Reduction In Number Of Platelets Research Articles

Impact of Shaking EDTA, Citrate, or MgSO4 Tubes on Platelet Count Results.

Background: In EDTA-induced pseudothrombopenia, citrate or MgSO4 are recommended for platelet counting. Pre-analytical conditions are poorly defined for tubes containing MgSO4 or citrate. In this study, we analyzed the impact of agitation of these tubes on platelet counts. Methods: K2EDTA, citrate, and MgSO4 tubes from 70 patients were gently agitated on a wheel rotating at 20 rpm. Platelets were analyzed on the Sysmex XN analyzer at different times, and the percentage of platelet deviation from T0 was assessed and compared with the desirable bias of the EFLM. Results: at 180 min in fluorescence, the relative variation of platelets after shaking is 1.17% for K2EDTA, -29.76% for citrate, and -33.18% for MgSO4, while for unshaken MgSO4 platelets the variation is -1.3%. The reduction in platelet numbers when citrate or MgSO4 tubes are shaken is linked to the appearance of platelet clusters. Conclusions: agitation of MgSO4 and especially citrate tubes led to a decrease in platelet counts due to the formation of platelet aggregates; on the other hand, platelet counts on EDTA are virtually stable. During transport, we recommend putting sodium citrate and MgSO4 tubes in an upright position and avoiding shaking them to avoid giving an erroneous platelet result.

Platelet Desialylation Is a Novel Mechanism and Therapeutic Target in Daboia siamensis and Agkistrodon halys Envenomation-Induced Thrombocytopenia.

Venom-induced thrombocytopenia (VIT) is one of the most important hemotoxic effects of a snakebite, which is often associated with venom-induced consumptive coagulopathy (VICC). Refractory thrombocytopenia without significant coagulation abnormalities has also been reported after envenomation by some viperid snakes; however, the mechanisms are not well understood and therapeutic strategies are lacking. Here, we found that patients injured by Daboia siamensis or Agkistrodon halys snakes, who were resistant to standard antivenom treatment, had developed coagulopathy-independent thrombocytopenia. Venoms from these viperid snakes, rather than from the elapid snake (Bungarus multicinctus), induced platelet surface expression of neuraminidase-1 (NEU-1), and significantly increased the desialylation of the glycoproteins on human platelets. The desialylated platelets caused by viperid snake venoms were further internalized by macrophages, which resulted in reduced platelet numbers in peripheral blood. Importantly, neuraminidase inhibitor significantly decreased viper venom-induced platelet desialylation, therefore inhibiting platelet phagocytosis by macrophages, and alleviating venom-induced thrombocytopenia. Collectively, these findings support an important role for desialylated platelet clearance in the progression of viper envenomation-induced, coagulopathy-independent thrombocytopenia. Our study demonstrates that the neuraminidase inhibitor may be a potential therapy or adjuvant therapy to treat snakebite-induced thrombocytopenia.

Cryopreservation of apheresis platelets treated with riboflavin and UV light

BackgroundPathogen reduction technology (PRT) is increasingly used in the preparation of platelets for therapeutic transfusion. As the Czech Republic considers PRT, we asked what effects PRT may have on the recovery and function of platelets after cryopreservation (CP), which we use in both military and civilian blood settings. Study design and methods16 Group O apheresis platelets units were treated with PRT (Mirasol, Terumo BCT, USA) before freezing; 15 similarly collected units were frozen without PRT as controls. All units were processed with 5–6% DMSO, frozen at − 80 °C, stored > 14 days, and reconstituted in thawed AB plasma. After reconstitution, all units were assessed for: platelet count, mean platelet volume (MPV), platelet recovery, thromboelastography, thrombin generation time, endogenous thrombin potential (ETP), glucose, lactate, pH, pO2, pCO2, HCO3, CD41, CD42b, CD62, Annexin V, CCL5, CD62P, and aggregates > 2 mm and selected units for Kunicki score. ResultsPRT treated platelet units had lower platelet number (247 vs 278 ×109/U), reduced thromboelastographic MA (38 vs 62 mm) and demonstrated aggregates compared to untreated platelets. Plasma coagulation functions were largely unchanged. ConclusionsSamples from PRT units showed reduced platelet number, reduced function greater than the reduced number would cause, and aggregates. While the platelet numbers are sufficient to meet the European standard, marked platelets activation with weak clot strength suggest reduced effectiveness.

Association and predictive value of biomarkers with severe outcomes in hospitalized patients with SARS-CoV-2 infection.

This study analyzed the levels at admission of biomarkers for their association with and ability to predict risk of severe outcomes, including admission to the ICU, need for invasive mechanical ventilation (IMV), need for vasopressor use (VU), and in-hospital mortality (IHM) in 700 patients hospitalized with COVID-19. Biomarker data split by outcomes was compared using Mann-Whitney U tests; frequencies of biomarker values were compared using Chi-square tests and multivariable logistic regression analysis was performed to look at the impact of biomarkers by outcome. Patients that suffered IHM were more likely to have reduced platelet numbers and high blood urea nitrogen (BUN) levels among patients admitted to the ICU. Risk factors for mortality were related to hyper-coagulability (low platelet count and increased D-dimer) and decreased respiratory (PaO2/FiO2 ratio) and kidney function (BUN). Association with risks of other severe outcomes were as follows: ICU with hyper-inflammation (IL-6) and decreased respiratory function; IMV with low platelet count, abnormal neutrophil–lymphocyte ratio with reduced respiratory function, VU with inflammatory markers (IL-6), and low platelet count with respiratory function. Our studies confirmed the association of biomarkers of hematological, inflammatory, coagulation, pulmonary and kidney functions with disease severity. Whether these biomarkers have any mechanistic or causal role in the disease progress requires further investigation.

Platelet and White Cell Reactivity to Top-Load Intravenous Perfluorocarbon Infusion in Healthy Sheep

BackgroundPerfluorocarbon emulsions (PFCs) are intravenous artificial oxygen carriers with enhanced gas solubility. As lipid micelle nanoparticle emulsions, PFCs may have a class effect that causes degrees of thrombocytopenia. Understanding the extent of the platelet effects, including mechanism and potential inflammation after PFC infusion, is important for safe human trials. MethodsNormal sheep (Dorper) were infused with 5 mL/kg of Oxygent (w/v 60% PFC) or Perftoran (w/v 20% PFC). Controls received 6% Hetastarch or were naive. Blood samples were analyzed from baseline, time 0 (the end of infusion), 3 and 24 hours, and 4 and 7 days. Platelet count, plateletcrit, mean platelet volume, platelet distribution width, and CD-62p (a platelet activation-dependent membrane protein) were measured. Neutrophils, monocytes, and total white blood cell counts were analyzed. ResultsIn these inflammatory cell lines, there were no consistent changes or cellular activation after PFC infusion. A decrease (<10% from baseline and naive controls) in platelet count was seen on day 4 after Oxygent infusion (3 g/kg), which recovered by day 7. No platelet effect was seen in Perftoran (1 g/kg). Plateletcrit, mean platelet volume, and platelet distribution width did not change significantly at any time point among the groups. CD-62p, ADP, and collagen aggregometry showed no significant change in platelet function. ConclusionThere was no evidence of overall reduction in platelet number, or any correlation with the change in platelet activation or inhibition. Therefore, the risk of increased thrombosis/bleeding after PFC intravenous infusion is low in this non-trauma sheep model.

Reduction of ARHGAP21 Alters Platelet Biogenesis in Vitro and Accelerates Hemostatic Response In Vivo

Megakaryocyte differentiation and platelet biogenesis require profound cytoskeleton rearrangement regulated by the Rho family of GTPases. ARHGAP21 is a RhoGAP protein that has been shown to negatively regulate the activity of RhoA, RhoC and Cdc42. We have previously demonstrated that ARHGAP21 knockdown in human common myeloid progenitors and in bipotent megakaryocyte-erythrocyte progenitors may bias the fate decision toward the megakaryocyte lineage. In addition, a mouse model with reduction of Arhgap21 expression (Arhgap21+/-) present a slight reduction in platelet number and increased platelet volume. However, the participation of ARHGAP21 in platelet biogenesis and hemostatic response has never been investigated. We studied the role of ARHGAP21 on cytoskeletal changes during megakaryocyte differentiation in HEL cell line. Differentiation was induced with 20ηM phorbol-13 myristate-12 acetate (PMA) for four days. ARHGAP21 protein expression was increased during the differentiation and was mostly detected in the protein cell fraction containing polymerized tubulin, in comparison with cell extracts containing soluble tubulin. ARHGAP21 co-localized (R ≥ 0.86 in all days of differentiation) and interacted with α-tubulin on day 2 of megakaryocyte differentiation, when ARHGAP21 expression was the highest. Silencing of ARHGAP21 with siRNA decreased the expression of Glu-tubulin and enhanced CDC42 activity on days 2 and 3 of differentiation. Increased cell size and spreading and alteration of the adhesion proteins p-p130Cas, vinculin, p-zyxin and p-FAK925 were also observed upon ARHGAP21 inhibition. There was no change in the acquisition of CD61, CD41 and CD42 megakaryocytic markers, neither in the polyploidy of HEL cells during differentiation. We further investigated the effects of ARHGAP21 inhibition on platelet morphology and on the hemostatic response in vivo, using the C57BL/6 Arhgap21 heterozygous mouse model (Arhgap21+/-). The study was approved by the Ethical Committee of our Institution. No differences were observed in CD61+CD41+ nor in CD41+CD42+ bone marrow cells from Arhgap21+/- and wild type (WT) mice. However, transmission electron microscopy revealed that Arhgap21+/- platelets presented increased alpha-granule size when compared to wild-type (WT). Tail bleeding time of Arhgap21+/- mice was decreased compared to WT (P= 0.0008). Intravital microscopy of carotid artery injured by FeCl3 showed increased adhesion of platelet and white blood cells on the vessel wall of Arhgap21+/-, which reflected in accelerated occlusion time (twice as fast) compared to WT (P= 0.0150). In conclusion, ARHGAP21 silencing may alter cell morphology and lead to increased microtubule dynamic instability during megakaryocyte differentiation in vitro, without compromising the acquisition of differentiation markers. In vivo, deficiency of Arhgap21 increases platelet granule size and accelerates hemostatic response. Together, these results indicate that ARHGAP21 may be a critical protein in the regulation of platelet production and function through the control of cytoskeletal rearrangement. This study was supported by São Paulo Research Foundation (FAPESP) National Council for Scientific and Technological Development (CNPq) and Coordination for the Improvement of Higher Education Personnel (CAPES). Disclosures No relevant conflicts of interest to declare.

Platelet Dysfunction and Persistent Fibrinolysis in Pediatric Scoliosis Surgery Patients Receiving Tranexamic Acid

High blood loss requiring transfusion is a common complication in pediatric scoliosis surgery. Bleeding in patients undergoing spinal surgery is exacerbated by the development of coagulopathy in response to extensive activation of the acute phase response (APR). Coagulopathy is characterized by consumption of coagulation factors, inflammation, and hyperfibrinolysis. Although the administration of antifibrinolytics (e.g. tranexamic acid, TXA) has significantly reduced transfusions, patients still experience high rates of blood loss, suggesting either that TXA dosing needs optimization or that plasmin-independent aspects of coagulopathy could be driving bleeding. Major orthopedic surgeries are characterized by a reduction in platelet counts indicating the coagulopathic conditions of surgery exert a tropism on the circulating platelet population. Indeed, platelet dysfunction concomitant with trauma-induced coagulopathy has been extensively described in literature. Platelet dysfunction during adult orthopedic surgery has been observed but has not been correlated with bleeding, and platelet function in pediatric spine surgery has yet to be assessed. To determine if pediatric patients also experience platelet dysfunction, we measured platelet activation by whole blood flow cytometry during the course of scoliosis surgery in 8 pediatric patients. In parallel, circulating markers of the APR (i.e. IL-6, IL-10, IL-8), products of fibrinolysis (D-dimer), and indicators of consumptive coagulopathy (Protein C) were measured by multiplex ELISA. Samples were collected preoperatively (Preop), intraoperatively (every 2 hours, Intraop 1 and Intraop 2), immediately after closure (Postop) and the following morning (POD1). Samples were stimulated with submaximal concentrations of thrombin, convulxin (GPVI agonist, collagen receptor), and ADP. Platelet integrin activation was measured by PAC1 binding (GPIIbIIIa activation) and platelet secretion was measured by CD62p binding (P-selectin surface expression). We observed a progressive and statistically significant reduction in the platelet response (GPIIbIIIa activation and P-selectin expression) to both thrombin and convulxin stimulation during the procedure but with distinct temporal patterns. The reduction in response to thrombin became significant at Postop, remaining down even at POD1 (PAC1 max reduction, Postop 1: 32.5%, p=0.0403; P-selectin max reduction, POD1: 33.9%, p=0.016). The reduction in response to convulxin, on the other hand developed almost immediately after the initiation of surgery, starting with Intraop1 and resolving by Postop (PAC1 max reduction, Intraop 1: 25.1%, p=0.0087; P-selectin max reduction, Intraop 1: 18.3%, p=0.0369). Intra-operative responses to thrombin (both PAC1 and CD62p binding) significantly correlated with estimates of blood loss based on red blood cell mass (Estimated Red Blood Cell Mass, ERCM def) and hemoglobin mass (Hg mass loss) (see Table I below). Although we observed a consistent and statistically significant reduction in platelet number it correlated with blood loss at POD1 only, suggesting platelet function is a better predictor of blood loss than platelet count. Despite all patients receiving a bolus of TXA with continuous low dose infusion, multiplex results indicated a progressive increase in circulating D-dimer, becoming statistically significant at Postop and persisting through POD 1. Elevations in D-dimer, the byproduct of plasmin degradation of fibrin, suggest elusive fibrinolysis despite administration of TXA. Significant intraoperative elevations in IL-6, IL-10, and IL-8 and consumption of Protein C were also noted that persisted through POD1, indicating the development of coagulopathy. In conclusion, pediatric scoliosis surgery is characterized by persistent fibrinolysis platelet dysfunction, activation of the APR, and coagulopathy despite administration of the anti-fibrinolytic TXA. Future studies will focus on altering the dose of TXA to determine if an optimal dosing can be achieved that effectively inhibits fibrinolysis and answer important questions about the dependence of the APR and platelet hypofunction on the persistent fibrinolysis seen in these patients. Figure Disclosures No relevant conflicts of interest to declare.

Oral Mucositis in Pediatric Oncology Patients: A Nested Case-Control to a Prospective Cohort.

This study aimed to evaluate the factors associated with the occurrence of severe oral mucositis (SOM) in pediatric oncology patients during the chemotherapeutic treatment. This is a nested case-control to a prospective cohort that monitored 105 patients for 10 consecutive weeks after the beginning of the chemotherapy treatment. Logistic regression was used to identify the factors associated with SOM, by group of malignancy (hematologic or solid tumors) (Sig.=5%). To patients with hematologic tumors were found factors associated with SOM in two weeks of treatment: in the 6th week (increase in frequency of chemotherapy doses (OR=3.02)) and in the 7th week (female sex (OR=21.28); and increase in frequency of chemotherapy doses (OR=2.51)); and to patients with solid tumors were found factors associated with SOM in five weeks of treatment: in the 1st week (female sex (OR=14.43); age increase (OR=1.24)); in the 2nd week (Miscellany (OR=6.39)); in the 5th week (Antimetabolites (OR=17.44); Miscellany (OR=45.42); and platelets reduction (OR=1.12)); in the 6th week (creatinine increase (OR=1.63)); and in the 7th week (creatinine increase (OR=2.39)). For patients with hematologic tumors, to be female, and the increase in the frequency of chemotherapy doses increased the risk for SOM and for patients with solid tumors, to be female, the increase in age and in level blood concentration of creatinine, the reduction in number of platelets and the use of chemotherapy with miscellany and antimetabolites agents were associated with an increase in risk for occurrence of SOM.

Platelet Transfusion in Perioperative Medicine.

Platelet transfusions aim to improve primary hemostasis and to prevent or treat bleeding in patients with reduced platelet numbers and/or platelet function. In this review, the authors address the role of platelet transfusions with a focus on perioperative medicine. They summarize different causes of thrombocytopenia in perioperative patients, describe general characteristics and potential adverse effects of different platelet concentrates, describe principles of perioperative platelet transfusion strategies, and highlight specific perioperative scenarios, for example, in patients undergoing antiplatelet therapy. The evidence for any transfusion threshold in perioperative patients based on platelet numbers is low. The evidence supporting prophylactic platelet transfusions in the perioperative setting is very low, and all recommended thresholds for preintervention platelet transfusions are based on weak evidence or expert opinion. Besides the platelet count, platelet function, additional risk factors for bleeding, and the pharmacokinetic properties of concomitant antiplatelet drugs are important criteria for the decision to transfuse or not to transfuse platelets. The few available prospective trials give at least a signal that a liberal platelet transfusion strategy might be associated with poorer outcomes compared with a restrictive platelet transfusion strategy in critically ill patients. Given the unknown risks for adverse outcomes, a therapeutic transfusion strategy during surgery (eventually guided by point of care testing in cardiac surgery, major liver surgery, and major trauma) may be most appropriate for interventions, in which intraoperative bleeding can be controlled until platelets are available, and during the postsurgery period.

The GABA receptor GABRR1 is expressed on and functional in hematopoietic stem cells and megakaryocyte progenitors

GABRR1 is a rho subunit receptor of GABA, the major inhibitory neurotransmitter in the mammalian brain. While most investigations of its function focused on the nervous system, its regulatory role in hematopoiesis has not been reported. In this study, we found GABRR1 is mainly expressed on subsets of human and mouse hematopoietic stem cells (HSCs) and megakaryocyte progenitors (MkPs). GABRR1-negative (GR-) HSCs led to higher donor-derived hematopoietic chimerism than GABRR1-positive (GR+) HSCs. GR+ but not GR- HSCs and MkPs respond to GABA in patch clamp studies. Inhibition of GABRR1 via genetic knockout or antagonists inhibited MkP differentiation and reduced platelet numbers in blood. Overexpression of GABRR1 or treatment with agonists significantly promoted MkP generation and megakaryocyte colonies. Thus, this study identifies a link between the neural and hematopoietic systems and opens up the possibility of manipulating GABA signaling for platelet-required clinical applications.

A novel therapy, using Ghrelin with pegylated G-CSF, inhibits brain hemorrhage from ionizing radiation or combined radiation injury.

Medical treatment becomes challenging when complicated injuries arise from secondary reactive metabolic and inflammatory products induced by initial acute ionizing radiation injury (RI) or when combined with subsequent trauma insult(s) (CI). With such detrimental effects on many organs, CI exacerbates the severity of primary injuries and decreases survival. Previously, in a novel study, we reported that ghrelin therapy significantly improved survival after CI. This study aimed to investigate whether brain hemorrhage induced by RI and CI could be inhibited by ghrelin therapy with pegylated G-CSF (i.e., Neulasta®, an FDA-approved drug). B6D2F1 female mice were exposed to 9.5 Gy 60Co-γ-radiation followed by 15% total-skin surface wound. Several endpoints were measured at several days. Brain hemorrhage and platelet depletion were observed in RI and CI mice. Brain hemorrhage severity was significantly higher in CI mice than in RI mice. Ghrelin therapy with pegylated G-CSF reduced the severity in brains of both RI and CI mice. RI and CI did not alter PARP and NF-κB but did significantly reduce PGC-1α and ghrelin receptors; the therapy, however, was able to partially recover ghrelin receptors. RI and CI significantly increased IL-6, KC, Eotaxin, G-CSF, MIP-2, MCP-1, MIP-1α, but significantly decreased IL-2, IL-9, IL-10, MIG, IFN-γ, and PDGF-bb; the therapy inhibited these changes. RI and CI significantly reduced platelet numbers, cellular ATP levels, NRF1/2, and AKT phosphorylation. The therapy significantly mitigated these CI-induced changes and reduced p53-mdm2 mediated caspase-3 activation. Our data are the first to support the view that Ghrelin therapy with pegylated G-CSF is potentially a novel therapy for treating brain hemorrhage after RI and CI.

Generation of two high affinity anti-mouse FcRn antibodies: Inhibition of IgG recycling in wild type mice and effect in a mouse model of immune thrombocytopenia

Primary immune thrombocytopenia (ITP) is an autoimmune disease characterized by pathogenic immunoglobulin G (IgG) autoantibodies that bind to platelets, causing their phagocytic removal and leading to reductions in platelet number. The neonatal Fc receptor (FcRn) selectively salvages and recycles IgG, including pathogenic IgG, thereby extending the half-life of IgG in plasma. Two anti-mouse FcRn monoclonal antibodies (mAb) (4470 and 4464) were generated to evaluate the effect of inhibiting IgG recycling. Statistically significant reductions in plasma IgG concentration were observed upon administration of 4470 (10, 30 and 100 mg/kg) in wild-type mice. In a passive mouse model of ITP, 4464 alleviated the reduction in platelet number and/or preserved newly produced platelets when dosed prophylactically as well as in a therapeutic dosing regimen once platelet numbers had already been reduced. These results support the investigation of anti-FcRn therapy as a potential treatment for ITP.

Bone marrow-derived mononuclear cell seeded bioresorbable vascular graft improves acute graft patency by inhibiting thrombus formation via platelet adhesion

BackgroundAcute thrombosis is a crucial cause of bioresorbable vascular graft (BVG) failure. Bone marrow-derived mononuclear cell (BM-MNC)-seeded BVGs demonstrated high graft patency, however, the effect of seeded BM-MNCs against thrombosis remains to be elucidated. Thus, we evaluated an antithrombotic effect of BM-MNC-seeding and utilized platelet-depletion mouse models to evaluate the contribution of platelets to acute thrombosis of BVGs. Methods and resultsBVGs were composed of poly(glycolic acid) mesh sealed with poly(l-lactideco-ε-caprolactone). BM-MNC-seeded BVGs and unseeded BVGs were implanted to wild type C57BL/6 mice (n = 10/group) as inferior vena cava interposition conduits. To evaluate platelet effect on acute thrombosis, c-Mpl−/− mice and Pf4-Cre+; iDTR mice with decreased platelet number were also implanted with unseeded BVGs (n = 10/group). BVG patency was evaluated at 2, 4, and 8 weeks by ultrasound. BM-MNC-seeded BVGs demonstrated a significantly higher patency rate than unseeded BVGs during the acute phase (2-week, 90% vs 30%, p = .020), and patency rates of these grafts were sustained until week 8. Similar to BM-MNC-seeded BVGs, C-Mpl−/− and Pf4-Cre+; iDTR mice also showed favorable graft patency (2-week, 90% and 80%, respectively) during the acute phase. However, the patency rate of Pf4-Cre+; iDTR mice decreased gradually after DTR treatment as platelet number recovered to baseline. An in vitro study revealed BM-MNC-seeding significantly inhibited platelet adhesion to BVGs compared to unseeded BVGs, (1.75 ± 0.45 vs 8.69 ± 0.68 × 103 platelets/mm2, p < .001). ConclusionsBM-MNC-seeding and the reduction in platelet number prevented BVG thrombosis and improved BVG patency, and those results might be caused by inhibiting platelet adhesion to the BVG.

Platelet glycoprotein VI aids in local immunity during pneumonia-derived sepsis caused by gram-negative bacteria

AbstractPlatelet collagen receptor glycoprotein VI (GPVI) and podoplanin receptor C-type lectin-like receptor 2 (CLEC2) are receptors implicated in platelet activation that both signal via an immunoreceptor tyrosine–based activation motif. Platelets are necessary for host defense and prevention of hemorrhage during sepsis, but the role of platelet GPVI and CLEC2 herein is unknown. To investigate this, we infected mice depleted of platelet GPVI or CLEC2 by antibody treatment or GPVI−/− mice with the common human sepsis pathogen Klebsiella pneumoniae via the airways to induce pneumonia-derived sepsis. The GPVI ligand collagen and the CLEC2 ligand podoplanin were constitutively present in the lung, whereas the GPVI ligands fibrin and histone were induced during pneumonia. During late-stage infection, both mice depleted of GPVI and GPVI−/− mice showed increased bacterial growth in lungs, and GPVI−/− mice also showed increased bacterial growth in distant body sites. Despite higher bacterial loads, GPVI-depleted mice showed reduced platelet numbers, platelet activation, and platelet–leukocyte complex formation in the bronchoalveolar space. Consistently, in human whole blood, GPVI stimulation of platelets increased platelet–leukocyte complex formation and leukocyte activation, which was accompanied by enhanced phagocytosis of Klebsiella. GPVI-depleted mice showed increased lung hemorrhage during infection, but not to the extent observed in platelet-depleted mice, and lung bleeding was not significantly different between GPVI−/− and wild-type mice. CLEC2 depletion did not affect any of the responses during pneumonia. These results suggest that platelet GPVI, but not CLEC2, contributes to local host defense during pneumonia-derived sepsis by enhancing leukocyte function.

Methodology for exposing avian embryos to quantified levels of airborne aromatic compounds associated with crude oil spills

Oil spills on birds and other organisms have focused primarily on direct effects of oil exposure through ingestion or direct body fouling. Little is known of indirect effects of airborne volatiles from spilled oil, especially on vulnerable developing embryos within the bird egg. Here a technique is described for exposing bird embryos in the egg to quantifiable amounts of airborne volatile toxicants from Deepwater Horizon crude oil. A novel membrane inlet mass spectrometry system was used to measure major classes of airborne oil-derived toxicants and correlate these exposures with biological endpoints. Exposure induced a reduction in platelet number and increase in osmolality of the blood of embryos of the chicken (Gallus gallus). Additionally, expression of cytochrome P4501A, a protein biomarker of oil exposure, occurred in renal, pulmonary, hepatic and vascular tissues. These data confirm that this system for generating and measuring airborne volatiles can be used for future in-depth analysis of the toxicity of volatile organic compounds in birds and potentially other terrestrial organisms.

Can Mean Platelet Volume Be Used as a Predictor of Vascular Disease? Problems and Pitfalls.

AbstractPlatelets play a pivotal role in both hemostasis and thrombosis. Increased platelet volume has several etiologies and may be secondary to a genetic variant, platelet activation, increased platelet turnover, or in response to inflammatory stimuli. There are several hereditary syndromes in which an increased mean platelet volume (MPV) occurs in association with a reduction in platelet number and function. An acquired increase in platelet size is often associated with increased platelet reactivity, shortened bleeding time, and increased platelet aggregation. Many studies have shown that high MPV is associated with increased risk of venous and arterial thrombosis, but genome-wide association analyses found a weak inverse association between the risk of coronary heart disease and MPV. The disease associations of MPV suggest that it could provide a useful contribution to risk assessment algorithms for both venous thromboembolism and arterial thrombosis. However, owing to the problems associated with reliably assessing MPV, it does not yet constitute a practically useful biomarker for this purpose. Before MPV can be adopted for wider clinical use, the methodological problems involved in obtaining accurate and interlaboratory comparable results must be resolved. In particular, a major degree of standardization is required, as are local reference ranges calculated with respect to specified time intervals from venipuncture to laboratory analysis. We discuss here the potential role of MPV in predicting the risk of acquired thrombotic disorders in addition to roles in congenital thrombocytopenic disorders, as well as the problems and pitfalls associated with use of MPV in these settings.

Tranexamic acid leads to paradoxical coagulation changes during cardiac surgery: a pilot rotational thromboelastometry study

BackgroundTranexamic acid (TXA) is increasingly used during major surgery with the goal to reduce excessive bleeding, transfusion requirements, and reexploration. Our aim was to examine the effect of TXA on coagulation at different times during cardiac surgery using rotational thromboelastometry. Materials and methodsNineteen adult males (EuroSCORE 4-5) were recruited consecutively for first-time cardiopulmonary bypass (CPB) surgery. Ten patients received TXA at anesthesia and nine received no TXA. Rotational thromboelastometry analysis occurred before anesthesia (baseline), after sternotomy, after CPB–heparinization and surgery, and after protamine administration–sternal closure. ResultsA median sternotomy had no effect on clot time (CT), formation, amplitude, or lysis in non-TXA patients. In contrast, TXA patients had twofold prolonged clotting time (all-tests) and ∼30% reduced FIBTEM (A5-30) and maximum clot firmness, indicating reduced thrombin generation and lower clot fibrinogen. After CPB, CTs in both groups were prolonged, possibly linked to overheparinization. In addition, TXA patients had significantly decreased EXTEM (A5-30), suggesting lower clot strength. After protamine–sternal closure, clotting time remained prolonged in both groups, and TXA patients had a persistently 25%-33% lower FIBTEM (A5-30) and maximum clot firmness. TXA patients also had significantly reduced platelet numbers (37% from baseline), which continued Days 1 and 2. Maximum clot lysis was <10% indicating little or no hyperfibrinolysis during cardiac surgery. ConclusionsIn this nonrandomized, nonblinded, observational trial, patients in the TXA group displayed prolonged CTs and clot fibrinogen (FIBTEM A5-30) after sternotomy, decreased clot strength (EXTEM) after CPB/surgery, and acute thrombocytopenia after protamine–sternal closure. There was no significant decrease in clot lysis, questioning the need for TXA in this medium-risk group.

Imetelstat, a telomerase inhibitor, differentially affects normal and malignant megakaryopoiesis.

Imetelstat (GRN163L) is a specific telomerase inhibitor that has demonstrated clinical activity in patients with myeloproliferative neoplasms (MPN) and in patients with solid tumors. The antitumor effects were associated with the development of thrombocytopenia, one of the common side effects observed in patients treated with imetelstat. The events underlying these adverse effects are not apparent. In this report, we investigated the potential mechanisms that account for imetelstat's beneficial effects in MPN patients and the manner by which imetelstat treatment leads to a reduction in platelet numbers. Using a well-established system of ex vivo megakaryopoiesis, we demonstrated that imetelestat treatment affects normal megakaryocyte (MK) development by exclusively delaying maturation of MK precursor cells. By contrast, additional stages along MPN MK development were affected by imetelstat resulting in reduced numbers of assayable colony-forming unit MK and impaired MK maturation. In addition, treatment with imetelstat inhibited the secretion of fibrogenic growth factors by malignant but not by normal MK. Our results indicate that the delay observed in normal MK maturation may account for imetelstat-induced thrombocytopenia, while the more global effects of imetelstat on several stages along the hierarchy of MPN megakaryopoiesis may be responsible for the favorable clinical outcomes reported in MPN patients.

Primary melanoma tumor inhibits metastasis through alterations in systemic hemostasis

Progression from a primary tumor to distant metastases requires extensive interactions between tumor cells and their microenvironment. The primary tumor is not only the source of metastatic cells but also can also modulate host responses to these cells, leading to an enhancement or inhibition of metastasis. Tumor-mediated stimulation of bone marrow can result in pre-metastatic niche formation and increased metastasis. However, a primary tumor can also inhibit metastasis through concomitant tumor resistance-inhibition of metastatic growth by existing tumor mass. Here, we report that the presence of a B16F10 primary tumor significantly restricted numbers and sizes of experimental lung metastases through reduction of circulating platelets and reduced formation of metastatic tumor cell-associated thrombi. Tumor-bearing mice displayed splenomegaly, correlated with primary tumor size and platelet count. Reduction in platelet numbers in tumor-bearing animals was responsible for metastatic inhibition, as restoration of platelet numbers using isolated platelets re-established both tumor cell-associated thrombus formation and experimental metastasis. Consumption of platelets due to a B16F10 primary tumor is a form of concomitant tumor resistance and demonstrates the systemic impact of a growing tumor. Understanding the interplay between primary tumors and metastases is essential, as clarification of concomitant tumor resistance mechanisms may allow inhibition of metastatic growth following tumor resection. Key messages Mice with a primary B16F10 tumor had reduced metastasis vs. mice without a primary tumor. Tumor-bearing mice had splenomegaly and fewer platelets and tumor-associated thrombi. Restoring platelets restored tumor-associated thrombi and increased metastasis. This work shows the impact that a primary tumor can have on systemic metastasis. Understanding these interactions may lead to improved ways to inhibit metastasis.

ARHGAP21 Is Upregulated and Triggers the Modulation of Rho Gtpase Signaling Pathways during Megakaryocytic Differentiation

Introduction: During differentiation, the megakaryocyte goes through profound changes in the cytoskeleton of actin and tubulin through Rho GTPase activity. Microtubules provide the elongation of proplatelets, whereas actin microfilaments mediate force to increase branching and release of platelets. ARHGAP21 is a RhoGAP for RhoA, RhoC and Cdc42, which has been shown to interact with α-tubulin in cancer cells. Moreover, arhgap21+/- mice exhibit significant reduction in platelet number and increased platelet volume.Aim: To evaluate ARHGAP21 function in the activity of Rho GTPase and their effectors during megakaryocytic differentiation.Materials and Methods: Megakaryocyte differentiation was stimulated in HEL cells through treatment with 20 nM of phorbol myristate acetate -13 -12 (PMA) for 4 days and was confirmed by the expression of CD41a, CD42b and CD61 and polyploidy using flow cytometry. Morphological changes were observed by optical microscopy. The localization of ARHGAP21, F-Actin and α-Tubulin cytoskeletal proteins was assessed by confocal microscopy. The expression of ARHGAP21, and the Rho GTPases RhoA, RhoC, Cdc42 and downstream proteins Rock1 e2, phospho-MLC2, MLC2, phospho-cofilin, cofilin and mDia1 were analyzed by western blotting. Rho GTPases activity was determined through pull down assays using Rhotekin-GST (RhoA, RhoB and RhoC) and WASP-GST (Cdc42) constructions. Tubulin polymerization was evaluated by soluble and insoluble tubulin precipitation assay. ARHGAP21 silencing was performed by siRNA, after PMA treatment for 2 and 3 days and was followed by the analysis of the expression and activity of Rho GTPases and their effectors, ploidy and differentiation markers.Results: Megakaryocytic differentiation of HEL cells was accompanied by intense rearrangement of the cytoskeleton, increased cell size, polyploidy and increased expression of the membrane receptors CD61, CD41a and CD42b. Interestingly, a gradual upregulation of ARHGAP21 was observed during differentiation, especially on days 2 and 3 of treatment (both 9.33-fold increase) and mainly in extracts containing polymerized tubulin. ARHGAP21 upregulation was concomitant with the reduction of RhoA and Cdc42 activities (92% decreased and 52% decreased, respectively), but not in RhoC. Silencing of ARHGAP21 by siRNA was confirmed by western blot. Downregulation of ARHGAP21 in HEL cells trigged increased phosphorylation on serine 19 of myosin light chain2 (MLC2) on the day 2. Moreover, mDia1, a common effector of RhoA and Cdc42, was also increased at the same point. ARHGAP21 silencing induced an increase in CD42b on day 3 (5% increased, P<0.015). No difference was observed in the expression of CD61 and CD41a and in the ploidy of ARHGAP21 silenced cells compared to control.Conclusion: Our results suggest that the upregulation of ARHGAP21 during megakaryocytic differentiation is important to control the dynamics of the cytoskeleton through the regulation of RhoA and Cdc42. Silencing of ARHGAP21 induces increased phosphorylation of MLC2 and the expression of mDia1, which may impair megakaryocytic differentiation. Furthermore, ARHGAP21 appears to regulate the acquisition of CD42b receptor, participating in the final stages of megakaryopoiesis. DisclosuresNo relevant conflicts of interest to declare.