The fatal contribution of serine protease-related genetic variants to COVID-19 outcomes.

Introduction: Serine proteases play a critical role during SARS-CoV-2 infection. Therefore, polymorphisms of transmembrane protease serine 2 (TMPRSS2) and serpine family E member 1 (SERPINE1) could help to elucidate the contribution of variability to COVID-19 outcomes. Methods: To evaluate the genetic variants of the genes previously associated with COVID-19 outcomes, we performed a cross-sectional study in which 1536 SARS-CoV-2-positive participants were enrolled. TMPRSS2 (rs2070788, rs75603675, rs12329760) and SERPINE1 (rs2227631, rs2227667, rs2070682, rs2227692) were genotyped using the Open Array Platform. The association of polymorphisms with disease outcomes was determined by logistic regression analysis adjusted for covariates (age, sex, hypertension, type 2 diabetes, and obesity). Results: According to our codominant model, the GA genotype of rs2227667 (OR=0.55; 95% CI = 0.36-0.84; p=0.006) and the AG genotype of rs2227667 (OR=0.59; 95% CI = 0.38-0.91; p=0.02) of SERPINE1 played a protective role against disease. However, the rs2227692 T allele and TT genotype SERPINE1 (OR=1.45; 95% CI = 1.11-1.91; p=0.006; OR=2.08; 95% CI = 1.22-3.57; p=0.007; respectively) were associated with a decreased risk of death. Similarly, the rs75603675 AA genotype TMPRSS2 had an OR of 1.97 (95% CI = 1.07-3.6; p=0.03) for deceased patients. Finally, the rs2227692 T allele SERPINE1 was associated with increased D-dimer levels (OR=1.24; 95% CI = 1.03-1.48; p=0.02). Discussion: Our data suggest that the rs75603675 TMPRSS2 and rs2227692 SERPINE1 polymorphisms are associated with a poor outcome. Additionally, rs2227692 SERPINE1 could participate in hypercoagulable conditions in critical COVID-19 patients, and this genetic variant could contribute to the identification of new pharmacological targets and treatment strategies to block the inhibition of TMPRSS2 entry into SARS-CoV-2.

Distinguishing Plasmin-Generating Microvesicles: Tiny Messengers Involved in Fibrinolysis and Proteolysis.

A number of stressors and inflammatory mediators (cytokines, proteases, oxidative stress mediators) released during inflammation or ischemia stimulate and activate cells in blood, the vessel wall or tissues. The most well-known functional and phenotypic responses of activated cells are (1) the immediate expression and/or release of stored or newly synthesized bioactive molecules, and (2) membrane blebbing followed by release of microvesicles. An ultimate response, namely the formation of extracellular traps by neutrophils (NETs), is outside the scope of this work. The main objective of this article is to provide an overview on the mechanism of plasminogen reception and activation at the surface of cell-derived microvesicles, new actors in fibrinolysis and proteolysis. The role of microvesicle-bound plasmin in pathological settings involving inflammation, atherosclerosis, angiogenesis, and tumour growth, remains to be investigated. Further studies are necessary to determine if profibrinolytic microvesicles are involved in a finely regulated equilibrium with pro-coagulant microvesicles, which ensures a balanced haemostasis, leading to the maintenance of vascular patency.

Circulating Levels of Urokinase-Type Plasminogen Activator Receptor and D-Dimer in Patients With Hematological Malignancies.

BACKGROUND: Patients with cancer exhibit changes in their hemostatic mechanisms. The D-dimer (D-D) is the most important subproduct of fibrinolysis, and urokinase plasminogen activator receptor (uPAR) is related to invasiveness and metastases, and is overexpressed in neoplastic cells. The objective of this study was to identify in patients with hematological neoplasia, the serum levels of uPAR and D-D, and to determine their effects on outcome. PATIENTS AND METHODS: A cross-sectional study was performed. Clinical and demographic data were obtained from the clinical chart. Determination of uPAR in serum (pg/L) was performed using an enzyme-linked immunosorbent assay, and D-D (µg/dL) using nephelometry. RESULTS: We included 42 patients (35 with lymphomas). Statistically significant differences were found in D-D (P < .001) and uPAR (P < .01) between patients and control participants. Response was an accumulated clinical outcome. We observed statistical differences between groups (P < .001). D-D was positive in 70% of cases. CONCLUSION: We found differences in D-D serum levels and soluble uPAR between control participants and patients with lymphoma. These results indicate that D-D serum levels and soluble uPAR should be considered biomarkers of response and survival in patients with lymphoma.

Reduction of no-reflow and reperfusion injury with the synthetic 17ß-aminoestrogen compound Prolame is associated with PI3K/Akt/eNOS signaling cascade.

A high proportion of primary percutaneous coronary interventions performed in the setting of acute myocardial infarction, concur with inadequate myocardial perfusion at the microvascular level. This phenomenon, known as "no-reflow" contributes to reperfusion injury, poor prognosis and to unfavorable clinical outcome. In this study, we evaluated the hypothesis that the synthetic 17ß-aminoestrogen Prolame, may confer cardioprotection and prevent against no-reflow. In an open-chest model of 30-min ischemia and 90-min reperfusion, male Wistar rats were randomly assigned to different groups: Control, Prolame, Prolame followed by the nitric oxide synthase inhibitor (L-NAME), and 17ß-estradiol. Areas of risk, infarct size and no-reflow were determined by planimetry with triphenyltetrazolium chloride and thioflavin-S stains. Structural damage of the vasculature was measured as capillary compression in clarified tissue after intra-atrial injection of Microfil. Hemodynamic function was obtained at the end of stabilization, ischemia and reperfusion; nitric oxide (NO·) content was determined indirectly using the Griess reaction. Activation of the eNOS signaling cascade was determined by western blot. Prolame reduced the infarcted area, decreased the zones of no-reflow and capillary compression by activating the PI3K/Akt/eNOS signaling pathway in correlation with NO· increase. Prolame also activated endothelial cells augmenting NO· production, which was inhibited by ICI182780 (a selective estrogen receptor down-regulator), supporting the notion that the cardioprotective effect of Prolame involves the preservation of endothelium through the activation of estrogen receptor downstream signaling. Our results provide evidence that Prolame has potential therapeutic application in patients with AMI, as it prevents from both vascular and cardiac tissue damage.

Distribution of ABCB1, CYP3A5, CYP2C19, and P2RY12 gene polymorphisms in a Mexican Mestizos population.

The aim of the present study was to establish the gene frequency of six polymorphisms of the ABCB1, CYP3A5, CYP2C19, and P2RY12 genes in a population resident of Mexico City. The proteins encoded by these genes have been associated with the absorption, and biotransformation of clopidogrel. The ABCB1 T3435C, CYP3A5 V3 A6986G, P2RY12 G52T, P2RY12 C34T, CYP2C19 V2 and V3 (positions G681A and G636A, respectively), polymorphisms were analyzed by 5' exonuclease TaqMan genotyping assays in a group of 269 healthy unrelated Mexican Mestizo individuals. The CYP2C19 V3 G636A polymorphism was not detected in the Mexican Mestizos population. However, the studied population presented significant differences (P < 0.05) in the distribution of the T3435C, A6986G, G681A, G52T and C34T polymorphisms when compared to reported frequencies of Amerindian of South America, Caucasian, Asian, and African populations. In summary, the distribution of the ABCB1, CYP3A5, CYP2C19, and P2RY12 gene polymorphisms distinguishes to the Mexican Mestizos population from other ethnic groups.

Mifepristone (RU 486) induces vasodilation and inhibits platelet aggregation: nongenomic and genomic action to cause hemorrhage.

BACKGROUND: The regimen mifepristone/misoprostol is an established and highly effective method for early termination of pregnancy. However, its side effects such as a significantly long bleeding time and hemorrhage have been scantly studied. STUDY DESIGN: Human umbilical artery (HUA) from pregnant women undergoing elective cesarean section at term and rat thoracic aorta (RTA) were isometrically recorded. The vasorelaxing effect of mifepristone was analyzed on the contractile responses induced by KCl or serotonin (5-HT); moreover, the potential response of mifepristone on adenosine diphosphate (ADP)-induced human platelet aggregation was also evaluated. RESULTS: This study describes that mifepristone elicits (1) rapid and reversible vasorelaxation on KCl- or 5-HT-induced contraction in HUA and RTA with and without endothelium and (2) immediate prevention of ADP-induced human platelet aggregation. CONCLUSIONS: These effects seem to be responsible for increased and prolonged hemorrhage. Since mifepristone-prevented platelet aggregation was observed in the anucleate platelets, and mifepristone-induced vasorelaxation remained unaffected in de-endothelized tissues, by inhibitors of transcription and translation and a nitric oxide (NO) synthase inhibitor, a nongenomic endothelium- and NO-independent mechanism was revealed. Additionally, the results indicated a blockade of voltage- and receptor-operated calcium channels. The antiglucocorticoid genomic action of mifepristone, by inducing an excess of NO, may also contribute to exacerbated hemorrhage.

The antithrombotic effect of the aminoestrogen prolame (N-(3-hydroxy-1,3,5(10)-estratrien-17B-YL)-3-hydroxypropylamine) is linked to an increase in nitric oxide production by platelets and endothelial cells.

OBJECTIVE: Women under hormone replacement therapy carry an increased risk of venous thromboembolism (VTE), mostly during the first year. Despite great efforts devoted to hormone therapy research, VTE remains a major drawback of estrogenic therapy, and the search for new compounds continues. We have synthesized and evaluated prolame, an aminoestrogen with anticoagulant properties. The aim of our work was to elucidate the anticoagulant mechanism of prolame. METHODS: We studied the effects of prolame on nitric oxide (NO) synthesis in cultured endothelial cells and platelets using flow cytometry, on NO metabolites using a modified Griess method, on NO formation in vivo using electron paramagnetic resonance spectroscopy, on participation of nuclear estrogen receptors using flow cytometry, and on endothelial NO synthase (eNOS) mRNA expression using RT-PCR. We also studied the impact of prolame-treated endothelial cells (EC) on ADP-induced platelet aggregation, as well as the ability to prevent occlusive thrombi in an in vivo mice thrombosis model. RESULTS: (a) Prolame induces NO production in ECs, platelets, and in a mouse model in vivo. (b) The NO-elevating effect of prolame can only be partially attributed to the nuclear estrogen receptors (ERs) since endothelial nitric oxide synthase (e-NOS) is slightly induced (37%) in ECs treated with prolame. (c) Platelets become 60% less responsive to aggregation induced by 10muM ADP when in suspension with prolame-treated ECs. (d) Prolame reduces the formation of thrombi in an in vivo thrombosis model. CONCLUSIONS: Prolame could be a preferred alternative to other estrogens because of its reduced thromboembolic risk.

[Pro-inflammatory and anti-inflammatory markers in coronary artery disease and acute ischemic coronary syndrome]. / Marcadores pro y antiinflamatorios en la enfermedad arterial coronaria y el síndrome isquémico coronario agudo.

Inflammation plays an important role in the development of atherosclerotic lesions, affecting several stages of the atheroma's development going from the initial leukocyte recruitment to the eventual rupture of the unstable atherosclerotic plaque. The inflammatory reactions within coronary atherosclerotic plaques influence the clinical outcome of acute coronary syndromes and coronary artery disease. Recent studies suggest that inflammation markers may reflect different aspects of the atherothrombotic process in relation to the stages of acute coronary syndrome. These markers play an important role in the risk of developing coronary artery disease, and may correlate with its severity. Some cytokines, acute phase proteins, acute phase reactants proteins, and adhesion molecules released from the inflammatory cells may reflect the inflammatory process in atherosclerotic plaques. However, it remains to be determined whether these pro- and anti-inflammation markers may confer risk or protection for cardiovascular disease, or simply reflect the underlying disease process. The analysis of the markers may be useful for the development of new strategies for coronary disease prevention and treatment. Therefore, we need a well-designed evaluation of these markers before their use in the clinical practice.

Platelet aggregation in whole blood, a new approach for understanding the antiplatelet effect of N-(3-hydroxy-1,3,5(10)-estratrien-17b-yl) butylamine (buame).

We have previously reported the effect of a compound derived from estradiol containing a radical amino butyl at the 17-beta position which has shown anticoagulant effects in whole blood and antiplatelet effects in light transmission aggregometry where platelets are isolated from other blood cells. In contrast, whole blood aggregometry includes the platelet interactions with blood elements such as erythrocytes and leukocytes. We examined the cooperative effect between leukocytes, erythrocytes and platelets and the antiplatelet effect of Buame in whole blood aggregometry, a tool to assess platelet function in its physiological environment. Buame (5-500 microM) dissolved in DMSO was tested in platelet aggregation induced by ADP (1.25 microM) or collagen (1 microg/mL) and the response recorded over 5 min. Controls were run with DMSO and the average control aggregation was taken as 100%. Results were obtained in both whole blood and platelet aggregometry. Buame was able to inhibit the secondary aggregation induced with ADP suggesting impairment in thromboxane A2 production. Also the first and second aggregation phases were inhibited when collagen-induced platelet activation was employed. This concentration-dependent pattern was shown in both whole blood and platelet aggregometry assays. When tested in light transmission aggregometry, a higher concentration of Buame was required in order to inhibit to the same degree ADP- or collagen-induced platelet aggregation (30 microM ,114 microM) than that required in the whole blood assay (IC50 84 microM, 191 microM). Interactions among different cell types in whole blood may modify the response of Buame-treated platelets to agonists suggesting a cooperative mechanism.

Palmitic acid in HDL is associated to low apo A-I fractional catabolic rates in vivo.

BACKGROUND: HDL becomes enriched with non-esterified fatty acids (NEFAs) in some pathologies, such as nephrotic syndrome, as well as after aerobic exercise. However, little is known about the impact of NEFAs on HDL metabolism. We investigated the effects of one NEFA, the palmitic acid, on HDL structure and catabolism. METHODS: HDL enrichment with palmitic acid (HDLPal) was performed by fusing phosphatidyl choline small unilamellar vesicles containing the NEFA with human HDL isolated from a pool of 5 normolipidemic plasma. HDL enriched only with phosphatidyl choline (HDLPhl) and native HDL (HDLCtrl) were included as controls. RESULTS: As expected, HDLPal surface charge density was higher than HDLPhl and HDLCtrl (2014.4+/-164.8 vs. 1682.7+/-149.5 and 1758.2+/-124.3-esu/cm2, respectively, p<0.05). Both, HDLPal and HDLPhl were better substrates for cholesteryl esters transfer protein (CETP) than HDLCtrl (% of transfer, 13.02+/-3.8 and 12.7+/-4.5 vs. 7.8+/-2.7% in 16 h, respectively, p<0.05). HDLPal apo A-I catabolism in vivo, as performed in New Zealand white rabbits by exogenous radiolabeling, was markedly lower than that of HDLPhl and HDLCtrl (fractional catabolic rate, 0.019+/-0.008 vs. 0.030+/-0.005 and 0.047+/-0.003 h-1, respectively, p<0.001), suggesting that negative charge is inversely related to HDL-apo A-I catabolism. CONCLUSIONS: Enrichment with palmitic acid increases the negative electric charge of HDL at physiological pH, contributes to decrease their catabolism, and is associated to an enhanced lipid transfer by CETP that has been related to the atherogenic process.

Beta1-adrenergic receptor gene polymorphisms in Mexican patients with idiopathic dilated cardiomyopathy.

The objective of the study was to evaluate the role of beta1-adrenergic receptor gene polymorphisms (Ser49Gly and Arg389Gly) as susceptibility markers for idiopathic dilated cardiomyopathy (IDC) in Mexican patients. The polymorphisms were analyzed in 47 patients with IDC and 93 ethnically matched healthy controls by polymerase chain reaction-restriction fragment length polymorphism. The Ser49Gly allele and genotype frequencies were similar in patients and healthy controls. On the other hand, the analysis of the Arg389Gly polymorphism showed an increased frequencies of the *Gly allele (pC = 0.022, OR = 2.16) and *Arg/*Gly genotype (pC = 0.027, OR = 2.70) in the group of IDC patients when compared to healthy controls. The data suggest that Arg389Gly polymorphism could be involved in the genetic susceptibility to develop IDC in Mexicans.

Cytochrome P4501A1 polymorphisms in the Amerindian and Mestizo populations of Mexico.

Several polymorphisms in the CYP1A1 locus have been identified and their genotypes appear to exhibit population frequencies that depend on ethnicity. We studied two CYP1A1 polymorphic sites (position 4889 and 6235) in a group of 212 unrelated healthy individuals belonging to three different Mexican populations (106 Mexican Mestizos, 52 Teenek and 54 Mayos). Comparison among Mexican populations showed increased frequency of the *Ile allele (A on position 4889) in Mexican Mestizos when compared to Amerindians (p < 0.05). The analysis of position 6235 showed increased frequencies of *m2 (C in this position) allele in Teenek when compared to Mestizos and Mayos (p < 0.05) and of *m2/*m2 genotype when compared to Mestizos (p < 0.05). Amerindian populations (from Mexico and South America) presented the lowest frequencies of *Ile (position 4889) and *m1 (position 6235) alleles, however these frequencies vary according to the ethnic group studied. Mexican Amerindian groups together with other South Amerindian populations showed the highest frequencies for *Val at position 4889 and the *m2 allele at position 6235. The present study corroborates the high frequencies of*Val and *m2 alleles in the Amerindian populations and detects some differences between Mexican populations that correlate with linguistic differences. Our data could be helpful in understanding the distribution of these polymorphisms and in clarifying their roles as genetic and evolution markers in Amerindian populations.

Intracellular Ca2+ stimulates the binding to androgen receptors in platelets.

In this study, we demonstrated that ADP-induced platelet aggregation activates the binding of testosterone (T) to its receptor. It is well known that binding of ADP to its receptors induced the release of Ca2+ ions from dense bodies into the cytosol of platelets. In this work, we compared the binding of testosterone or dihydrotestosterone to their receptors using cytosol obtained from ADP-treated and non-treated platelets. These experiments were repeated using EGTA (a calcium chelator) or U73122 (a phospholipase C enzymatic activity inhibitor) to the ADP-treated platelets. In addition, we also developed a competition analysis for the androgen receptors (AR) using [3H]DHT, non-radioactive T, DHT or cyproterone acetate from ADP-treated platelets cytosol. The results from this study indicate that the cytosol obtained from non-ADP-treated platelets did not show any binding to [3H]T or [3H]DHT, whereas cytosol from ADP-treated platelets binds to the radio-labeled androgens. Furthermore cytosol from ADP plus U73122-treated platelets did not show binding to [3H]T or [3H]DHT. These data suggest that intracellular Ca2+ ions stimulates the binding of androgens to their receptors in platelets cytosol. The competition analysis shows that T and DHT have high affinities for the androgen receptors with similar IC50 values, whereas cyproterone acetate shows a lower affinity. The results from these data clearly indicate the presence of androgen receptors in platelets.

Increased fibrin polymerization rate in patients with primary antiphospholipid syndrome and systemic lupus erythematosus.

The main event in blood coagulation is the thrombincatalyzed conversion of fibrinogen into fibrin. This singular transformation of a soluble protein into an insoluble polymeric network occurs with faultless precision. Abnormalities of fibrin polymerization can lead to hemorragic and thrombotic disorders. Increased fibrinogen plasma concentration (Fg) and fibrin polymerization rate (FPR) could be additional risk factors associated with atherothrombosis in antiphospholipid syndrome (APS) and in systemic lupus erythematosus (SLE). Our objective was to investigate Fg and FPR in consecutive patients with APS and SLE. Thirty-nine patients and 31 age- and gender-matched healthy controls were studied. Sixteen patients had primary APS, 13 patients had SLE, and 10 patients had SLE plus APS. The mean of the FPR was significantly increased (0.2799 +/- 0.091) in patients with APS plus SLE as compared with the control group (0.2052 +/- 0.055) (p < 0.05). Fg was higher in APS plus SLE (3.15 g/L +/- 0.43) and in primary APS (3.03 g/L +/- 0.29) than in controls (2.87 g/L +/- 0.49). Our results demonstrated an increased FPR in patients with APS plus SLE. This phenomenon could be an additional risk factor for thrombosis in these autoimmune diseases.

Eficacia del clopidogrel como inhibidor de la agregación plaquetaria dependiente de ADP. un estudio en individuos con enfermedad arteral coronaria / Eficacy of clopidogrel as inhibitor of ADP-Induced platelet aggregation. A study in patients with coronary artery disease

El ácido acetil-salicílico, al inhibir la producción de tromboxano A2, reduce entre 20 y 25 por ciento el riesgo de eventos oclusivos arteriales. La ticlopidina inhibe la agregación plaquetaria dependiente de adenosín-difosfato, y reduce el mismo riesgo en 30 a 35 por ciento, pero tiene efectos indeseables. Su derivado, el clopidogrel, tiene el mismo mecanismo de acción y disminuye indirectamente la expresión del receptor de fibrinógeno en las plaquetas. Conserva la eficacia clínica de la ticlopidina, con menor frecuencia de efectos adversos. En este estudio, evaluamos la acción de 75 mg diarios de clopidogrel sobre la función plaquetaria de 33 individuos con enfermedad arterial coronaria; se les practicó agregometría plaquetaria inducida con adenosín-difosfato 5 mM y colágena 20 mg/mL, tiempo de hemorragia y fibrinógeno, antes del tratamiento y a las semanas 6 y 12. La agregación plaquetaria inducida con adenosín-difosfato fue de 90.7 por ciento ñ 13.2, 54.6 por ciento ñ 23.2 y 49.2 por ciento ñ 23.7 en las muestras basal y a las semanas 6 y 12, lo que representó una reducción significativa de 38.6 por ciento y 44.4 por ciento. La agregación plaquetaria inducida con colágena no disminuyó significativamente. El tiempo de hemorragia se prolongó de 4.1 ñ 1.6 a 15.43 ñ 13.1 y 14.6 ñ 14.4 minutos (3.7-3.5 veces). No se observaron modificaciones en la concentración de fibrinógeno. No se presentaron complicaciones hemorrágicas. Ocurrieron molestias digestivas con una frecuencia menor al 3 por ciento. Se concluye que el clopidogrel reduce eficazmente la agregación plaquetaria dependiente de adenosin-difosfato, y prolonga el tiempo de hemorragia. El perfil de seguridad clínico y de laboratorio son adecuados.