Diagnostic methods for Chagas disease rely primarily on serological and molecular tests to detect anti-T cruzi antibodies or parasite-derived DNA. However, these tests do not predict clinical outcomes or treatment responses, as serological results may take years to turn negative, making them impractical for monitoring treatment progress. Molecular tests, though highly sensitive, can identify parasites but cannot rule out the presence of dormant forms or low levels of parasitemia, which may later reactivate. Due to the lack of reliable biomarkers for disease prognosis and treatment monitoring, there has been growing interest in host-derived markers as alternatives. One area of focus is the hypercoagulability observed in T. cruzi-infected individuals, which increases the risk of thrombotic events due to factors like chronic inflammation, dysregulation of immunothrombosis, and vasculitis. In addition, extracellular vesicles (EVs), i.e., double-membrane-enclosed particles found in various biological fluids, have emerged as a potential source of biomarkers for disease detection, prognosis, and monitoring. EVs contain proteins, lipids, and nucleic acids that can provide valuable molecular information about a patient's condition. This chapter outlines procedures for detecting host-derived biomarkers of Chagas disease, particularly those related to hypercoagulability, such as F1 + 2 and endogenous thrombin potential assays, and the isolation and characterization of EVs as biomarkers for response to treatment and disease progression.

Mathematical models of thrombin generation (TG) that have been developed are based on a systems biology approach. Although this approach provides important information about the coagulation system, its clinical applicability is limited by its complexity and number of input variables required. The aim of this study was to develop a semimechanistic model able to describe TG in trauma and control patients. A dataset containing longitudinal data of TG assays and coagulation factors from 40 trauma patients and 20 control patients was used for model building. The model considered three fundamental processes: the degradation of tissue factor (TF) through a first-order process, the activation of factor II by the TF through a first-order process, and the degradation of thrombin through a first-order process. Model fitting was performed using a nonlinear mixed-effects approach. The condition of the patient (trauma and control) and coagulation factors were modelled as covariates. Model building demonstrated the presence of two additional processes that improved the predictive capacity of the model: the activation of factor II by TF governed by a second-order constant and, a mechanism of factor II activation by TF characterized by a 7-compartment transit chain governed by a second-order constant. In the covariate model only the inclusion of patient condition was significant. Model evaluation demonstrated excellent performance in describing the temporal pattern of TG in trauma and control patients. Thrombin generation can be adequately modelled using a semimechanistic approach. Its application in practice could help to better assess the risk of hemorrhage and/or thrombosis in different settings.

Platelets are highly specialized anucleate cells that play crucial roles in haemostasis, inflammation and disease. Recent data suggest that mRNA translational control is the primary mechanism of gene regulation in platelets. This study unveils a novel pathway to control mRNA translation and protein expression in platelets via the mRNA binding protein CPEB4. We found high CPEB4 expression levels in human and mouse platelets, colocalizing with cytoskeletal proteins. In vivo loss-of-function studies, using the megakaryocytic lineage-specific Pf4-Cre transgenic mice crossed with a strain containing loxP-flanked CPEB4 (Pf4+:Cpeb4–/– mice), revealed a significant reduction of platelet activation and aggregation. These effects were specific to mature platelets, as CPEB4 deficiency did not significantly impact megakaryocytopoiesis. Functional assays, including tail bleeding and collagen-induced embolism, demonstrated impaired coagulation in Pf4+:Cpeb4–/– mice compared to controls. Furthermore, proteomics analysis of resting and activated platelets from Pf4–:Cpeb4+/+ and Pf4+:Cpeb4–/– mice identified a molecular signature of proteins and networks regulated by CPEB4, including those involved in haemostasis and platelet clearance. Our study reveals CPEB4-mediated mRNA translational regulation as a novel fine-tuning mechanism for modulating protein synthesis in platelets, with significant functional consequences in both physiological and pathological conditions.

Venous thromboembolism (VTE) can present different challenging situations for which high-quality evidence to guide optimal preventive and therapeutic management is lacking and for which clinical practice guidelines have not established solid recommendations. The aim of this article is to achieve consensus on a proposal of action for the clinical management of complex, clinically relevant situations with a low level of evidence which generate great uncertainty-the duration of VTE treatment and the role of thrombus recanalization, the prevention of VTE within the context of pregnancy, management of anticoagulant treatment in patients with VTE and special characteristics, such as renal insufficiency and obesity, the therapeutic management of pluripathological and polymedicated older patients with VTE, and finally, primary ambulatory thromboembolic prevention in cancer patients. This consensus article arose from a collaboration of experts in VTE from different medical specialties.

BACKGROUND:Although there is a biological basis for it, there is scarce evidence on the effect of heparin in ameliorating placental insufficiency and maximizing gestational age at delivery among fetal growth restriction pregnancies. OBJECTIVE:To explore the effectiveness of treatment using low molecular weight heparin at a prophylactic dose started at the time of diagnosis in prolonging gestation in pregnancies with early-onset fetal growth restriction. STUDY DESIGN:This was a phase III, multicenter, triple-blind, parallel-arm randomized clinical trial conducted in 2 university hospitals in Spain. Singleton pregnancies qualifying for early-onset placental FGR according to the adapted Delphi consensus (20+0-31+6 weeks at diagnosis with umbilical artery Doppler with absent/reversed diastolic flow; or estimated fetal weight <10th percentile plus pulsatility index of umbilical artery Doppler >95th percentile; or estimated fetal weight <10th percentile plus mean pulsatility index of uterine artery Doppler >95th) were randomized to receive either subcutaneous treatment with bemiparin 3500 IU/0.2 mL/d or a placebo from inclusion at diagnosis to the time of delivery. The primary outcomes were prolongation of pregnancy from inclusion to live birth (days) and gestational age at live birth (days). RESULTS:Forty-nine patients were included (23 in the low molecular weight heparin group and 26 in the placebo group). In the low molecular weight heparin group, the median prolongation of pregnancy was 42 days, while in the placebo group it was 41.5 days (median difference 0.5 days [95% confidence interval -22.7 to 6.3] (P=.667)) and in the low molecular weight heparin group, the median gestational age at delivery was 35.1 weeks, while in the placebo group, it was 34.6 weeks (median difference 0.5 weeks [95% confidence interval -3.4 to 1.2] (P=.639)). CONCLUSION:The use of prophylactic dose low molecular weight heparin started at the time of diagnosis does not prolong pregnancy in individuals with early-onset fetal restriction.

Objective: Several studies have reported a high prevalence of autoimmune diseases such as systemic lupus ery-thematosus (SLE) in endometriosis patients. The aim of this study was to evaluate the SLE autoimmune antibody profile in patients with deep (DE) and non-deep endometriosis (Non-DE).Materials and methods: Four groups of premenopausal patients were evaluated: patients with DE (n = 50); pa-tients with ovarian endometriomas (Non-DE; n = 50); healthy patients without endometriosis (C group; n = 45); and SLE patients without endometriosis (SLE group; N = 46). Blood samples were obtained and the standard SLE autoimmune profile was evaluated in all patients. Pain symptoms related to endometriosis and clinical SLE manifestations were also recorded.Results: The DE group presented a statistically significant higher proportion of patients with antinuclear anti-bodies (ANA) (20%) compared to the Non-DE group (4%) and C group (2.2%). Levels of complement were more frequently lower among DE and Non-DE patients although differences did not reach statistical significance. Similarly, anti-dsDNA antibodies and anticoagulant lupus were positive in more patients of the DE group but did not reach statistical significance. The DE group complained of more arthralgia and asthenia compared to the Non-DE and C groups.Conclusions: The results of this study showed higher positivity of ANA and greater arthralgia and asthenia in patients with DE compared with Non-DE patients and healthy controls, suggesting that they may have a higher susceptibility to autoimmune diseases and present more generalized pain.

VWF Changes in primary hemostasis have been implicated in thrombotic and bleeding complications in myeloproliferative neoplasms (MPN), so their evaluation could be useful in the management of these patients. We studied primary hemostasis in 248 patients with essential thrombocythemia-type MPN (TE n=129), polycythemia vera (PV n=71) and myelofibrosis (MF n=48). Von Willebrand antigen (VWF:Ag) (normal: 60-160 U/dL), von Willebrand factor (VWF) activity (VWF:GPIbM) (normal: 60-160 U/dL) and closure time with the platelet function analyzer (PFA-200) (pathological: collagen-Epinephrine channel > 200 seconds; collagen-ADP channel > 140 seconds) were assessed. VWF:GPIbM < 60 U/dL and a VWF:GPIbM / VWF:Ag ratio < 0.7 were considered to be suggestive of acquired von Willebrand disease (aVWD). In antiplatelet patients with acetyl salicílic acid (ASA), we defined in vitro resistance as a PFA-200 Col-Epi time < 200 seconds. The results were correlated with the diagnosis, hematological values, conventional or NGS genotype (n=176) and treatment at the time of the study. Patients with thrombocytopenia < 50x10 9/L were excluded from the PFA-200 analysis. Table 1 describes the clinical variables of the patients. GPIbM was decreased in 14 cases, increased in 85 cases, and normal in the rest. We identified a direct correlation with age (β=0.29, R2=0.08, p=0.001), and an inverse correlation with the number of platelets (β=-0.45, R2=0.20, p<0.001), independent of the genotype. . We found elongated PFA-200 in the Col-Epi and Col-ADP channels in 129 (54%) and 63 (26%) cases, respectively. In 10 (4%) patients we found parameters suggestive of avWD. All of them had thrombocytosis. Elevated platelet count (>600x109/L) was associated with VWF values suggestive of aVWD, with a sensitivity and specificity of 90% and 83%, respectively (AUC: 0.938, 95%CI: 0.896-0.980). Of the 99 patients not on antiplatelet therapy, 31 (31%) had an elongated PFA-200. Elongated PFA-200 was associated with lower Hb, higher leukocytes, higher platelets, higher JAK2 mutational load, lower VWF:GPIbM, and CALR genotype. This alteration was observed in 71% of patients with the CALR genotype. In multivariate analysis, decreased VWF:GPIbM (OR: 0.98, 95%CI: 0.97-0.99, p=0.001) and CALR mutation (OR: 0.98, 95%CI: 0.97- 0.99, p=0.001), maintained their value. Of the 130 patients receiving ASA, in vitro resistance was found in 31 cases (24%) (PV 30%, TE 25%, MF 0%). ASA resistance was associated with a higher platelet count and higher VWF:GPIbM. 50% of the patients with increased VWF:GPIbM (>160 U/dL) had resistance to ASA, showing VWF:GPIbM as an independent variable in the multivariate analysis.

There has been increasing interest in the study of new pathogenic mechanisms in endometriosis (END), including the coagulation/fibrinolysis system and its link with inflammation and tissue remodeling. It has been suggested that END patients, especially with deep-infiltrating (DE) forms, could present a hypercoagulable state revealing higher levels of proinflammatory and procoagulant markers, such as total circulating microparticles (cMPs) and cMP-TF (tissue factor), released by cells in response to damage, activation, or apoptosis. However, no previous study has assessed the effect of END hormonal treatments on cMP and cMP-TF levels. Therefore, the aim of this study was to evaluate the impact of these treatments on cMP and cMP-TF levels in DE patients. Three groups were compared: DE patients receiving a continuous combined oral contraceptive regimen (CCOCR) (n = 41), DE patients without CCOCR (n = 45), and a control group (n = 43). cMP and cMP-TF levels were evaluated in platelet-free plasma. A significant decrease in the total cMP levels was found in the DE group with CCOCR versus the group without CCOCR, reflecting a higher chronic inflammatory status in DE patients that decreased with the treatment. cMP-TF levels were higher in DE patients receiving CCOCR versus those not receiving CCOCR, suggesting that treatments containing estrogens play a predominant role in suppressing the inhibitory pathway of TF.

Thromboelastometry is considered the best method to assesses hemostasis in liver disease. Diagnostic performance could be improved by adding protein C activators such as thrombomodulin or Protac®. We assessed changes in ROTEM parameters after the addition of Protac® in patients with acute-on-chronic liver failure (ACLF), acute decompensation (AD), and healthy individuals (HI) to define different hemostasis patterns, considering standard and velocity ROTEM parameters, and assess whether Protac® can improve the definition of the pattern. Pre-test, we investigated whether diluted EXTEM reagent improved the effect of Protac® on the clotting time (CT)-ratio with and without Protac®. Ten ACLF and 20 AD patients and 21 HI were included in the main study. Standard EXTEM was used in the main study. INTEM CFT, INTEM A5 (inverse), and INTEM TPI (inverse) were the parameters that best differentiated liver disease from HI (ROC AUC, 0.921, 0.906, and 0.928, respectively; all P-values < 0.001). Combining INTEM CFT with EXTEM LI60-ratio only slightly improved the diagnostic performance (ROC AUC, 0.948; P < 0.001). EXTEM LI60 and INTEM maxV-t were the parameters that best differentiated between ACLF and AD patients (ROC AUC, 0.743, P = 0.033; and 0.723, P = 0.050; respectively). Combining EXTEM LI60 + INTEM maxV-t moderately improved the diagnostic performance (ROC AUC, 0.81, P < 0.001). ROTEM velocity, fibrinolysis parameters and the indices calculated improve the diagnosis in combination with standard parameters (e.g., CFT and A5). Ratios calculated with and without Protac® (e.g., EXTEM LI60-ratio) only slightly increased the diagnostic performance in discriminating hemostasis patterns.

Antivitamin K agent (AVK) reversal in patients with cirrhosis awaiting liver transplantation (LT) is not defined in guidelines. We investigated the effect of reversion with prothrombin complex concentrate (PCC) on intraoperative transfusion, bleeding, and safety in LT patients on AVK.

AbstractThe Spanish Acquired Hemophilia A (AHA) Registry is intended to update the status of AHA in Spain. One hundred and fifty-four patients were included and retrospectively followed for a median of 12 months. Patients were predominantly male (56.3%), with median age at diagnosis of 74 years. AHA was more frequently idiopathic (44.1%) and autoimmune disorder-associated (31.7%). Thirty-four percent of patients were on antithrombotic therapy at diagnosis. Hemostatic treatment was used in 70% of patients. Recombinant activated factor VII was more frequently infused (60.3% vs 20.6% activated prothrombin complex concentrate). Only 1 patient did not achieve control of hemorrhage. Complete remission (CR) was achieved by 84.2% of cases after immunosuppressive therapy. Steroids alone were less efficient than the other strategies (68.2% vs 87.2%, P = .049), whereas no differences existed among these (steroids/cyclophosphamide, 88.5%, vs steroids/calcineurin inhibitors, 81.2%, vs rituximab-based regimens, 87.5%). Female sex and high inhibitor levels influenced CR negatively. Thirty-six deaths (23.8%) were reported. Main causes of death were infection (15 patients, 9.9%) and hemorrhage (5 patients, 3.3%). All hemorrhage-related and half the infection-related deaths occurred within 2 months of diagnosis. Prior antithrombotic therapy was inversely associated with survival, irrespective of age. Median age of nonsurvivors was significantly higher (79 vs 73 years in survivors). Patients dying of infection were older than the other nonsurvivors (85 vs 78 years). In summary, fatal infection in the first months is common in our series. Antithrombotic therapy is associated with mortality. Particular care should be taken to avoid misdiagnosis.

Objective The objective of this paper is to assess the prevalence of the main clinical manifestations and laboratory features at disease onset and during the ensuing 10 years of a large cohort of patients with antiphospholipid syndrome (APS) from a single center. Methods The study included all consecutive APS patients followed longitudinally in our center from 2003 to 2013. Descriptive statistics for demographics, clinical and laboratory features and mortality were performed. Results A total of 160 patients were included. Most of them, 128 (78.8%), were women and the mean (SD) age at diagnosis was 39.1 (14.0) years. The majority of them, 104 (65.0%), had primary APS, 36 (22.5%) had APS associated with systemic lupus erythematous, and 20 (12.5%) had APS associated with other autoimmune disease. During the study period, thrombotic events occurred in 27 (16.9%) patients, the most common being strokes, nonbacterial thrombotic endocarditis and deep venous thrombosis. Regarding obstetric morbidity, 18 women (14.3%) became pregnant and 90% of pregnancies succeeded in having live births. The most common obstetric complication was early pregnancy loss (15% of pregnancies). Prematurity (11.1% of live births) and intrauterine growth restriction (5.6% of live births) were the most frequent fetal morbidities. Ten (6.3%) patients died and the most frequent causes of death were severe thrombosis, hemorrhage, and cancer. Three (0.9%) cases of catastrophic APS occurred. The survival probability at 10 years was 93.8%. Conclusions Patients with APS develop significant morbidity and mortality despite current treatment. It is imperative to identify prognostic factors and therapeutic measures to prevent these complications.

COVID-19 coagulopathy linked to increased D-dimer levels has been associated with high mortality (Fei Z et al. in Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study. Lancet (London, England) 395(10229):1054–62, 2020). While D-dimer is accepted as a disseminated intravascular coagulation marker, rotational thromboelastometry (ROTEM) also detects fibrinolysis (Wright FL et al. in Fibrinolysis shutdown correlates to thromboembolic events in severe COVID-19 infection. J Am Coll Surg (2020). Available from https://pubmed.ncbi.nlm.nih.gov/32422349/ [cited 14 Jun 2020]; Schmitt FCF et al. in Acute fibrinolysis shutdown occurs early in septic shock and is associated with increased morbidity and mortality: results of an observational pilot study. Ann Intensive Care 9(1):19, 2019). We describe the ROTEM profile in severely ill COVID-19 patients and compare it with the standard laboratory coagulation test. Adult patients diagnosed with COVID-19 admitted to the ICU were prospectively enrolled after Ethics Committee approval (HCB/2020/0371). All patients received venous thromboembolism prophylaxis; those on therapeutic anticoagulation were excluded. The standard laboratory coagulation test and ROTEM were performed simultaneously at 24–48 h after ICU admission. Sequential organ failure assessment (SOFA), disseminated intravascular coagulation (DIC) and sepsis-induced coagulopathy (SIC) scores were calculated at sample collection. Nineteen patients were included with median SOFA-score of 4 (2–6), DIC-score of 1 (0–3) and SIC-score of 1.8 (0.9). Median fibrinogen, D-dimer levels and platelet count were 6.2 (4.8–7.6 g/L), 1000 (600–4200 ng/ml) and 236 (136–364 109/L), respectively. Clot firmness was above the normal range in the EXTEM and FIBTEM tests while clot lysis was decreased. There was no significant correlation between ROTEM or D-dimer parameters and the SOFA score. In COVID-19 patients, the ROTEM pattern was characterized by a hypercoagulable state with decreased fibrinolytic capacity despite a paradoxical increase in D-dimer levels. We suggest that, in COVID-19 patients, the lungs could be the main source of D-dimer, while a systemic hypofibrinolytic state coexists. This hypothesis should be confirmed by future studies.

Microparticles (MPs) have been associated with inflammatory and thrombotic disease. High levels of MPs have been identified in patients with systemic lupus erythematosus (SLE) and associated with cardiovascular disease. We analyzed the procoagulant activity of MPs and its correlation with arteriosclerosis and arterial thrombosis in SLE patients. Eighty-seven patients with SLE were included: 22 (25.3%) with associated antiphospholipid syndrome (APS), 32 (36.8%) without antiphospholipid antibodies (aPL) and 33 (37.9%) with aPL but without APS. Subclinical arteriosclerosis, defined as the presence and number of plaques, was evaluated by ultrasonography of carotid arteries. Thrombotic events were confirmed by objective methods. The procoagulant activity of MPs was determined by a functional assay with annexin V. Subclinical arteriosclerosis was found in 19 (21.8%) patients. Thirteen episodes of arterial thrombosis and eight of venous thrombosis were recorded. The procoagulant activity of MPs was greater in patients with arterial thrombosis (17.28 ± 8.29 nM vs 12.96 ± 7.90 nM, p < 0.05). In patients without arterial thrombosis, greater procoagulant activity of MPs was identified in patients with multiple (≥ 2) carotid plaques (17.26 ± 10.63 nM vs 12.78 ± 7.15 nM, p = 0.04). In the multivariate analysis, the procoagulant activity of MPs was independently associated with multiple (≥ 2) carotid plaques and arterial thrombosis [OR = 1.094 (95%CI 1.010–1.185), p = 0.027 and OR = 1.101 (95%CI 1.025–1.182), p = 0.008; respectively]. In conclusion, the procoagulant activity of MPs is associated with arteriosclerosis burden and arterial thrombosis in patients with SLE.

The most severe clinical symptomatology of Chagas disease affects ~30% of those chronically infected with the Trypanosoma cruzi parasite. The pathogenic mechanisms that lead to life-threatening heart and gut tissue disruptions occur "silently" for a longtime in a majority of cases. As a result, despite there are several serological and molecular methods available to diagnose the infection in its acute and chronic stages, diagnosis is often achieved only after the onset of clinical symptoms in the chronic phase of the disease. Furthermore, although there are two drugs to treat it, the assessment of their performance is impractical with current parasite-derived diagnostics, and therapeutic efficacy cannot be acknowledged in a timely manner.In this chapter we present two procedures to measure host-derived molecules as surrogates of therapeutic response against chronic T. cruzi infection. Their outputs relate to the generation and activity of thrombin, a major component of the blood coagulation cascade. This is due to the fact that a hypercoagulability state has been described to occur in chronic Chagas disease patients and revert after treatment with benznidazole.