim of this study was to explore whether CoQ10 affects platelet function and αIIbβ3 outside-in signalling and thus inhibiting the progress of atherosclerosis in vivo and the underlying mechanisms in vitro.Methods:In vitro study, The murine platelet rich plasma (PRP) from C57BL/6J wild-type (WT) mice or human PRP and gel-filtered platelets were incubated with different concentrations (1, 10 or 100 μM) of CoQ10 or the vehicle control for 50 min. Platelet aggregation, spreading on fibrinogen (Fg) and clot retraction were determined. In addition, the effects of CoQ10 on platelet integrin αIIbβ3 inside-out signalling (e.g., talin-1 and kindlin-3 binding to integrin β3) were determined by immunoprecipitation, and outside-in signalling (e.g., phosphorylation of sarcoma tyrosine-protein kinase (c-Src), focal adhesion kinase (FAK), and β3 cytoplasmic tail, myosin light chain (MLC)) were determined by Western blotting. The levels of platelet ATP and cAMP were measured by ELISA assays. In vivo study, male homozygous apolipoprotein E-deficient (apoE-/-) mice (C57BL/6 genetic background) were fed either a standard normal AIN-93G diet (NC group), a Western-type diet (HFD group) or a Western-type diet supplemented with CoQ10 (1800 mg/kg diet) (CoQ10 group) for 12 weeks. Platelet aggregation, granule secretion, platelet spreading, clot retraction, integrin αIIbβ3 outside-in signalling, platelet-leukocyte interactions and carotid artery plaque area were also examined. In our randomized, double-blind, placebo-controlled trial, 101 hypercholesterolemic subjects were randomly administrated to 120 mg CoQ10 or placebo daily for 24 weeks. Platelet intracellular CoQ10 levels, platelet aggregation in PRP, platelet platelet factor 4 (PF-4) and C-C motif ligand 5 (CCL5) release, and platelet integrin αIIbβ3 outside-in signalling were also evaluated before and after 24 weeks of intervention.Results: We found that CoQ10 inhibited human and WT mouse platelet aggregation, platelet spreading, granule secretion, and clot retraction in vitro and apoE-/- mice on a high fat diet. CoQ10 also reduced atherosclerosis and platelet-monocyte aggregation in apoE-/- mice. The inhibitory effects of CoQ10 is mediated by attenuated αIIbβ3 outside-in signalling pathway (e.g., attenuation of phosphorylation of c-Src, FAK, and β3 cytoplasmic tail, and MLC in thrombin-activated platelets or platelets exposed to immobilized Fg), which requires up-regulation of the cAMP/PKA pathway, where CoQ10 inhibited phosphodiesterase 3A activity and activated the A2A adenosine receptor. However, CoQ10 did not affect platelet integrin αIIbβ3 inside-out signalling pathway, platelet cellular ATP, or platelet apoptosis (the mitochondrial membrane potential and phosphatidylserine exposure). Moreover, our clinical trial in dyslipidemic patients demonstrated that CoQ10 supplementation attenuated platelet aggregation, which was positively correlated with the increased platelet CoQ10 concentrations, inhibited αIIbβ3 outside-in signalling and decreased platelet PF-4 and CCL5 secretion.Conclusions: We present new data to suggest that CoQ10 plays a novel role in attenuating platelet function and integrin αIIbβ3 outside-in signalling though targeting cAMP/PKA signalling cascade and thus inhibiting the progress of atherosclerosis. CoQ10 is therefore a promising agent for the prevention and/or treatment for cardiovascular disease. DisclosuresNo relevant conflicts of interest to declare.

Tannic Acid Inhibits Protein Disulfide Isomerase, Platelet Activation and Thrombus Formation

Tannic acid (TA) was a polyphenol that harbors anti-oxidant capacity. A recent report implied that surface coating with TA might blunt thrombosis via altering the structure of fibrinogen. However, the effect of TA on platelet function and in vivo thrombus formation has not been reported. In this study, we showed that TA inhibits PDI activity and attenuates platelet activation.To explore the effects of TA on platelet aggregation, gel-filtered human platelets from healthy human donors were pretreated with TA (10/30/50 μM) or vehicle (0.9% sodium chloride) before being stimulated by various agonists. Turbidity analyses on a Chronolog aggregometer showed that TA dose-dependently inhibited platelet aggregation induced by thrombin, SFLLRN, GYQGQV, collagen, CRP, U46619, and ristocetin. Next, we employed flow cytometry (FACS) to determine the role of TA in platelet activation, including α-granule secretion and integrin activation. Pretreatment of platelets with TA led to significant reductions in surface P-selectin expression and soluble fibrinogen binding, supporting the inhibition of diverse platelet activation pathways. Supportively, platelet spreading on immobilized fibrinogen was significantly suppressed by TA treatment. In addition, cell viability assay with Almar blue agent showed no detrimental impact of TA on the survival of platelets.To ask whether the antiplatelet role of TA might be translated into an antithrombotic efficacy, we tested the effect of TA in both ex vivo and in vivo thrombosis models. Calcein-labeled human whole blood was perfused through microfluidic channels coated with collagen, and adherent platelets were visualized under a fluorescent microscopy. However, treatment with TA suppressed the number of adherent platelets under flow conditions. Moreover, in laser-induced mouse cremaster muscle arteries, administration of TA (5mg/kg) significantly reduced the size of forming thrombi compared with the vehicle. Verification of bleeding risk using tail truncation assay indicated no prolongation of bleeding time in mice receiving TA. Thus, TA shows an antiplatelet effect and may also attenuate thrombus formation.To gain a mechanistic insight to the role of TA in platelet function, we performed a molecular docking screen of the structure of TA and platelet surface proteins using the Autodock Vina software, which displayed the binding of TA with protein disulfide isomerase at the enzymatic active center. We then measured the impact of TA on PDI reductase activity with the dieosin glutathione disulfide assay in vitro (di-GSSG), showing that TA significantly inhibited PDI activity in a concentration-dependent manner. The results were verified in platelets using the 3-(N-Maleimidylpropionyl) biocytin (MPB) labeling, which showed that TA abrogated thrombin-stimulated free thiol formation on platelet surface. Supportively, FACS demonstrated that TA significantly suppressed the binding of fluorescent-labeled PDI to Mn2+-activated platelet integrin β3. Taken together, our findings demonstrated that TA inhibits PDI activity and may become a novel antithrombotic agent. DisclosuresNo relevant conflicts of interest to declare.

Functional redundancy between RAP1 isoforms in murine platelet production and function

AbstractRAP GTPases, important regulators of cellular adhesion, are abundant signaling molecules in the platelet/megakaryocytic lineage. However, mice lacking the predominant isoform, RAP1B, display a partial platelet integrin activation defect and have a normal platelet count, suggesting the existence of a RAP1-independent pathway to integrin activation in platelets and a negligible role for RAP GTPases in megakaryocyte biology. To determine the importance of individual RAP isoforms on platelet production and on platelet activation at sites of mechanical injury or vascular leakage, we generated mice with megakaryocyte-specific deletion (mKO) of Rap1a and/or Rap1b. Interestingly, Rap1a/b-mKO mice displayed a marked macrothrombocytopenia due to impaired proplatelet formation by megakaryocytes. In platelets, RAP isoforms had redundant and isoform-specific functions. Deletion of RAP1B, but not RAP1A, significantly reduced α-granule secretion and activation of the cytoskeleton regulator RAC1. Both isoforms significantly contributed to thromboxane A2 generation and the inside-out activation of platelet integrins. Combined deficiency of RAP1A and RAP1B markedly impaired platelet aggregation, spreading, and clot retraction. Consistently, thrombus formation in physiological flow conditions was abolished in Rap1a/b-mKO, but not Rap1a-mKO or Rap1b-mKO, platelets. Rap1a/b-mKO mice were strongly protected from experimental thrombosis and exhibited a severe defect in hemostasis after mechanical injury. Surprisingly, Rap1a/b-mKO platelets were indistinguishable from controls in their ability to prevent blood–lymphatic mixing during development and hemorrhage at sites of inflammation. In summary, our studies demonstrate an essential role for RAP1 signaling in platelet integrin activation and a critical role in platelet production. Although important for hemostatic/thrombotic plug formation, platelet RAP1 signaling is dispensable for vascular integrity during development and inflammation.

99 - Investigation of redox modifications of platelet integrin GPIIb-IIIa in diabetes

Diabetes is a global health problem, affecting 382 million people worldwide. Critically, up to 70% of deaths from diabetes are due to the development of occlusive blood clots (thrombosis), leading to heart attack and stroke. This is due to both accelerated atherosclerosis and an exaggerated thrombotic response. Platelets are central to the initiation and promotion of thrombus formation. Importantly, in diabetic individuals these cells are ‘hyperactive’ resulting in larger, more occlusive thrombi. Moreover, currently used anti-platelet agents have reduced efficacy in diabetic patients. The underlying mechanisms responsible for platelet hyperactivity and antiplatelet resistance in diabetes is incompletely understood. Our recent work has uncovered a new mechanism of enhanced platelet aggregation and adhesion in diabetes, which is mediated by biomechanical activation of the major platelet integrin, GPIIb-IIIa. How diabetes promotes enhanced sensitivity to biomechanical stimuli in GPIIb-IIIa is an important remaining question. Chronic oxidative stress is a well-defined risk factor for cardiovascular disease and diabetes. Herein, we explored the possibility that chronic oxidative stress may promote redox-mediated modifications of GPIIb-IIIa in diabetic platelets. The reduction/oxidation state of cysteines in GPIIb-IIIa have been reported to mediate the conformation, and thus ligand binding affinity, of this adhesion receptor. Here we employed multiple techniques to probe the redox state of these cysteines to understand whether chronic oxidative stress can post-translationally modify GPIIb-IIIa to account for the increased reactivity of this integrin in diabetic platelets. Understanding the role of oxidative stress in modifying the platelet integrin GPIIb-IIIa could have important implications in how we approach developing strategies to treat platelet hyperactivity in diabetes.

Loss of the mitochondrial kinase PINK1 does not alter platelet function

PTEN-induced putative kinase (PINK) 1 is regarded as a master regulator of cellular mitophagy such that loss of function mutations contribute to early onset Parkinson’s disease, through aberrant mitochondrial control and function. Mitochondrial function is key to platelet procoagulant activity, controlling the haemostatic response to vessel injury, but can also predispose blood vessels to thrombotic complications. Here, we sought to determine the role of PINK1 in platelet mitochondrial health and function using PINK1 knockout (KO) mice. The data largely show an absence of such a role. Haematological analysis of blood counts from KO mice was comparable to wild type. Quantification of mitochondrial mass by citrate synthase activity assay or expression of mitochondrial markers were comparable, suggesting normal mitophagy in KO platelets. Analysis of mitochondrial permeability transition pore opening, changes in mitochondrial membrane potential and calcium signalling to platelet activation were unaffected by loss of PINK1, whereas subtle enhancements of activation-induced reactive oxygen species were detected. Platelet aggregation, integrin activation, α- and dense granule secretion and phosphatidylserine exposure were unaltered in KO platelets while mouse tail bleeding responses were similar to wild type. Together these results demonstrate that PINK1 does not regulate basal platelet mitophagy and is dispensable for platelet function.

Cobimetinib and trametinib inhibit platelet MEK but do not cause platelet dysfunction

The MEK inhibitors cobimetinib and trametinib are used in combination with BRAF inhibitors to treat metastatic melanoma but increase rates of hemorrhage relative to BRAF inhibitors alone. Platelets express several members of the MAPK signalling cascade including MEK1 and MEK2 and ERK1 and ERK2 but their role in platelet function and haemostasis is ambiguous as previous reports have been contradictory. It is therefore unclear if MEK inhibitors might be causing platelet dysfunction and contributing to increased hemorrhage. In the present study we performed pharmacological characterisation of cobimetinib and trametinib in vitro to investigate potential for MEK inhibitors to cause platelet dysfunction.We report that whilst both cobimetinib and trametinib are potent inhibitors of platelet MEK activity, treatment with trametinib did not alter platelet function. Treatment with cobimetinib results in inhibition of platelet aggregation, integrin activation, alpha-granule secretion and adhesion but only at suprapharmacological concentrations. We identified that the inhibitory effects of high concentrations of cobimetinib are associated with off-target inhibition on Akt and PKC. Neither inhibitor caused any alteration in thrombus formation on collagen under flow conditions in vitro.Our findings demonstrate that platelets are able to function normally when MEK activity is fully inhibited, indicating MEK activity is dispensable for normal platelet function. We conclude that the MEK inhibitors cobimetinib and trametinib do not induce platelet dysfunction and are therefore unlikely to contribute to increased incidence of bleeding reported during MEK inhibitor therapy.

Leu33Pro (PlA) polymorphism of integrin beta3 modulates platelet Src pY418 and focal adhesion kinase pY397 phosphorylation in response to abnormally high shear stress.

Shear stress can activate platelet integrin-mediated signaling that leads to shear-induced platelet aggregation (SIPA) and eventually contribute to acute myocardial infarction. The major platelet integrin αIIbβ3 is polymorphic at residue 33 [Leu33Pro (PlA) polymorphism]. The Pro33 isoform has been shown to have a prothrombotic phenotype. In this work, we studied the impact of Leu33/Pro33 polymorphism on the shear-induced integrin-mediated Src and FAK activation in platelets. Platelets of both genotypes were placed on immobilized fibrinogen or heat activated BSA and were exposed to physiological (500/s) or abnormally high (5000/s) shear rates for 2-10 min. Platelets after exposure to shear were analysed for Src pY418 and FAK pY397 activities. Whereas physiological shear stress does not affect platelet signaling, abnormally high-shear stress considerably elevates Src and FAK phosphorylation in both Pro33 and Leu33 platelets. Both under static and flow conditions, Pro33 platelets exhibited a significantly higher Src and FAK activities than Leu33 platelets. Interestingly, even in the absence of the αIIbβ3-fibrinogen interaction, we could detect a shear-induced integrin-mediated signaling of Src and FAK in platelets. In parallel experiments in which platelets were pretreated with abciximab, an integrin αIIbβ3 antagonist, activation of both kinases by shear was inhibited. Taken together, our data indicates an important role of αIIbβ3 and shows that Leu33Pro polymorphism modulates the integrin-mediated Src and FAK signaling in platelets in response to shear stress.

Optimized processing for pathogen inactivation of double-dose buffy-coat platelet concentrates: maintained invitro quality over 7-day storage.

Efficient pathogen inactivation (PI) offers the possibility of increasing the number of buffy coats per pool without the concurrent increased risk of pathogen transmission. Here, we describe the findings of invitro analyses of platelets from pools of eight buffy coats treated with amotosalen and UVA light (INTERCEPT Blood System for Platelets) using INTERCEPT disposable processing sets with plastic materials sourced from alternate suppliers and split afterwards to obtain two therapeutic transfusion doses. Double-dose platelet concentrates were prepared from pools of eight buffy coats in additive solution (SSP+) using either previous 6-lead or new 8-lead pooling sets and PI processing sets in previous or alternate supplier sourced plastics (AS). Platelets were treated with the INTERCEPT Blood System then stored for up to 7days and tested for invitro quality. All tested units (n=30) were in conformity with European guidelines. Using AS sets more effectively maintained glucose reserves (P<0·01), reduced lactate production (P<0·01), reduced CD62P expression (P<0·01) and downregulated levels of surface CD42b (P<0·01) overtime. AS set maintained JC-positive cells (NS) between day 2 and day 7 and sustained platelet integrin activation (PAC-1) between day 2 and day 7 (NS). Overall sCD40L and PGDF accumulated in an equivalent way (P<0·01) within series. In summary, our data demonstrate that PI treatment using AS sets, in combination with the new pooling set for double-dose platelet preparation, maintained the platelet invitro quality over 7days of storage.

Association of Oxidative Stress and Platelet Receptor Glycoprotein GPIbα and GPVI Shedding During Nonsurgical Bleeding in Heart Failure Patients With Continuous-Flow Left Ventricular Assist Device Support.

Nonsurgical bleeding (NSB) in heart failure (HF) patients with continuous-flow left ventricular assist device (CF-LVAD) support is the most common clinical complication. The aim of this study was to investigate the association between oxidative stress and platelet glycoproteins GPIbα and GPVI shedding on the incidence of NSB in CF-LVAD patients. Fifty-one HF patients undergoing CF-LVAD implantation and 11 healthy volunteers were recruited. Fourteen patients developed NSB (bleeder group) during 1 month follow-up duration, while others were considered nonbleeder group (n = 37). Several biomarkers of oxidative stress were quantified at baseline and weekly intervals in all patients. Surface expression and plasma elements of platelet receptor glycoproteins GPIbα and GPVI were measured. Oxidative stress biomarkers and platelet GPIbα and GPVI receptor-shedding (decreased surface expression and higher plasma levels) were found to be preexisting conditions in baseline samples of both groups of HF patients when compared with healthy volunteers. Significantly elevated oxidative stress biomarkers and platelet glycoprotein receptor shedding were observed in postimplant bleeder group temporarily when compared with nonbleeder group. Strong significant associations between biomarkers of oxidative stress and platelet glycoprotein receptor shedding were observed, suggesting a possible role of oxidative stress in platelet integrin shedding leading to NSB in CF-LVAD patients. Receiver operating characteristic analyses of GPIbα and GPVI indicated that the likelihood of NSB had a predictive power of bleeding complication in CF-LVAD patients. In conclusion, elevated oxidative stress may play a role in GPIbα and GPVI shedding in the event of NSB. Thus, oxidative stress and GPIbα and GPVI shedding may be used as potential biomarkers for bleeding risk stratification in those patients.