Soluble urokinase Plasminogen Activator Receptor (suPAR) levels are predictive of COVID-19 severity: an Italian experience.

BACKGROUND: The soluble urokinase Plasminogen Activator Receptor (suPAR) has been identified as a reliable marker of COVID-19 severity, helping in personalizing COVID-19 therapy. This study aims to evaluate the correlation between suPAR levels and COVID-19 severity, in relation to the traditional inflammatory markers. METHODS: Sera from 71 COVID-19 patients were tested for suPAR levels using Chorus suPAR assay (Diesse Diagnostica Senese SpA, Italy). suPAR levels were compared with other inflammatory markers: IL-1ß, IL-6, TNF-α, circulating calprotectin, neutrophil and lymphocyte counts, and Neutrophil/Lymphocytes Ratio (NLR). Respiratory failure, expressed as P/F ratio, and mortality rate were used as indicators of disease severity. RESULTS: A positive correlation of suPAR levels with IL-6 (r = 0.479, p = 0.000), TNF-α (r = 0.348, p = 0.003), circulating calprotectin (r = 0.369, p = 0.002), neutrophil counts (r = 0.447, p = 0.001), NLR (r = 0.492, p = 0.001) has been shown. Stratifying COVID-19 population by suPAR concentration above and below 6 ng/mL, we observed higher levels of circulating calprotectin (10.1 µg/mL, SD 7.9 versus 6.4 µg/mL, SD 7.5, p < 0.001), higher levels of P/F ratio (207.5 IQR 188.3 vs 312.0 IQR 127.8, p = 0.013) and higher mortality rate. Median levels of suPAR were increased in all COVID-19 patients requiring additional respiratory support (Nasal Cannula, Venturi Mask, BPAP and CPAP) (6.5 IQR = 4.9) compared to the group at room air (4.6 IQR = 4.2). CONCLUSION: suPAR levels correlate with disease severity and survival rate of COVID-19 patients, representing a promising prognostic biomarker for the risk assessment of the disease.

Cardiovascular safety, cancer and Jak-inhibitors: Differences to be highlighted.

Rheumatoid arthritis (RA) is a chronic inflammatory disease whose natural history leads to articular and extra-articular damage. Both cardiovascular risk and malignancy risk results higher in RA patients, compared to general population. Janus kinase inhibitors (JAKis) are oral targeted synthetic disease modifying antirheumatic drugs (tsDMARDs) that disrupt cytokine cascade and exert anti-inflammatory effects by interfering with signaling through the JAK-STAT intracellular pathways. A recent RCT comparing tofacitinib 5 mg twice daily, tofacitinib 10 mg twice daily and anti-TNF in rheumatoid arthritis demonstrated an increased risk of MACE HR 1.33 and cancer HR 1.49 at a follow-up of 4 years. This has led the FDA to class warnings for tofacitinib, baricitinib and upadacitinib. Cumulative RCT data, RCT extension data demonstrated a safety profile for Jak inhibitors. Conflicting data results from real life registries; the different selectivity for JAKs (JAK1, JAK2, JAK3 and Tyk2) probably determines differences in efficacy and safety profiles among the members of this group which should actually be evaluated. In order to better understand the cardiovascular and neoplastic risk linked to these class of drugs, we aim to provide a literature review on existing evidence of the safety of Jak-Inhibitors in rheumatoid arthritis.

The role of neutralizing antibodies by sVNT after two doses of BNT162b2 mRNA vaccine in a cohort of Italian healthcare workers.

OBJECTIVES: Evaluating anti-SARS-CoV-2 antibody levels is a current priority to drive immunization, as well as to predict when a vaccine booster dose may be required and for which priority groups. The aim of our study was to investigate the kinetics of anti-SARS-CoV-2 Spike S1 protein IgG (anti-S1 IgG) antibodies and neutralizing antibodies (NAbs) in an Italian cohort of healthcare workers (HCWs), following the Pfizer/BNT162b2 mRNA vaccine, over a period of up to six months after the second dose. METHODS: We enrolled 57 HCWs, without clinical history of COVID-19 infection. Fluoroenzyme-immunoassay was used for the quantitative anti-S1 IgG antibodies at different time points T1 (one month), T3 (three months) and T6 (six months) following the second vaccine shot. Simultaneously, a commercial surrogate virus neutralization test (sVNT) was used for the determination of NAbs, expressed as inhibition percentage (% IH). RESULTS: Median values of anti-S1 IgG antibodies decreased from T1 (1,452 BAU/mL) to T6 (104 BAU/mL) with a percent variation of 92.8% while the sVNT showed a percent variation of 34.3% for the same time frame. The decline in anti-S1 IgG antibodies from T1 to T6 was not accompanied by a loss of the neutralizing capacity of antibodies. In fact at T6 a neutralization percentage <20% IH was observed only in 3.51% of HCWs. CONCLUSIONS: Our findings reveal that the decrease of anti-S1 IgG levels do not correspond in parallel to a decrease of NAbs over time, which highlights the necessity of using both assays to assess vaccination effectiveness.

Assessing T-Cell Immunity in Kidney Transplant Recipients with Absent Antibody Production after a 3rd Dose of the mRNA-1273 Vaccine.

The vulnerable population of kidney transplant recipients (KTRs) are low responders to COVID-19 vaccines, so specific immune surveillance is needed. The interferon-gamma (IFN-γ) release assay (IGRA) is effective in assessing T cell-mediated immunity. We assessed SARS-CoV-2-directed T cell responses in KTRs with absent antibody production after a third dose of the mRNA-1273 vaccine, using two different IGRAs. A cohort of 57 KTRs, who were actively followed up, received a third dose of the mRNA-1273 vaccine. After the evaluation of humoral immunity to SARS-CoV-2, 14 seronegative patients were tested with two commercial IGRAs (SD Biosensor and Euroimmun). Out of 14 patients, one and three samples were positive by IGRAs with Euroimmun and SD Biosensor, respectively. The overall agreement between the two assays was 85.7% (κ = 0.444). In addition, multivariate linear regression analysis showed no statistically significant association between the IFN-γ concentration, and the independent variables analyzed (age, gender, years since transplant, total lymphocytes cells/mcl, CD3+ cells/mcl, CD3+ CD4+ cells/mcl, CD3+ CD8+ cells/mcl, CD19+ cells/mcl, CD3-CD16+CD56+ cells/mcl) (p > 0.01). In a vulnerable setting, assessing cellular immune response to complement the humoral response may be advantageous. Since the two commercial IGRAs showed a good agreement on negative samples, the three discordant samples highlight the need for further investigations.

Closing the serological gap in the diagnostic testing for COVID-19: The value of anti-SARS-CoV-2 IgA antibodies.

During coronavirus disease 2019 (COVID-19) pandemic, the early diagnosis of patients is a priority. Serological assays, in particular immunoglobulin (Ig)M and IgG anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), have today several applications but the interpretation of their results remains an open challenge. Given the emerging role of the IgA isotype in the COVID-19 diagnostics, we aimed to identify the SARS-CoV-2 IgA antibodies in a COVID-19 population seronegative for IgM. A total of 30 patients hospitalized in San Giovanni di Dio Hospital (Florence, Italy) for COVID-19, seronegative for IgM antibodies, have been studied for anti-SARS-CoV-2 antibodies. They all had a positive oro/nasopharyngeal swab reverse transcription-polymerase chain reaction result. Assays used were a chemiluminescent assay measuring SARS-CoV-2 specific IgM and IgG (S + N) and an ELISA, measuring specific IgG (S1) and IgA antibodies against SARS-CoV-2. Among the 30 patients, eight were positive for IgA, seven were positive for IgG (N + S), and two for IgG (S1), at the first point (5-7 days from the onset of symptoms). The IgA antibodies mean values at the second (9-13 days) and third (21-25 days) time points were even more than twice as high as IgG assays. The agreement between the two IgG assays was moderate (Cohen's K = 0.59; SE = 0.13). The inclusion of the IgA antibodies determination among serological tests of the COVID-19 diagnostic is recommended. IgA antibodies may help to close the serological gap of the COVID-19. Variations among anti-SARS-CoV-2 IgG assays should be considered in the interpretation of results.

Diagnostic accuracy of an automated chemiluminescent immunoassay for anti-SARS-CoV-2 IgM and IgG antibodies: an Italian experience.

A pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been spreading throughout the world. Though molecular diagnostic tests are the gold standard for COVID-19, serological testing is emerging as a potential surveillance tool, in addition to its complementary role in COVID-19 diagnostics. Indubitably quantitative serological testing provides greater advantages than qualitative tests but today there is still little known about serological diagnostics and what the most appropriate role quantitative tests might play. Sixty-one COVID-19 patients and 64 patients from a control group were tested by iFlash1800 CLIA analyzer for anti-SARS CoV-2 antibodies IgM and IgG. All COVID-19 patients were hospitalized in San Giovanni di Dio Hospital (Florence, Italy) and had a positive oro/nasopharyngeal swab reverse-transcription polymerase chain reaction result. The highest sensitivity with a very good specificity performance was reached at a cutoff value of 10.0 AU/mL for IgM and of 7.1 for IgG antibodies, hence near to the manufacturer's cutoff values of 10 AU/mL for both isotypes. The receiver operating characteristic curves showed area under the curve values of 0.918 and 0.980 for anti-SARS CoV-2 antibodies IgM and IgG, respectively. iFlash1800 CLIA analyzer has shown highly accurate results for the anti-SARS-CoV-2 antibodies profile and can be considered an excellent tool for COVID-19 diagnostics.

Evaluation of a New Multiparametric Microdot Array-Based Immunoassay Panel for Systemic Autoimmune Disease Diagnosis.

BACKGROUND: The early reliable detection and quantification of autoantibodies play an important role in autoimmune disease diagnosis and in disease-course monitoring. New technologies, such as the multiplexed determination of autoantibodies, have recently been introduced and are being adopted more frequently. The aim of this study was to evaluate the ability of a new microdot array-based multiparametric assay (ZENIT AMiDot CTD panel, A. Menarini Diagnostics, Firenze, Italy) to correctly classify patients with autoimmune rheumatic diseases (ARDs) and compare it to a fluorescence enzyme immunoassay (FEIA) for the detection of anti-ENAs. METHODS: The study included 69 consecutive samples from patients with ARDs that were analyzed using two different methods (FEIA and AMiDot) to detect anti-CENP B and six anti-ENA antibodies: anti-Scl-70, anti-SSB/La, anti-Jo-1, anti-U1-RNP, anti-Ro52, and anti-Ro60. The control group sera came from sixty-eight blood donors. Tests were run on the automated slide processor ZENIT FLOW, and then the slides were imaged and analyzed using ZENIT fast. RESULTS: Since the samples were selected for at least one antibody positivity with an ARD diagnosis, we did not calculate clinical sensitivity but only specificity, which was 98.53%, ranging from 90% for anti-SSB/La antibodies to 100% for anti-CENP B ones. Mean agreement among the methods assessed by Cohen's kappa was 0.816 ± 0.240. CONCLUSIONS: The assay demonstrated good clinical performance and may be considered a valuable aid in detecting ARD patients, offering an alternative to methods such as FEIA which are largely in use today.

Do Ultrasound Lung Abnormalities Correlate to Biomarkers and Male Gender in Rheumatoid Arthritis Patients? A Monocentric Cross-Sectional Study.

Background: Lung ultrasound (LUS) is a tool of growing interest in Rheumatoid Arthritis (RA) oligo- symptomatic ILD to avoid. Objective: We aimed to evaluate (i) the prevalence of pleural (PLUS) and parenchymal (PAUS) abnormalities in LUS in the RA population and their possible correlation to biomarkers; (ii) the predictivity of gender, smoking habits, previous infections (past COVID-19 tuberculosis), and treatments; (iii) the differences in LUS between sexes. Methods: We collected the data of 155 (15 early and 140 late) RA patients with mild respiratory symptoms, evaluating PLUS and PAUS, in fourteen lung areas and also summing the scores (LUS-T). Results: Only 13/155 (8.4%) were completely negative; LUS correlated to age (all parameters p 0.0001), rheumatoid factor IgM (PLUS p 0.0006, PAUS p 0.02, LUS-T p 0.001) and ACPA (p 0.001, 0.006, 0.001, respectively), and PLUS also correlated to IL6 (p 0.02). The male gender was predictive of all LUS evaluations (p 0.001, 0.05, 0.001, respectively), which were higher than in women (p 0.001, 0.01, 0.001, respectively). Other potential risk factors were independent, except biological treatments, which showed a low predictivity to PLUS (p < 0.05). Conclusions: We can conclude that LUS is a useful technique in RA low respiratory symptoms and correlates with age, the most important RA biomarkers, and male sex.

Real-Life Comparison of Four JAK Inhibitors in Rheumatoid Arthritis (ELECTRA-i Study).

Background: Real-world evidence of the efficacy and adverse events of JAK inhibitor treatment (Tofacitinib, Baricitinib, Upadacitinib, and Filgotinib) in rheumatoid arthritis is still limited. Methods: We studied 115 patients from the Rheumatology Unit of S. Giovanni di Dio Hospital affected by D2T-RA, according to the 2010 EULAR criteria. Out of the 115 patients, 17 had been treated with Baricitinib 8 mg/daily, 32 with Filgotinib 200 mg/daily, 21 with Tofacitinib 10 mg/daily, and 45 with Upadacitinib 15 mg/daily. We evaluated the clinical response after 3, 6, and 12 months of treatment and the follow-up from September 2022 to September 2023. All patients were evaluated according to the number of tender joints (NTJs), number of swollen joints (NSJs), visual analog scale (VAS), global assessment (GA), health assessment questionnaire (HAQ), Disease Activity Score (DAS28), and CDAI. Furthermore, laboratory parameters of efficacy and tolerability were evaluated. Results: All treatments demonstrated a statistically significant decrease in the DAS28 and CDAI scores, tender and swollen joint counts, VAS, HAQ, and patient global assessment (PGA) after 3, 6, and 12 months of treatment. All treatments showed similar behavior, and statistically significant decreases in circulating calprotectin, TNFα, and IL-6 were observed for all drugs after 12 months of treatment. In addition, soluble urokinase plasminogen activator receptor (suPAR) values showed significant differences at baseline and after 12 months of treatment for Filgotinib: 4.87 ± 4.53 vs. 3.61 ± 0.9 (0.009) and Upadacitinib: 6.64 ± 7.12 vs. 4.06 ± 3.61 (0.0003), while no statistically significant differences were found for Baricitinib: 3.4 ± 0.1 vs. 3.78 ± 0.1 and Tofacitinib: 3.95 ± 1.77 vs. 2.58 ± 0.1. The TC/HDL-C ratio (atherogenic index) showed significant differences when comparing Baricitinib vs. Filgotinib (0.0012), Filgotinib vs. Tofacitinib (0.0095), and Filgotinib vs. Upadacitinib (0.0001); furthermore, the LDL-C/HDL-C ratio in the Filgotinib group did not change (2.37 ± 0.45 vs. 2.35 ± 2.13 (NS)) after 12 months of treatment. Venous Thrombotic Events (VTEs) and major adverse cardiovascular events (MACEs) accounted for 1% of adverse events after treatment with Baricitinib. Herpes zoster reactivation accounted for 1% of adverse events after treatment with Filgotinib and Tofacitinib, while non-melanoma skin cancer (NMSC) accounted for 1% of adverse events after Upadacitinib treatment. Conclusions: Our real-world data from patients with RA show differences in some laboratory parameters and in the impact of lipid metabolism in JAK inhibitor treatment.

Role of booster with BNT162b2 mRNA in SARS-CoV-2 vaccination in patients with rheumatoid arthritis.

Only case reports and small clinical series report the effects of booster vaccination with BNT162b2 in patients with rheumatoid arthritis (RA). We studied 200 patients with RA in clinical remission evaluated with the DAS28. All patients were vaccinated for SARS CoV-2 with the BNT162b2 mRNA vaccine. The value of anti-SARS-CoV 2 Spike RBD IgG antibodies was determined at T1 (3 weeks after first vaccination) and T2 (3 weeks after booster). In addition, patients underwent assessment of lymphocyte subpopulations by flow cytometry analysis before starting the vaccination cycle (T0). Furthermore, the serum antibody levels of 96 health care workers (HCWs) were analyzed for comparison. DAS28 values at T0, T1, and T2 indicated remission or low disease activity in all patients. Levels of anti-SARS CoV-2 IgG at T1 were higher in HCWs than in patients' groups: 1562.00 BAU WHO/mL [975.00-1632.00] vs 416.00 BAU WHO/mL [110.00, 1581.00], p <0.001. Anti-SARS COV2 IgG levels at T1 and at T2 were slightly lower in patients taking b/tsDMARDs than in patients under csDMARDs. Regression analysis evidenced age, treatment with abatacept (ABA), JAK inhibitors, and rituximab (RTX) as negative predictors of higher anti-SARS CoV-2 IgG levels at T1. Moreover, treatment with anti-IL6, anti-JAK, and anti-tumor necrosis factor (TNF) emerged as positive predictors of higher levels of anti-SARS CoV-2 IgG at T2. Our data show that despite the booster vaccine with BNT162b2, seroconversion in patients with rheumatoid arthritis is influenced by the background therapy, particularly for patients being treated with ABA and RTX.

Performance evaluation of four surrogate Virus Neutralization Tests (sVNTs) in comparison to the in vivo gold standard test.

BACKGROUND: Several commercial surrogate Virus Neutralization Tests (sVNTs) have been developed in the last year. Neutralizing anti-SARS-CoV-2 antibodies through interaction with Spike protein Receptor Binding Domain (S-RBD) can block the virus from entering and infecting host cells. However, there is a lack of information about the functional activity of SARS-CoV-2 antibodies that may be associated with protective responses. For these reasons, to counteract viral infection, the conventional virus neutralization test (VNT) is still considered the gold standard. The aim of this study was to contribute more and detailed information about sVNTs' performance, by determining in vitro the anti-SARS-CoV-2 neutralizing antibody concentration using four different commercial assays and then comparing the obtained data to VNT. METHODS: Eighty-eight samples were tested using two chemiluminescence assays (Snibe and Mindray) and two ELISA assays (Euroimmun and Diesse). The antibody titers were subsequently detected and quantified by VNT. RESULTS: The overall agreement between each sVNT and VNT was 95.45% for Euroimmun and 98.86% for Diesse, Mindray and Snibe. Additionally, we investigated whether the sVNTs were closer to the gold standard than traditional anti-SARS-CoV-2 antibody assays S-RBD or S1 based, finding a higher agreement mean value for sVNTs (98.01 ± 1.705% vs 95.45 ± 1.921%; p < 0.05). Furthermore, Spearman's statistical analysis for the correlation of sVNT versus VNT showed r = 0.666 for Mindray; r = 0.696 for Diesse; r = 0.779 for Mindray and r = 0.810 for Euroimmun. CONCLUSIONS: Our data revealed a good agreement between VNT and sVNTs. Despite the VNT still remains the gold standard, the sVNT might be a valuable tool for screening wider populations.

Vaccination for SARS-CoV-2 in Patients With Psoriatic Arthritis: Can Therapy Affect the Immunological Response?

Background: A few studies on vaccination in patients with rheumatic diseases, including arthritis, connective tissue diseases, vasculitis, and psoriatic arthropathy (PsA), demonstrated reduced production of neutralizing antibodies to SARS-CoV-2 Spike RBD (receptor-binding domain contained in the N-terminal of the S1 globular head region) when compared to the general population. Objective: The aim of our study was to observe whether different therapies for PsA [methotrexate, anti-TNF antibodies, soluble TNF receptor (etanercept) or IL-17 inhibitors] have a different impact on SARS-CoV-2 vaccination in a homogeneous population of patients. Methods: We enrolled 110 PsA patients in remission, assessed with Disease Activity in PSoriatic Arthritis (DAPSA). Of these: 63 were in treatment with anti-TNF-α therapy (26 etanercept, 15 certolizumab, 5 golimumab, 17 adalimumab); 37 with anti-IL17 secukinumab; 10 with methotrexate. All patients underwent vaccination for SARS-CoV-2 with mRNA BNT162b2 vaccine. Assessment of absolute and percentage lymphocyte subsets and anti-SARS-CoV-2 Spike RBD IgG antibody value 3 weeks after the second vaccine dose were performed. In addition, the serum antibody levels of 96 healthy healthcare workers (HCW) were analyzed. Results: The mean disease activity assessed with DAPSA score was 2.96 (SD = 0.60) with no significant differences between patients under different medications (p = 0.779). Median levels of neutralizing antibodies to SARS-CoV-2 Spike RBD were 928.00 binding antibody unit (BAU)/mL [IQR 329.25, 1632.0]; 1068.00 BAU/ml [IQR 475.00, 1632.00] in patients taking MTX, 846.00 BAU/ml [IQR 125.00, 1632.00] in patients taking etanercept, 908.00 BAU/mL [IQR 396.00, 1632.00] in patients taking anti-IL17 and 1148.00 BAU/ml [IQR 327.00, 1632.00] in patients taking TNF-α inhibitors, without statistically significant differences between these groups. Mean serum antibody level of HCW group was 1562.00 BAU/ml [IQR 975.00, 1632.00], being significantly higher than in the patient group (p = 0.000816). Absolute and percentage count of lymphocyte subsets were not statistically different between the subgroups under different treatments and when compared with HCW. Conclusions: As for other rheumatic diseases on immunomodulatory treatment, our data showed a reduced humoral response in PsA patients compared to the control group. However, antibody response did not significantly differ between groups treated with different medications.

The WHO International Standard for COVID-19 serological tests: towards harmonization of anti-spike assays.

BACKGROUND AND AIMS: SARS-CoV-2 antibody assays are relevant in managing the COVID-19 pandemic, providing valuable data on the immunization status of the population. However, current serology tests are highly variable, due to their different characteristics and to the lack of reference materials. The aim of the World Health Organization (WHO) first International Standard (IS) for anti-SARS-CoV-2 immunoglobulin is to harmonize humoral immune response assessment after natural infection or vaccination, and recommend reporting the results for binding activity in Binding Antibody Units (BAU). MATERIALS AND METHODS: This study analyzed six commercial quantitative anti-SARS-CoV-2 S-protein assays in a head-to-head comparison, using the manufacturers' conversion factors for the WHO IS to obtain BAU/mL values. RESULTS: Our data showed good alignment up to 1000 BAU/mL, then began to disperse, exhibiting some discrepancies. Moreover, correlations among methods varied with Cohen's Kappa ranging from 0.580 to 1.00, with the lowest agreement values for kits using different target antigens or different antibody isotypes, making it clear that the laboratory report should include this information. Values expressed as BAU/ml showed a reduced between-assays variability compared to AU/ml (median coefficients of variation 0.38 and 0.68, respectively; p < 0.001). CONCLUSION: On the basis of these data at present anti-SARS CoV-2 serological assays' results are not interchangeable, and, more importantly, individual immune monitoring should be performed with the same method.

High levels of homocysteine, lipoprotein (a) and plasminogen activator inhibitor-1 are present in patients with abdominal aortic aneurysm.

Over the last few years,there has been increasing interest in the investigation of the pathogenesis of AAA, and a role for some novel risk factors, in particular thrombophilic risk factors, has been suggested. The aim of this study was to evaluate a number of thrombophilic parameters in a large group of patients with AAA. In 438 patients with AAA, and in 438 healthy subjects, selected to be comparable for age and gender with patients and without instrumental evidence of AAA, a pattern of thrombophilic parameters [homocysteine (Hcy), lipoprotein (a) [Lp(a)], plasminogen activator inhibitor-1 (PAI-1), anticardiolipin antibodies (ACA), MTHFR C677T polymorphism, prothrombin gene G20210A variant and FactorV Leiden mutation] has been evaluated. A significant difference for Hcy, PAI-1 and Lp(a) plasma levels has been observed between patients and controls. After adjustment for the traditional cardiovascular risk factors, a significant increased risk of having AAA has been observed for high levels of Hcy (OR: 7.8; p<0.0001), Lp(a) (OR: 2.4; p<0.0001) and PAI-1 (OR: 3.2; p<0.0001). The association has been confirmed after exclusion of patients with other localization of atherosclerosis. Moreover, a significant association between larger abdominal aortic diameters and the number of thrombophilic parameters has been reported (r = 0.13; p = 0.005). In conclusion, a significant association between abnormal levels of some metabolic parameters related to thrombosis such as Hcy, Lp(a) and PAI-1 and AAA has been observed.

Thrombophilic risk factors for symptomatic peripheral arterial disease.

OBJECTIVE: Peripheral arterial disease (PAD) is a common manifestation of systemic atherosclerosis. Over the last years, several novel mediators relevant to the process of atherogenesis have been identified, but few and conflicting data are available on the possible association with PAD symptoms. The aim of this study was to determine an extended thrombophilic risk profile of patients with symptomatic PAD. METHODS: Two hundred eighty patients with symptomatic PAD admitted to the Department of Vascular Surgery of the University of Florence were compared with 280 control subjects without PAD, matched for age and gender. The following metabolic and genetic risk factors were evaluated: lipoprotein(a), homocysteine, antiphospholipid antibodies, plasminogen activator inhibitor-1, factor V Leiden mutation, prothrombin variant, and 5,10-methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism. RESULTS: Multivariate logistic regression analysis, adjusted for traditional cardiovascular risk factors, showed a significant association between PAD symptoms and prothrombin variant, altered levels of homocysteine, lipoprotein(a), plasminogen activator inhibitor-1, and antiphospholipid antibodies. Moreover, the presence of high levels of lipoprotein(a) and another metabolic risk factor raised the likelihood of PAD symptoms (dyslipidemia and elevated lipoprotein[a]: odds ratio [OR], 29; 95% confidence interval [CI], 6.2 to 136.2; P <.0001; hyperhomocysteinemia and elevated lipoprotein[a]: OR, 37.7; 95% CI, 3.7 to 381.5; P <.0001). A significant correlation between the number of altered thrombophilic parameters and the Fontaine stage was observed ( R = 0.16; P = .006). CONCLUSION: There is an independent association between altered levels of important thrombophilic risk factors and PAD symptoms. The clinical significance of this association needs to be tested in prospective population-based trials.