Syntaxin 12 and COMMD3 are new factors that function with VPS33B in the biogenesis of platelet α-granules.

Platelet α-granules regulate hemostasis and myriad other physiological processes, but their biogenesis is unclear. Mutations in only 3 proteins are known to cause α-granule defects and bleeding disorders in humans. Two such proteins, VPS16B and VPS33B, form a complex mediating transport of newly synthesized α-granule proteins through megakaryocyte (MK) endosomal compartments. It is unclear how the VPS16B/VPS33B complex accomplishes this function. Here we report VPS16B/VPS33B associates physically with Syntaxin 12 (Stx12), a SNARE protein that mediates vesicle fusion at endosomes. Importantly, Stx12-deficient MKs display reduced α-granule numbers and overall levels of α-granule proteins, thus revealing Stx12 as a new component of the α-granule biogenesis machinery. VPS16B/VPS33B also binds CCDC22, a component of the CCC complex working at endosome exit sites. CCDC22 competes with Stx12 for binding to VPS16B/VPS33B, suggesting a possible hand-off mechanism. Moreover, the major CCC form expressed in MKs contains COMMD3, one of 10 COMMD proteins. Deficiency of COMMD3/CCDC22 causes reduced α-granule numbers and overall levels of α-granule proteins, establishing the COMMD3/CCC complex as a new factor in α-granule biogenesis. Furthermore, P-selectin traffics through the cell surface in a COMMD3-dependent manner and depletion of COMMD3 results in lysosomal degradation of P-selectin and PF4. Stx12 and COMMD3/CCC deficiency cause less severe phenotypes than VPS16B/VPS33B deficiency, suggesting Stx12 and COMMD3/CCC assist but are less important than VPS16B/VPS33B in α-granule biogenesis. Mechanistically, our results suggest VPS16B/VPS33B coordinates the endosomal entry and exit of α-granule proteins by linking the fusogenic machinery with a ubiquitous endosomal retrieval complex that is repurposed in MKs to make α-granules.

Beyond eosinophilia: inflammatory patterns in patients with asthma.

Background: Recently, inflammatory cell ratios have gained importance as useful indicators in the categorization of asthma.Objective: We compared the concentration of white blood cells in peripheral blood, as well as their respective inflammatory cell ratios, between patients with asthma and a healthy control group.Methods: We performed cross-sectional analyses of the data obtained from 53 adult patients with asthma and 109 adult controls. In our study, we estimated and compared the following inflammatory cell ratios: Neutrophil-Lymphocyte Ratio (NLR), Eosinophil-Lymphocyte Ratio (ELR), Eosinophil-Neutrophil Ratio (ENR), Eosinophil-Monocyte Ratio (EMR), and Platelet-Lymphocyte Ratio (PLR). The magnitude of association was quantified with the odds ratio.Results: In both groups, the average age was 33 years. In asthmatic patients, we obtained the following results: eosinophils ≥ 400 cells/µl, accounted for 37.7%; basophils ≥ 110 cells/µl, comprised 37.7%; and monocytes < 320 cells/µl, reached 11.3%. In the control group, the results were as follows: 4.6%, 9.2% and 0.9%, respectively. When compared to the control group, asthmatic patients had higher odds of eosinophils ≥ 400 cells/µl (OR = 12.61, p < 0.0001); higher odds of basophils ≥ 110 cells/µl (OR = 6.00, p < 0.0001); and increased odds of monocytes < 320 cells/µl (OR = 13.79, p = 0.017). NLR did not differ between our two groups; however, ELR, ENR, EMR and PLR were significantly higher in the asthma group.Conclusions: Overall, patients with asthma have a higher concentration of eosinophils and basophils, fewer monocytes in their blood, and higher ratios of increased chronic inflammation.

First day postoperative values of the neutrophil-to-lymphocyte ratio, platelet-to-lymphocyte ratio, and C-reactive protein as complication predictors following gastric oncologic surgery.

INTRODUCTION: The neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and C-reactive protein (CRP) level are simple laboratory test parameters that can provide us with information on the inflammatory status of the organism. CRP has been shown to be a predictor of postoperative complications, whereas NLR and PLR have shown greater usefulness in the prognosis of oncologic pathologies. AIM: To evaluate the associations of NLR and PLR with postoperative complications following gastric oncologic surgery and compare them with CRP. MATERIALS AND METHODS: A prospective study was conducted on 66 patients that underwent oncologic gastric surgery, within the time frame of January 2014 and March 2019. The variables analyzed were sociodemographic data, surgical technique, tumor extension, and NLR, PLR, and CRP levels from the first day after surgery, as well as postoperative complications. RESULTS: Seventeen patients (25.8%) presented with grade III-V complications, utilizing the Clavien-Dindo classification system. Mean NLR value was 11.30 and was associated with the appearance of major complications, with statistical significance (p = 0.009). Mean PLR was266.05 and was not significantly associated with complications (p = 0.149). Fifty-four patients had a mean CRP level of 143.24 and it was not related to the appearance of major complications (p = 0.164). CONCLUSIONS: The NLR is a simple and inexpensive parameter, which measured on postoperative day one, predicted the appearance of major postoperative complications in our study sample and appears to be a better predictive parameter than CRP for said complications. Further studies to confirm that trend need to be carried out.

Ex Vivo Manufacture of Megakaryocytes and Platelets from Stem Cells: Recent Advances Toward Transfusion in Humans.

The generation of ex vivo functional megakaryocytes (MK) and platelets is an important issue in transfusion medicine as donor dependence implies in limitations, such as shortage of eligible volunteers. Indeed, platelet transfusion is still a procedure that saves the lives of patients with defective platelet production. Recent technological development has enabled the isolation and expansion of stem cells that can be used as a source for the production of functional platelets for transfusion. In this review, we discuss recent approaches of in vitro or ex vivo production of MK and platelets, suggesting that, in the near future, donor-independent sources may become a possibility. The feasibility of using these cells in the clinic may be safer, and in vitro manipulation could generate universally compatible products, solving problems related to platelet refractoriness. However, functionality and survival testing of these products in human beings are scarce; therefore, additional studies are needed to consolidate this purpose.

Asociación entre los niveles séricos de vitamina D y marcadores inflamatorios en pacientes en hemodiálisis / Association between vitamin D serum levels and inflammatory markers in patients on hemodialysis

Resumen Introducción: La relación entre 25-OH-vitamina D y el sistema inmune en pacientes con enfermedad renal crónica es objeto de atención. Objetivos: Evaluar la prevalencia de la deficiencia de vitamina D en pacientes en hemodiálisis e investigar la asociación entre la vitamina D y proteína C reactiva ultrasensible (PCRus), índice neutrófilo/linfocito (INL) e índice plaqueta/linfocito (IPL). Método: Estudio transversal de 80 pacientes en hemodiálisis, divididos en dos grupos: un nivel sérico de 25-OH-vitamina D < 20 ng/mL se consideró como deficiencia de vitamina D y ≥ 20 ng/mL, como normal. Con el análisis de correlación de Spearman se definió la relación entre los parámetros. Resultados: 40 % de los pacientes presentó deficiencia de vitamina D. Hubo diferencias significativas entre los grupos en PCRus (p = 0.047), INL (p = 0.039), IPL (p = 0.042) y tratamiento con análogos de vitamina D (p = 0.022). La vitamina D tuvo una correlación negativa significativa con PCRus (p = 0.026), INL (p = 0.013) e IPL (p = 0.022). Conclusiones: La deficiencia de vitamina D fue de 40 %. Los niveles de PCRus, INL e IPL fueron significativamente más altos ante deficiencia de vitamina D. Se encontró correlación inversa significativa entre vitamina D y PCRus, INL e IPL.

Abstract Introduction: The relationship between 25-OH-vitamin D and the immune system in patients with chronic kidney disease is a subject of attention. Objectives: To assess the prevalence of vitamin D deficiency in patients on hemodialysis and to investigate the association between vitamin D, ultra-sensitive C-reactive protein (US-CRP), neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR). Method: Cross-sectional study of 80 patients on hemodialysis, divided into two groups: a serum 25-OH-vitamin D level < 20 ng/mL was considered to be vitamin D deficiency and a serum level ≥ 20 ng/mL was regarded as normal. The relationship between the parameters was defined with Spearman’s correlation analysis. Results: 40 % of the patients had vitamin D deficiency. There were significant differences between groups in US-CRP (p = 0.047), NLR (p = 0.039), PLR (p = 0.042) and treatment with vitamin D analogues (p = 0.022). Vitamin D had a significant negative correlation with US-CRP (p = 0.026), NLR (p = 0.013) and PLR (p = 0.022). Conclusions: The prevalence of vitamin D deficiency was 40 %. The values of US-CRP, NLR and PLR were significantly higher in the presence of vitamin D deficiency. A significant inverse correlation was found between vitamin D levels and US-CRP, NLR and PLR.

Platelet disturbances correlate with endothelial cell activation in uncomplicated Plasmodium vivax malaria.

Platelets drive endothelial cell activation in many diseases. However, if this occurs in Plasmodium vivax malaria is unclear. As platelets have been reported to be activated and to play a role in inflammatory response during malaria, we hypothesized that this would correlate with endothelial alterations during acute illness. We performed platelet flow cytometry of PAC-1 and P-selectin. We measured platelet markers (CXCL4, CD40L, P-selectin, Thrombopoietin, IL-11) and endothelial activation markers (ICAM-1, von Willebrand Factor and E-selectin) in plasma with a multiplex-based assay. The values of each mediator were used to generate heatmaps, K-means clustering and Principal Component analysis. In addition, we determined pair-wise Pearson's correlation coefficients to generate correlation networks. Platelet counts were reduced, and mean platelet volume increased in malaria patients. The activation of circulating platelets in flow cytometry did not differ between patients and controls. CD40L levels (Median [IQ]: 517 [406-651] vs. 1029 [732-1267] pg/mL, P = 0.0001) were significantly higher in patients, while P-selectin and CXCL4 showed a nonsignificant trend towards higher levels in patients. The network correlation approach demonstrated the correlation between markers of platelet and endothelial activation, and the heatmaps revealed a distinct pattern of activation in two subsets of P. vivax patients when compared to controls. Although absolute platelet activation was not strong in uncomplicated vivax malaria, markers of platelet activity and production were correlated with higher endothelial cell activation, especially in a specific subset of patients.

Does periodontitis affect mean platelet volume(MPV) and plateletcrit (PCT) levels in healthy adults?

OBJECTIVE: Periodontitis may stimulate infectious and immune response and cause the development of atherogenesis, coronary heart disease, and myocardial infarction. The aim of this study was to compare the plateletcrit (PCT) and mean platelet volume (MPV) levels derived from complete blood count (CBC) tests in patients suffering from stage 3 periodontitis with those of healthy individuals without periodontal disease. METHODS: The study included 57 patients (28 females and 29 males) with Stage 3 Periodontitis and 57 volunteering individuals (31 females and 26 males) who were periodontally healthy. The age of study participants ranged from 18 to 50 years. Their periodontal condition was investigated with probing depth (PD), clinical attachment level, bleeding on probing, and plaque index. Leukocyte (WBC) and erythrocyte count (RBC), hemoglobin (Hb) and hematocrit (HCT) levels, mean corpuscular volume (MCV) and red cell distribution width (RDW), thrombocyte count, mean platelet volume (MPV), plateletcrit (PCT ), and neutrophil and lymphocyte counts were evaluated based on the CBC test results of the study participants. RESULTS: PCT, WBC, Neutrophil, and MPV values were found to be significantly higher in the periodontitis group (p<0.05). There were no significant differences in RBC counts, Hb, HCT, MCV, RDW, and platelet and lymphocyte counts between the two study groups (p>0.05). CONCLUSIONS: PCT and MPV levels may be a more useful marker to determine an increased thrombotic state and inflammatory response in periodontal diseases.

Does periodontitis affect mean platelet volume(MPV) and plateletcrit (PCT) levels in healthy adults?

SUMMARY OBJECTIVE Periodontitis may stimulate infectious and immune response and cause the development of atherogenesis, coronary heart disease, and myocardial infarction. The aim of this study was to compare the plateletcrit (PCT) and mean platelet volume (MPV) levels derived from complete blood count (CBC) tests in patients suffering from stage 3 periodontitis with those of healthy individuals without periodontal disease. METHODS The study included 57 patients (28 females and 29 males) with Stage 3 Periodontitis and 57 volunteering individuals (31 females and 26 males) who were periodontally healthy. The age of study participants ranged from 18 to 50 years. Their periodontal condition was investigated with probing depth (PD), clinical attachment level, bleeding on probing, and plaque index. Leukocyte (WBC) and erythrocyte count (RBC), hemoglobin (Hb) and hematocrit (HCT) levels, mean corpuscular volume (MCV) and red cell distribution width (RDW), thrombocyte count, mean platelet volume (MPV), plateletcrit (PCT ), and neutrophil and lymphocyte counts were evaluated based on the CBC test results of the study participants. RESULTS PCT, WBC, Neutrophil, and MPV values were found to be significantly higher in the periodontitis group (p<0.05). There were no significant differences in RBC counts, Hb, HCT, MCV, RDW, and platelet and lymphocyte counts between the two study groups (p>0.05). CONCLUSIONS PCT and MPV levels may be a more useful marker to determine an increased thrombotic state and inflammatory response in periodontal diseases.

RESUMO OBJETIVO A periodontite pode estimular a resposta infecciosa e imunitária e causar o desenvolvimento da aterogênese, doença coronária e infarto do miocárdio. O objetivo deste estudo foi comparar os níveis de plaquetócrito (PCT) e de volume médio de plaquetas (VMP) derivados dos testes de hemograma completo (CBC) em doentes que sofrem de periodontite de fase 3 com os de indivíduos saudáveis, sem doença periodontal. MÉTODOS O estudo incluiu 57 doentes (28 mulheres e 29 homens) com periodontite de fase 3 e 57 voluntários (31 mulheres e 26 homens) que eram periodontalmente saudáveis. A idade dos participantes do estudo variou de 18 a 50 anos. A condição periodontal dos participantes do estudo foi investigada com profundidade de sonda (PD), nível de ligação clínica, hemorragia na sonda e índice de placas. Contagem de leucócitos (WBC) e eritrócitos (RBC), níveis de hemoglobina (Hb) e hematócrito (HCT), volume corpuscular médio (VCM) e largura de distribuição das células vermelhas (RDW), contagem de trombócitos, volume plaquetário médio (MPV), plaquetócrito (PCT) e contagem de neutrófilos e linfócitos foram avaliados com base nos resultados do teste CBC dos participantes do estudo. RESULTADO Verificou-se que os valores de PCT, WBC, neutrófilos e MPV eram significativamente mais elevados no grupo da periodontite (p<0,05). Não houve diferenças significativas nas contagens de glóbulos vermelhos, Hb, HCT, MCV, RDW; nem nas contagens de plaquetas e linfócitos entre os dois grupos estudados (p>0, 05). CONCLUSÃO Os níveis de PCT e MPV podem ser um marcador mais útil para determinar um estado trombótico aumentado e a resposta inflamatória em doenças periodontais.

The immunosuppressive mechanisms of mesenchymal stem cells are differentially regulated by platelet poor plasma and fetal bovine serum supplemented media.

PURPOSE: Mesenchymal Stem Cells (MSCs) can interact with and modulate the functions of all immune cells, suppressing both the innate and adaptive immune responses. Currently, most of the in vitro studies which have led to the description of MSC properties have resulted from MSC culture in the presence of fetal bovine serum (FBS), in spite of the recognition of FBS limitations and attempts to substitute this component from the MSC media. METHODS: Herein, we compare FBS and Platelet Poor Plasma (PPP) as MSC media supplements, according to Adipose-derived MSC (AMSC) phenotype, proliferation and immunoregulatory mechanisms. RESULTS: Interestingly, despite maintaining the classic phenotypic profile of MSCs, PPP cultured AMSCs showed impaired proliferative potential. Furthermore, our results clearly show that AMSC culture with PPP leads to decreased expression of soluble immunosuppressive factors, which resulted in decreased capacity of inducing regulatory T-cells (Tregs) generation by these cells. In contrast, PPP supplementation promoted enhanced VCAM-1 and ICAM-1 expression on AMSC surface. Therefore, AMSCs cultured with PPP showed limited potential to produce soluble immunomodulatory factors, indicating a reduced capacity to control the immune system thought paracrine activity. CONCLUSION: Overall, our data sheds light on the importance of culture media supplementation for MSC immunomodulatory behavior, as well as serving as an alert regarding the complexity of replacing FBS in MSC culture.

Association between vitamin D serum levels and inflammatory markers in patients on hemodialysis.

INTRODUCTION: The relationship between 25-OH-vitamin D and the immune system in patients with chronic kidney disease is a subject of attention. OBJECTIVES: To assess the prevalence of vitamin D deficiency in patients on hemodialysis and to investigate the association between vitamin D, ultra-sensitive C-reactive protein (US-CRP), neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR). METHOD: Cross-sectional study of 80 patients on hemodialysis, divided into two groups: a serum 25-OH-vitamin D level < 20 ng/mL was considered to be vitamin D deficiency and a serum level ≥ 20 ng/mL was regarded as normal. The relationship between the parameters was defined with Spearman's correlation analysis. RESULTS: 40 % of the patients had vitamin D deficiency. There were significant differences between groups in US-CRP (p = 0.047), NLR (p = 0.039), PLR (p = 0.042) and treatment with vitamin D analogues (p = 0.022). Vitamin D had a significant negative correlation with US-CRP (p = 0.026), NLR (p = 0.013) and PLR (p = 0.022). CONCLUSIONS: The prevalence of vitamin D deficiency was 40 %. The values of US-CRP, NLR and PLR were significantly higher in the presence of vitamin D deficiency. A significant inverse correlation was found between vitamin D levels and US-CRP, NLR and PLR. INTRODUCCIÓN: La relación entre 25-OH-vitamina D y el sistema inmune en pacientes con enfermedad renal crónica es objeto de atención. OBJETIVOS: Evaluar la prevalencia de la deficiencia de vitamina D en pacientes en hemodiálisis e investigar la asociación entre la vitamina D y proteína C reactiva ultrasensible (PCRus), índice neutrófilo-linfocito (INL) e índice plaqueta-linfocito (IPL). MÉTODO: Estudio transversal de 80 pacientes en hemodiálisis, divididos en dos grupos: un nivel sérico de 25-OH-vitamina D < 20 ng/mL se consideró como deficiencia de vitamina D y ≥ 20 ng/mL, como normal. Con el análisis de correlación de Spearman se definió la relación entre los parámetros. RESULTADOS: 40 % de los pacientes presentó deficiencia de vitamina D. Hubo diferencias significativas entre los grupos en PCRus (p = 0.047), INL (p = 0.039), IPL (p = 0.042) y tratamiento con análogos de vitamina D (p = 0.022). La vitamina D tuvo una correlación negativa significativa con PCRus (p = 0.026), INL (p = 0.013) e IPL (p = 0.022). CONCLUSIONES: La deficiencia de vitamina D fue de 40 %. Los niveles de PCRus, INL e IPL fueron significativamente más altos ante deficiencia de vitamina D. Se encontró correlación inversa significativa entre vitamina D y PCRus, INL e IPL.

Association of platelet to lymphocyte and neutrophil to lymphocyte ratios with in-hospital mortality in patients with type A acute aortic dissection

Abstract Objective: To evaluate the relationship between neutrophil to lymphocyte ratio (NLR) and platelet to lymphocyte ratio (PLR) with in-hospital mortality in type A acute aortic dissection (AAD). Methods: A total of 96 patients who presented to the emergency department between January 2013 and June 2018 with a diagnosis of type A AAD were enrolled in this study. White blood cell count subtypes such as NLR and PLR were calculated at the time of admission. The end point was in-hospital mortality. Results: Of the 96 type A AAD patients included in this analysis, 17 patients (17.7%) died during hospitalization. NLR and PLR were significantly elevated in patients with type A AAD (P<0.001 and <0.001, respectively). Based on the receiver operating characteristic curve, the best NLR cut-off value to predict in-hospital mortality was 9.74, with 70.6% sensitivity and 76.8% specificity, whereas the best PLR cut-off value was 195.8, with 76.5% sensitivity and 78.1% specificity. Conclusion: Admission NLR and PLR levels were important risk factors and independently associated with in-hospital mortality of type A AAD patients.

Platelet-to-lymphocyte ratio (PLR) and Plateletcrit (PCT) in young patients with morbid obesity.

OBJECTIVE: To compare the complete blood counts, namely the plateletcrit (PCT) and Platelet-To-Lymphocyte Ratio (PLR) of healthy subjects and those with morbid obesity in the young population. METHODS: We included 45 patients with morbid obesity (body mass index -BMI - greater than or equal to 45 kg/m2) and 45 healthy subjects (BMI less than or equal to 25 kg/m2) in our study. Blood samples were obtained from the participants following a 12-hour fasting period. Then we evaluated the levels of hemoglobin (Hb), hematocrit (HCT), red cell distribution width (RDW), mean platelet volume (MPV), white blood cell (WBC), PLR, platelet counts, and PCT in the complete blood count. RESULTS: The morbid obesity group had significantly higher platelet counts and PCT values (p<0.001), and PLR values (p=0.033). The value of WBC was also higher in the obese group (p=0.001). MPV was lower in the obesity group but not statistically significant (p=0.815). No significant difference was found between hemoglobin and hematocrit values in these groups; but RDW valuewere higher and statistically significant in the obese group (p=0.001). CONCLUSION: PLR or PCT may be more useful as a marker in determining an increased thrombotic state and inflammatory response in morbid obesity.

SASP-Dependent Interactions between Senescent Cells and Platelets Modulate Migration and Invasion of Cancer Cells.

Alterations in platelet aggregation are common in aging individuals and in the context of age-related pathologies such as cancer. So far, however, the effects of senescent cells on platelets have not been explored. In addition to serving as a barrier to tumor progression, cellular senescence can contribute to remodeling tissue microenvironments through the capacity of senescent cells to synthesize and secrete a plethora of bioactive factors, a feature referred to as the senescence-associated secretory phenotype (SASP). As senescent cells accumulate in aging tissues, sites of tissue injury, or in response to drugs, SASP factors may contribute to increase platelet activity and, through this mechanism, generate a microenvironment that facilitates cancer progression. Using in vitro models of drug-induced senescence, in which cellular senescence was induced following exposure of mammary epithelial cells (MCF-10A and MCF-7) and gastric cancer cells (AGS) to the CDK4/6 inhibitor Palbociclib, we show that senescent mammary and gastric cells display unique expression profiles of selected SASP factors, most of them being downregulated at the RNA level in senescent AGS cells. In addition, we observed cell-type specific differences in the levels of secreted factors, including IL-1ß, in media conditioned by senescent cells. Interestingly, only media conditioned by senescent MCF-10A and MCF-7 cells were able to enhance platelet aggregation, although all three types of senescent cells were able to attract platelets in vitro. Nevertheless, the effects of factors secreted by senescent cells and platelets on the migration and invasion of non-senescent cells are complex. Overall, platelets have prominent effects on migration, while factors secreted by senescent cells tend to promote invasion. These differential responses likely reflect differences in the specific arrays of secreted senescence-associated factors, specific factors released by platelets upon activation, and the susceptibility of target cells to respond to these agents.

Association of Platelet to Lymphocyte and Neutrophil to Lymphocyte Ratios with In-Hospital Mortality in Patients with Type A Acute Aortic Dissection.

OBJECTIVE: To evaluate the relationship between neutrophil to lymphocyte ratio (NLR) and platelet to lymphocyte ratio (PLR) with in-hospital mortality in type A acute aortic dissection (AAD). METHODS: A total of 96 patients who presented to the emergency department between January 2013 and June 2018 with a diagnosis of type A AAD were enrolled in this study. White blood cell count subtypes such as NLR and PLR were calculated at the time of admission. The end point was in-hospital mortality. RESULTS: Of the 96 type A AAD patients included in this analysis, 17 patients (17.7%) died during hospitalization. NLR and PLR were significantly elevated in patients with type A AAD (P<0.001 and <0.001, respectively). Based on the receiver operating characteristic curve, the best NLR cut-off value to predict in-hospital mortality was 9.74, with 70.6% sensitivity and 76.8% specificity, whereas the best PLR cut-off value was 195.8, with 76.5% sensitivity and 78.1% specificity. CONCLUSION: Admission NLR and PLR levels were important risk factors and independently associated with in-hospital mortality of type A AAD patients.

Platelet-to-lymphocyte ratio (PLR) and Plateletcrit (PCT) in young patients with morbid obesity

SUMMARY OBJECTIVE To compare the complete blood counts, namely the plateletcrit (PCT) and Platelet-To-Lymphocyte Ratio (PLR) of healthy subjects and those with morbid obesity in the young population. METHODS We included 45 patients with morbid obesity (body mass index -BMI - greater than or equal to 45 kg/m2) and 45 healthy subjects (BMI less than or equal to 25 kg/m2) in our study. Blood samples were obtained from the participants following a 12-hour fasting period. Then we evaluated the levels of hemoglobin (Hb), hematocrit (HCT), red cell distribution width (RDW), mean platelet volume (MPV), white blood cell (WBC), PLR, platelet counts, and PCT in the complete blood count. RESULTS The morbid obesity group had significantly higher platelet counts and PCT values (p<0.001), and PLR values (p=0.033). The value of WBC was also higher in the obese group (p=0.001). MPV was lower in the obesity group but not statistically significant (p=0.815). No significant difference was found between hemoglobin and hematocrit values in these groups; but RDW valuewere higher and statistically significant in the obese group (p=0.001). CONCLUSION PLR or PCT may be more useful as a marker in determining an increased thrombotic state and inflammatory response in morbid obesity.

RESUMO OBJETIVO Comparar as contagens sanguíneas completas, nomeadamente o plateletcrit (PCT) e a razão plaquetas/linfócitos (PPL) de indivíduos saudáveis com aqueles que têm obesidade mórbida na população jovem. MÉTODOS Incluímos 45 pacientes com obesidade mórbida (índice de massa corporal superior a 45 kg/m2) e 45 indivíduos saudáveis (índice de massa corporal inferior a 25 kg/m2) em nosso estudo. Foram obtidas amostras de sangue dos participantes após um período de jejum de 12 horas. Depois, avaliamos os níveis de hemoglobina, hematócrito, largura de distribuição dos glóbulos vermelhos, volume médio de plaquetas, glóbulos brancos, razão plaquetas/linfócitos, contagem de plaquetas e plateletcrit no hemograma completo. RESULTADOS O grupo de obesidade mórbida teve contagens plaquetárias e valores plateletcrit significativamente mais elevados (p<0, 001), e valores razão plaquetas/linfócitos (p=0, 033). O valor dos glóbulos brancos também foi maior no grupo obeso (p=0, 001). O volume médio dos plateletes foi inferior no grupo da obesidade, mas não estatisticamente significativo (p=0, 815). Não foi encontrada diferença significativa entre os valores de hemoglobina e hematócrito nesses grupos, mas os valores da largura de distribuição dos glóbulos vermelhos foram mais elevados no grupo obeso e estatisticamente significativos (p=0, 001). CONCLUSÃO Relação plaquetas-linfócitos e valores de plateletcrit podem ser mais úteis como marcadores na determinação de um estado trombótico aumentado e da resposta inflamatória na obesidade mórbida.

Lipid Metabolism and Signaling in Platelet Function.

Modern society has changed its diet composition, transitioning to a higher intake of saturated fat with a 50% increase of cardiovascular risk (CVD). Within the context of increased CVD, there is an induction of a prothrombotic phenotype mainly due to increased platelet reactivity as well as decreased platelet response to inhibitors. Platelets maintain haemostasis through both blood components and endothelial cells that secrete inhibitory or stimulatory molecules to regulate thrombus formation. There exist a correlation between platelets' polyunsaturated fatty acid (PUFA) and the increase in platelet reactivity. The aim of this chapter is to review the metabolism of the main PUFAs involved in platelet function associated with the role that their enzyme-derived oxidized metabolites exert in platelet function and fate. Finally, how lipid metabolism in the organism affect platelet aggregation and activation and the pharmacological modulation of these processes will also be discussed.

Infused factor VIII-expressing platelets or megakaryocytes as a novel therapeutic strategy for hemophilia A.

B-domainless factor VIII (FVIII) ectopically expressed in megakaryocytes (MKs) is stored in α granules of platelets (pFVIII) and is capable of restoring hemostasis in FVIIInull mice, even in the presence of circulating inhibitors. However, our prior studies have shown that this ectopically expressed pFVIII can injure developing MKs. Moreover, the known risks of prolonged thrombocytopenia after bone marrow transplantation are significant challenges to the use of this strategy to treat individuals with severe hemophilia A and particularly those with intractable clinically relevant inhibitors. Because of these limitations, we now propose the alternative therapeutic pFVIII strategy of infusing pFVIII-expressing MKs or platelets derived from induced pluripotent stem cells (iPSCs). pFVIII-expressing iPSC-derived MKs, termed iMKs, release platelets that can contribute to improved hemostasis in problematic inhibitor patients with hemophilia A. As proof of principle, we demonstrate that hemostasis can be achieved in vitro and in vivo with pFVIII-expressing platelets and show prolonged efficacy. Notably, pFVIII-expressing platelets are also effective in the presence of inhibitors, and their effect was enhanced with recombinant FVIIa. Human pFVIII-expressing iMKs improved hemostasis in vitro, and derived platelets from infused human pFVIII-expressing iMKs improved hemostasis in FVIIInull mice. These studies indicate the potential therapeutic use of recurrent pFVIII-expressing MK or platelet infusions with prolonged hemostatic coverage that may be additive with bypassing agents in hemophilia A patients with neutralizing inhibitors.

The signaling effects of ATP on melanoma-like skin cancer.

Melanoma is a type of skin cancer originated by the malignant transformation of melanocytes. Increasing incidence and mortality require efforts focused on studies and research about this cancer. Its microenvironment is rich in extracellular ATP, but there are no studies evaluating the ectonucleotidases and ATP effects on tumor-derived melanoma cells with known amounts of ATP. This way, the objective of this work was to evaluate the purinergic signaling in the pathophysiology of in vivo melanoma and the in vitro effects of ATP signaling. We found increased and effective extracellular ATP hydrolysis in platelets and a significant decrease of extracellular ATP levels and adenosine hydrolysis. In addition, we cultured PBMCs of melanoma patients and used ATP salt with specific concentrations to evaluate its signaling effects. The enzymatic activity analysis revealed that even with higher ATP doses cells metabolize adenine nucleotides less efficiently, and present low ATP, ADP and AMP hydrolytic activity in CM compared to CT cells. In summary, we showed for the first time important data about the purinergic signaling in the pathophysiology of melanoma and ATP signaling exercising immunosuppressive effects. Therefore, as already shown for other tumors, the purinergic signaling should be considered a potential target for melanoma management and treatment and could offer novel therapeutic prospects.

Platelets inhibit apoptotic lung epithelial cell death and protect mice against infection-induced lung injury.

Thrombocytopenia is associated with worse outcomes in patients with acute respiratory distress syndrome, which is most commonly caused by infection and marked by alveolar-capillary barrier disruption. However, the mechanisms by which platelets protect the lung alveolar-capillary barrier during infectious injury remain unclear. We found that natively thrombocytopenic Mpl -/- mice deficient in the thrombopoietin receptor sustain severe lung injury marked by alveolar barrier disruption and hemorrhagic pneumonia with early mortality following acute intrapulmonary Pseudomonas aeruginosa (PA) infection; barrier disruption was attenuated by platelet reconstitution. Although PA infection was associated with a brisk neutrophil influx, depletion of airspace neutrophils failed to substantially mitigate PA-triggered alveolar barrier disruption in Mpl -/- mice. Rather, PA cell-free supernatant was sufficient to induce lung epithelial cell apoptosis in vitro and in vivo and alveolar barrier disruption in both platelet-depleted mice and Mpl -/- mice in vivo. Cell-free supernatant from PA with genetic deletion of the type 2 secretion system, but not the type 3 secretion system, mitigated lung epithelial cell death in vitro and lung injury in Mpl -/- mice. Moreover, platelet releasates reduced poly (ADP ribose) polymerase cleavage and lung injury in Mpl -/- mice, and boiling of platelet releasates, but not apyrase treatment, abrogated PA supernatant-induced lung epithelial cell cytotoxicity in vitro. These findings indicate that while neutrophil airspace influx does not potentiate infectious lung injury in the thrombocytopenic host, platelets and their factors protect against severe pulmonary complications from pathogen-secreted virulence factors that promote host cell death even in the absence of overt infection.

Injectable platelet rich fibrin: cell content, morphological, and protein characterization.

OBJECTIVES: The aim of the present study was to evaluate the blood cell content, morphological aspects, gene expression of type I collagen, and release of growth factors on an injectable platelet rich fibrin (i-PRF). MATERIALS AND METHODS: Blood samples were collected from 15 volunteers to prepare i-PRF samples. Peripheral blood was used as a control group. Blood clot and i-PRF samples were cultured for 10 days. The supernatant of the samples was collected for ELISA immunoassay quantification of PDGF and VEGF growth factors over periods of 1, 8, 24, 72, and 240 h. I-PRF and blood clot samples were biologically characterized using histological and immunohistochemistry analysis for IL-10, osteocalcin, and TGF-ß. Scanning electron microscopy (SEM) was used to inspect the fibrin network and distribution of blood platelets and leukocytes. Reverse transcriptase polymerase chain reaction (RT-PCR) method was used to evaluate gene expression for type I collagen. RESULTS: A higher concentration of platelets and lymphocytes was recorded in i-PRF than in peripheral blood (p < 0.05). The release of VEGF was higher in blood clot samples (1933 ± 704) than that for i-PRF (852 ± 376; p < 0.001). Immunohistochemistry showed upregulation of TGF-B, IL-10, and osteocalcin in the i-PRF group. RT-PCR showed increased type I collagen gene expression in i-PRF (p < 0.05). SEM images revealed agglomeration of platelets in some regions, while a fibrin networking was noticeable in the entire i-PRF sample. CONCLUSIONS: Injectable platelet rich fibrin becomes a good approach for soft and mineralized tissue healing considering the formation of a three-dimensional fibrin network embedding platelets, leukocytes, type I collagen, osteocalcin, and growth factors. Indeed, the injectable platelet rich fibrin can be indicated in several medical applications regarding bioactivity, simplied technique, and flowable mixing with other biomaterials. CLINICAL RELEVANCE: Morphological, cell, and protein characterization of platelet rich fibrin provides a better understanding of the clinical effects and improvement of clinical guidelines for several medical applications. Once well physicochemical and biologically characterized, the use of an injectable platelet rich fibrin can be extended to other applications in the field of orthopedics, periodontics, and implant dentistry on the repairing process of both soft and mineralized tissues.