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Standard platelet concentrates (PCs) stored at 22°C have a limited shelf life of 5 days. Because of the storage temperature, bacterial contamination of PCs can result in life-threatening infections in transfused patients. The potential of blood components to cause infections through contaminating pathogens or transmitting blood-borne diseases has always been a concern. The current safety practice to prevent pathogen transmission through blood transfusion starts with a stringent screening of donors and regulated testing of blood samples to ensure that known infections cannot reach transfusion products. Pathogen reduction technologies (PRTs), initially implemented to ensure the safety of plasma products, have been adapted to treat platelet products. In addition to reducing bacterial contamination, PRT applied to PCs can extend their shelf life up to 7 days, alleviating the impact of their shortage, while providing an additional safety layer against emerging blood-borne infectious diseases. While a deleterious action of PRTs in quantitative and qualitative aspects of plasma is accepted, the impact of PRTs on the quality, function, and clinical efficacy of PCs has been under constant examination. The potential of PRTs to prevent the possibility of new emerging diseases to reach cellular blood components has been considered more hypothetical than real. In 2019, a coronavirus-related disease (COVID-19) became a pandemic. This episode should help when reconsidering the possibility of future blood transmissible threats. The following text intends to evaluate the impact of different PRTs on the quality, function, and clinical effectiveness of platelets within the perspective of a developing pandemic.

BACKGROUND: Treatment with triple antithrombotic therapies increases bleeding risks. AIM: We evaluated the antithrombotic and prohemorrhagic actions of different concentrations of apixaban (APIX) in healthy donors and patients exposed to currently used antiplatelet regimens. METHODS: 25 healthy subjects and 53 patients treated with aspirin (ASA, n=21), ASA and clopidogrel (ASA+CLOPI, n=11), or ASA and ticagrelor (ASA+TICA, n=21) participated in the study. Blood samples from participants were spiked ex vivo with apixaban 0 (APIX0), 40 (APIX40), and 160 ng/mL (APIX160). We assessed the effects of APIX on 1) clot formation, by ROTEM thromboelastometry; 2) thrombin generation primed by platelets; and 3) platelet and fibrin interactions with a thrombogenic surface, in a microfluidic model with circulating blood. RESULTS: APIX caused dose-related prolongations of clotting time with minimal impact on other ROTEM parameters. Thrombin generation was significantly inhibited by APIX160 and moderately affected by the antiplatelets, with ASA+TICA actions being the strongest among them (pCONCLUSIONS: APIX potentiated the antithrombotic effects of current antiplatelet regimens. APIX at 40 ng/mL, enhanced the antithrombotic action of single or dual antiplatelet regimens but appears more conservative for hemostasis than the concentrations of 160 ng/mL that corresponds to the Cmax reached after the standard dose for thromboprophylaxis.

Despite major advancements in the development of safer and more effective anticoagulant agents, bleeding complications remain a significant concern in the treatment of thromboembolic diseases. Improvements in our understanding of the coagulation pathways highlights the notion that the contact pathway—specifically factor XI (FXI)—has a greater role in the etiopathogenesis of thrombosis than in physiological hemostasis. As a result, a number of drugs targeting FXI are currently in different stages of testing and development. This article aims to review the different strategies directed towards FXI-inhibition with a brief summation of the agents in clinical development, and to comment on the therapeutic areas that could be explored for potential indications. Therapeutics targeting FXI/FXIa inhibition have the potential to usher in a new era of anticoagulation therapy.

Post-transplantation cyclophosphamide (PTCy) has decreased GVHD incidence. Endothelial damage in allo-HCT is caused by multiple factors, including conditioning treatments and some immunosupressants, and underlies HCT-complications as GVHD. Nevertheless, the specific impact of PTCy on the endothelium remains unclear. We evaluated the effect of mafosfamide (MAF), an active Cy analog, on endothelial cells (ECs) vs. cyclosporine A (CSA), with known damaging endothelial effect. ECs were exposed to MAF and CSA to explore changes in endothelial damage markers: i) surface VCAM-1, ii) leukocyte adhesion on ECs, iii) VE-cadherin expression, iv) production of VWF, and v) activation of intracellular signaling proteins (p38MAPK, Akt). Results obtained (expressed in folds vs. controls) indicate that both compounds increased VCAM-1 expression (3.1 ± 0.3 and 2.8 ± 0.6, respectively, p

Background: Endotheliopathy is a key element in COVID-19 pathophysiology, contributing to both morbidity and mortality. Biomarkers distinguishing different COVID-19 phenotypes from sepsis syndrome remain poorly understood. Objective: To characterize circulating biomarkers of endothelial damage in different COVID-19 clinical disease stages compared with sepsis syndrome and normal volunteers. Methods: Patients with COVID-19 pneumonia (n = 49) were classified into moderate, severe, or critical (life-threatening) disease. Plasma samples were collected within 48 to 72 h of hospitalization to analyze endothelial activation markers, including soluble Vascular Cell Adhesion Molecule-1 (sVCAM-1), von Willebrand Factor (VWF), A disintegrin-like and metalloprotease with thrombospondin type 1 motif no. 13 (ADAMTS-13) activity, thrombomodulin (TM), and soluble TNF receptor I (sTNFRI); heparan sulfate (HS) for endothelial glycocalyx degradation; C5b9 deposits on endothelial cells in culture and soluble C5b9 for complement activation; circulating dsDNA for neutrophil extracellular traps (NETs) presence, and alpha 2-antiplasmin and PAI-1 as parameters of fibrinolysis. We compared the level of each biomarker in all three COVID-19 groups and healthy donors as controls (n = 45). Results in critically ill COVID-19 patients were compared with other intensive care unit (ICU) patients with septic shock (SS, n = 14), sepsis (S, n = 7), and noninfectious systemic inflammatory response syndrome (NI-SIRS, n = 7). Results: All analyzed biomarkers were increased in COVID-19 patients versus controls (P < 0.001), except for ADAMTS-13 activity that was normal in both groups. The increased expression of sVCAM-1, VWF, sTNFRI, and HS was related to COVID-19 disease severity (P < 0.05). Several differences in these parameters were found between ICU groups: SS patients showed significantly higher levels of VWF, TM, sTNFRI, and NETS compared with critical COVID-19 patients and ADAMTS-13 activity was significantly lover in SS, S, and NI-SIRS versus critical COVID-19 (P < 0.001). Furthermore, alpha 2-antiplasmin activity was higher in critical COVID-19 versus NI-SIRS (P < 0.01) and SS (P < 0.001), whereas PAI-1 levels were significantly lower in COVID-19 patients compared with NI-SIRS, S, and SS patients (P < 0.01). Conclusions: COVID-19 patients present with increased circulating endothelial stress products, complement activation, and fibrinolytic dysregulation, associated with disease severity. COVID-19 endotheliopathy differs from SS, in which endothelial damage is also a critical feature of pathobiology. These biomarkers could help to stratify the severity of COVID-19 disease and may also provide information to guide specific therapeutic strategies to mitigate endotheliopathy progression.

Patients with COVID-19 present a wide spectrum of disease severity, from asymptomatic cases in the majority to serious disease leading to critical care and even death. Clinically, four different scenarios occur within the typical disease timeline: first, an incubation and asymptomatic period; second, a stage with mild symptoms due mainly to the virus itself; third, in up to 20% of the patients, a stage with severe symptoms where a hyperinflammatory response with a cytokine storm driven by host immunity induces acute respiratory distress syndrome; and finally, a post-acute sequelae (PASC) phase, which present symptoms that can range from mild or annoying to actually quite incapacitating. Although the most common manifestation is acute respiratory failure of the lungs, other organs are also frequently involved. The clinical manifestations of the COVID-19 infection support a key role for endothelial dysfunction in the pathobiology of this condition. The virus enters into the organism via its interaction with angiotensin-converting enzyme 2-receptor that is present prominently in the alveoli, but also in endothelial cells, which can be directly infected by the virus. Cytokine release syndrome can also drive endothelial damage independently. Consequently, a distinctive feature of SARS-CoV-2 infection is vascular harm, with severe endothelial injury, widespread thrombosis, microangiopathy, and neo-angiogenesis in response to endothelial damage. Therefore, endothelial dysfunction seems to be the pathophysiological substrate for severe COVID-19 complications. Biomarkers of endothelial injury could constitute strong indicators of disease progression and severity. In addition, the endothelium could represent a very attractive target to both prevent and treat these complications. To establish an adequate therapy, the underlying pathophysiology and corresponding clinical stage should be clearly identified. In this review, the clinical features of COVID-19, the central role of the endothelium in COVID-19 and in other pathologies, and the potential of specific therapies aimed at protecting the endothelium in COVID-19 patients are addressed.

Stent thrombosis (ST) is a catastrophic event and efforts to reduce its incidence by altering blood-stent interactions are longstanding. A new electret coating technology that produces long-lasting negative charge on stent surface could make them intrinsically resistant to thrombosis. We assessed the thrombogenicity of stents using an annular perfusion model with confocal microscopy, and determined the efficacy of electret coating technology to confer thrombo-resistant properties to standard stents. Using an annular perfusion chamber, Bare Metal Stent (BMS), standard uncoated DES (DES), and Electret-coated DES (e-DES) were exposed to human blood under arterial flow conditions. Deposits of fibrinogen and platelets on the stent surface were analyzed using immunofluorescence staining and confocal microscopy. Surface coverage by fibrinogen and platelets and the deposit/aggregate size were quantified using computerized morphometric analysis. The experimental methodology produced consistent, quantifiable results. Area of stent surface covered by fibrinogen and platelets and the average size of the deposits/aggregates were lowest for e-DES and highest on BMS, with DES in the middle. The size of fibrinogen-deposits showed no differences between the stents. The testing methodology used in our study successfully demonstrated that electret coating confers significant antithrombotic property to DES stents. These findings warrant confirmation in a larger study.

BackgroundChimeric antigen receptor (CAR)-T cell-based immunotherapy constitutes a revolutionary advance for treatment of relapsed/refractory hematological malignancies. Nevertheless, cytokine release and immune effector cell-associated neurotoxicity syndromes are life-threatening toxicities in which the endothelium could be a pathophysiological substrate. Furthermore, differential diagnosis from sepsis, highly incident in these patients, is challenging. Suitable laboratory tools could be determinant for their appropriate management.MethodsSixty-two patients treated with CAR-T cell immunotherapy for hematological malignancies (n=46 with CD19-positive diseases, n=16 with multiple myeloma) were included. Plasma samples were obtained: before CAR-T cell infusion (baseline); after 24–48 hours; at suspicion of any toxicity onset and 24–48 hours after immunomodulatory treatment. Biomarkers of endothelial dysfunction (soluble vascular cell adhesion molecule 1 (sVCAM-1), soluble TNF receptor 1 (sTNFRI), thrombomodulin (TM), soluble suppression of tumorigenesis-2 factor (ST2), angiopoietin-2 (Ang-2)), innate immunity activation (neutrophil extracellular traps (NETs), soluble C5b-9 (sC5b-9)) and hemostasis/fibrinolysis (von Willebrand Factor antigen (VWF:Ag), ADAMTS-13 (A13), α2-antiplasmin (α2-AP), plasminogen activator inhibitor-1 antigen (PAI-1 Ag)) were measured and compared with those in cohorts of patients with sepsis and healthy donors.ResultsPatients who developed CAR-T cell toxicities presented increased levels of sVCAM-1, sTNFRI and ST2 at the clinical onset versus postinfusion values. Twenty-four hours after infusion, ST2 levels were good predictors of any CAR-T cell toxicity, and combination of ST2, Ang-2 and NETs differentiated patients requiring intensive care unit admission from those with milder clinical presentations. Association of Ang-2, NETs, sC5b-9, VWF:Ag and PAI-1 Ag showed excellent discrimination between severe CAR-T cell toxicities and sepsis.ConclusionsThis study provides relevant contributions to the current knowledge of the CAR-T cell toxicities pathophysiology. Markers of endotheliopathy, innate immunity activation and hemostatic imbalance appear as potential laboratory tools for their prediction, severity and differential diagnosis.

Identification of qualitative variants of von Willebrand disease (VWD) can be a diagnostic challenge because of discrepant results obtained in the multiple laboratory tests available for its appropriate classification. We report two cases of infrequent inherited variants of VWD with unclear preliminary results with the test panel available at the time of first consultation and that were finally diagnosed as a VWD type 2A/IID with a c.8318 G C, p.Cys2773Ser mutation and a VWD type 2M with c.4225 T G, p.Val1409Phe mutation, respectively. The description of these two cases highlights that despite the limited diagnostic panel for the evaluation of von Willebrand Factor (VWF) functionality, the multimeric analysis and genetic family studies were fundamental tools to achieve the final diagnosis.

Background An evolving strategy in the setting of percutaneous coronary intervention (PCI) involves withdrawal of acetylsalicylic acid (ASA), or aspirin, while maintaining P2Y12 inhibition. However, the pharmacodynamic effects of this approach on blood thrombogenicity and platelet reactivity remain unknown. Objectives This study sought to compare the antithrombotic potency of ticagrelor alone versus ticagrelor plus ASA among high-risk patients undergoing PCI with drug-eluting stents. Methods This was a mechanistic substudy within the TWILIGHT (Ticagrelor With Aspirin or Alone in High-Risk Patients After Coronary Intervention) trial, which randomized patients undergoing PCI to ticagrelor plus placebo versus ticagrelor plus ASA following 3 months of dual antiplatelet therapy. Substudy participants were enrolled after randomization, at which time ex vivo assays to quantify thrombus size under dynamic flow conditions and platelet reactivity were performed. Pharmacodynamic assessments were repeated 1 to 6 months thereafter. The primary endpoint was thrombus size at the post-randomization visit with platelet reactivity following stimuli to arachidonic acid, collagen, adenosine diphosphate, and thrombin as secondary endpoints. Results were analyzed using analysis of covariance. Results A total of 51 patients were enrolled, among whom 42 underwent perfusion assays at baseline and follow-up with a median time between studies of 1.5 months. The adjusted mean difference in post-randomization thrombus area was similar between groups: −218.2 μm2 (95% confidence interval [CI]: −575.9 to 139.9 μm2; p = 0.22). Markers sensitive to cyclo-oxygenase-1 blockade, including platelet reactivity in response to arachidonic acid (mean difference: 10.9 U; 95% CI: 1.9 to 19.9 U) and collagen (mean difference: 9.8 U; 95% CI: 0.8 to 18.8 U) stimuli were higher among patients receiving placebo, whereas levels of platelet reactivity were similar with adenosine diphosphate and thrombin. Conclusions Among high-risk patients receiving drug-eluting stents, the antithrombotic potency of ticagrelor monotherapy is similar to that of ticagrelor plus ASA with respect to ex vivo blood thrombogenicity, whereas markers sensitive to cyclo-oxygenase-1 blockade are increased in the absence of ASA. (Platelet Substudy of the TWILIGHT Trial; NCT04001374).

Circulating platelet microparticles (PMP) are the most abundant in bloodstream, are highly procoagulant and contribute to cross-talk with inflammatory cells. The aim of the present study was to investigate the interactions of PMP with platelets and explore the involvement of toll-like receptor 4 (TLR-4).PMP were separated by ultracentrifugation of expired platelet concentrates and added to: i) washed platelets, to confirm uptake, by flow cytometry and confocal and transmission electron microscopy, ii) platelet rich plasma (PRP), to assess changes in platelet function due to uptake by aggregometry in response to ADP; and iii) whole blood, to evaluate heterotypic aggregate (HA) formation by flow cytometry. Moreover, whole blood previously enriched with platelets with internalized PMP was used to explore modifications in thromboelastometry parameters (ROTEM). The inhibitory action of anti-TLR-4 was investigated.Confocal and ultrastructural microscopy studies revealed PMP internalization by platelets. Flow cytometry showed PMP-platelet association (p 0.01 vs controls, at different PMP dilutions). PMP, at 1/20 dilution, increased HA (p 0.05 vs controls), the percentage of maximal platelet aggregation to ADP (p 0.05 vs controls), and accelerated clotting and clot formation times (p 0.05 vs controls). Incubation of platelets with anti-TLR-4 prior to exposure to PMP reduced PMP-platelet association (p 0.05 vs absence of the antibody), prevented HA formation, reduced maximal platelet aggregation and normalized ROTEM parameters.Platelets exhibit internalization ability towards their own PMP, a process that potentiates their thrombogenicity and is partially mediated by the innate immunity receptor TLR-4.

Background The vascular endothelium plays a key role in sepsis pathophysiology and the associated organ dysfunction. Methods We evaluated endothelial function in an experimental in vitro model of sepsis, using endothelial cells grown in the presence of serum from patients with septic syndromes (sepsis, severe sepsis, and septic shock), noninfectious systemic inflammatory response syndrome (NI-SIRS) and healthy volunteers. Experiments were performed in the absence and presence of defibrotide (DF) (100 mu g/ml) to evaluate its potential protective effect. Results After exposure to patients' sera, there was a progressive endothelial cell activation in correlation with sepsis severity, with a proinflammatory and prothrombotic phenotype, exhibiting significantly increased expression of adhesion receptors at the surface (intercellular adhesion molecule-1, p < .05 and vascular cell adhesion molecule-1, p < .05); higher production and release to the extracellular matrix (ECM) of von Willebrand factor (p < .001); augmented thrombogenicity of the ECM toward platelets (p < .001); and increased phosphorylation of intracellular p38MAPK. DF prevented these changes in all groups. Conclusions Markers of endothelial damage increased progressively in association with the severity of septic syndromes. The endothelium is therefore an important therapeutic target to prevent complications of sepsis. DF shows promising potential to modulate the endothelial damage associated with sepsis and may constitute a pharmacological tool to decrease its sequelae including multiorgan failure.

Background: Clinical and analytical data on patients suffering from coronavirus disease-2019 (COVID-19) indicate that endothelial damage plays a key role in the pathophysiology of the disease and is responsible for the pulmonary complications and the thrombotic microangiopathy affecting multiple organs, which contribute directly to mortality (Ackerman et al. N Engl J Med 2020). Detection of biomarkers of endothelial injury in circulating blood may provide critical diagnostic and prognostic information on the disease course (Goshua et al. Lancet Haematology 2020). Endothelial injury is also a cornerstone of pathobiology in other septic and potentially life-threatening inflammatory syndromes. Objectives: To identify circulating markers of endothelial damage in COVID-19 patients, and compare their levels with those observed in other septic syndromes. Methods: Plasma samples from non-critically ill patients with confirmed COVID-19 pneumonia (positive nasopharyngeal swab and confirmatory radiological chest imaging) requiring admission (n=42) were collected during the first 36h of hospitalization. Endothelial damage was evaluated by measuring in plasma: i) markers of endothelial function and activation (sVCAM-1, VWF, ADAMTS-13 activity, Protein C and α2-antiplasmin as a marker of fibrinolysis); ii) heparan sulfate (HS) levels, as indicators of endothelial glycocalyx degradation and loss of endothelial barrier function; and iii) C5b9 deposits on endothelial cells in culture, and soluble C5b9 (sC5b9) levels, to measure complement activation. Circulating dsDNA was analyzed as an indicator of the presence of neutrophil extracellular traps (NETs). ELISA tests were used for sVCAM-1, Protein C, HS, and sC5b9 levels. ADAMTS-13 activity was evaluated by FRETS. VWF, Protein C, and α2-antiplasmin were measured at the Atellica COAG 360 (Siemens Healthineers). C5b9 deposits were assessed by immunofluorescence and dsDNA levels by Quant-iT PicoGreen assay kit. Results were compared with those obtained in healthy donors (controls, n=45), and patients with non-infectious systemic inflammatory response syndrome (NI-SIRS, n=8) and septic shock (SS, n=8). Results: Levels of sVCAM-1 were significantly higher in COVID-19 patients vs. controls, NI-SIRS and SS (159±12 vs. 79±4, 57±8 and 80±10 ng/mL, respectively, p Conclusions: Our data clearly demonstrate the presence of endothelial stress products in the circulating blood of non-critically ill COVID-19 patients. These biomarkers of endothelial injury are suggestive indicators of different aspects of the disease: specifically, release of acute phase reactants, degradation of the endothelial cell glycocalyx, and activation of the complement system. Furthermore, this profile of biomarkers in COVID-19 appears specific, with a differential behavior in comparison with septic shock, in which endothelial damage is also known to be critical. Additional studies are needed to validate these biomarkers as diagnostic and prognostic tools of the endothelial complications in COVID-19 patients, both in early disease and later, as well as supporting specific forms of therapeutic intervention. Figure Disclosures Carreras: Jazz Pharmaceuticals: Research Funding, Speakers Bureau; German Jose´ Carreras Leukaemia Foundation: Research Funding. Carlo-Stella:Boehringer Ingelheim and Sanofi: Consultancy; ADC Therapeutics and Rhizen Pharmaceuticals: Research Funding; Bristol-Myers Squibb, Merck Sharp & Dohme, Janssen Oncology, AstraZeneca: Honoraria; Servier, Novartis, Genenta Science srl, ADC Therapeutics, F. Hoffmann-La Roche, Karyopharm, Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Moraleda:Sandoz: Consultancy, Other: Travel Expenses; Novartis: Consultancy, Other: Travel Expenses; Gilead: Consultancy, Other: Travel Expenses; Jazz Pharmaceuticals: Consultancy, Research Funding; Takeda: Consultancy, Other: Travel Expenses. Richardson:Celgene/BMS, Oncopeptides, Takeda, Karyopharm: Research Funding. Diaz-Ricart:German Jose Carreras Leukaemia Foundation: Research Funding; Jazz Pharmaceuticals: Honoraria, Research Funding.

Chronic kidney disease (CKD) associates with inflammatory and prothrombotic phenotypes, resulting in higher cardiovascular risk. Factor Xa displays functions beyond coagulation, exhibiting proinflammatory effects. The aim of the present study was to investigate whether a direct FXa inhibitor protects from the endothelial dysfunction (ED) caused by uremia. Macro (HUVEC) and microvascular (HMEC) endothelial cells (ECs) were exposed to serum from uremic patients or healthy donors, in absence and presence of apixaban (60 ng/ml). We evaluated changes in surface VCAM-1 and ICAM-1, intracellular eNOS, reactive oxygen species (ROS), and von Willebrand Factor (VWF) production by immunofluorescence, reactivity of the extracellular matrix (ECM) towards platelets, and intracellular signaling. ECs exposed to uremic serum triggered dysregulation of all the parameters. Presence of apixaban resulted in decreased expression of VCAM-1 (178 ± 14 to 89 ± 2% on HMEC and 324 ± 71 to 142 ± 25% on HUVEC) and ICAM-1 (388 ± 60 to 111 ± 10% on HMEC and 148 ± 9% to 90 ± 7% on HUVEC); increased eNOS (72 ± 8% to 95 ± 10% on HMEC); normalization of ROS levels (173 ± 21 to 114 ± 13% on HMEC and 165 ± 14 to 127 ± 7% on HUVEC); lower production of VWF (168 ± 14 to 92 ± 4% on HMEC and 151 ± 22 to 99 ± 11% on HUVEC); and decreased platelet adhesion onto ECM (134 ± 22 to 93 ± 23% on HMEC and 161 ± 14 to 117 ± 7% on HUVEC). Apixaban inhibited p38MAPK and p42/44 activation in HUVEC (139 ± 15 to 48 ± 15% and 411 ± 66 to 177 ± 57%, respectively) (p < 0.05 vs control for all parameters). Anti-FXa strategies, such as apixaban, prevented ED caused by the uremic milieu, exhibiting anti-inflammatory and antioxidant properties and modulating the reactivity of the ECM.

Multiple myeloma induction treatment includes proteasome inhibitors (PI) and immunomodulatory agents at present. The incidence of engraftment syndrome, a transplant complication potentially related to endothelium, has increased in the last years. Our aim was to investigate whether bortezomib (Velcade, V), thalidomide (T), and dexamethasone (D) affect the endothelium, and explore defibrotide (DF) as protective agent. Endothelial cells (ECs) in culture were exposed to the compounds separately or in combination, without (VTD) and with DF (VTD + DF). Changes in markers of: (i) inflammation (ICAM-1 expression and leukocyte adhesion), (ii) VWF production, (iii) cell permeability (VE-cadherin expression and cell monolayer integrity), and (iv) oxidative stress (ROS production and eNOS expression) were measured. ICAM-1 and VWF expression increased significantly in VTD but were similar to controls in VTD + DF. Separately, bortezomib was the main deleterious agent whereas dexamethasone showed no harmful effect. Leukocyte adhesion showed similar trends. VE-cadherin expression was lower in VTD and normalized in VTD + DF. EC permeability increased only with bortezomib. No changes were observed in oxidative stress markers. Our results demonstrate that bortezomib damages the endothelium, and DF prevents this effect. A better knowledge of the induction drugs impact will allow the design of measures to protect the endothelium.

The aim of the present study was to explore whether there is enhanced endothelial dysfunction in patients developing acute GvHD (aGvHD) after allogeneic hematopoietic cell transplantation (allo-HCT) and to identify biomarkers with predictive and/or diagnostic value. In in vitro experiments, endothelial cells (ECs) were exposed to serum from patients with (aGvHD, n = 31) and without (NoGvHD, n = 13) aGvHD, to evaluate changes in surface adhesion receptors, the reactivity of the extracellular matrix by measuring the presence of Von Willebrand factor (VWF) and platelet adhesion, and the activation of intracellular signaling proteins. Plasma levels of VWF, ADAMTS-13, TNF receptor 1 (TNFR1), soluble vascular cell adhesion molecule 1 and soluble intercellular adhesion molecule 1 were also measured. In vitro results showed a more marked proinflammatory and prothrombotic phenotype in ECs in association with aGvHD. Regarding circulating biomarkers, levels of VWF and TNFR1 above an optimal cutoff score, taken independently or combined, at day 7 after allo-HCT, would be able to positively predict that around 90% of patients will develop aGvHD. Our results demonstrate that endothelial damage is aggravated in those allo-HCT recipients developing aGvHD, and that VWF and TNFR1 are promising predictive aGvHD biomarkers. These findings could contribute to improve the understanding of the pathophysiology of aGvHD.

Immobilization of bioactive peptide sequences on CoCr surfaces is an effective route to improve endothelialization, which is of great interest for cardiovascular stents. In this work, we explored the effect of physical and covalent immoblization of RGDS, YIGSR and their equimolar combination peptides on endothelial cells (EC) and smooth muscle cell (SMC) adhesion and on thrombogenicity. We extensively investigated using RT-qPCR, the expression by ECs cultured on functionalised CoCr surfaces of different genes. Genes relevant for adhesion (ICAM-1 and VCAM-1), vascularization (VEGFA, VEGFR-1 and VEGFR-2) and anti-thrombogenicity (tPA and eNOS) were over-expressed in the ECs grown to covalently functionalized CoCr surfaces compared to physisorbed and control surfaces. Pro-thrombogenic genes expression (PAI-1 and vWF) decreased over time. Cell co-cultures of ECs/SMCs found that functionalization increased the amount of adhered ECs onto modified surfaces compared to plain CoCr, independently of the used peptide and the strategy of immobilization. SMCs adhered less compared to ECs in all surfaces. All studied peptides showed a lower platelet cell adhesion compared to TCPS. Covalent functionalization of CoCr surfaces with an equimolar combination of RGDS and YIGSR represented prevailing strategy to enhance the early stages of ECs adhesion and proliferation, while preventing SMCs and platelet adhesion. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 973-983, 2017.

Background Serotonin reuptake inhibitors (SSRIs) may impair platelet function. Thrombin is a strong platelet agonist causing irreversible aggregation, release of granules' contents, cytoskeletal rearrangement and activation of signalling pathways. We investigated the effects of the SSRI escitalopram (SCIT) on thrombin-induced platelet response. Methods Isolated platelets were exposed to SCIT and activated with thrombin. We evaluated (1) platelet response by aggregometry and flow cytometry; (2) modifications in cytoskeleton proteins and signalling pathways by electrophoresis and Western blot; and (3) ultrastructural changes in platelets by electron microscopy. Results SCIT inhibited platelet response to thrombin, measured as platelet aggregation and expression of activation markers CD62-P and CD63 from platelet granules. Platelet aggregation decreased in a dose-dependent manner, reaching statistical significance with SCIT ≥32 µg/mL (65.4 ± 6.8% vs. 77.7 ± 2.5% for controls; p Conclusions Our data indicate that SCIT inhibits thrombin-induced platelet response and interferes with cytoskeletal assembly and related signalling pathways, thus resulting in compromised release of granules' contents, reduced platelet activation and aggregation. These mechanisms may explain the antithrombotic benefits observed in patients treated with this SSRI, and could become new therapeutic targets for future antithrombotic strategies.

Objectives Pathogen reduction technologies are implemented to increase the safety of blood products. We previously showed that the UVB alone significantly contributes to the storage lesions observed in platelets treated with riboflavin/UVB using a home-made illuminator. The present study aims at confirming these observations using the commercial Mirasol® technology. Methods A three-arm study (untreated, UV−, Mirasol®-treated platelets) was conducted to investigate the platelet storage lesions throughout storage (n = 4). A two-arm study was then designed to compare Intersol and T-PAS+ additive solutions (n = 3). Phenotype and functional platelet characteristics were assessed using flow cytometry, aggregometry, antioxidant assays and metabolic parameters. Results Mirasol®-treated platelets exhibit enhanced storage lesions compared to controls (increase of activation markers and glycolysis rate, lower hypotonic shock and double-agonist activation responses, and decrease of total antioxidant capacity). Here, we also confirmed that the UV radiation alone is causing platelet lesions. Riboflavin tends to have an intracellular protective role while it decreases the extracellular antioxidant defenses. Furthermore, benefits of platelet additive solutions containing potassium and magnesium were confirmed as it reduces the extent of storage lesions. Conclusions The photosensitizer, UV illumination and composition of the platelet additive solutions are key parameters influencing the platelet storage lesion. The clinical relevance of these findings is not fully understood and recent published clinical studies could not show increase in bleeding in patients receiving Mirasol-treated platelets. New developments in storage solutions might help to improve storage conditions of PRT-treated platelets and should be prioritised as research subject in the future.

Developments during the past few years have resulted in multiple kinds of platelet products for transfusion. This involves different collection methods, containers, preservative solutions, modifications of storage temperatures and durations, and additional treatments such as pathogen reduction. Much experience has been obtained testing these processes in vitro to seek indications of in vivo effectiveness. Availability of an in vitro method that correlated with in vivo effectiveness would be extremely valuable for these different kinds of platelet products and as more innovation in platelet preparation occurs in the future. This report reviews the methods for in vitro platelet testing with a view to their in vivo implications and whether such testing could be helpful in projecting the clinical effectiveness of different platelet products.

BACKGROUND Comparative studies on the restoration of hemostasis with different reversal agents after dabigatran therapy have not been performed. We compared the efficacy and prothrombotic potential of the specific antidote idarucizumab with that of previously recommended non-specific procoagulant concentrates. STUDY DESIGN AND METHODS We explored the in vitro effects of dabigatran (184 ng/mL) on fibrin and platelet-aggregate formation onto a damaged vessel under flow conditions (600 s-1 ). The reversal mechanisms and efficacy of idarucizumab (0.3-3 mg/mL) were compared with that of the non-specific procoagulant concentrates aPCC (25-75 U/Kg), PCC (70 U/Kg), or rFVIIa (120 μg/Kg). Generation of thrombin and prothrombin fragment (F1 + 2), and thromboelastometry parameters of clot formation were measured. RESULTS Dabigatran caused pronounced reductions in fibrin (87%) and platelet interactions (36%) with damaged vessels (p

In the case of vascular injury, a complex process (of clotting) starts, involving mainly platelets and coagulation factors. This process in healthy humans is known as hemostasis, but when it is deregulated (thrombosis), it can be the cause of important cardiovascular diseases. Nowadays, the aging of the population and unhealthy lifestyles increase the impact of thrombosis, and therefore there is a need for tools to provide a better understanding of the hemostasis mechanisms, as well as more cost-effective diagnosis and control devices. This study proposes a novel microflow chamber, with interchangeable biomimetic surfaces to evaluate global hemostasis, using reduced amounts of blood sample and reagents, and also a minimized time required to do the test. To validate the performance of this novel device, a study on the new oral anticoagulant Apixaban (APIX) has been performed and compared to previous conventional techniques. The test shows an excellent agreement, while the amount of the required sample has been reduced (only 100 &micro, L is used), and the amount of reagent as well. An imprinted electrode embedded in the chamber in order to measure the impedance during the coagulation process. This approach distinguishes the impedance behavior of plasma poor in platelets (PPP) and plasma rich in platelets (PRP) for the first time.

The short shelf life of platelet concentrates (PCs) presents logistic problems in providing timely PC transfusions for patients highly alloimmunized by HLA/HPA antibodies, and when long or major transportation barriers exist (e.g. rural care settings, severe weather, military operations). Several strategies have been tried to circumvent the limited storage time of PC. Among them, the cryopreservation of human platelets using dimethyl sulfoxide, the freeze-drying of outdated human platelets and even the manufacture of artificial phospholipids that might act as artificial platelets. Several in vitro techniques are currently available to characterize the structure and function of those products. Among other we can use, complete blood count, flow cytometry combined to monoclonal antibodies, platelet aggregation, studies under flow conditions and thromboelastography/thromboelastometry. The combined use of those in vitro techniques provides valuable information about the structure and functional haemostatic capacities of those platelet components. Depending on the results, the decision can be made if the product can be or cannot be tested on human volunteers and patients. As for instance was the case of artificial phospholipids that after observing a very high procoagulant activity in vitro, this approach has not gone further in its development.

Platelets are important in hemostasis, but also detect particles and pathogens in the circulation. Phagocytic and endocytic activities of platelets are widely recognized; however, receptors and mechanisms involved remain poorly understood. We previously demonstrated that platelets internalize and store phospholipid microvesicles enriched in human tissue factor (TF+MVs) and that platelet-associated TF enhances thrombus formation at sites of vascular damage. Here, we investigate the mechanisms implied in the interactions of TF+MVs with platelets and the effects of specific inhibitory strategies. Aggregometry and electron microscopy were used to assess platelet activation and TF+MVs uptake. Cytoskeletal assembly and activation of phosphoinositide 3-kinase (PI3K) and RhoA were analyzed by western blot and ELISA. Exposure of platelets to TF+MVs caused reversible platelet aggregation, actin polymerization and association of contractile proteins to the cytoskeleton being maximal at 1 min. The same kinetics were observed for activation of PI3K and translocation of RhoA to the cytoskeleton. Inhibitory strategies to block glycoprotein IIb-IIIa (GPIIb-IIIa), scavenger receptor CD36, serotonin transporter (SERT) and PI3K, fully prevented platelet aggregation by TF+MVs. Ultrastructural techniques revealed that uptake of TF+MVs was efficiently prevented by anti-CD36 and SERT inhibitor, but only moderately interfered by GPIIb-IIIa blockade. We conclude that internalization of TF+MVs by platelets occurs independently of receptors related to their main hemostatic function (GPIIb-IIIa), involves the scavenger receptor CD36, SERT and engages PI3-Kinase activation and cytoskeletal assembly. CD36 and SERT appear as potential therapeutic targets to interfere with the association of TF+MVs with platelets and possibly downregulate their prothrombotic phenotype.

Hemophilia A is an X-linked bleeding disorder caused by defects in the gene encoding factor VIII (FVIII). Routine prophylaxis with exogenous FVIII requires frequent intravenous injections. One of the most challenging issues in the treatment of hemophilia A is the development of alloantibodies against infused FVIII. Presence of inhibitors results in an ineffective factor replacement therapy and increases the risk of morbidity and mortality in these patients. Therefore, there is growing interest in the development of new strategies for the prophylaxis and prevention of bleeding in patients with hemophilia to circumvent these drawbacks. Emicizumab (ACE-910; Roche, Genentech and Chugai Pharmaceutical) is a recombinant humanized bispecific antibody that restores the function of missing FVIII by bridging activated FIX and FX, simulating the cofactor function of FVIII.

Patients with HIV have higher thrombogenicity that correlates with markers of inflammation; both respond to clopidogrel treatment., Persons with HIV infection (PWH) have increased risk for cardiovascular disease (CVD), but the underlying mechanisms remain unclear. Coronary thrombosis is known to provoke myocardial infarctions, but whether PWH have elevated thrombotic propensity is unknown. We compared thrombogenicity of PWH on antiretroviral therapy versus matched controls using the Badimon chamber. Measures of inflammation, platelet reactivity, and innate immune activation were simultaneously performed. Enrolled PWH were then randomized to placebo, aspirin (81 mg), or clopidogrel (75 mg) for 24 weeks to assess treatment effects on study parameters. Thrombogenicity was significantly higher in PWH and correlated strongly with plasma levels of D-dimer, soluble TNF receptors 1 and 2, and circulating classical and nonclassical monocytes in PWH. Clopidogrel significantly reduced thrombogenicity and sCD14. Our data suggest that higher thrombogenicity, interacting with inflammatory and immune activation markers, contributes to the increased CVD risk observed in PWH. Clopidogrel exhibits an anti-inflammatory activity in addition to its antithrombotic effect in PWH.