Targeted genotyping of COVID-19 patients reveals a signature of complement C3 and factor B coding SNPs associated with severe infection.

We have attempted to explore further the involvement of complement components in the host COVID-19 (Coronavirus disease-19) immune responses by targeted genotyping of COVID-19 adult patients and analysis for missense coding Single Nucleotide Polymorphisms (coding SNPs) of genes encoding Alternative pathway (AP) components. We have identified a small group of common coding SNPs in Survivors and Deceased individuals, present in either relatively similar frequencies (CFH and CFI SNPs) or with stark differences in their relative abundance (C3 and CFB SNPs). In addition, we have identified several sporadic, potentially protective, coding SNPs of C3, CFB, CFD, CFH, CFHR1 and CFI in Survivors. No coding SNPs were detected for CD46 and CD55. Our demographic analysis indicated that the C3 rs1047286 or rs2230199 coding SNPs were present in 60 % of all the Deceased patients (n = 25) (the rs2230199 in 67 % of all Deceased Males) and in 31 % of all the Survivors (n = 105, p = 0.012) (the rs2230199 in 25 % of all Survivor Males). When we analysed these two major study groups using the presence of the C3 rs1047286 or rs2230199 SNPs as potential biomarkers, we noticed the complete absence of the protective CFB rs12614 and rs641153 coding SNPs from Deceased Males compared to Females (p = 0.0023). We propose that in these individuals, C3 carrying the R102G and CFB lacking the R32W or the R32Q amino acid substitutions, may contribute to enhanced association dynamics of the C3bBb AP pre-convertase complex assembly, thus enabling the exploitation of the activation of the Complement Alternative pathway (AP) by SARS-CoV-2.

Platelet, Fibrinolytic and Other Coagulation Abnormalities in Newly-Diagnosed Patients with Chronic Thromboembolic Pulmonary Hypertension.

The pathophysiological background of chronic thromboembolic pulmonary hypertension (CTEPH) has not been fully elucidated. Evidence suggests that abnormal platelet function and ineffective fibrinolysis may play a key role in the development of the disease. The purpose of this study was to evaluate platelet and coagulation function in CTEPH, using non-conventional global coagulation assays, and platelet activation and endothelial dysfunction laboratory markers. A total of 40 newly-diagnosed CTEPH patients were studied, along with 35 healthy controls. Blood samples from CTEPH patients were taken directly from the pulmonary artery. All subjects were assessed with platelet function analyzer-100, light transmission aggregometry, thromboelastometry, endogenous thrombin potential. von Willebrand antigen and activity, p-selectin, thromboxane A2 and serotonin levels were also assessed. The results showed that CTEPH patients present diminished platelet aggregation, presence of disaggregation, decreased rate of fibrinolysis, defective thrombin generation and increased levels of thromboxane A2, p-selectin, von Willebrand antigen and activity. Serotonin levels did not present any differences between the two groups. The results of this study suggest that CTEPH patients present platelet function, fibrinolytic, thrombin generation and other clot formation abnormalities. Well-designed clinical studies are needed to further evaluate the complex hemostatic abnormalities in the CTEPH setting and assess their potential clinical applications.

Genetic prediction of ICU hospitalization and mortality in COVID-19 patients using artificial neural networks.

There is an unmet need of models for early prediction of morbidity and mortality of Coronavirus disease-19 (COVID-19). We aimed to a) identify complement-related genetic variants associated with the clinical outcomes of ICU hospitalization and death, b) develop an artificial neural network (ANN) predicting these outcomes and c) validate whether complement-related variants are associated with an impaired complement phenotype. We prospectively recruited consecutive adult patients of Caucasian origin, hospitalized due to COVID-19. Through targeted next-generation sequencing, we identified variants in complement factor H/CFH, CFB, CFH-related, CFD, CD55, C3, C5, CFI, CD46, thrombomodulin/THBD, and A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS13). Among 381 variants in 133 patients, we identified 5 critical variants associated with severe COVID-19: rs2547438 (C3), rs2250656 (C3), rs1042580 (THBD), rs800292 (CFH) and rs414628 (CFHR1). Using age, gender and presence or absence of each variant, we developed an ANN predicting morbidity and mortality in 89.47% of the examined population. Furthermore, THBD and C3a levels were significantly increased in severe COVID-19 patients and those harbouring relevant variants. Thus, we reveal for the first time an ANN accurately predicting ICU hospitalization and death in COVID-19 patients, based on genetic variants in complement genes, age and gender. Importantly, we confirm that genetic dysregulation is associated with impaired complement phenotype.

Haemostatic profile of riboflavin-treated apheresis platelet concentrates.

BACKGROUND: The haemostatic activity of platelet concentrates (PCs) treated with pathogen reduction technology (PRT) remains a subject of debate. Our aim was to investigate the effect of Mirasol PRT on the haemostatic properties of PCs stored in plasma. MATERIAL AND METHODS: Untreated and Mirasol-treated platelets stored in plasma and derived from ten split double-dose apheresis PCs were evaluated in vitro on days 1, 3 and 5 post collection for functionality, microparticle procoagulation activity (MPA), endogenous thrombin potential (ETP), and haemostatic profile using rotational thromboelastometry (ROTEM). RESULTS: P-selectin expression was significantly higher in Mirasol-treated platelets compared with untreated counterparts on days 3 and 5 (p=0.003 and p=0.002, respectively). Clot strength, as shown by EXTEM maximum clot firmness (MCF), was significantly lower in the Mirasol-treated platelets at all time points (days 1, 3, 5) than in untreated platelets (p=0.009, p<0.001, p<0.001, respectively). There was a considerable increase in MPA over time (p<0.001) and this was significantly higher in the Mirasol-treated platelets on day 5 (p=0.015). A notable acceleration of decrease in ETP values was observed for Mirasol-treated PCs over time (p<0.001), with significant differences between PRT-treated and untreated PCs on days 3 and 5 (p=0.038 and p=0.019, respectively). Clot strength attenuation was significantly associated with pH reduction (p<0.001, Spearman's rho: 0.84), increased microparticle procoagulant activity (p<0.001, Spearman's rho: -0.75), and with decreased ETP (p<0.032, Spearman's rho: 0.41). DISCUSSION: Increased platelet activation induced by PRT treatment leads to a decrease in in vitro haemostatic capacity as seen by reduced clot strength and thrombin generation capacity over time. The clinical relevance of this needs to be investigated.

Genetic justification of severe COVID-19 using a rigorous algorithm.

Recent studies suggest excessive complement activation in severe coronavirus disease-19 (COVID-19). The latter shares common characteristics with complement-mediated thrombotic microangiopathy (TMA). We hypothesized that genetic susceptibility would be evident in patients with severe COVID-19 (similar to TMA) and associated with disease severity. We analyzed genetic and clinical data from 97 patients hospitalized for COVID-19. Through targeted next-generation-sequencing we found an ADAMTS13 variant in 49 patients, along with two risk factor variants (C3, 21 patients; CFH,34 patients). 31 (32%) patients had a combination of these, which was independently associated with ICU hospitalization (p = 0.022). Analysis of almost infinite variant combinations showed that patients with rs1042580 in thrombomodulin and without rs800292 in complement factor H did not require ICU hospitalization. We also observed gender differences in ADAMTS13 and complement-related variants. In light of encouraging results by complement inhibitors, our study highlights a patient population that might benefit from early initiation of specific treatment.

The haemostatic profile in critically ill COVID-19 patients receiving therapeutic anticoagulant therapy: An observational study.

Hypercoagulability and thrombosis remain a challenge in severe coronavirus disease 2019 (COVID-19) infections. Our aim is to investigate the hemostatic profile of critically ill COVID-19 patients on therapeutic anticoagulant treatment.Forty one patients were enrolled into the study. We recruited 11 consecutive, COVID-19, patients who received therapeutic anticoagulant treatment on intensive care unit (ICU) admission. Disease severity indexes, biochemical, hematological and haemostatic parameters, endogenous thrombin potential (ETP), plasminogen activator inhibitor-1 (PAI-1) activity and extrinsically activated rotational thromboelastometry assay (EXTEM) were recorded on days 1, 3, 7. We also enrolled 9 ICU non-COVID-19, 21 non-ICU COVID-19 patients and 20 healthy blood donors as control populations.Critically ill COVID-19 patients demonstrated a more hypercoagulable and hypofibrinolytic profile related to those with COVID-19 mild illness, based on EXTEM amplitude at 10 min (A10), maximum clot firmness (MCF) and lysis index at 60 min (LI60) variables (p = 0.020, 0.046 and 0.001, respectively). Similarly, a more hypercoagulable state was detected in COVID-19 ICU patients related to non-COVID-19 ICU patients based on A10 and MCF parameters (p = 0.03 and 0.04, respectively). On the contrary, ETP and EXTEM (clotting time) CT values were similar between patients with severe and mild form of the COVID-19 infection, probably due to anticoagulant treatment given.Critically ill COVID-19 patients showed a hypercoagulable profile despite the therapeutic anticoagulant doses given. Due to the small sample size and the study design, the prognostic role of the hypercoagulability in this clinical setting remains unknown and further research is required in order to be assessed.

Coagulation Profiles of Pulmonary Arterial Hypertension Patients, Assessed by Non-Conventional Hemostatic Tests and Markers of Platelet Activation and Endothelial Dysfunction.

Many pathophysiologic processes of pulmonary arterial hypertension (PAH), namely, excess vasoconstriction, vascular remodeling and in situ thrombosis, involve the coagulation cascade, and more specifically, platelets. The aim of this study was to globally assess coagulation processes in PAH, by using non-conventional hemostatic tests, along with markers of platelet activation and endothelial dysfunction. We studied 44 new PAH patients (22 with idiopathic PAH and 22 with connective tissue disease) and 25 healthy controls. The following tests were performed: platelet function analyzer-100 (PFA-100), light transmission aggregometry (LTA), rotational thromboelastometry (ROTEM), endogenous thrombin potential (ETP), serotonin, thromboxane A2 and p-selectin plasma levels, and von Willebrand antigen (VWF:Ag) and activity (VWF:Ac). Our results showed that PAH patients had diminished platelet aggregation, presence of disaggregation, defective initiation of the clotting process and clot propagation, and diminished thrombin formation capacity. Serotonin, thromboxane A2 and p-selectin levels were increased, and VWF:Ag and VWF:Ac decreased in the same population. The results of this study suggest that the platelets of PAH patients are activated and present functional abnormalities. The procoagulant activity, in general, appears to be impaired probably due to a sustained and prolonged activation of the procoagulant processes. Larger observational studies are warranted to confirm these laboratory findings.

Laboratory Assessment of the Anticoagulant Activity of Apixaban in Patients With Nonvalvular Atrial Fibrillation.

Our aim is to determine the most appropriate laboratory tests, besides anti-factor Xa (anti-FXa) chromogenic assays, to estimate the degree of anticoagulation with apixaban and compare it with that of rivaroxaban in real-world patients. Twenty patients with nonvalvular atrial fibrillation treated with apixaban 5 mg twice daily and 20 patients on rivaroxaban 20 mg once daily were studied. Conventional coagulation tests, thrombin generation assay (TGA), and thromboelastometry (nonactivated TEM [NATEM] assay) were performed in the 40 patients and 20 controls. The anti-FXa chromogenic assays were used to measure apixaban and rivaroxaban plasma levels. The NATEM measurements showed no significant difference between the 2 groups of patients. Concerning TGA, endogenous thrombin potential (ETP) was significantly decreased in patients on rivaroxaban as compared to those treated with apixaban (P < .003). A statistically significant, strong inverse correlation between apixaban plasma concentrations and ETP (P < .001) was observed. Apixaban significantly reduces ETP compared to controls, but to a lesser extent than rivaroxaban. Thrombin generation assay might provide additional information on apixaban exposure, which is required in order to individualize treatment especially for patients with a high bleeding risk. Our findings have to be further investigated in studies with larger sample sizes, in the entire range of apixaban exposure, with other direct oral anticoagulants, and in relation to clinical outcomes.

The effect of four hemostatic gene polymorphisms on the outcome of septic critically ill patients.

Genetic variants of hemostatic factors leading to prothrombotic phenotypes of hypercoagulability and hypofibrinolysis might affect prognosis of septic critically ill patients. Our aim was to evaluate the effect of four hemostatic genetic variants, namely fibrinogen-beta-455G/A, factor XIII (FXIII) V34L, plasminogen activator inhibitor-1 (PAI-1) 4G/5G polymorphisms and factor V Leiden (FVL) mutation on survival of critically ill patients with severe sepsis or septic shock. A prospective, observational study in an 18-bed general ICU included 73 patients with severe sepsis or septic shock. Epidemiological, laboratory data and comorbidities along with severity scores were recorded. Genotyping for fibrinogen-beta-455G/A, FXIII V34L and PAI-1 4G/5G polymorphism and FVL mutation was carried out in all patients. The primary outcomes were the 28-day and the 90-day survival. Age, septic shock, severity indexes, prior steroid use and arterial pH were identified as predictors of the 28-day and 90-day survival in both the univariate and the multivariate models. On the contrary, none of the examined polymorphisms was found to significantly affect either the 28-day or the 90-day survival. Our data suggest that the importance of these hemostatic polymorphisms as predictors of the prognosis of sepsis in critically ill patients is probably very small.

The impact of the PAI-1 4G/5G polymorphism on the outcome of patients with ALI/ARDS.

INTRODUCTION: Increased levels of plasminogen activator inhibitor-1 (PAI-1) have been associated with worse outcome in ALI/ARDS. A single guanosine insertion/deletion (4G/5G) polymorphism in the promoter region of the PAI-1 gene, may play an important role in the regulation of PAI-1 expression. The objective of the study was to evaluate the effect of this polymorphism on the outcome of critically ill patients with ALI/ARDS. MATERIALS AND METHODS: 52 consecutive ventilated patients with ALI/ARDS were studied. Bronchoalveolar lavage was performed within 48 hours from diagnosis. Measurement of plasma and BALF PAI-1 activity and D-dimers levels, and 4G/5G genotyping of PAI-1 were carried out. The primary outcome was 28-day mortality, and secondary outcomes included organ dysfunction and ventilator-free days. RESULTS: 17 patients were homozygotes for the 4G allele. Severity scores were not different between subgroups upon study enrollment. 28-day mortality was 70.6% and 42.9% for the 4G-4G and the non-4G-4G patients, respectively (p=0.06). PAI-1 activity levels and D-dimer in plasma and BALF were not significantly different between the 4G-4G and the non-4G-4G subgroups. In the multivariate analysis, genotype 4G/4G was the only variable independently associated with 28-day mortality (Odds Ratio=9.95, 95% CI: 1.79-55.28, p=0.009). Furthermore, genotype 4G/4G and plasma PAI-1 activity levels were independently negatively associated with ventilator free days (p=0.033 and p=0.008, respectively). CONCLUSIONS: ALI/ARDS patients, homozygous for the 4G allele of the PAI-1 gene, experienced higher 28-day mortality. This genotype was associated with a reduction in the number of days of unassisted ventilation and was inversely associated with the number of days without organ failure.

Monitoring aspirin treatment in patients with thrombocytosis: comparison of the platelet function analyzer (PFA)-100 with optical aggregometry.

Aspirin provides satisfactory protection against thrombotic episodes in essential thrombocythemia (ET), but at higher platelet counts has been less effective. Our aim was to compare the platelet function analyzer (PFA)-100 with optical aggregometry in order to determine a reliable method in monitoring aspirin's influence on platelet function in patients with thrombocytosis. We studied 36 patients with thrombocytosis. Sixteen of them, receiving aspirin, composed group A, while group B consisted of 20 patients not taking aspirin. In all patients, we compared the platelet function measured by classic optical aggregation tests with closure times (CT) obtained by the PFA-100. The definition of platelet responses as normal or pathological showed that PFA-100 collagen and/or epinephrine (CEPI) CTs and epinephrine-induced aggregometry is the pair of methods with the higher agreement in monitoring of platelet dysfunction due to ASA treatment (a=94%). Satisfactory results were also obtained for group B (a=81%). The comparison between PFA-100 CEPI CTs and arachidonic acid-induced aggregometry exhibited moderate agreement both in the total number of patients and in group A (a=79% and 94%, respectively). PFA-100 collagen and/or ADP (CADP) CTs and ADP-induced aggregometry were not concordant. The PFA-100 system appears to be a reliable and rapid method in the assessment of aspirin's antiplatelet effect in patients with thrombocytosis. Regarding aggregometry, the selection of the inducer, its concentration and cut-off points is crucial in defining the response to antiaggregating agents. It still remains to determine whether there is any relevance between the measurements obtained by these methods and clinical outcome in thrombocythemic patients.

Association between the plasminogen activator inhibitor-1 4G/5G polymorphism and venous thrombosis. A meta-analysis.

The effect of the 675 insertion/deletion (4G/5G) polymorphism of plasminogen activator inhibitor-1 (PAI-1) gene on the risk of venous thromboembolism (VTE) remains controversial. In this study, we performed a meta-analysis of published data regarding this issue. A comprehensive electronic search was carried out up until September 2006. A total of 22 articles were included in the analysis that was performed using random effects models. Eighteen papers, concerning patients without another known risk factor, comprised 2,644 cases and 3,739 controls. The alleles contrast (4G vs. 5G allele) yielded a statistically significant odds ratio (OR) of 1.153 (95% confidence interval [CI]: 1.068-1.246). In a sub-analysis of five studies that included 256 cases with another genetic risk factor and 147 controls, the combined per-allele OR was still significant (OR: 1.833,95% CI: 1.325-2.536). On the contrary, the analysis of five studies regarding cases with a non-genetic risk factor for VTE (antiphospholipid antibody syndrome, Behcet disease) provided insignificant results in all aspects. There was no evidence for heterogeneity and publication bias in all analyses. Based on our findings, the 4G allele appears to increase the risk of venous thrombosis, particularly in subjects with other genetic thrombophilic defects. Recommendation for detection of this polymorphism in evaluating thrombophilia in such patients might be considered.