Inflammatory and Prothrombotic Biomarkers Contribute to the Persistence of Sequelae in Recovered COVID-19 Patients.

The presence of long COVID (LC) following SARS-CoV-2 infection is a common condition that affects the quality of life of patients and represents a diagnostic challenge due to the diversity of symptoms that may coexist. We still do not have accurate information regarding the pathophysiological pathways that generate the presence of LC, and so it is important to know the inflammatory and immunothrombotic biomarker profiles and their implications in order to characterize risk subgroups and establish early therapeutic strategies. We performed the determination of inflammatory and immunothrombotic biomarkers in volunteers with previous diagnoses of SARS-CoV-2. The inflammatory biomarkers were analyzed in plasma by flow cytometry, and we analyzed the von Willebrand factor (vWF) in the plasma samples using ELISA. The clinical variables and the presence or absence of long COVID symptoms were then analyzed. IL-6, sCD40L, p-Selectin, PSGL-1, PAI-1, tPA, D-Dimer, TF, and Factor IX levels were elevated in the groups with LC, especially in the subgroup of patients with metabolic syndrome (MetS). VWF levels were found to be increased in patients with sequelae and MetS. Our results confirmed the persistence of an active immunothrombotic state, and so it is important to identify the population at risk in order to provide adequate clinical follow-up.

Platelet Reactivity and Inflammatory Phenotype Induced by Full-Length Spike SARS-CoV-2 Protein and Its RBD Domain.

A state of immunothrombosis has been reported in COVID-19. Platelets actively participate in this process. However, little is known about the ability of SARS-CoV-2 virus proteins to induce platelet activity. Platelet-rich plasma (PRP) was incubated with spike full-length protein and the RBD domain in independent assays. We evaluated platelet activation through the expression of P-selectin and activation of glicoprotein IIbIIIa (GP IIbIIIa), determined by flow cytometry and the ability of the proteins to induce platelet aggregation. We determined concentrations of immunothrombotic biomarkers in PRP supernatant treated with the proteins. We determined that the spike full-length proteins and the RBD domain induced an increase in P-selectin expression and GP IIbIIIa activation (p < 0.0001). We observed that the proteins did not induce platelet aggregation, but favored a pro-aggregating state that, in response to minimal doses of collagen, could re-establish the process (p < 0.0001). On the other hand, the viral proteins stimulated the release of interleukin 6, interleukin 8, P-selectin and the soluble fraction of CD40 ligand (sCD40L), molecules that favor an inflammatory state p < 0.05. These results indicate that the spike full-length protein and its RBD domain can induce platelet activation favoring an inflammatory phenotype that might contribute to the development of an immunothrombotic state.

Temperature-dependent secretion of Zika virus envelope and non-structural protein 1 in mammalian cells for clinical applications.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus associated with congenital Zika syndrome and Guillain-Barré syndrome in adults. The recombinant ZIKV envelope (E) antigen can be useful for serodiagnosis of ZIKV infection and for monitoring immune responses during preclinical and clinical ZIKV vaccine development. In this study, we describe production of ZIKV E using the modified polyethyleneimine (PEI) transfection in HEK293 cells to improve cost-effective large-scale production. We show that the secretion of ZIKV E in HEK293 cells is dependent on cell culture incubation temperatures where incubation at a low temperature of 28 °C improved protein secretion of both, E-CD4 and E, whereas a substantial decrease in secretion was observed at 37 °C. The resulting E-CD4 produced at low temperature yielded similar binding profiles in ELISAs in comparison with a commercially available E protein using human seropositive sera to ZIKV. We also show that ZIKV NS1 and NS1 ß-ladder antigens produced in HEK293 cells, have similar binding profiles in ELISA which suggests that both NS1 or NS1 ß-ladder can be used for serodiagnosis of ZIKV. In conclusion, we propose a cost-effective production of the ZIKV E and NS1, suitable for both, clinical and research applications in endemic countries.

Inflammatory and Prothrombotic Biomarkers Associated With the Severity of COVID-19 Infection.

Among COVID-19 hospitalized patients, high incidence of alterations in inflammatory and coagulation biomarkers correlates with a poor prognosis. Comorbidities such as chronic degenerative diseases are frequently associated with complications in COVID-19 patients. The aim of this study was to evaluate inflammatory and procoagulant biomarkers in COVID-19 patients from a public hospital in Mexico. Blood was sampled within the first 48 h after admission in 119 confirmed COVID-19 patients that were classified in 3 groups according to oxygen demand, evolution and the severity of the disease as follows: 1) Non severe: nasal cannula or oxygen mask; 2) Severe: high flow nasal cannula and 3) Death: mechanical ventilation eventually leading to fatal outcome. Blood samples from 20 healthy donors were included as a Control Group. Analysis of inflammatory and coagulation biomarkers including D-dimer, interleukin 6, interleukin 8, PAI-1, P-selectin and VWF was performed in plasma. Routine laboratory and clinical biomarkers were also included and compared among groups. Concentrations of D-dimer (14.5 ± 13.8 µg/ml) and PAI-1 (1223 ± 889.6 ng/ml) were significantly elevated in severe COVID-19 patients (P < 0.0001). A significant difference was found in interleukin-6, PAI-1 and P-selectin in non-severe and healthy donors when compared to Severe COVID-19 and deceased patients (P < 0.001). VWF levels were also significantly different between severe patients (153.5 ± 24.3 UI/dl) and non-severe ones (133.9 ± 20.2 UI/dl) (P < 0.0001). WBC and glucose levels were also significantly elevated in patients with Severe COVID-19. Plasma concentrations of all prothrombotic biomarkers were significantly higher in patients with a fatal outcome.

Production and Purification of Zika Virus NS1 Glycoprotein in HEK293 Cells.

In this chapter, we describe production and purification of the Zika virus NS1 glycoprotein in human embryonic kidney (HEK293T) cells at small, research laboratory scale. The expression of secreted NS1 (sNS1) and the C-terminal ß-ladder domain in HEK293T cells were tested in a small-scale transfection before scaling up to a larger-scale transfection using roller bottles. Two different purification approaches have been applied to obtain purified NS1 (sNS1) and the C-terminal ß-ladder domain ready for clinical applications.

Development of an E2 ELISA Methodology to Assess Chikungunya Seroprevalence in Patients from an Endemic Region of Mexico.

Chikungunya fever is a debilitating disease caused by Chikungunya virus (CHIKV) that can result in long-lasting arthralgias. The early diagnosis of CHIKV relies on PCR during the acute infection phase to allow differential diagnosis with other co-circulating arboviruses such as dengue and Zika. Alternatively, serology can support diagnosis and provide epidemiological information on current and past outbreaks. Many commercial serological ELISA assays are based on the inactivated whole CHIKV, but their sensitivity and specificity show great variability. We produced recombinant CHIKV E2 that is suitable for ELISA assays, which was used for the serodiagnosis of CHIKV infections occurring in an arbovirus endemic Mexican region within Michoacán state. A cross-sectional study was conducted in 2016-2017; sera was obtained from 15 healthy donors and 68 patients presenting undifferentiated febrile illness. Serum samples were screened by RT-PCR and by our in-house ELISA assay. Our results indicate that IgM and IgG anti-CHIKV E2 antibodies were detected with our ELISA assay with higher sensitivity than a commercially available CHIKV ELISA kit. Our simple and sensitive ELISA assay for the serodiagnosis of CHIKV infections can be applied to population-based seroprevalence surveys and has potential for monitoring vaccine immunogenicity in CHIKV vaccine clinical trials.

Optimization of Zika virus envelope protein production for ELISA and correlation of antibody titers with virus neutralization in Mexican patients from an arbovirus endemic region.

BACKGROUND: Zika virus (ZIKV) has become a global threat with immediate need for accurate diagnostics, efficacious vaccines and therapeutics. Several ZIKV envelope (Env)-based vaccines have been developed recently. However, many commercially available ZIKV Env are based on the African lineage and produced in insect cells. Here, we sought to produce Asian-lineage ZIKV Env in mammalian cells for research and clinical applications. METHODS: We designed various gene expression constructs to optimize the production of ZIKV using prM-Env and full or C-terminal truncations of Env; with or without a rat CD4 fusion partner to allow large-scale production of soluble protein in mammalian HEK293 cells. Protein expression was verified by mass spectrometry and western-blot with a pan-flavivirus antibody, a ZIKV Env monoclonal antibody and with immune sera from adenoviral (ChAdOx1) ZIKV Env-vaccinated mice. The resulting Env-CD4 was used as a coating reagent for immunoassay (ELISA) using both mouse and human seropositive sera. RESULTS: Replacement of the C-terminus transmembrane Env domain by a rat CD4 and addition of prM supported optimal expression and secretion of Env. Binding between the antigens and the antibodies was similar to binding when using commercially available ZIKV Env reagents. Furthermore, antibodies from ZIKV patients bound ZIKV Env-CD4 in ELISA assays, whereas sera from healthy blood donors yielded minimal OD background. The serological outcomes of this assay correlated also with ZIKV neutralisation capacity in vitro. CONCLUSIONS: Results obtained from this study indicate the potential of the Asian-lineage Zika Env-CD4 and Env proteins in ELISA assays to monitor humoral immune responses in upcoming clinical trials as well as a sero-diagnostic tool in ZIKV infection.

Tailoring a Plasmodium vivax Vaccine To Enhance Efficacy through a Combination of a CSP Virus-Like Particle and TRAP Viral Vectors.

Vivax malaria remains one of the most serious and neglected tropical diseases, with 132 to 391 million clinical cases per year and 2.5 billion people at risk of infection. A vaccine against Plasmodium vivax could have more impact than any other intervention, and the use of a vaccine targeting multiple antigens may result in higher efficacy against sporozoite infection than targeting a single antigen. Here, two leading P. vivax preerythrocytic vaccine candidate antigens, the P. vivax circumsporozoite protein (PvCSP) and the thrombospondin-related adhesion protein (PvTRAP) were delivered as a combined vaccine. This strategy provided a dose-sparing effect, with 100% sterile protection in mice using doses that individually conferred low or no protection, as with the unadjuvanted antigens PvTRAP (0%) and PvCSP (50%), and reached protection similar to that of adjuvanted components. Efficacy against malaria infection was assessed using a new mouse challenge model consisting of a double-transgenic Plasmodium berghei parasite simultaneously expressing PvCSP and PvTRAP used in mice immunized with the virus-like particle (VLP) Rv21 previously reported to induce high efficacy in mice using Matrix-M adjuvant, while PvTRAP was concomitantly administered in chimpanzee adenovirus and modified vaccinia virus Ankara (MVA) vectors (viral-vectored TRAP, or vvTRAP) to support effective induction of T cells. We examined immunity elicited by these vaccines in the context of two adjuvants approved for human use (AddaVax and Matrix-M). Matrix-M supported the highest anti-PvCSP antibody titers when combined with Rv21, and, interestingly, mixing PvCSP Rv21 and PvTRAP viral vectors enhanced immunity to malaria over levels provided by single vaccines.

Prevalence of malnutrition-inflammation complex syndrome and its correlation with thyroid hormones in chronic haemodialysis patients. / Prevalencia del síndrome complejo de malnutrición e inflamación y su correlación con las hormonas tiroideas en pacientes en hemodiálisis crónica.

INTRODUCTION: Low levels of thyroid hormones, total triiodothyronine (T3) and free triiodothyronine (FT3) in haemodialysis patients is a marker of malnutrition and inflammation and are predictors of mortality. The aim of this study was to determine the prevalence of malnutrition-inflammation complex syndrome in haemodialysis and its relationship with the thyroid hormones thyrotropin, T3, FT3 and free thyroxine (FT4), as well as to evaluate the prevalence of low FT3 syndrome and its correlation with nutritional and inflammatory markers. MATERIALS AND METHODS: Cross-sectional, analytical and comparative study that enrolled 128 haemodialysis patients: 50.8% females; mean age 45.05±17.01 years; mean time on haemodialysis 45.4±38.8 months; 29.7% diabetics; 79.7% with hypertension. Serum thyroid hormones thyrotropin, T3, FT3 and FT4 concentrations were measured and Malnutritition-Inflammation Score (MIS) was applie to diagnostic. RESULTS: Mean thyroid hormone values were: thyroid hormones thyrotropin 2.48±1.8 mIU/ml (range: 0.015-9.5), T3 1.18±0.39 ng/ml (range 0.67-2.64), FT3 5.21±0.96pmol/l (range: 3.47-9.75); FT4 1.35±0.4 ng/ml (range: 0.52-2.57). Malnutrition-inflammation complex syndrome prevalence was 53.9%; 11.7% presented low FT3 levels. Serum T3 and FT3 concentrations inversely correlated with Malnutritition-Inflammation Score (MIS), while FT4 correlated positively with Malnutrition-Inflammation Score. In the linear regression analysis, low FT3 was associated with IL-6 (ß= 0.265, p=.031), C-reactive protein (CRP) (ß= -0.313, p=.018) and albumin (ß= 0.276, p=.002). CONCLUSION: Low T3 and FT3 levels are correlated with malnutrition and inflammation parameters. Malnutrition-inflammation complex syndrome can affect serum concentrations of thyroid hormones.

Von Willebrand Factor: Multimeric Structure and Functional Activity in Patients With Atrial Fibrillation With and Without Oral Anticoagulation.

von Willebrand factor (vWF) is a multimeric glycoprotein present in blood plasma. It is synthesized in megakaryocytes and endothelial cells, secreted into circulation in the form of high-molecular-weight multimers (HMWMs), and cleaved into shorter, less active multimers by ADAMTS13. It is essential for platelet adhesion and aggregation. Previous studies have investigated the relationship between vWF levels and thromboembolic events with little regard to vWF multimeric structure. Patients with atrial fibrillation (AF) exhibit higher plasma vWF and lower ADAMTS13 levels. One hundred seven patients with AF, 51 anticoagulated and 56 nonanticoagulated, were eligible for the study. Plasma samples were analyzed for vWF antigen, vWF activity, and ADAMTS13; vWF multimers were analyzed by Western blot in 1% to 1.3% sodium dodecyl sulfate agarose gel electrophoresis. Patients with AF without oral anticoagulation (OAC) had significantly higher vWF plasma levels (154.00 [75-201] UI/dL) and vWF activity (60.00% [20%-210%]) compared to patients with OAC (133.50 [90-192] UI/dL, P = <.001; 50.00% [20%-160%], P = .02). Both were specially decreased in patients treated with acenocumarin. Patients without OAC also showed lower ADAMTS13 levels and presence of vWF HMWMs. Patients with AF show higher plasma levels and vWF activity. Moreover, treatment with traditional OAC (acenocumarin) significantly reduced vWF levels. Patients without OAC might have an increased risk of thrombotic events showing lower ADAMTS13 and higher vWF levels. Patients with stroke had higher plasma levels, vWF activity, and HMWMs. Our study suggests that increased vWF levels and presence of HMWMs could be related to cerebrovascular disease and may represent useful biomarkers for stroke in AF.

Evaluation of Plasmodium vivax Cell-Traversal Protein for Ookinetes and Sporozoites as a Preerythrocytic P. vivax Vaccine.

Four different vaccine platforms, each targeting the human malaria parasite Plasmodium vivax cell-traversal protein for ookinetes and sporozoites (PvCelTOS), were generated and assessed for protective efficacy. These platforms consisted of a recombinant chimpanzee adenoviral vector 63 (ChAd63) expressing PvCelTOS (Ad), a recombinant modified vaccinia virus Ankara expressing PvCelTOS (MVA), PvCelTOS conjugated to bacteriophage Qß virus-like particles (VLPs), and a recombinant PvCelTOS protein expressed in eukaryotic HEK293T cells (protein). Inbred BALB/c mice and outbred CD-1 mice were immunized using the following prime-boost regimens: Ad-MVA, Ad-VLPs, and Ad-protein. Protective efficacy against sporozoite challenge was assessed after immunization using a novel chimeric rodent Plasmodium berghei parasite (Pb-PvCelTOS). This chimeric parasite expresses P. vivax CelTOS in place of the endogenous P. berghei CelTOS and produces fully infectious sporozoites. A single Ad immunization in BALB/c and CD-1 mice induced anti-PvCelTOS antibodies which were boosted efficiently using MVA, VLP, or protein immunization. PvCelTOS-specific gamma interferon- and tumor necrosis factor alpha-producing CD8+ T cells were induced at high frequencies by all prime-boost regimens in BALB/c mice but not in CD-1 mice; in CD-1 mice, they were only marginally increased after boosting with MVA. Despite the induction of anti-PvCelTOS antibodies and PvCelTOS-specific CD8+ T-cell responses, only low levels of protective efficacy against challenge with Pb-PvCelTOS sporozoites were obtained using any immunization strategy. In BALB/c mice, no immunization regimens provided significant protection against a Pb-PvCelTOS chimeric sporozoite challenge. In CD-1 mice, modest protective efficacy against challenge with chimeric P. berghei sporozoites expressing either PvCelTOS or P. falciparum CelTOS was observed using the Ad-protein vaccination regimen.

Thyroid disorders among dialysis patients / Alteraciones tiroideas en pacientes en diálisis

The thyroid gland and the kidney are closely related. Thyroid hormones (TH) contribute to the homeostasis of the human being through complex interactions of fluids and electrolytes, protein synthesis, etc. The effects on the kidney of TH may be pre renal or direct actions. Decreasing glomerular filtration (GF) this balance especially in advanced stages of chronic kidney disease (CKD) is altered. CKD is linked to alterations in TH levels and/or metabolism, resulting in a high prevalence of subclinical hypothyroidism and low free triiodothyronine (FT3) syndrome. These alterations are linked in micro inflammation, endothelial damage, cardiac abnormalities, and high mortality. In this study, we describe the most common thyroid abnormalities reported in CKD with dialytic stage approach.

La glándula tiroides y el riñón están estrechamente relacionados. Las hormonas tiroideas (HT) contribuyen en la homeostasis del ser humano a través de complejas interacciones de líquidos y electrolitos, síntesis de proteínas, etc. Los efectos de las HT sobre el riñón pueden ser pre-renales o directos. Está demostrado que al disminuir la filtración glomerular (FG) se altera este equilibrio, sobre todo en estadios avanzados de la enfermedad renal crónica (ERC). La ERC está vinculada con alteraciones en los niveles de HT y/o su metabolismo, lo que resulta en una alta prevalencia de hipotiroidismo subclínico y el síndrome de T3 libre baja. Estas alteraciones están relacionadas con micro inflamation, daño endotelial, alteraciones cardiacas y alta mortalidad. El presente estudio, describe las alteraciones tiroideas más frecuentes reportadas en ERC con enfoque en la etapa dialítica.

Retracted: FLT3: beyond good and evil.

The above article, published online on 29 July 2016 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the Journal Editor in Chief, Journal Production Manager, and John Wiley & Sons, Ltd. The retraction has been agreed due to 48% similar significant between this article and an article published in Nature Reviews Cancer journal.

[Pro-inflammatory serum cytokines in diabetic retinopathy]. / Citoquinas séricas proinflamatorias en retinopatía diabética.

BACKGROUND: Pro-inflammatory cytokines play an important role in diabetic retinopathy. There is conflicting evidence about their serum elevation in this condition and that they also may be possible serum inflammatory biomarkers of diabetic retinopathy. OBJECTIVE: To evaluate the presence of serum pro-inflammatory cytokines and acute phase reactants in the serum of patients with and without diabetic retinopathy. MATERIAL AND METHODS: Comparative case series with 36 patients divided into three groups were included: 12 patients with diabetes mellitus and diabetic retinopathy (group 1), 12 diabetic patients without diabetic retinopathy (group 2), and 12 healthy patients as a control group. Serum levels of the following pro-inflammatory cytokines were measured in all patients: TNF-α, IL-1ß and IL-6. Pro-inflammatory biomarkers measurements were also performed, such as erythrocyte sedimentation rate and C-reactive protein. RESULTS: The levels of TNF-α and IL-6 were higher in group 1 (TNF-α: 19.4 ± 10.9 pg/ml, IL-6: 5.75 ± 7 pg/ml) compared to the other two groups, although the difference was statistically significant only in the case of TNF-α (group 1: 19.4 ± 10.9 pg/ml, group 2: 14 ± 4.3 pg/ml and control: 8.49 ± 3.69 pg/ml, p = 0.001). There were no differences among pro-inflammatory biomarkers such as erythrocyte sedimentation rate and C reactive protein. among the three groups (p > 0.05). CONCLUSIONS: Pro-- inflammatory serum cytokine levels were higher in the diabetes mellitus with diabetic retinopathy group. Larger studies are warranted to establish the real impact of this finding.

[Determination of von Willebrand factor multimers in Mexican population]. / Determinación de los multímeros del factor von Willebrand en población mexicana.

BACKGROUND: Von Willebrand disease is an inherited disease in which the structure, function, and concentration of von Willebrand factor are altered, as well as the platelet von Willebrand factor endothelium interaction. In Mexico there are no epidemiological records of the disease. Only a few isolated studies have been reported from the clinical and hematological standpoint. METHODS: We studied 155 Mexican Mestizos: 75 with presumptive diagnosis of von Willebrand disease, 15 with suspected diagnosis ofhemophilia A and 65 healthy donors (controls). Basic coagulation tests, special tests and classification test (analysis of multimeric composition) were carried out. RESULTS: There were 15 patients with clinical diagnosis of hemophilia A, 75 patients with suspected von Willebrand disease of which 50 were diagnosed as the following types and subtypes: Type 1 (62%), Type 2 (22%) [subtypes: 2A (14%), 2B (2%), and 2N (6%)] and Type 3 (16%). CONCLUSION: It has been reported that analysis of von Willebrand factor is a method that meets the characteristics for the diagnosis of von Willebrand disease. It is necessary to implement this methodology to study and improve the specific diagnoses.

Antecedentes: la enfermedad de von Willebrand es un padecimiento hereditario en el que la estructura, función y concentración del factor de von Willebrand están alteradas y, en consecuencia, también la interacción plaqueta-factor de von Willebrand-endotelio. En México no hay registros epidemiológicos de la enfermedad, sólo se han efectuado algunos estudios aislados desde el punto de vista clínico y hematológico. Material y métodos: estudio retrospectivo efectuado en 155 mexicanos mestizos, 75 de ellos con diagnóstico presuntivo de enfermedad de von Willebrand, 15 con sospecha de hemofilia A y 65 donadores sanos (testigos). Se realizaron pruebas: básicas de coagulación, especiales y de clasificación: análisis de la composición multimérica. Resultados: 15 pacientes se diagnosticaron con hemofilia A; de los 75 sujetos con sospecha de enfermedad de von Willebrand se diagnosticaron 50 de la manera siguiente: tipo 1 (62%), tipo 2 (22%) [subtipos: 2A (14%), 2B (2%) y 2N (6%)] y tipo 3 (16%). Conclusión: el análisis de los multímeros del factor de von Willebrand es un método que cumple con las características adecuadas para el diagnóstico de la enfermedad de von Willebrand, por lo que es necesario implementar esta metodología para su estudio y mejorar su diagnóstico específico.

Von Willebrand Factor Plasma Levels Variability In Nonvalvular Atrial Fibrillation.

Atrial Fibrillation (AF) is the most common cardiac arrhythmia of clinical significance; it increases the risk of mortality due to stroke. The mechanisms behind cerebral thromboembolism in AF are associated with a prothrombotic state, demonstrated by higher levels of von Willebrand Factor (vWF), a multimeric glycoprotein that plays a crucial role in platelet adhesion and aggregation and it has been proposed as a biomarker of endothelial dysfunction. Plasma vWF levels are elevated in patients with nonvalvular Atrial Fibrillation (NVAF) associated to the presence of cardiovascular risk factors. The variability in vWF plasma levels in healthy subjects has a wide distribution, but there is no description available of the variability in AF patients and among types of AF. The aim of this study was to determine the variability of vWF plasma concentrations in patients with NVAF, associated to cardiovascular risk factors. Search strategy included PubMed and Ovid. Keywords used were "Atrial Fibrillation" and "von Willebrand Factor". It includes original articles, with analysis of plasma vWF levels by ELISA, without acute stroke. Review articles and meta-analysis were excluded. Reviewed studies include 22 trials and 6542 patients with nonvalvular AF associated to cardiovascular disease risk factors: age, sex, hypertension, heart failure, diabetes mellitus, prior stroke, coronary artery disease. Variability in vWF plasma levels was wide, with minimum values of 77 IU/dl and maximum values of 245 IU/dl and a mean of 146 IU/dl. Age of patients ranged between 54 and 78 years, and the percentage of males ranged between 23% and 80%. According to type of AF vWF levels were as follows, in paroxysmal AF: 92-264 IU/dl; persistent AF: 76-234 IU/dl; permanent AF: 91-247 IU/dl. The variability in vWF plasma levels is affected by risk factors and the AF type, however vWF levels in AF patients are higher when compared with healthy subjects.