Cryo-EM structure and functional basis of prothrombin recognition by a type I antiprothrombin antiphospholipid antibody.

ABSTRACT: Antiprothrombin antibodies are found in antiphospholipid patients, but how they interact with prothrombin remains elusive. Prothrombin adopts closed and open forms. We recently discovered type I and type II antibodies and proposed that type I recognizes the open form. In this study, we report the discovery and structural and functional characterization in human plasma of a type I antibody, POmAb (prothrombin open monoclonal antibody). Using surface plasmon resonance and single-molecule spectroscopy, we show that POmAb interacts with kringle-1 of prothrombin, shifting the equilibrium toward the open form. Using single-particle cryogenic electron microscopy (cryo-EM), we establish that the epitope targeted by POmAb is in kringle-1, comprising an extended binding interface centered at residues R90-Y93. The 3.2-Å cryo-EM structure of the complex reveals that the epitope overlaps with the position occupied by the protease domain of prothrombin in the closed state, explaining the exclusive binding of POmAb to the open form. In human plasma, POmAb prolongs phospholipid-initiated and diluted Russell's viper venom clotting time, which could be partly rescued by excess phospholipids, indicating POmAb is an anticoagulant but exerts a weak lupus anticoagulant effect. These studies reveal the structural basis of prothrombin recognition by a type I antiphospholipid antibody and uncover an exciting new strategy to achieve anticoagulation in human plasma.

Anti-ß2-glycoprotein I and anti-phosphatidylserine/prothrombin antibodies interfere with cleavage of factor V(a) by activated protein C.

BACKGROUND: The acquired thrombotic risk factor known as lupus anticoagulant (LA) interferes with laboratory clotting assays and can be caused by autoantibodies against ß2-glycoprotein I (ß2GPI) and prothrombin. LA is associated with activated protein C (APC) resistance, which might contribute to thrombotic risk in patients with antiphospholipid syndrome. How antibodies against ß2GPI and prothrombin cause APC resistance is currently unclear. OBJECTIVES: To investigate how anti-ß2GPI and antiphosphatidylserine/prothrombin (PS/PT) antibodies induce APC resistance. METHODS: The effects of anti-ß2GPI and anti-PS/PT antibodies on APC resistance were studied in plasma (of patients with antiphospholipid syndrome) and with purified coagulation factors and antibodies. RESULTS: APC resistance was observed in LA-positive patients with anti-ß2GPI or anti-PS/PT antibodies and in normal plasma spiked with monoclonal anti-ß2GPI or anti-PS/PT antibodies with LA activity. Analysis of factor (F)V cleavage patterns after APC incubation indicated that anti-ß2GPI antibodies attenuated APC-mediated FV cleavage at R506 and R306. APC-mediated cleavage at R506 is required for FV cofactor activity during inactivation of FVIIIa. Assays with purified coagulation factors confirmed that anti-ß2GPI antibodies interfered with the cofactor function of FV during FVIIIa inactivation but not with FVa inactivation. Anti-PS/PT antibodies attenuated APC-mediated FVa and FVIIIa inactivation. Analysis of FV(a) cleavage patterns after APC incubation indicated that anti-PS/PT antibodies interfere with APC-mediated cleavage of FV at positions R506 and R306. CONCLUSION: Anti-ß2GPI antibodies with LA activity contribute to a procoagulant state by causing APC resistance via interference with the cofactor function of FV during FVIIIa inactivation. LA-causing anti-PS/PT antibodies interfere with the anticoagulant function of APC by preventing FV(a) cleavage.

'Non-criteria antiphospholipid antibodies': bridging the gap between seropositive and seronegative antiphospholipid syndrome.

OBJECTIVE: We aimed to analyse the prevalence of non-criteria anti-phospholipid (aPL) antibodies and their role in the diagnosis, treatment and prognosis in a cohort of patients with clinical features consistent with a diagnosis of antiphospholipid syndrome (APS), but persistently negative for criteria aPL - anti-cardiolipin antibodies (aCL), anti-ß2-glycoprotein I antibodies (aß2-GPI) and lupus anticoagulant (LA) - named seronegative APS (SN-APS). METHODS: Sera from SN-APS patients were tested for aCL by TLC-immunostaining, anti-vimentin/cardiolipin (aVim/CL) and anti-phosphatidylserine/prothrombin (anti-PS/PT) by ELISA. Control groups of our study were APS patients and healthy controls. RESULTS: We enrolled 114 consecutive SN-APS patients, 69 (60.5%) resulted positive for at least one non-criteria test in two occasions 12 weeks apart. Among the persistently positive patients to these tests, 97% resulted positive for aCL by TLC-immunostaining, 52.3% for aVim/CL and 17.4% for aPS/PT. SN-APS patients with double positivity (aCL by TLC-immunostaining and aVim/CL) showed a likelihood positive ratio of 8 to present mixed thrombotic and obstetrical features. Among SN-APS patients tested positive, after the therapeutic changes, three cases of recurrent thrombosis were observed [median follow-up 41 months (IQR 39.5)]. Twenty pregnancies were recorded in 17 SN-APS patients after the detection of unconventional aPL and 12 of them (60%) experienced a good outcome under conventional treatment for APS. CONCLUSIONS: This is the largest monocentric study demonstrating that aCL tested by TLC-immunostaining and aVim/CL can detect aPL positivity in SN-APS. It may encourage clinicians to monitor and provide adequate targeted therapy, which improve SN-APS prognosis.

Anti-prothrombin autoantibodies enriched after infection with SARS-CoV-2 and influenced by strength of antibody response against SARS-CoV-2 proteins.

Antiphospholipid antibodies (aPL), assumed to cause antiphospholipid syndrome (APS), are notorious for their heterogeneity in targeting phospholipids and phospholipid-binding proteins. The persistent presence of Lupus anticoagulant and/or aPL against cardiolipin and/or ß2-glycoprotein I have been shown to be independent risk factors for vascular thrombosis and pregnancy morbidity in APS. aPL production is thought to be triggered by-among other factors-viral infections, though infection-associated aPL have mostly been considered non-pathogenic. Recently, the potential pathogenicity of infection-associated aPL has gained momentum since an increasing number of patients infected with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been described with coagulation abnormalities and hyperinflammation, together with the presence of aPL. Here, we present data from a multicentric, mixed-severity study including three cohorts of individuals who contracted SARS-CoV-2 as well as non-infected blood donors. We simultaneously measured 10 different criteria and non-criteria aPL (IgM and IgG) by using a line immunoassay. Further, IgG antibody response against three SARS-CoV-2 proteins was investigated using tripartite automated blood immunoassay technology. Our analyses revealed that selected non-criteria aPL were enriched concomitant to or after an infection with SARS-CoV-2. Linear mixed-effects models suggest an association of aPL with prothrombin (PT). The strength of the antibody response against SARS-CoV-2 was further influenced by SARS-CoV-2 disease severity and sex of the individuals. In conclusion, our study is the first to report an association between disease severity, anti-SARS-CoV-2 immunoreactivity, and aPL against PT in patients with SARS-CoV-2.

Anti-Phosphatidylserine/Prothrombin Antibodies at Two Points: Correlation With Lupus Anticoagulant and Thrombotic Risk.

Antibodies to phospholipids (aPL) and associated proteins are a hallmark in the diagnosis of anti-phospholipid syndrome (APS). Those included in the classification criteria are the lupus anticoagulant (LA) and the IgG and IgM isotypes of anticardiolipin (aCL) and anti-beta-2 glycoprotein I (ß2GPI) antibodies. Non-classification criteria markers such as autoantibodies that recognize the phosphatidylserine/prothrombin (aPS/PT) complex have been proposed as biomarkers for APS. Studies of aPS/PT antibodies have shown a strong correlation to clinical manifestations and LA. We aimed to study the value and the persistence of aPS/PT IgG and IgM antibodies in a cohort of consecutive patients with clinical suspicion of APS and their utility as thrombotic risk markers. Our study, with 103 patients, demonstrates that persistently positive results for aPS/PT IgG antibodies were significantly associated with APS classification, thrombosis, triple aPL positivity, LA positive result, and the Global APS Score (GAPSS) > than 9 points (p < 0.01, for each condition). On the other hand, no association was seen with pregnancy morbidity (p = 0.56) and SLE (p = 0.07). Persistence of aPS/PT antibodies, defined according to the current laboratory classification criteria, likely improves the diagnosis and clinical assessment of patients with APS.

A Novel ELISA Assay for the Detection of Anti-Prothrombin Antibodies in Antiphospholipid Syndrome Patients at High Risk of Thrombosis.

Autoantibodies targeting prothrombin (aPT) can be found in antiphospholipid syndrome (APS) patients. However, their detection has proven difficult to standardize. Here, we developed a new ELISA assay to improve the identification of aPT and compared its performance with currently available anti-phosphatidylserine/prothrombin antibodies (aPS/PT) and autoantibodies targeting prothrombin bound to the plastic plate (aPT-A) assays using a cohort of 27 APS patients at high risk of thrombosis. We generated a novel prothrombin variant, ProTS525A-Biot, carrying an artificial tag at the C-terminus suitable for site-specific biotinylation and added the mutation S525A to improve stability. ProTS525A-Biot was immobilized to neutravidin-coated plates at the desired density and with a defined orientation, i.e., pointing the N-terminal fragment-1 toward the solvent. Antibodies against ProTS525A-Biot (aPT-Bio) were found in 24 out of 27 triple-positive APS patients (88%). When compared to aPS/PT and aPT-A, aPT-Bio showed an excellent linear correlation with aPS/PT (R2 = 0.85) but not with aPT-A (R2 = 0.40). Since aPS/PT but not aPT-A are an emerging biomarker of thrombosis in APS, this method may find utility for detecting pathogenic aPT in APS but also other prothrombotic conditions such as COVID-19.

Anti-phosphatidyl-serine/prothrombin antibodies (aPS/PT) in isolated lupus anticoagulant (LA): is their presence linked to dual test positivity?

OBJECTIVES: Anti phosphatidylserine/prothrombin antibodies (aPS/PT) are often present in patients with antiphospholipid syndrome (APS) and might be relevant in the pathogenesis of this condition. They are major determinant of lupus anticoagulant (LA) in triple-positive antiphospholipid (aPL) profile. Whether they are present and pathogenic in patients with isolated LA [negative anticardiolipin (aCL) and anti ß2-glycoprotein I (aß2GPI) antibodies] is a matter of debate. METHODS: We measured aPS/PT in a large number of isolated LA with the aim to ascertain whether there is a link between the way isolated LA is assessed and the presence of these antibodies. APS/PT were measured in 86 patients with isolated LA (aCL- and abeta2GPI-). LA was assessed by two test systems, the dilute Russell Viper Venom Time (dRVVT) and the Silica Clotting Time (SCT). RESULTS: Sixty-six (77%) individuals with isolated LA were positive for aPS/PT (IgM 44, IgG and IgM 15, IgG in 7). Diagnosis of LA was made based on positive results in both dRVVT and SCT in 40 patients (Group 1) and based on only one positive test in the remaining 46 patients (Group 2). The rate of positive aPS/PT antibodies was significantly higher in Group 1 (OR=7.2, 95% CI 1.9-27.0, p<0.002). Moreover, the titre of IgM aPS/PT was significantly increased in Group 1 as compared to Group 2 (137 U, IQR 64-179 vs. 43 U, IQR 11-120, p=0.008). CONCLUSIONS: These data indicate an association between LA based on two positive coagulation tests and the presence of aPS/PT antibodies, especially of IgM isotype.

Cerebrovascular events in patients with isolated anti-phosphatidyl-serine/prothrombin antibodies.

The interest of extra-criteria antiphospholipid antibodies is growing, especially in patients negative for conventional antibodies. In this study we aimed to assess the clinical utility of anti-phosphatidyl-serine/prothrombin antibodies (aPS/PT) testing in patients negative for Beta2-Glycoprotein 1(ß2GPI)-dependent tests, for identifying antiphospholipid syndrome (APS) patients that developed cerebrovascular events (CVE). When screening APS patients attending our center, out of 119 aPS/PT IgG/IgM-positive patients, thus patients negative for aß2GPI and aCL, 42 patients (35%) tested negative for ß2GPI-dependent tests and were tested with thrombin generation assay (TGA). Ten patients (24%), with isolated aPS/PT IgG/IgM, had a history of CVE. Lupus anticoagulant (LA)-positive test was more frequently observed in patients with CVE (8/22 vs. 2/20; p = 0.045). Out of the 10 patients who experienced CVE, 3 patients were aPS/PT IgG positive (all LA positive), and 8 patients were aPS/PT IgM positive (6/8 LA positive). One patient was positive for both aPS/PT IgG and IgM. LA-positive patients had only high titers of aPS/PT IgG/IgM, all of them being ≥ 80 U/ml, while the 2 LA-negative patients were aPS/PT IgM positive with medium titers [40-60 U/ml]. LA-positive patients had significantly altered TGA profile when compared to those who were LA negative, considering all TGA parameters. LA-positive patients had significantly higher tLag (8.4 ± 3.3 min vs. 6.6 ± 1.8 min; p = 0.046), higher tPeak (14 ± 4.3 min vs. 11 ± 2.7 min; p = 0.015) and lower Peak (207 ± 152 nM vs. 356.3 ± 104.7 nM; p < 0.001) and lower AUC (2109.7 ± 1006.9 nM vs. 2772.5 ± 776.8 nM; p = 0.033). The use of aPS/PT might be of help in identifying patients with CVE and APS, as also confirmed by TGA testing.

Anti-Phospholipid Antibodies in COVID-19 Are Different From Those Detectable in the Anti-Phospholipid Syndrome.

Background: Critically ill patients with coronavirus disease 2019 (COVID-19) have a profound hypercoagulable state and often develop coagulopathy which leads to organ failure and death. Because of a prolonged activated partial-thromboplastin time (aPTT), a relationship with anti-phospholipid antibodies (aPLs) has been proposed, but results are controversial. Functional assays for aPL (i.e., lupus anticoagulant) can be influenced by concomitant anticoagulation and/or high levels of C reactive protein. The presence of anti-cardiolipin (aCL), anti-beta2-glycoprotein I (anti-ß2GPI), and anti-phosphatidylserine/prothrombin (aPS/PT) antibodies was not investigated systematically. Epitope specificity of anti-ß2GPI antibodies was not reported. Objective: To evaluate the prevalence and the clinical association of aPL in a large cohort of COVID-19 patients, and to characterize the epitope specificity of anti-ß2GPI antibodies. Methods: ELISA and chemiluminescence assays were used to test 122 sera of patients suffering from severe COVID-19. Of them, 16 displayed major thrombotic events. Results: Anti-ß2GPI IgG/IgA/IgM was the most frequent in 15.6/6.6/9.0% of patients, while aCL IgG/IgM was detected in 5.7/6.6% by ELISA. Comparable values were found by chemiluminescence. aPS/PT IgG/IgM were detectable in 2.5 and 9.8% by ELISA. No association between thrombosis and aPL was found. Reactivity against domain 1 and 4-5 of ß2GPI was limited to 3/58 (5.2%) tested sera for each domain and did not correlate with aCL/anti-ß2GPI nor with thrombosis. Conclusions: aPL show a low prevalence in COVID-19 patients and are not associated with major thrombotic events. aPL in COVID-19 patients are mainly directed against ß2GPI but display an epitope specificity different from antibodies in antiphospholipid syndrome.

Antiphospholipid antibodies in patients with COVID-19: A relevant observation?

BACKGROUND: High incidence of thrombosis in COVID-19 patients indicates a hypercoagulable state. Hence, exploring the involvement of antiphospholipid antibodies (aPL) in these patients is of interest. OBJECTIVES: To illustrate the incidence of criteria (lupus anticoagulant [LAC], anticardiolipin [aCL] immunoglobulin G [IgG]/IgM, antibeta2-glycoprotein I antibodies [aß2GPI] IgG/IgM) and noncriteria (anti-phosphatidyl serine/prothrombin [aPS/PT], aCL, and aß2GPI IgA) aPL in a consecutive cohort of critically ill SARS-CoV-2 patients, their association with thrombosis, antibody profile and titers of aPL. PATIENTS/METHODS: Thirty-one consecutive confirmed COVID-19 patients admitted to the intensive care unit were included. aPL were measured at one time point, with part of the aPL-positive patients retested after 1 month. RESULTS: Sixteen patients were single LAC-positive, two triple-positive, one double-positive, one single aCL, and three aCL IgG and LAC positive. Seven of nine thrombotic patients had at least one aPL. Sixteen of 22 patients without thrombosis were aPL positive, amongst them two triple positives. Nine of 10 retested LAC-positive patients were negative on a second occasion, as well as the double-positive patient. Seven patients were aPS/PT-positive associated to LAC. Three patients were aCL and aß2GPI IgA-positive. CONCLUSION: Our observations support the frequent single LAC positivity during (acute phase) observed in COVID-19 infection; however, not clearly related to thrombotic complications. Triple aPL positivity and high aCL/aß2GPI titers are rare. Repeat testing suggests aPL to be mostly transient. Further studies and international registration of aPL should improve understanding the role of aPL in thrombotic COVID-19 patients.

Global antiphospholipid syndrome score and anti-ß2-glycoprotein I domain I for thrombotic risk stratification in antiphospholipid syndrome: A four-year prospective study.

OBJECTIVE: This study aimed to assess prospectively the role of anti-ß2-glycoprotein I domain I antibody (aß2GPI-DI) and the Global Antiphospholipid Syndrome Score (GAPSS) in identifying antiphospholipid syndrome (APS) patients at higher risk of a new event. METHODS: Thrombotic APS patients were followed from May 2013 to July 2017. At baseline, we measured lupus anticoagulant, IgG/IgM anticardiolipin, anti-ß2-glycoprotein I, antiphosphatidylserine-prothrombin (aPS/PT) and IgG aß2GPI-DI, and calculated GAPSS for each patient. RESULTS: A total of 44 patients (age 43 ± 10 years, 89% female, 73% primary APS) were followed for 39 months (range 9-46 months). Four new thromboses occurred, two of them after vitamin K antagonist interruption. Recurrent patients presented higher GAPSS (median 20) and were triple and aß2GPI-DI positive; non-recurrent patients had lower GAPSS (median 10.5, range 0-20) and lower ratio of triple (33%) and aß2GPI-DI positivities (38%). aß2GPI-DI was associated with higher GAPSS (median 19 vs. 7, p < 0.001; Pearson correlation 0.82, p < 0.001) and had a greater proportion of triple (83% vs. 4%, p < 0.001) and aPS/PT positivity (94% vs. 50%, p = 0.002). CONCLUSION: Our data show a significant correlation between a validated risk score such as GAPSS and the novel antiphospholipid antibody aß2GPI-DI. Future studies are needed. However, one could speculate a role of aß2GPI-DI as a risk-stratifying tool for thrombotic events in APS.

Antiphosphatidylserine/Prothrombin Antibodies: An Update on Their Association with Clinical Manifestations of Antiphospholipid Syndrome.

OBJECTIVE: The aim of the study is to perform a systematic review on the recent available evidence on antiphosphatidylserine/prothrombin (aPS/PT) antibodies and their association with clinical manifestations of the antiphospholipid syndrome (APS). METHODS: A detailed literature search was applied a priori to Ovid MEDLINE, In-Process and Other Non-Indexed Citation 2012 to present and to abstract from EULAR and ACR/ARHP Annual Meetings (2012-2019). RESULTS: Data from 2,901 patients, 587 diseases controls and 559 healthy controls included in 15 retrieved studies was analyzed. The patient population included 1,219 patients classified as APS according to the Sidney criteria, 285 patients with isolated persistently positive antiphospholipid antibodies (aPL) and 1,397 patients with a clinical suspicion of APS. Twelve studies, including 1,888 patients, analyzed the association between aPS/PT antibodies and thrombosis. We observed a statistically significant association between aPS/PT IgG/IgM positivity and thrombotic events (mean odds ratio [OR]: 6.8 [95% CI: 3.18-16.4], p < 0.05), confirmed when analyzing aPS/PT IgG (mean OR: 6.7 [95% CI: 3.04-21.6], p < 0.05) and aPS/PT IgM (mean OR: 4.35 [95% CI: 1.54-17.77], p < 0.05) separately. Seven studies, including 1,388 patients, evaluated the association between aPS/PT antibodies and PM. When pooled together, we found a statistically significant association between any PM and aPS/PT IgG/IgM positivity (mean OR: 10.6 [95% CI: 3.54-35.38], p < 0.05), particularly aPS/PT IgG positivity (mean OR: 6.7 [95% CI: 3.04-21.6], p < 0.05). CONCLUSION: Our results highlight the strong association between aPS/PT and the clinical manifestations of APS. With the available level of evidence, aPS/PT testing can be considered as a robust test applicable in the investigation of patients suspected for APS, also beyond the research settings.

Relationship between lysosomal-associated membrane protein-2 and anti-phosphatidylserine/prothrombin complex antibody in the pathogenesis of cutaneous vasculitis.

OBJECTIVES: We investigated the relationship between lysosomal-associated membrane protein-2 (LAMP-2) and anti-phosphatidylserine/prothrombin (PS/PT) antibody in the pathogenesis of cutaneous vasculitis. METHODS: Cell surface LAMP-2 expression of human neutrophils was measured using flow cytometry. Twenty inbred wild-type Wistar-King-Aptekman-Hokudai (WKAH) rats were divided into four groups: Group 1, rabbit IgG injection only as negative control (n=5); Group 2, both histone and rabbit IgG injection (n=5); Group 3, anti-LAMP-2 antibody injection only (n=5); and Group 4, both histone and anti-LAMP-2 antibody injection (n=5). Ten WKAH rats were divided into two groups: Group A, histone, anti-PS/PT antibody, and anti-LAMP-2 antibody injection (n=5), and Group B, histone, anti-PS/PT antibody, and rabbit IgG injection as control (n=5). RESULTS: LAMP-2 expression on human neutrophils was induced by cell-free histone exposure in a dose- and time-dependent manner. Histopathological examination revealed the recruitment of neutrophils in cutaneous small vessels in all Group 4 rats. These observations were not evident in systemic organs other than the skin. LAMP-2 expression on the surface of vascular endothelial cells was evident in Group 2, exclusively in the skin, but not in Group 1. Thrombi were detected in various organs in all Groups A and B rats. However, no apparent thrombi were observed in the skin. CONCLUSIONS: Anti-PS/PT and anti-LAMP-2 antibodies are responsible for independent effector mechanisms in the rats given intravenous injection of cell-free histones. We considered that undetermined factors other than cell-free histones could be required for the induction of cutaneous vasculitis by anti-PS/PT and anti-LAMP-2 antibodies.

Interest of IgG and IgM antiprothrombin autoantibodies in the exploration of antiphospholipid syndrome: a 5-year retrospective study.

OBJECTIVES: Non-conventional aPL have been described in patients presenting clinical manifestations of antiphospholipid syndrome but negative for conventional markers. Among them, detection of autoantibodies against prothrombin has been proposed to improve diagnosis and management of these patients. However autoantibodies against prothrombin are heterogeneous and their use in clinical practice still remains unclear. The aim of this study was to evaluate the interest of IgG and IgM autoantibodies directed against the prothrombin only (aPT). METHODS: We retrospectively studied IgM and IgG aPT results, conventional antiphospholipid syndrome markers and clinical data of a large cohort of 441 patients referred for antiphospholipid syndrome exploration with aPT detection over a period of 5 years. RESULTS: We observed a total prevalence of 17% of aPT-positive patients (75/441). A significant association was found between aPT and thrombosis (P = 0.035), with 70% of patients having unexplained thrombosis, aPT representing the sole aPL detected. aPT positivity was significantly more frequent in venous thrombosis than in arterial thrombosis (P = 0.004). Interestingly, we demonstrated for the first time that aPT IgG levels were higher in recurrent thrombosis than in isolated thrombosis (P = 0.013), leading us to propose a predictive level of recurrence for thrombosis. CONCLUSION: Our results show that aPT are associated with thrombosis and demonstrate the interest of assessing both IgG and IgM aPT, in particular in venous thrombosis when conventional markers are negative. Quantification of aPT could predict recurrence of thrombosis and influence subsequent treatment strategy. Prospective clinical studies are now required to confirm these results.

Diagnostic performance of aPS/PT antibodies in neuropsychiatric lupus and cardiovascular complications of systemic lupus erythematosus.

Background: Systemic lupus erythematosus (SLE) is associated with a constellation of complications affecting multiple organs, including neuropsychiatric manifestations (NPSLE) and ischaemic events, leading to increased long-term morbidity. Antiphospholipid antibodies (aPL) are a major determinant of vascular inflammation and thromboembolic risk. The diagnostic role of anti-phosphatidylserine/prothrombin (aPS/PT) antibodies in this setting is incompletely defined.Aim: To verify whether aPS/PT add to diagnostics and disease stratification in patients with SLE with or without other aPL.Methods: 131 consecutive patients were studied, including 20 patients with SLE and secondary antiphospholipid syndrome (APS). aPS/PT IgG and IgM were assessed through ELISA and patients were stratified based on the presence of other aPL, on their clinical and laboratory features at time of blood sampling and on their clinical history. Synthetic indices of disease activity, chronic damage and cardiovascular risk were calculated at time of venipuncture.Results: Fifty-one (38.9%) patients with SLE had aPS/PT and 15 (11.5%) patients had aPS/PT as the only aPL (aPS/PT-only). aPS/PT-only patients had a significantly higher prevalence of NPSLE than quadruple aPL-negative patients (p = .007). Patients with aPS/PT were more likely to have a history of ischaemia, thrombocytopenia and Libman-Sacks' endocarditis. The presence of aPS/PT also associated with previous accrual of at least one damage item (p = .043), but had limited predictive values for damage progression in the short term.Conclusion: aPS/PT antibodies provide non-redundant information that could contribute to risk assessment and stratification of patients with SLE.

Splenic infarction associated with transient anti-prothrombin antibodies is a rare manifestation of acute Mycoplasma pneumoniae infection.

We report the first paediatric case of splenic infarction following acute Mycoplasma pneumoniae infection with induction of anti-prothrombin (aPT) antibodies. A 12-year-old boy was admitted to the paediatric emergency department for a left pleuropneumonia and a splenic infarction. aPT antibodies were transitorily detected. The patient recovered fully after antibiotic therapy and a 3-month course of anticoagulation treatment. Antiphospholipid (aPL) antibodies induced by acute infections have already been reported but cases of clinically relevant thrombosis remain rare. The pathogenicity of aPT antibodies is discussed here. We hypothesize that these antibodies were involved in this symptomatic hypercoagulable state.

Laboratory Evaluation of Antiphospholipid Syndrome.

OBJECTIVES: Anti-ß2 glycoprotein I domain I (anti-domain I) and anti-phosphatidylserine/prothrombin (aPS/PT) antibodies are present in patients with antiphospholipid syndrome (APS); however, their use in evaluation remains unclear. METHODS: Diagnostic attributes of lupus anticoagulant (LAC), anti-domain I IgG, anti-cardiolipin, anti-ß2 glycoprotein I (anti-ß2GPI), and aPS/PT IgG and IgM antibodies were assessed in 216 patients evaluated for APS. RESULTS: LAC had the best odds ratio (OR, 14.2) while that for anti-domain 1 IgG was comparable to anti-ß2GPI IgG (OR, 8.3 vs 9.4) but higher than all others. Significant correlations were observed for thrombosis (P = .03) and pregnancy-related morbidity (P = .001) with anti-domain IgG and for any thrombosis with aPS/PT IgG (P = .006). Use of noncriteria antiphospholipid with or without criteria markers did not significantly increase the probability to diagnose APS. CONCLUSIONS: Noncriteria tests can contribute to diagnosis and stratification of APS but do not improve diagnostic yield. Optimal strategies for implementation require prospective investigation.

Antiphosphatidylserine/prothrombin complex antibodies as a determinant of prothrombotic plasma fibrin clot properties in patients with antiphospholipid syndrome.

BACKGROUND: Antiphosphatidylserine/prothrombin complex (aPS/PT) antibodies are recognized as a marker for antiphospholipid syndrome (APS). Dense and poorly lysable fibrin clots occur in thrombotic APS. Compact clots predict thromboembolism, but determinants of the unfavorable clot phenotype remain unknown in APS. We hypothesized that elevated aPS/PT antibodies determine unfavorable clot features. METHODS: In a cohort study involving 124 consecutive patients with thrombotic APS, we measured at baseline plasma fibrin clot permeability (Ks ), efficiency of fibrinolysis (clot lysis time, CLT), and turbidity (off anticoagulation) along with immunoglobulin (Ig)G/IgM aPS/PT. During follow-up, symptomatic thromboembolic events were recorded. RESULTS: Elevated IgG and IgM aPS/PT antibodies >30 international enzyme units (UI) were detected in 54.8% and 42.7% of APS patients, including 76.2% and 54% of lupus anticoagulant- (LA, n = 63) positive patients, respectively. Elevated IgG and IgM aPS/PT antibodies predicted low Ks (lower quartile, <6 × 10-9  cm2 ; odds ratio [OR] = 5.93, 95% confidence interval [CI] 2.09-16.82 and OR = 11.79, 95% CI 4.10-33.92) and prolonged CLT (top quartile, ≥116 min; OR = 4.85, 95% CI 2.42-25.07 and OR = 6.04, 95% CI 2.42-15.07). No such associations were observed for anticardiolipin or ß2-glycoprotein I antibodies or LA presence. During follow-up (median 72.5, range 66-83 months), thromboembolic events observed in 32 (26.7%, 4.6%/year) patients were independently predicted by IgG aPS/PT antibodies >30 UI (hazard ratio [HR] = 3.04, 95% CI 1.20-8.88) and low Ks (HR = 3.00, 95% CI 1.41-6.50). CONCLUSIONS: We identified aPS/PT antibodies as a determinant of denser and poorly lysable plasma fibrin clot formation in APS patients. The association of elevated aPS/PT antibodies with thromboembolism in APS could be at least in part mediated by prothrombotic clot properties.

Lupus anticoagulant-hypoprothrombinemia syndrome and similar diseases: experiences at a single center in Japan.

Patients with lupus anticoagulant (LA), a thrombotic risk factor, along with decreased prothrombin (FII) activity are classified as lupus anticoagulant-hypoprothrombinemia syndrome (LAHPS) and occasionally show bleeding symptoms, although this is not essential for diagnosis. We treated 20 cases of LAHPS over a 3-year period. Median FII activity was 20.9% and the anti-prothrombin antibody (anti-II Ab), shown by ELISA findings, was detected in 55%. Bleeding symptoms were observed in 20%, although that finding was not correlated with FII activity or anti-FII Ab quantity. We also observed 21 LA cases with decreased activity of coagulation factors other than FII, which we have designated LAHPS-like syndrome (LLS). Among LLS patients, anti-FII Ab and bleeding symptoms were seen in 47.6% and 14.3%, respectively. Our findings suggest that bleeding in LAHPS and LLS cannot be explained only by FII activity decreased by anti-FII Ab. Low FVIII activity and the anti-FVIII antibody (anti-FVIII Ab) were detected in some LAHPS and LLS patients, making it difficult to distinguish those from acquired hemophilia A cases. Detection of anti-FVIII Ab quantity by ELISA may be useful for accurate determination, as that was not performed in our LAHPS or LLS patients.