P-selectin Exposure Research Articles

Impediment of Gpibα-Mediated Platelet Clearance By an Anti-Gpibβ Antibody Derivative

Background:Glycoprotein (GP)Ib-IX complex plays a critical role in mediating platelet activation and platelet clearance. Recently, we identified the mechanosensory domain (MSD) in the GPIbα subunit, and demonstrated that unfolding of the MSD and subsequent exposure of the Trigger sequence (residues 473-483) therein activates GPIb-IX and induces rapid platelet clearance. This mechanism could explain acute thrombocytopenia induced by activated VWF, anti-GPIbα antibodies, neuraminidase, and ectodomain shedding of GPIbα. Consistently, platelets in IL4R-IbαTg mice, a transgenic strain in which the entire extracellular domain of human GPIbα except the Trigger sequence was replaced with that of the α-subunit of interleukin-4 receptor, exhibit constitutively more filopodia and are cleared much faster than the wild type.Previously, an anti-GPIbβ antibody RAM.1 was developed. RAM.1 significantly inhibits GPIb-IX-mediated filopodia formation and Ca 2+ signaling in platelets. In addition, it could inhibit GPIb-dependent thrombin generation. These results suggest that targeting GPIbβ could inhibit activation of GPIb-IX induced by MSD unfolding.Objectives:To investigate whether targeting GPIbβ with RAM.1 can impede rapid platelet clearance induced by exposed Trigger sequence and ameliorate related thrombocytopenia.Methods:Spontaneous filopodia in platelets and transfected Chinese hamster ovary (CHO) cells were visualized by fluorescence staining of actin and confocal microscopy. Images were quantified by ImageJ. Platelet signaling events, like P-selectin exposure, β-galactose exposure, and Ca 2+ influx, were measured by flow cytometry. Endogenous platelet life span was tracked by Alexa 488-labeled anti-mouse GPIX antibody.Results:CHO cells stably expressing the same mutant GPIb-IX complex in IL4R-IbαTg mouse platelets have been successfully obtained. Like IL4R-IbαTg platelets, these IL4R-IbαTg CHO cells exhibited spontaneous filopodia in the absence of any GPIbα ligands. RAM.1 could inhibit spontaneous filopodia formation in these CHO cells and IL4R-IbαTg platelets (Fig. 1, 2). Compared to wild-type mouse platelets, IL4R-IbαTg platelets constitutively exhibited increased P-selectin exposure, increased β-galactose exposure, and elevated intracellular Ca 2+, all of which could be inhibited by treatment of RAM.1 (Fig. 3). Recombinant RAM.1-GCN4 protein (rRAM.1-GCN4), in which the Fc region of RAM.1 heavy chain was replaced with the GCN4 coiled coil dimerizing sequence, has been generated and used as an alternative of the divalent RAM.1-Fab2. It retained the ability of RAM.1 antibody to inhibit GPIb-IX signaling. Injecting rRAM.1-GCN4 into IL4R-IbαTg mice dramatically improved the life span of endogenous IL4R-IbαTg platelets (Fig. 4).Conclusion:These results demonstrate that the exposed Trigger sequence is sufficient to activate GPIb-IX in transfected CHO cells, and that RAM.1 derivatives can impede GPIbα-mediated rapid platelet clearance. Targeting GPIbβ may be a novel approach to treat GPIb-related thrombocytopenia. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Introducing a Novel Biophysical Platelet Function Panel to Investigate Disorders of Primary Hemostasis and Bleeding of Unknown Cause

A subset of patients with chronic bleeding remain undiagnosed even after extensive diagnostic evaluation are labeled as “bleeding of unknown cause” (BUC). The key barrier to treating these patients is that they have a clinical bleeding tendency in the presence of normal diagnostic tests, and optimal methods for monitoring and treating patients with BUC remain unknown. While patients with BUC have symptoms of a primary hemostatic disorder, there is no diagnostic test or biomarker that can accurately identify which patients are at risk for bleeding such as those with mild Von Willebrand Disease (VWD) which comprise a broad spectrum of patients with varying degrees of bleeding. In order to fill this diagnostic gap in disorders of primary hemostasis, there is a clinical need for more assays of platelet function.To that end, we have engineered multiple new biophysical assays to assess disorders of primary hemostasis and apply this panel of platelet function testing to potentially define new bleeding disorders, characterize platelet phenotypes in patients with BUC, and refine the definition of mild VWD. Our panel of platelet function tests (Fig 1) collectively enables us to simultaneously assess different facets of primary hemostasis from the microscopic level of single-platelet physiology to hemostatic plug formation, thereby capturing various aspects of platelet function with a single blood sample. Our platelet function panel ranges from platelet adhesion and bulk clot contraction assays to spatially-regulated platelet granule secretion assay, single-platelet contraction cytometry, microfluidics, and a microengineered vascularized bleeding model. As such, we are leveraging these biophysical assays to correlate platelet function with bleeding phenotype severity and establish the dynamic range of this diagnostic panel. We have now established that our assays can be utilized to study blood samples from patients with disorders including hemophilia A, Hermansky-Pudlak Syndrome, FLI-1 mutation, and sickle cell disease among others (Fig 2), demonstrating the clinical utility of our platelet function panel.Our panel can also be used to assess the effects of novel therapeutics on different aspects of platelet function simultaneously. To investigate how crizanlizumab (p-selectin inhibitor) affects hemostatic plug formation, healthy human blood was treated with crizanlizumab. Platelet α-granule secretion enables exposure of P-selectin, and with crizanlizumab we observed restricted platelet filopodial extension and diminished α-granule exocytosis, and an overall decrease in adhered platelets to the fibrinogen micropattern (Fig 3A). The adhesion assay demonstrated a decrease in spreading and adhesion of platelets to collagen and fibrinogen with treatment (Fig 3B). Using the bleeding model, hemostasis was achieved within the normal established range and platelets contracted normally. This suggests that p-selectin has a limited role in the setting of minor injury. Utilizing the bulk contraction assay, we exhibited increased contraction early in clot formation, however over time the treated platelets contracted similarly to the control (Fig 3C). Interestingly, the effect of crizanlizumab-induced restriction of filopodial extension did not correlate with impaired bulk clot contraction or time to form hemostatic plug. Our work suggests crizanlizumab affects platelet spreading at the single-cell level but does not impair platelet function in achieving primary hemostasis at the whole blood level.Here we demonstrate the translational utility of our platelet function panel in providing a deeper understanding of platelet biophysics as it relates to hematologic conditions, with implications for investigation to include pharmaceutical applications. The versatility of this novel panel in capturing platelet function from single-platelet contraction to providing in vitro models with the bleeding device provides multiple dimensions to platelet investigation for primary hemostatic disorders and BUC that have not yet been elucidated. Ongoing research is being conducted using our comprehensive platelet function panel to investigate platelet properties in BUC and mild VWD and correlate these biophysics with bleeding phenotypes. Using this approach we aim to provide novel diagnostic testing with clinical relevance for disorders that have been incompletely characterized until now. [Display omitted] DisclosuresMeeks: National Institutes of Health: Research Funding; Hemophilia of Georgia: Research Funding; National Hemophilia Foundation: Research Funding; Spark Therapeutics: Consultancy; Sangamo Therapeutics: Consultancy; Pfizer: Consultancy; Sanofi: Consultancy; CSL Behring: Consultancy; Genentech: Consultancy; Takeda: Consultancy. Lam: Sanguina, Inc.: Current holder of individual stocks in a privately-held company.

The possible role of mutated endothelial cells in myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPN) are chronic, clonal hematologic malignancies characterized by myeloproliferation and a high incidence of vascular complications (thrombotic and bleeding). Although MPN-specific driver mutations have been identified, the underlying events that culminate in these clinical manifestations require further clarification. We reviewed the numerous studies performed during the last decade identifying endothelial cell (EC) dysregulation as a factor contributing to MPN disease development. The JAK2V617F MPN mutation and other myeloid-associated mutations have been detected not only in hematopoietic cells but also in EC and their precursors in MPN patients, suggesting a link between mutated EC and the high incidence of vascular events. To date, however, the role of EC in MPN continues to be questioned by some investigators. In order to further clarify the role of EC in MPN, we first describe the experimental strategies used to study EC biology and then analyze the available evidence generated using these assays which implicate mutated EC in MPN-associated abnormalities. Mutated EC have been reported to possess a pro-adhesive phenotype as a result of increased endothelial Pselectin exposure, secondary to degranulation of Weibel-Palade bodies, which is further accentuated by exposure to pro-inflammatory cytokines. Additional evidence indicates that MPN myeloproliferation requires JAK2V617F expression by both hematopoietic stem cells and EC. Furthermore, the reports of JAK2V617F and other myeloid malignancy- associated mutations in both hematopoietic cells and EC in MPN patients support the hypothesis that MPN driver mutations may first appear in a common precursor cell for both EC and hematopoietic cells.

Activation of Platelet NLRP3 Inflammasome in Crohn's Disease.

Patients with Crohn’s disease (CD) are inclined to have platelet hyperactivity and an increased risk of intestinal micro-thrombosis. However, the mechanisms underlying platelet hyperactivity in CD are not well understood. We investigated the assembly of platelet NLRP3 inflammasome in patients with active CD and its correlation with platelet hyperactivity. In this study, Real-time PCR and western blotting analyses uncovered that ASC, NLRP3, and active caspase-1 were significantly upregulated in platelets from patients with active CD compared with healthy subjects. As revealed by flow cytometry (FCM) and ELISA analyses, the levels of interleukin-1β in both serum and isolated platelets were elevated in patients with active CD. Co-immunoprecipitation and immunofluorescence experiments revealed an increased assembly of NLRP3 inflammasome in platelets from patients with active CD. In addition, higher levels of intracellular reactive oxygen species (ROS) were observed in these platelets by FCM. Furthermore, elevated levels of platelet P-selectin exposure and fibrinogen binding were demonstrated in patients with active CD by FCM. They were positively correlated with the protein levels of NLRP3 inflammasome components. Collectively, our results indicate that the ROS-NLRP3 inflammasome-interleukin-1β axis may contribute to platelet hyperactivity in active CD.

GPVI Intracellular Signaling Is Activated in Platelets from Patients with Essential Thrombocythemia

GPVI Intracellular Signaling Is Activated in Platelets from Patients with Essential Thrombocythemia

A CRISPR-Cas9 Generated Etv6 mutant Mouse Exhibits Decreased Platelet Counts and Functional Platelet Defects

We and others recently described families with autosomal dominant thrombocytopenia, variable platelet function defects, and a predisposition to leukemia caused by heterozygous germline mutations in ETV6 . One of these mutations in ETV6, encoding an amino acid change at position 214 (P214L), was described in five independent families. The P214L mutation, located in the central domain of ETV6, was shown to disrupt nuclear localization of the ETV6 protein and to block megakaryocyte maturation of human cells overexpressing the mutant protein in vitro . Additionally, RNA seq of platelets from patients with the ETV6-P214L mutation showed downregulation of cytoskeletal transcripts and transcripts involved in platelet production and function. To further study the effect of the P214L mutation on ETV6 and on platelet production and function, we have generated a CRISPR-Cas9 knock-in mouse with the equivalent mutation to the human P214L in the mouse Etv6 ortholog (in the corresponding amino acid P216 in mice). CRISPR-Cas9 knock-in mice were generated by designing short guide RNAs specific to the site of Etv6 encoding the proline to be mutated. Additionally, a ssDNA oligo was designed encoding the mutation (cca>cta, P>L) and flanking homologous sequence to provide a template for recombination. Heterozygous and homozygous mice were viable. Heterozygous by heterozygous crosses yielded ratios not significantly different from expected Mendelian ratios: wild type (WT) 18.0%, heterozygous 63.0%, and homozygous 19.1% (p=0.18, Chi-squared test). Complete blood counts were obtained by retro-orbital bleeds and showed a mild but significant decrease in platelet counts in homozygous mice (WT 967.3 x 103/uL, heterozygous 840 x 103/uL, homozygous 807.5 x 103/uL, p=0.03). Western blot indicated normal expression of Etv6 protein in mouse bone marrow. Immunofluorescence and confocal imaging of megakaryocytes from these mice showed an increased percentage of small, immature megakaryocytes in homozygous mice compared to WT mice. Flow cytometry analysis suggested normal distribution of the hematopoietic stem cell compartment. These data are consistent with the patient phenotype and in vitro data, which showed that the P214L mutation resulted in variable thrombocytopenia, immature megakaryocytes, and normal ETV6 protein expression.Considering the downregulation of transcripts related to platelet function in patients with the P214L mutation and the variable platelet function defects reported in patients with germline ETV6 mutations, we characterized the platelet function of Etv6 mutant mice. Platelets from homozygous mice showed decreased platelet activation upon high (10 ug/ml) and low (2 ug/ml) collagen stimulation as measured by light transmission aggregometry (p=0.041 and 0.019, respectively). Additionally, homozygous mice had decreased markers of platelet activation (thrombin and convulxin) including granule secretion (P-selectin exposure) and aIIbb3 activation (JONA binding).In conclusion, we have generated a CRISPR-Cas9 knock-in mouse with the orthologous mutation to the human P214L recurrent germline mutation. Mice homozygous for this mutation recapitulate the human phenotype of decreased platelet counts and impaired megakaryocyte maturation. Additionally, mice homozygous for this mutation show a significant defect in platelet activation, further supporting that Etv6 is important for platelet function. Although complete knock-outs of Etv6 are embryonic lethal and Etv6 is expressed in the hematopoietic stem cell, we did not find a significant hematopoietic stem cell defect in our mutant mice. This finding suggests that the most important function of Etv6 is further downstream in hematopoiesis. The Etv6 P214L mouse represents a reliable model for studying the role of Etv6 in megakaryocyte biology, platelet function, and potentially future studies in leukemogenesis. DisclosuresNo relevant conflicts of interest to declare.

Towards a Closed System for Extracting Lymphocytes from Whole Blood

Introduction: Cellular therapies promise new treatment options and potentially curative efficacy for a wide array of acute and chronic diseases. The ability to obtain viable, uncontaminated lymphocyte concentrates from peripheral blood is in high demand. A major barrier for translating lymphocyte-based therapies into clinical practice is the poor scalability of manual, labor- and time-consuming ‘open system’ methods (e.g. Ficoll density gradient centrifugation) currently being used for isolating lymphocytes. To address the well-known limitations of existing separation methods, we developed a two-stage cell isolation approach that enables highly-efficient extraction of lymphocytes from whole blood (WB) without the use of centrifugation or any open-air manipulations by (1) depleting ~99% of red blood cells (RBCs) from the sample via rapid sedimentation induced by HES (6% hydroxyethyl starch in 0.9% sodium chloride), and then (2) passing the leukocyte-rich supernatant through a high-throughput microfluidic device (based on our newly-developed approach of ‘controlled incremental filtration,‘ CIF) to separate lymphocytes from platelets and residual RBCs.Materials and Methods: WB was collected from healthy consenting volunteers under an IRB approved protocol. An optimization study was performed to identify parameters of the sedimentation protocol with the highest lymphocyte yield and minimal contamination. This optimal protocol was then used to compare performance of (i) the standard Ficoll density gradient separation method, and (ii) our new two-stage separation approach. Each WB sample was divided into two equal parts - one half was processed via the conventional density gradient method, and the other half was first mixed with HES, allowed to sediment, and then the supernatant was passed through a CIF-based microfluidic device (designed for high-precision separation of lymphocytes from smaller cells, and ~15-fold reduction of the sample volume) at a flow rate of 0.5 mL/min. For both separation methods, product yield and purity were quantified by performing CBC with a 5-part differential on all samples. Flow cytometry was used to measure viability of isolated lymphocytes, and activation level of residual platelets via P-selectin and phosphatidylserine (PS) exposure. Separated lymphocytes were cultured for 4 days using standard proliferation techniques. Total number of viable cells was determined using Trypan Blue exclusion dye and manual counting with a hemocytometer.Results and Discussion: The optimized sedimentation protocol, which involved mixing WB with 10% HES (v/v) and waiting 30 min to complete, yielded the best combination of lymphocyte recovery (72.1±2.2%) and RBC depletion (99.48±0.07%) for the first stage of our separation approach. In the split-sample study, our two-stage method overall recovered 70.4±0.7% of lymphocytes from the initial WB sample, with 98.2±0.6% viability, while depleting 92.4±0.4% of platelets and 99.62±0.1% of RBCs. In comparison, the Ficoll density gradient centrifugation method removed 99.95±0.2% of RBCs and 94.2±0.1% of platelets, while recovering 74.5±2.40% of lymphocytes, with 98.3±0.7% viability. Lymphocytes produced by our method showed a significnatly higher prolifiration capacity than cells separated via density gradient centrifugation (Fig. 1). Markers of activation for residual platelets in the lymphocyte concentrate produced by our two-stage method were several fold lower than for Ficoll density gradient separation: 1.9±0.1% (our method) vs. 8.4±7.0% (Ficoll) for P-selectin, and 2.0±1.5% (our method) vs. 4.4±1.5% (Ficoll) for PS exposure. Importantly, lymphocyte isolation using our two-stage method was 3-fold faster than the Ficoll density graduate separation for the same amount of WB sample: <60 min (our method) vs. >180 min (Ficoll).Conclusion: We developed an easy-to-use approach for extracting lymphocytes from peripheral blood with purity, viability and yield meeting the current standard for cellular therapies. Since our new approach is scalable, compatible with ‘closed system’ operation, produces lymphocytes with higher proliferation capacity, and is much faster than conventional density gradient separation, it could have a potentially transformative impact by accelerating research into new cellular therapies, and simplifying translation of these treatments into clinical practice. [Display omitted] DisclosuresGifford:Halcyon Biomedical Incorporated: Employment, Equity Ownership. Shevkoplyas:Halcyon Biomedical Incorporated: Employment, Equity Ownership, Patents & Royalties: U.S. Patent Appl. 61/929,357, Research Funding; New Health Sciences, Inc.: Consultancy, Research Funding.

Distinctive Platelet Features and Lymphocyte Sets Distribution in Immune Thrombocytopaenia Patients Responders to Thrombopoietin Receptor Agonists

Background: The heterogeneous pathophysiology of immune thrombocytopaenia (ITP) may explain the wide variation in the presentation of the disease, the clinical course and the response to therapeutic treatments. Among them, agonists of thrombopoietin receptor (TPO-RA) were incorporated as a second line agent for the management of ITP patients who do not respond to other treatments. We hypothesized that platelet and white cells from these patients might present some characteristics different from those observed in ITP patients who don't need to be treated and those responders to a first line therapeutic agent. In order to test this hypothesis we designed this study to evaluate platelet function and distribution of lymphocyte´s compartments in three ITP patient's groups: 1) untreated (UT) and treated with 2) TPO-RA or 3) corticoids.Materials and Methods: We recruited 33 UT ITP patients (30% male), 35 treated with TPO-RA (45% male), 15 with corticoids (25 % male) and 96 healthy controls (42% male).The La Paz Universitary Hospital Ethics Committee approved the experimental protocol.Platelet-activation, granule's content release, surface exposure of phosphatidylserine (PS) and activities of caspase-3, -8 or -9 were assessed by flow cytometry (respectively performed with PAC1, anti P-selectin and anti-CD63 antibodies, from BD; Annexin V from BD Pharmingen and Millipore kit).The frequency of T (CD3+, CD4+, CD8+) and B (CD19+) lymphocytes, natural killer (NK) CD56bright and CD56dim and regulatory T (Treg) [CD4+CD25+ forkhead box protein 3(FoxP3)+] were determined by multi-parametric flow cytometry panels (BD Biosciences).The statistical analysis of the experimental data was using SPSS 9.0 software (SPSS Inc., Chicago, Illinois, USA).Results: Platelet count were similar in all ITP groups but lower than controls (p<0.001). Platelet activation, evaluated by binding of PAC1 (antibody that only recognizes the active form of fibrinogen receptor) after the stimulation with TRAP (an agonist for PAR-1 receptors), were equally reduced in all ITP groups. Platelet activation with ADP was only reduced in the TPO-RA ITP patients. A diminished exposure of P-selectin and CD63 was also observed in UT and TPO-RA ITP patients after platelet stimulation with TRAP (Table 1). No differences were observed in surface expression of CD41, CD61, CD42a and CD42b between ITP patients and healthy controls. Platelet apoptosis evaluation showed that ITP patients tended to have an increased activity of caspases -3,7,-8 and -9 activities, but this observation was significant only in the ITP group of treated with TPO-RA (Table 1).Regarding lymphocytes, no differences were observed in LT subsets among all groups. Intermediate (IM, CD14++CD16+), but not non-classical (NCM, CD14+CD16++) and classical (CM, CD14++CD16-) monocytes were increased in UT and TPO-RA ITP patients. CD56brightCD16- NKand CD56brightCD16+NK, abundant cytokine producers with a weak cytotoxic activity, were increased only in TPO-RA ITP patients. In this group a diminution was observed in memory LB cells. Regarding LTreg population, their count was reduced in UT and corticoid-treated patients, whereas their values reach normality in the TPO-RA treated group (Table 2).Conclusions: All ITP patients, untreated or responders to treatment with TPO-RA and corticoids recovered platelet count but not its function. TPO-RA-responders patients showed an increased apoptosis compared to the other ITP patients recruited. Nevertheless, TPO-RA seems to have a beneficial regulatory effect on immune system that might counteract platelet destruction.Work supported by grant from FIS-FEDER PI15/01457. NVB holds a Miguel Servet II (FIS-FEDER CP14/00024). [Display omitted] DisclosuresÁlvarez-Roman:Shire: Consultancy; Novo Nordisk: Consultancy. Jimenez-Yuste:Novo Nordisk: Consultancy, Honoraria, Research Funding; Roche: Consultancy.

The GPIbα intracellular tail - role in transducing VWF- and collagen/GPVI-mediated signaling.

The GPIbα-VWF A1 domain interaction is essential for platelet tethering under high shear. Synergy between GPIbα and GPVI signaling machineries has been suggested previously, however its molecular mechanism remains unclear. We generated a novel GPIbα transgenic mouse (GpIbaΔsig/Δsig) by CRISPR-Cas9 technology to delete the last 24 residues of the GPIbα intracellular tail that harbors the 14-3-3 and phosphoinositide-3 kinase binding sites. GPIbαΔsig/Δsig platelets bound von Willebrand factor (VWF) normally under flow. However, they formed fewer filopodia on VWF/botrocetin in the presence of a aIIbb3 blocker, demonstrating that despite normal ligand binding, VWF-dependent signaling is diminished. Activation of GPIbαΔsig/Δsig platelets with ADP and thrombin was normal, but GPIbαΔsig/Δsig platelets stimulated with collagenrelated- peptide (CRP) exhibited markedly decreased P-selectin exposure and aIIbb3 activation, suggesting a role for the GpIba intracellular tail in GPVI-mediated signaling. Consistent with this, while hemostasis was normal in GPIbαΔsig/Δsig mice, diminished tyrosine-phosphorylation, (particularly pSYK) was detected in CRP-stimulated GPIbαΔsig/Δsig platelets as well as reduced platelet spreading on CRP. Platelet responses to rhodocytin were also affected in GPIbαΔsig/Δsig platelets but to a lesser extent than those with CRP. GPIbαΔsig/Δsig platelets formed smaller aggregates than wild-type platelets on collagen-coated microchannels at low, medium and high shear. In response to both VWF and collagen binding, flow assays performed with plasma-free blood or in the presence of aIIbb3- or GPVI-blockers suggested reduced aIIbb3 activation contributes to the phenotype of the GPIbαΔsig/Δsig platelets. Together, these results reveal a new role for the intracellular tail of GPIbα in transducing both VWF-GPIbα and collagen-GPVI signaling events in platelets.

Antiplatelet activity and chemical analysis of leaf and fruit extracts from Aristotelia chilensis.

Aristotelia chilensis (Mol.) Stuntz, also known as maqui, is a plant native to Chile without chemical characterization and quantification of the bioactive compounds present in it. HPLC-UV and HPLC-MS/MS studies have shown the presence, at different concentrations, of phenolic and anthocyanin compounds in fruit and leave extracts of the domesticated maqui clones Luna Nueva, Morena, and Perla Negra. The extracts from leaves and unripe fruits of Luna Nueva and Morena clones significantly inhibit platelet aggregation induced by several agonists; the extracts inhibit platelet granule secretion by decreasing the exposure of P-selectin and CD63 at the platelet membrane. Reactive oxygen species formation in platelets is lower in the presence of maqui extracts. Statistical Pearson analysis supports the levels of phenolic and anthocyanin compounds being responsible for the antiaggregant maqui effects. This work is the first evidence of antiplatelet activity from Aristotelia chilensis giving added value to the use of leaves and unripe fruits from this species.

Protein Disulphide Isomerase and NADPH Oxidase 1 Cooperate to Control Platelet Function and Are Associated with Cardiometabolic Disease Risk Factors.

Background: Protein disulphide isomerase (PDI) and NADPH oxidase 1 (Nox-1) regulate platelet function and reactive oxygen species (ROS) generation, suggesting potentially interdependent roles. Increased platelet reactivity and ROS production have been correlated with cardiometabolic disease risk factors. Objectives: To establish whether PDI and Nox-1 cooperate to control platelet function. Methods: Immunofluorescence microscopy was utilised to determine expression and localisation of PDI and Nox-1. Platelet aggregation, fibrinogen binding, P-selectin exposure, spreading and calcium mobilization were measured as markers of platelet function. A cross-sectional population study (n = 136) was conducted to assess the relationship between platelet PDI and Nox-1 levels and cardiometabolic risk factors. Results: PDI and Nox-1 co-localized upon activation induced by the collagen receptor GPVI. Co-inhibition of PDI and Nox-1 led to additive inhibition of GPVI-mediated platelet aggregation, activation and calcium flux. This was confirmed in murine Nox-1−/− platelets treated with PDI inhibitor bepristat, without affecting bleeding. PDI and Nox-1 together contributed to GPVI signalling that involved the phosphorylation of p38 MAPK, p47phox, PKC and Akt. Platelet PDI and Nox-1 levels were upregulated in obesity, with platelet Nox-1 also elevated in hypertensive individuals. Conclusions: We show that PDI and Nox-1 cooperate to control platelet function and are associated with cardiometabolic risk factors.

Deficiency of ARHGAP21 alters megakaryocytic cell lineage responses and enhances platelet hemostatic function

BackgroundMicrotubules, actin and Rho GTPase proteins are essential players in the megakaryocyte biology for platelet formation and function. ObjectivesTo investigate the role of ARHGAP21, a RhoGAP protein, in megakaryocytic differentiation and platelet function. MethodsCytoskeletal proteins were investigated in HEL cells silenced for ARHGAP21 and submitted to megakaryocyte differentiation. The role of Arhgap21 in platelet function was accessed using haploinsufficient (Arhgap21+/−) mice. Arhgap21+/− platelet aggregation and p-selectin exposure were evaluated in response to thrombin. Vessel occlusion time and thrombus formation were detected after injury of the carotid artery. Platelet morphology was accessed by electronic microscopy. ResultsARHGAP21 was upregulated during megakaryocytic differentiation of the cell line and primary mouse cells. In the HEL cell model, ARHGAP21 was detected in the cytoplasmic protrusions, colocalized and associated with α-tubulin and was mostly detected in the protein cell fraction containing the polymerized tubulin. Silencing of ARHGAP21 decreased the expression of Glu-tubulin, suggesting microtubule instability, and enhanced cell spreading. Platelets from Arhgap21+/− mice presented enhanced thrombin-induced aggregation and p-selectin exposure associated with increased size of α-granules. Arhgap21+/− mice also showed increased CDC42 and RHOA activities, shorter tail-bleeding and accelerated thrombus formation. ConclusionsOur results indicate that ARHGAP21 may be a critical protein in the regulation of platelet production and function through the control of cytoskeletal rearrangement.

Platelet-derived extracellular vesicles express NADPH oxidase-1 (Nox-1), generate superoxide and modulate platelet function

BackgroundPlatelets release platelet-derived extracellular vesicles (PDEVs) upon activation – in a process that is regulated by generation of reactive oxygen species (ROS). Platelet NADPH oxidase-1 (Nox-1) contributes to ROS generation and thrombus formation downstream of the collagen receptor GPVI. ObjectivesWe aimed to investigate whether PDEVs contain Nox-1 and whether this is relevant for PDEV-induced platelet activation. MethodsPDEVs were isolated through serial centrifugation after platelet activation with thrombin receptor agonist TRAP-6 (activated PDEVs) or in the absence of agonist (resting PDEVs). The physical properties of PDEVs were analyzed through nanoparticle tracking analysis. Nox-1 levels, fibrinogen binding and P-selectin exposure were measured using flow cytometry, and protein levels quantified by immunoblot analysis. ROS were quantified using DCF fluorescence and electron paramagnetic resonance. ResultsNox-1 was found to be increased on the platelet outer membrane upon activation and was present in PDEVs. PDEVs induced platelet activation, while co-addition of GPVI agonist collagen-related peptide (CRP) did not potentiate this response. PDEVs were shown to be able to generate superoxide in a process at least partially mediated by Nox-1, while Nox-1 inhibition with ML171 (also known as 2-APT) did not influence PDEV production. Finally, inhibition of Nox-1 abrogated PDEV-mediated platelet activation. ConclusionsPDEVs are able to generate superoxide, bind to and activate platelets in a process mediated by Nox-1. These data provide novel mechanisms by which Nox-1 potentiates platelet responses, thus proposing Nox-1 inhibition as a feasible strategy to treat and prevent thrombotic diseases.

Loss of the exocyst complex component EXOC3 promotes hemostasis and accelerates arterial thrombosis

AbstractThe exocyst is an octameric complex comprising 8 distinct protein subunits, exocyst complex components (EXOC) 1 to 8. It has an established role in tethering secretory vesicles to the plasma membrane, but its relevance to platelet granule secretion and function remains to be determined. Here, EXOC3 conditional knockout (KO) mice in the megakaryocyte/platelet lineage were generated to assess exocyst function in platelets. Significant defects in platelet aggregation, integrin activation, α-granule (P-selectin and platelet factor 4), dense granule, and lysosomal granule secretion were detected in EXOC3 KO platelets after treatment with a glycoprotein VI (GPVI)-selective agonist, collagen-related peptide (CRP). Except for P-selectin exposure, these defects were completely recovered by maximal CRP concentrations. GPVI surface levels were also significantly decreased by 14.5% in KO platelets, whereas defects in proximal GPVI signaling responses, Syk and LAT phosphorylation, and calcium mobilization were also detected, implying an indirect mechanism for these recoverable defects due to decreased surface GPVI. Paradoxically, dense granule secretion, integrin activation, and changes in surface expression of integrin αIIb (CD41) were significantly increased in KO platelets after protease-activated receptor 4 activation, but calcium responses were unaltered. Elevated integrin activation responses were completely suppressed with a P2Y12 receptor antagonist, suggesting enhanced dense granule secretion of adenosine 5′-diphosphate as a critical mediator of these responses. Finally, arterial thrombosis was significantly accelerated in KO mice, which also displayed improved hemostasis determined by reduced tail bleeding times. These findings reveal a regulatory role for the exocyst in controlling critical aspects of platelet function pertinent to thrombosis and hemostasis.

Removal of citrate from PAS‐III additive solution improves functional and biochemical characteristics of buffy‐coat platelet concentrates stored for 7 days, with or without INTERCEPT pathogen reduction

Deterioration in quality of platelet concentrates (PCs) during storage results from the appearance of storage lesions affecting the hemostatic functions and posttransfusion survival of platelets. These lesions depend on the preparation and pathogen inactivation methods used, duration of storage, and platelet additive solutions (PASs) present in storage bags. We investigated the effects of citrate contained in third-generation PAS (PAS-III) on storage lesions in buffy-coat PCs with or without photochemical (amotosalen-ultraviolet A) treatment over 7 days. Platelet counts were conserved in all groups during storage, as was platelet swirling without appearance of macroscopic aggregates. Glycoprotein (GP) IIbIIIa and GPVI expression remained stable, whereas GPIbα declined similarly in all groups during storage. Removal of citrate from PAS-III, resulting in global reduction of citrate from 11 to 5 mM, led to a significant decrease in glucose consumption, which largely countered a modest deleterious effect of photochemical treatment. Citrate reduction also resulted in decreased lactate generation and better maintenance of pH during storage, while photochemical treatment had no impact on these parameters. Moreover, citrate-free storage significantly reduced exposure of P-selectin and the apoptosis signal phosphatidylserine, thereby abolishing the activating effect of photochemical treatment on both parameters. Citrate reduction benefited platelet aggregation to various agonists up to Day 7, whereas PCT had no impact on these responses. Removal of citrate from PAS-III has a beneficial impact on platelet metabolism, spontaneous activation, and apoptosis, and improves platelet aggregation, irrespective of photochemical treatment, which should allow transfusion of platelets with better and longer-lasting functional properties.

Droplet Microfluidics with Reagent Micromixing for Investigating Intrinsic Platelet Functionality.

Precision mapping of the functional structure of platelet populations holds great promise for the identification of hyper-reactive subtypes that are likely to be disease drivers, having value in prognostics and as therapeutic targets. However, the ability to measure the intrinsic functional capacity of individual platelets is confounded by potent paracrine cross-talk, resulting in phenotypic remodeling of the entire platelet population, and in doing so obscuring the identity of hyper-reactive platelets. To address this we have developed a droplet microfluidics strategy for single platelet confinement to exclude paracrine signaling. Consideration of the Poisson distribution was used for high throughput single platelet encapsulation and the preparation of minimal platelet collectives serving as digital models for understanding the role of hyper-reactive platelets coordinating system-level behavior by paracrine signaling. Platelets are retrieved from the droplets for phenotyping using standard flow cytometry. In addition, we have incorporated a staggered herringbone micromixing element for accurate agonist and antibody dispensing in droplets. The methodology was used for characterizing sensitivity distributions from healthy blood donors in response to convulxin (agonist of the GPVI receptor, the major platelet receptor for collagen). P-selectin exposure and α IIb β 3 integrin activation were used as analytical end-points to demonstrate the existence of hyper-reactive platelets that direct 20-fold gains in system level sensitivity. The analytical workflow represents an enabling tool for the accurate classification of platelet subtypes and description of their underlying biology. The online version of this article (10.1007/s12195-020-00665-6) contains supplementary material, which is available to authorized users.

Galectin-1 and platelet factor 4 (CXCL4) induce complementary platelet responses in vitro

Galectin-1 (gal-1) is a carbohydrate-binding lectin with important functions in angiogenesis, immune response, hemostasis and inflammation. Comparable functions are exerted by platelet factor 4 (CXCL4), a chemokine stored in the α-granules of platelets. Previously, gal-1 was found to activate platelets through integrin αIIbβ3. Both gal-1 and CXCL4 have high affinities for polysaccharides, and thus may mutually influence their functions. The aim of this study was to investigate a possible synergism of gal-1 and CXCL4 in platelet activation. Platelets were treated with increasing concentrations of gal-1, CXCL4 or both, and aggregation, integrin activation, P-selectin and phosphatidyl serine (PS) exposure were determined by light transmission aggregometry and by flow cytometry. To investigate the influence of cell surface sialic acid, platelets were treated with neuraminidase prior to stimulation. Gal-1 and CXCL4 were found to colocalize on the platelet surface. Stimulation with gal-1 led to integrin αIIbβ3 activation and to robust platelet aggregation, while CXCL4 weakly triggered aggregation and primarily induced P-selectin expression. Co-incubation of gal-1 and CXCL4 potentiated platelet aggregation compared with gal-1 alone. Whereas neither gal-1 and CXCL4 induced PS-exposure on platelets, prior removal of surface sialic acid strongly potentiated PS exposure. In addition, neuraminidase treatment increased the binding of gal-1 to platelets and lowered the activation threshold for gal-1. However, CXCL4 did not affect binding of gal-1 to platelets. Taken together, stimulation of platelets with gal-1 and CXCL4 led to distinct and complementary activation profiles, with additive rather than synergistic effects.

Response by Xu et al to Letter Regarding Article, “Branched-Chain Amino Acid Catabolism Promotes Thrombosis Risk by Enhancing Tropomodulin-3 Propionylation in Platelets”

HomeCirculationVol. 142, No. 24Response by Xu et al to Letter Regarding Article, “Branched-Chain Amino Acid Catabolism Promotes Thrombosis Risk by Enhancing Tropomodulin-3 Propionylation in Platelets” Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyRedditDiggEmail Jump toFree AccessLetterPDF/EPUBResponse by Xu et al to Letter Regarding Article, “Branched-Chain Amino Acid Catabolism Promotes Thrombosis Risk by Enhancing Tropomodulin-3 Propionylation in Platelets” Yanyan Xu, PhD, Haojie Jiang, PhD and Junling Liu, PhD Yanyan XuYanyan Xu Department of Biochemistry and Molecular Cell Biology, Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao Tong University School of Medicine, China (Y.X., H.J., J.L.). Search for more papers by this author , Haojie JiangHaojie Jiang Department of Biochemistry and Molecular Cell Biology, Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao Tong University School of Medicine, China (Y.X., H.J., J.L.). Search for more papers by this author and Junling LiuJunling Liu Department of Biochemistry and Molecular Cell Biology, Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao Tong University School of Medicine, China (Y.X., H.J., J.L.). The Cyrus Tang Hematology Center, Collaborative Innovation Center of Hematology, Soochow University, Suzhou, China (J.L.). Search for more papers by this author Originally published14 Dec 2020https://doi.org/10.1161/CIRCULATIONAHA.120.051367Circulation. 2020;142:e452–e453In Response:We thank Dr Liu and colleagues for their interest in our article, “Branched-Chain Amino Acid Catabolism Promotes Thrombosis Risk by Enhancing Tropomodulin-3 Propionylation in Platelets.”1We fully agree with Dr Liu that further exploration of the functions of leucine and mammalian target of rapamycin pathway will strengthen the understanding of the mechanism of branched-chain amino acid (BCAA)–induced platelet activation. In our previous study, we studied the individual functions of valine, isoleucine, leucine, and their catabolites on platelet activation. We found that valine/α-ketoisovaleric acid has the most significant promoting effect on platelet activation among BCAAs and branched-chain α-keto acids. Propionyl coenzyme A, as the end product of valine and isoleucine metabolism, promotes platelet aggregation and the TMOD3 (tropomodulin 3) propionylation. Leucine catabolism eventually leads to the formation of acetoacetyl coenzyme A and acetyl coenzyme A, but not propionyl coenzyme A. We found that leucine and its metabolite α-ketoisocaproic acid enhance agonist-induced platelet aggregation and P-selectin exposure. These suggested that there must be some other mechanisms between BCAAs and thrombosis formation. It has been reported that leucine participates in the mammalian target of rapamycin signaling pathway in T cells, macrophages, and other cells.2 It is necessary to further investigate the relationship between leucine and mammalian target of rapamycin in the process of platelet activation and thrombosis.We agree with Dr Liu that the age, medication, and diet of patients with type 2 diabetes mellitus should be taken into consideration to demonstrate the relationship between BCAA levels and platelet activation. In our study, the average age of inpatients with type 2 diabetes mellitus was 57.5±3.7 years and all patients had taken medication to control blood glucose level. Apparently, the number of patients in our study was not enough to perform the analysis of the correlation between the age, medication, diet, and the levels of BCAAs and platelet activation in patients. Larger scaled experiments are required to resolve this issue.As Dr Liu pointed out, BCAAs and branched-chain α-keto acids are supplied to patients who have chronic renal failure with other essential amino acids and their ketoanalogues to decrease protein intake as much as possible and maintain protein balance, which may increase the risk of thrombosis.3,4 Our unpublished data showed that P2Y12 inhibitor ticagrelor and thromboxane A2 production blocker aspirin significantly inhibited the enhancing effects of BCAAs on the aggregation of platelets in response to different agonists in vitro, which suggested that utilization of current antiplatelet drugs might reduce the risk of thrombosis in the therapy of patients with chronic renal failure. However, further basic and clinical studies must be performed before applying the findings to the supplement of BCAAs in patients.DisclosuresNone.

Prospective Study Reveals Increased Platelet Function Associated with Multiple Myeloma and Its Treatment

Background: Multiple Myeloma (MM) is a rare incurable bone marrow cancer characterised by a malignant proliferation of plasma cells. MM is usually preceded by a premalignant and benign Monoclonal Gammopathy of Undetermined Significance (MGUS). The incidence of arterial and venous thrombosis in MM is substantially higher than in the normal population, however the cause of this increased thrombosis risk and the impact of MM on platelet function is unclear. Treatments for both newly diagnosed and relapsed/refractory patients with MM include Immunomodulatory drugs (IMiDs) such as thalidomide/lenalidomide-based combinations. These treatments improve considerably patient outcomes, however iMiD treatment also increases the risk of thrombotic complications in these patients. Aims: In this prospective study we explored the impact of MM and its treatment on platelet function. Methods: High throughput functional analysis was performed using platelets from normal healthy controls (n=31) and patients with MGUS (n=18), smouldering multiple myeloma (SMM, n= 20), and MM (26). The MM group was further divided into 3 treatment cohorts; (1) no treatment, (2) treatment with proteasome inhibitor (PI) and dexamethasone (Dex), and (3) treatment with PI, Dex, immunomodulatory drug (iMiD) and direct oral anticoagulant. Platelet aggregation and activation (fibrinogen binding and P-selectin exposure) were measured in response to a concentration range of agonists including ADP, the thrombin receptor agonist TRAP-6, collagen, collagen-related peptide (CRP), a thromboxane receptor agonist U46619 and epinephrine. Cereblon protein was detected in platelet protein extracts by immunoblot analysis. Results: Consistent with previous reports, modestly increased VWF and factor VIII levels were detected in MM patients, but no additional differences in coagulation parameters were detected in patient groups compared to normal healthy controls (other than expected due to anticoagulant usage). Platelet aggregation in response to each agonist was increased significantly in the MM patient group compared to the normal healthy controls, suggesting that platelet reactivity is elevated in MM patients through a common mechanism that is shared by different activation pathways or the involvement of multiple mechanisms. P-selectin exposure on platelets from MM patients was not significantly different from normal healthy donors, indicating that enhanced platelet reactivity in MM is specifically through modulation of integrin αIIbβ3 activation, fibrinogen binding and therefore enhanced aggregation. The effects of treatment on platelet function in patients on iMiD vs. non iMiD treatment were assessed. In the iMiD treatment group, patient platelets aggregated in response to lower concentrations of ADP, collagen, epinephrine and CRP in samples taken post-treatment compared to those taken before and during treatment. This demonstrates an increased sensitivity to platelet activation in these patients induced by treatment. Immunoblot analysis revealed that platelets contain cereblon, a therapeutic target of lenalidomide. The potential direct effects of iMiDs on platelets in vitro was therefore explored. Lenalidomide treatment (10mM) increased the ability of platelets to aggregate in response to low concentrations of each agonist tested when compared to normal controls. Conclusions: Platelet reactivity is increased in multiple myeloma and increased further upon iMiD treatment. The presence of the key therapeutic target for iMiDs in platelets and the ability of lenalidomide to modulate platelet function directly, reveals new avenues for investigation to determine the underlying mechanism of action. Disclosures Laffan: CSL: Consultancy; Pfizer: Consultancy; Sobi: Consultancy; Roche: Consultancy; LFB: Consultancy; Shire: Consultancy; Octapharma: Consultancy; Bayer: Speakers Bureau; Roche-Chugai: Speakers Bureau; Takeda: Speakers Bureau; Leo-Pharma: Speakers Bureau; Pfizer: Speakers Bureau. Shapiro:Bayer: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; NovoNordisk: Consultancy, Speakers Bureau; Sobi: Consultancy, Speakers Bureau; Chugai/Roche: Consultancy, Speakers Bureau; Shire/Takeda: Consultancy, Speakers Bureau. Thakurta:Oxford University: Other: visiting professor; Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Ramasamy:Takeda: Research Funding; Janssen: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Research Funding; Amgen: Research Funding; Amgen: Honoraria; Takeda: Honoraria; Sanofi: Honoraria; Oncopeptides: Honoraria; Takeda: Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria; Bristol Myers Squibb: Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees; Bristol Myers squibb: Membership on an entity's Board of Directors or advisory committees. Gibbins:Bristol Myers Squibb: Research Funding; Arena Pharmaceuticals: Research Funding.

Platelet Storage Temperature Determines Recovery of GPVI-Function In Vivo

Background: Platelets (PLTs) are currently stored at 22°C (RT, room temperature) for clinical purposes. This approach ensures long circulation time but has numerous downsides, including limited storage time due to the risk of bacterial growth and increased costs due to bacterial testing or pathogen reduction processing. PLTs stored at 4°C were the standard of care in the 1960s and 1970s. In our previous study with healthy volunteers, we showed that humans who received cold-stored PLTs have a significantly weaker response to collagen (an agonist that acts predominantly via GPVI) compared to RT-stored PLTs. If and how cold-stored PLTs recover their function in vivo is poorly understood. Methods: We obtained human PLTs by an apheresis collection and sampled either at baseline (fresh) or after five days at RT or 4°C. To test the response to GPVI-dependent agonists, we stimulated platelet-rich plasma or washed PLTs with collagen and the GPVI-specific agonist convulxin (CVX) and tested for activated integrin and α-degranulation by flow cytometry. Platelet aggregation, in response to GPVI-dependent agonists, was tested by aggregometry. We checked for GPVI expression levels by flow cytometry and for signaling events downstream of GPVI by immunoblotting. To allow for recovery of function in vitro, we incubated either 4°C-stored, or RT-stored PLTs with fresh, platelet-depleted blood for 15min, and perfused the reconstituted whole blood through a microfluidic block and post device to quantify the contractile forces of platelet aggregates. Additionally, we performed platelet force measurements at the single cell level using a traction force microscopy approach. To validate a murine model of platelet storage and transfusion, we replicated functional studies in vitro by testing mouse PLTs for integrin activation and α-degranulation by flow cytometry. Platelet aggregation in response to collagen, CVX, and the GPVI-specific antibody JAQ-1 with crosslinking anti-IgG was also tested. To evaluate the platelet function after transfusion, we obtained whole blood from UbiC-GFP mice and isolated platelet-rich plasma followed by storage for 24 hours at either 4°C or RT. To allow tracking of stored PLTs in vivo, we transfused the UbiC-GFP PLTs into wild-type C57BL/6J mice and tested for integrin activation of endogenous and transfused PLTs. Results: In human PLTs, we found a significantly increased integrin response in 4°C-stored PLTs stimulated with collagen in flow cytometry studies in vitro. Similarly, the aggregation response of 4°C-stored PLTs to collagen was significantly increased compared to RT-stored PLTs in vitro. In line with these findings, we observed more PLCγ2 phosphorylation and Syk phosphorylation at baseline in 4°C-stored PLTs compared to RT-stored PLTs, suggesting more pre-activation downstream of GPVI. However, no differences in PLCγ2 phosphorylation or Syk-phosphorylation were found between RT and 4°C-stored PLTs after stimulation with CVX, and no significant differences in surface expression levels of GPVI were detected between RT and 4°C. Stored platelets in plasma showed superior function after 4°C-storage in aggregation and flow cytometry assays. In contrast, we found similar contractile forces of platelet aggregates when RT-stored or 4°C-stored PLTs were added to platelet-depleted fresh blood. Additionally, at the single cell level, we found a similar magnitude of platelet forces in RT-stored and 4°C-stored PLTs. Similar to human PLTs, mouse PLTs showed significantly more integrin activation, P-selectin exposure, and aggregation in 4°C-stored PLTs compared to RT. To test the recovery of function of stored mouse platelets in vivo, we transfused GFP-positive PLTs into GFP-negative wild-type mice. Contrary to our pre-transfusion results, we found a significantly lower integrin activation response to CVX in 4°C-stored platelets after transfusion, consistent with our previous results in healthy human volunteers. Summary: The in vivo recovery of function of stored PLTs is an underappreciated phenomenon in platelet storage biology, and most studies are solely based on functional in vitro data. Based on our post-transfusion results, storage temperature affects the ability to recover function in vivo significantly in human and mouse platelets. Whether these differences lead to differences in clinical outcomes needs to be investigated in clinical trials. Disclosures Sniadecki: Stasys Medical Corporation: Current equity holder in private company, Other: Co-founder; Curi Bio: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees.