T-TAS® 01 as a new tool for the evaluation of hemostasis in thrombocytopenic patients after platelet transfusion.

BACKGROUND: Current laboratory tests fail to evaluate the hemostatic function of platelets in patients with thrombocytopenia. We investigated the use of the Total Thrombus-Formation Analysis System (T-TAS® 01 [Fujimori Kogyo Co, Tokyo, Japan]) to evaluate hemostasis under conditions of experimental thrombocytopenia, and in patients before and after platelet transfusion. MATERIALS AND METHODS: Specific T-TAS 01 chips, for thrombocytopenic conditions, were used. The area under the curve (AUC) and occlusion time (OT, min) were measured in: (i) experimentally induced thrombocytopenia (183±15 to 6.3±1.2×103 platelets/µL) in blood samples from healthy donors (No.=13), and (ii) blood from oncohematological thrombocytopenic patients (No.=48), before and after platelet transfusion. The influences of hematocrit and number of transfusions were analyzed in these patients. RESULTS: Progressive reductions of AUC and prolongations of OT related significantly to decreasing platelet counts (p<0.05 for all) in experimental thrombocytopenia. In samples from thrombocytopenic patients, platelet counts, AUC and OT were, respectively, 10.8±0.6×103/µL, 175.2±59, and 27.2±1 min before transfusion; and 22±1.5×103/µL, 400.8±83 and 22.9±1.5 min after platelet transfusion (p<0.01 for all). A hematocrit below 25% or exposure to ten or more previous platelet transfusions had a negative impact on the T-TAS 01 performance in patients. In vitro correction of the hematocrit improved the hemostatic response in thrombocytopenic patients. DISCUSSION: T-TAS 01 measurements were sensitive to low platelet counts in the experimental setting. The technology was sensitive to evaluate the hemostatic capacity of platelet transfusions. Exposure to multiple medications, repeated platelet transfusions and lower hematocrits may interfere with the hemostatic performance in oncohematological patients with thrombocytopenia.

Easix Score Correlates With Endothelial Dysfunction Biomarkers and Predicts Risk of Acute Graft-Versus-Host Disease After Allogeneic Transplantation.

Plasma biomarkers of endothelial dysfunction have been postulated for the diagnosis and prognosis of acute graft-versus-host disease (aGVHD). However, their use is not validated in clinical practice yet. The endothelial activation and stress index (EASIX), a simple score based on routine laboratory parameters, is considered to be an indirect marker of endothelial damage. High value of EASIX was correlated with worse non-relapse mortality (NRM) and overall survival (OS) and a high risk of sinusoidal obstructive syndrome and transplant-associated thrombotic microangiopathy (TA-TMA). This study investigates the predictive value of plasma biomarkers and the EASIX score for the prediction of aGVHD. We assessed vascular cell adhesion molecule-1 (VCAM-1), tumor necrosis factor receptor 1 (TNFR1), and VWF:Ag plasma levels and the EASIX score before allogeneic hematopoietic stem cell transplantation (allo-HSCT) and on days 0, 3, 7, 14, and 21 in an experimental cohort (n = 33). EASIX was transformed to a base-2 logarithm to perform the analysis. For the most relevant biomarkers, we estimate the optimal cutoff values and the discriminatory ability to differentiate patients with high-risk of aGVHD. The conclusions obtained in the experimental cohort were validated in a large cohort of 321 patients at the same institution. Plasma biomarkers and EASIX showed similar post-transplantation dynamics consisting of a progressive increase. Multivariate analysis showed an association between high TNFR1 levels and Log-2 EASIX score on day 7 after transplantation with an increased likelihood of developing aGVHD (hazard ratio [HR] = 1, P = .002; HR = 2.31, P = .013, respectively). Patients with TNFR1 ≥1300 ng/mL (HR = 7.19, P = .006) and Log2-EASIX ≥3 (HR = 14.7, P <.001) at day 7 after transplantation were more likely to develop aGVHD with high predictive accuracy (C-index of 74% and 81%, respectively). In the validation cohort, patients with Log2-EASIX ≥3 on day 7 after transplantation presented a significantly higher incidence of grade II-IV aGVHD (HR = 1.94, P = .004) independent of GVHD prophylaxis (HR = 0.38, P = .004), conditioning regimen (HR = 0.59, P =.02) and type of donor (HR = 2.38, P = .014). Differential degree of endothelial damage can be measured using both EASIX score and plasma biomarkers in the early post-transplantation period. Patients at risk of developing aGVHD could be easily identified by a high EASIX score. Both indicators of endothelial activation represent a promising approach to predict aGVHD.

Functionalization of 3D printed polymeric bioresorbable stents with a dual cell-adhesive peptidic platform combining RGDS and YIGSR sequences.

Biomimetic surface modification with cell-adhesive peptides is a promising approach to improve endothelialization of current bioresorbable stents (BRS). Among them, RGDS and YIGSR sequences have been reported to mediate adhesion and migration of endothelial cells (ECs) while preventing platelet activation. This work presents the functionalization of novel 3D-printed poly-L-lactic acid (PLLA) and poly(L-lactic-co-ε-caprolactone) (PLCL) BRS with linear RGDS and YIGSR sequences, as well as a dual platform (PF) containing both motifs within a single biomolecule. Functionalized surfaces were characterized in terms of static contact angle, biomolecule distribution under confocal fluorescence microscopy and peptide quantification via detachment from the surface, showing a biomolecule density in the range of 0.5 to 3.5 nmol cm-2. Biological evaluation comprised a cell adhesion test on functionalized films with ECs and a blood perfusion assay on functionalized stents to assess ECs response and device hemocompatibility, respectively. Cell adhesion assays evidenced significantly increased cell number and spreading onto functionalized films with respect to control samples. Regarding stents' hemocompatibility, platelet adhesion onto PLCL stents was severely decreased with respect to PLLA. In addition, functionalization with RGDS, YIGSR and the PF rendered BRS stents displaying even further reduced platelet adhesion. In conclusion, the combination of intrinsically less prothrombogenic materials such as PLCL and its functionalization with EC-discriminating adhesive biomolecules paves the way for a new generation of BRS based on accelerated re-endothelialization approaches.

Differences and similarities in endothelial and angiogenic profiles of preeclampsia and COVID-19 in pregnancy.

BACKGROUND: COVID-19 presents a spectrum of signs and symptoms in pregnant women that might resemble preeclampsia. Differentiation between severe COVID-19 and preeclampsia is difficult in some cases. OBJECTIVE: To study biomarkers of endothelial damage, coagulation, innate immune response, and angiogenesis in preeclampsia and COVID-19 in pregnancy in addition to in vitro alterations in endothelial cells exposed to sera from pregnant women with preeclampsia and COVID-19. STUDY DESIGN: Plasma and sera samples were obtained from pregnant women with COVID-19 infection classified into mild (n=10) or severe (n=9) and from women with normotensive pregnancies as controls (n=10) and patients with preeclampsia (n=13). A panel of plasmatic biomarkers was assessed, including vascular cell adhesion molecule-1, soluble tumor necrosis factor-receptor I, heparan sulfate, von Willebrand factor antigen (activity and multimeric pattern), α2-antiplasmin, C5b9, neutrophil extracellular traps, placental growth factor, soluble fms-like tyrosine kinase-1, and angiopoietin 2. In addition, microvascular endothelial cells were exposed to patients' sera, and changes in the cell expression of intercellular adhesion molecule 1 on cell membranes and von Willebrand factor release to the extracellular matrix were evaluated through immunofluorescence. Changes in inflammation cell signaling pathways were also assessed by of p38 mitogen-activated protein kinase phosphorylation. Statistical analysis included univariate and multivariate methods. RESULTS: Biomarker profiles of patients with mild COVID-19 were similar to those of controls. Both preeclampsia and severe COVID-19 showed significant alterations in most circulating biomarkers with distinctive profiles. Whereas severe COVID-19 exhibited higher concentrations of vascular cell adhesion molecule-1, soluble tumor necrosis factor-α receptor I, heparan sulfate, von Willebrand factor antigen, and neutrophil extracellular traps, with a significant reduction of placental growth factor compared with controls, preeclampsia presented a marked increase in vascular cell adhesion molecule-1 and soluble tumor necrosis factor-α receptor I (significantly increased compared with controls and patients with severe COVID-19), with a striking reduction in von Willebrand factor antigen, von Willebrand factor activity, and α2-antiplasmin. As expected, reduced placental growth factor, increased soluble fms-like tyrosine kinase-1 and angiopoietin 2, and a very high soluble fms-like tyrosine kinase-1 to placental growth factor ratio were also observed in preeclampsia. In addition, a significant increase in C5b9 and neutrophil extracellular traps was also detected in preeclampsia compared with controls. Principal component analysis demonstrated a clear separation between patients with preeclampsia and the other groups (first and second components explained 42.2% and 13.5% of the variance), mainly differentiated by variables related to von Willebrand factor, soluble tumor necrosis factor-receptor I, heparan sulfate, and soluble fms-like tyrosine kinase-1. Von Willebrand factor multimeric analysis revealed the absence of von Willebrand factor high-molecular-weight multimers in preeclampsia (similar profile to von Willebrand disease type 2A), whereas in healthy pregnancies and COVID-19 patients, von Willebrand factor multimeric pattern was normal. Sera from both preeclampsia and severe COVID-19 patients induced an overexpression of intercellular adhesion molecule 1 and von Willebrand factor in endothelial cells in culture compared with controls. However, the effect of preeclampsia was less pronounced than the that of severe COVID-19. Immunoblots of lysates from endothelial cells exposed to mild and severe COVID-19 and preeclampsia sera showed an increase in p38 mitogen-activated protein kinase phosphorylation. Patients with severe COVID-19 and preeclampsia were statistically different from controls, suggesting that both severe COVID-19 and preeclampsia sera can activate inflammatory signaling pathways. CONCLUSION: Although similar in in vitro endothelial dysfunction, preeclampsia and severe COVID-19 exhibit distinctive profiles of circulating biomarkers related to endothelial damage, coagulopathy, and angiogenic imbalance that could aid in the differential diagnosis of these entities.

An endothelial proinflammatory phenotype precedes the development of the engraftment syndrome after autologous Hct.

Engraftment syndrome (ES) is a common complication after autologous hematopoietic cell transplantation (auto-HCT) whose pathophysiological substrate remains unclear. We investigated whether endothelial damage could contribute to ES. Circulating ECs-damage biomarkers were measured in plasma from patients with (ES; n = 14) or without ES (non-ES; n = 20), collected at different time points: before HCT, 5 (S5) and 10 days (S10) after HCT, and at either the ES onset (SON) or the discharge day (SDIS). Also, cultured endothelial cells (ECs) were exposed to serum samples, obtained at the same points, to evaluate changes in ECs-activation (ICAM-1, VE-Cadherin) biomarkers, the reactivity of ECs towards leukocytes, and activation of intracellular signaling proteins related to inflammation (p38MAPK) and proliferation (Erk1/2). Results showed that circulating VWF, sTNFR1 and sVCAM-1 levels were higher in ES patients at all the points assessed, especially at SON. In vitro results showed an increased ICAM-1 expression on ECs exposed to ES samples vs. non-ES samples, especially to S5, with elevated leukocyte adhesion. Also, a lower VE-Cadherin expression and an increased phosphorylation of p38MAPK and Erk1/2 proteins were observed in ECs exposed to ES vs. non-ES samples. Our results indicate that endothelial activation precedes ES development and could be one of its pathophysiological substrates.

Nucleosome Dynamics: a new tool for the dynamic analysis of nucleosome positioning.

We present Nucleosome Dynamics, a suite of programs integrated into a virtual research environment and created to define nucleosome architecture and dynamics from noisy experimental data. The package allows both the definition of nucleosome architectures and the detection of changes in nucleosomal organization due to changes in cellular conditions. Results are displayed in the context of genomic information thanks to different visualizers and browsers, allowing the user a holistic, multidimensional view of the genome/transcriptome. The package shows good performance for both locating equilibrium nucleosome architecture and nucleosome dynamics and provides abundant useful information in several test cases, where experimental data on nucleosome position (and for some cases expression level) have been collected for cells under different external conditions (cell cycle phase, yeast metabolic cycle progression, changes in nutrients or difference in MNase digestion level). Nucleosome Dynamics is a free software and is provided under several distribution models.

BioExcel Building Blocks, a software library for interoperable biomolecular simulation workflows.

In the recent years, the improvement of software and hardware performance has made biomolecular simulations a mature tool for the study of biological processes. Simulation length and the size and complexity of the analyzed systems make simulations both complementary and compatible with other bioinformatics disciplines. However, the characteristics of the software packages used for simulation have prevented the adoption of the technologies accepted in other bioinformatics fields like automated deployment systems, workflow orchestration, or the use of software containers. We present here a comprehensive exercise to bring biomolecular simulations to the "bioinformatics way of working". The exercise has led to the development of the BioExcel Building Blocks (BioBB) library. BioBB's are built as Python wrappers to provide an interoperable architecture. BioBB's have been integrated in a chain of usual software management tools to generate data ontologies, documentation, installation packages, software containers and ways of integration with workflow managers, that make them usable in most computational environments.

Role of sodium tungstate as a potential antiplatelet agent.

PURPOSE: Platelet inhibition is a key strategy in the management of atherothrombosis. However, the large variability in response to current strategies leads to the search for alternative inhibitors. The antiplatelet effect of the inorganic salt sodium tungstate (Na2O4W), a protein tyrosine phosphatase 1B (PTP1B) inhibitor, has been investigated in this study. METHODS: Wild-type (WT) and PTP1B knockout (PTP1B(-/-)) mice were treated for 1 week with Na2O4W to study platelet function with the platelet function analyzer PFA-100, a cone-and-plate analyzer, a flat perfusion chamber, and thrombus formation in vivo. Human blood aliquots were incubated with Na2O4W for 1 hour to measure platelet function using the PFA-100 and the annular perfusion chamber. Aggregometry and thromboelastometry were also performed. RESULTS: In WT mice, Na2O4W treatment prolonged closure times in the PFA-100 and decreased the surface covered (%SC) by platelets on collagen. Thrombi formed in a thrombosis mice model were smaller in animals treated with Na2O4W (4.6±0.7 mg vs 8.9±0.7 mg; P<0.001). Results with Na2O4W were similar to those in untreated PTP1B(-)/(-) mice (5.0±0.3 mg). Treatment of the PTP1B(-)/(-) mice with Na2O4W modified only slightly this response. In human blood, a dose-dependent effect was observed. At 200 µM, closure times in the PFA-100 were prolonged. On denuded vessels, %SC and thrombi formation (%T) decreased with Na2O4W. Neither the aggregating response nor the viscoelastic clot properties were affected. CONCLUSION: Na2O4W decreases consistently the hemostatic capacity of platelets, inhibiting their adhesive and cohesive properties under flow conditions in mice and in human blood, resulting in smaller thrombi. Although Na2O4W may be acting on platelet PTP1B, other potential targets should not be disregarded.

Distinct deleterious effects of cyclosporine and tacrolimus and combined tacrolimus-sirolimus on endothelial cells: protective effect of defibrotide.

Endothelial dysfunction seems to be a key factor in the development of several complications observed early after hematopoietic stem cell transplantation (HSCT). The conditioning regimen and many other factors associated with the procedure are responsible for this endothelial damage. The effects of immunosuppressive agents on endothelial function have not been explored in detail. We evaluated the effects of 3 drugs commonly used in HSCT: 2 calcineurin inhibitors, cyclosporine A (CSA) and tacrolimus (TAC), and an inhibitor of mTOR, sirolimus (SIR). We also evaluated the effect of the combination of TAC and SIR (TAC+SIR), which is used increasingly in clinical practice. Microvascular endothelial cells (HMEC-1) were exposed to these drugs to evaluate changes in (1) intercellular adhesion molecule (ICAM)-1 expression on the cell surface, assessed by immunofluorescence labeling and expressed as the mean gray value (MGV); (2) reactivity of the extracellular matrix (ECM) toward platelets, upon exposure of the ECM to circulating blood; and (3) whole-blood clot formation, assessed by thromboelastometry. Studies were conducted in the absence and presence of defibrotide (DF) to assess its possible protective effect. The exposure of HMEC-1 to CSA and TAC+SIR significantly increased the expression of ICAM-1 (157.5 ± 11.6 and 153.4 ± 9.5 MGV, respectively, versus 105.7 ± 6.5 MGV in controls [both P < .05]). TAC applied alone increased ICAM-1 slightly (120.3 ± 8.2 MGV), and SIR had no effect (108.9 ± 7.4 MGV). ECM reactivity increased significantly only in response to CSA (surface covered by platelets of 41.2% ± 5.4% versus 30.1% ± 2.0%, P < .05). DF attenuated all these changes. No significant changes in the viscoelastic properties of clot formation were observed in any condition with blood samples incubated in vitro. In conclusion, CSA and TAC+SIR had a proinflammatory effect, but only CSA exhibited an additional prothrombotic effect. Interestingly, DF exerted clear protective anti-inflammatory and antithrombotic effects on the endothelium.

Quantitative and qualitative analysis of coagulation factors in cryoprecipitate prepared from fresh-frozen plasma inactivated with amotosalen and ultraviolet A light.

BACKGROUND: There were no previous studies about the quality of cryoprecipitate prepared from fresh-frozen plasma (FFP) inactivated with amotosalen and ultraviolet A (UVA) light. The aim of this study was to analyze the quantity and quality of coagulation factors in cryoprecipitate prepared from FFP treated with amotosalen and UVA light. STUDY DESIGN AND METHODS: FFP was obtained from whole blood donations and inactivated with amotosalen and UVA light according to the manufacturer's instructions. Fibrinogen, factor VIII (FVIII), von Willebrand factor antigen (VWF:Ag) and activity (VWF:RCo), the von Willebrand factor cleavage protease activity (ADAMTS-13), and the multimeric structure of VWF were analyzed. RESULTS: The content of fibrinogen, FVIII, and ADAMTS-13 was lower in cryoprecipitates prepared from amotosalen-treated plasma when compared with cryoprecipitates prepared from nontreated plasma (35, 40, and 18% loss, respectively). The quantity and quality of VWF as well as VWF multimer patterns were not affected by the inactivation method. CONCLUSION: Cryoprecipitates prepared from amotosalen-treated FFP contained significantly reduced levels of fibrinogen, FVIII, and ADAMTS-13. However, the VWF quantity and quality was well preserved.

Preanalytical treatment of EDTA-anticoagulated blood to ensure stabilization of the mean platelet volume and component measured with the ADVIA counters.

The mean platelet component (MPC) parameter calculated by the ADVIA blood cell analyzers provides direct information on density, or granularity, of platelets and could become a useful biomarker to detect in vivo platelet activation. Unfortunately, it is largely affected by time and storage conditions in standard anticoagulants based on EDTA. The present study was designed to improve the stability of the MPC in blood specimens to facilitate a more standardized use in different laboratories. Blood from healthy controls was collected into EDTA plus additives, and stored at different conditions. MPC and the mean platelet volume (MPV) were assessed at 30 min and at 1, 3, 6 and 24hours after blood drawing on the ADVIA 2120 system. Flow cytometry was used to evaluate platelet-activation proteins. Ultrastructural morphology of platelets was assessed using electron microscopy. Storage in EDTA increased MPV, decreased MPC, reduced the number of alpha-granules, and induced changes in the phosphorylation patterns of platelet proteins. A solution based on EDTA containing wortmanin and tyrphostin (ED-WORTY), both inhibitors of signaling pathways, provided good stability for most of the parameters tested up to 6 hours at room temperature. Storage at lower temperatures produced more favorable results. ED-WORTY solutions preserved adequate morphology and had minimal influence on other parameters provided by the ADVIA 2120 system. Thus, the additives included in ED-WORTY may be useful for maintaining the stability of MPC for prolonged periods and to facilitate the transport and exchange of samples among institutions and laboratories.

Effects on primary haemostasis of an anti-inflammatory agent with 5-lipoxygenase and cyclooxygenase inhibitory activity.

OBJECTIVE: Licofelone ([2,2-dimethyl-6-(4-chlorophenyl)-7-phenyl-2,3-dihydro-1H-pyrrolizine-5-yl]-acetic acid) has been demonstrated to inhibit cyclooxygenase (COX)-1, COX-2, and 5-lipoxygenase. The aim of this study was to investigate the in-vitro effects of licofelone on platelet function. Effects observed were compared with those produced by the classic COX-1 inhibitor aspirin (ASA). METHODS: Platelet aggregation was assessed by a turbidimetric method. Platelet haemostatic performance was studied with the platelet function analyser (PFA-100), using collagen epinephrine and collagen ADP cartridges. Interaction of platelets with thrombogenic surfaces was analysed by perfusion experiments performed under flow conditions using both parallel and annular chambers. RESULTS: Licofelone prolonged the lag time of platelet aggregation induced by arachidonic acid and reduced maximal platelet aggregation induced by ADP or collagen. Studies using PFA-100 demonstrated that licofelone (0.1, 1 and 10 muM) significantly prolonged closure times (P < 0.05) with both types of cartridges. In studies with the parallel chamber exposing purified collagen, both licofelone and ASA significantly reduced (P < 0.05) overall platelet interaction with the thrombogenic surface. In studies performed in annular chamber exposing a highly thrombogenic vessel surface, licofelone reduced height and area of the platelet masses deposited (7.0 +/- 0.5 mum; P < 0.005 and 80.2 +/- 17.3 mum; P < 0.05 vs. control 10.6 +/- 0.9 mum and 194.8 +/- 44.7 mum, respectively). ASA also impaired thrombus formation but differences did not reach the levels of statistical significance. CONCLUSIONS: Under our experimental in-vitro conditions, licofelone interfered with platelet function as demonstrated by a diminished platelet aggregation, being more powerful than ASA and reducing the interaction of platelets with thrombogenic surfaces.

Effects of beta2-glycoprotein I and monoclonal anticardiolipin antibodies in platelet interaction with subendothelium under flow conditions.

OBJECTIVE: To evaluate whether the effect of human monoclonal anticardiolipin antibodies (aCL) on platelet interaction with the subendothelium under flow conditions is dependent on beta(2)-glycoprotein I (beta(2)GPI). METHODS: Three monoclonal IgM aCL with anti-beta(2)GPI activity (TM1B3, GR1D5, and EY2C9) obtained from patients with antiphospholipid syndrome, a monoclonal aCL with lupus anticoagulant activity but without anti-beta(2)GPI activity (FRO) obtained from a patient with a splenic lymphoma, and a control monoclonal IgM without aCL activity were used. TM1B3, GR1D5, EY2C9, FRO, and control IgM (30 microg/ml) were added to reconstituted blood containing gel-filtered platelets (200 x 10(9)/liter), factor VIII (100 units/dl), and fibrinogen (1.5 gm/liter). Samples were perfused (wall-shear rate 800 seconds(-1)), with and without the addition of purified beta(2)GPI (20 microg/ml), through annular chambers containing collagen-rich denuded vascular segments, and the percentages of surface covered by platelets and by thrombi were evaluated. RESULTS: No differences in the percentages of surface covered by platelets and by thrombi were observed among samples with TM1B3, GR1D5, EY2C9, FRO, and control IgM added when reconstituted blood samples without beta(2)GPI were used. However, a significant increase in the percentage of surface covered by platelets was observed in the presence of TM1B3, GR1D5, and EY2C9 but not in the presence of FRO when samples containing beta(2)GPI were used. Increased thrombi formation was induced by TM1B3 and GR1D5 but not by EY2C9 or FRO in samples with added beta(2)GPI. CONCLUSION: Monoclonal aCL require anti-beta(2)GPI activity to promote platelet interaction with the subendothelium under flow conditions.