Frequency and Clinical Significance Anti-PS/PT Antibodies in Patients with Antiphospholipid Syndrome—Single Centre Observational Study in the United Kingdom

Antiphospholipid syndrome (APS) is an acquired autoimmune disease, defined by the presence of persistently positive antiphospholipid antibodies (aPLs) on two or more occasions at least 12 weeks apart, in association with the clinical occurrence of thrombotic and/or obstetric complications.[1] Although current classification criteria for APS,[1] often also used as surrogate diagnostic criteria, include only lupus anticoagulant (LA), IgG and IgM anticardiolipin antibodies (aCL) and anti-β2-glycoprotein I (anti-β2GPI) antibodies, a broad group of autoantibodies against various targets are present in APS patients, which may variably contribute to pathogenesis of the disorder. Of these numerous autoantibodies, anti-phosphatidylserine/prothrombin antibodies (anti-PS/PT) that target the anionic phospholipid phosphatidylserine and the procoagulant plasma protein prothrombin, have recently become of particular interest.[2] [3] Similar to cardiolipin that forms complexes with β2GPI, PS/PT is a complex composed of phosphatidylserine and prothrombin. Several studies have shown an enhanced performance of anti-PS/PT compared with antibodies against prothrombin alone (aPT) in the diagnosis of APS[4] [5] and inclusion of anti-PS/PT may contribute to more accurate risk stratification in this heterogeneous disorder.

LA is a class of heterogeneous pro-thrombotic antibodies that can prolong phospholipid-dependent clotting times in vitro.[6] Several studies have shown that anti-PS/PT antibodies mediate LA activity, which is associated with a high risk of thrombosis, recurrent early miscarriages and premature delivery, independent of classical aCL and anti-β2GPI.[7] [8] [9] Some studies have also documented a correlation between anti-PS/PT and clinical features of APS.[6] [7] [8] [9] [10] Anticoagulant treatment, especially with direct oral anticoagulants (DOACs), can compromise in vitro phospholipid-dependent clotting testing leading to false positive results. Therefore, detection or confirmation of LA may require interruption of anticoagulation, a problem that does not affect aPL solid phase immunoassays.[11]

Although LA has a strong association with thrombosis, the pattern of aPL is also a strong predictor of thrombotic risk in APS, with triple antibody positive patients being at the greatest risk, demonstrating a 30% risk of recurrence within 10 years, despite otherwise adequate anticoagulation.[12] Presence of anti-PS/PT has been shown to be especially common in APS patients who are triple positive for the traditional aPL, creating a so called “tetra-positive” group.[3]

There remains a need to find more reliable biomarkers that correlate more closely with the clinical manifestations and thrombotic risk of APS. This study aims to investigate the ability of anti-PS and anti-PS/PT antibodies in combination with traditional aPL to improve this by assessing their frequency and clinical significance in a cohort of patients from a dedicated APS tertiary referral center.

All patients attending a dedicated APS clinic at a tertiary referral center at Hammersmith Hospital, Imperial College Healthcare NHS Trust, London, United Kingdom between June 2021 and March 2022, were considered for this study. Patients with a confirmed diagnosis of APS, according to the revised Sapporo (“Sydney”) criteria[1] were included in the study cohort. Blood samples were obtained from all patients included in the study at routine clinic visits, following informed written consent. Samples were double centrifuged using 2,000 g for 15 minutes, plasma separated and stored at −80°C, until analysis.

Patient demographics, clinical manifestations including thrombotic and/or obstetric complications, and specific clinical management were collected using the electronic patient record system (Cerner). Routine laboratory parameters including standard laboratory testing for the diagnosis of APS, complement levels, and full blood counts were collated. [Supplementary Table S1] (available in the online version only) details the clinical and laboratory data collected from the patient electronic records.

Presence of aPL including IgG and IgM anti-PS/PT antibodies was assessed as per manufacturer protocols using commercial enzyme-linked immunosorbent assays (ELISAs) kits from Werfen United Kingdom (Quanta Lite anti-PS/PT ELISA Kits). Standard aPL assays (IgG and IgM aCL and anti-β2GPI antibodies) were performed from all patients as part of the routine laboratory testing when patients attended their regular clinic visits. Values above 20GPL/MPL for aCL IgG/IgM/IgA and anti-β2GPI IgG/IgM/IgA were considered positive, calculated by the 99th centile of healthy subjects as per manufacturer instructions. Values >30 units were considered as positive for anti-PS/PT IgG/IgM as recommended by the manufacturer. LA was tested as per ISTH SSC guidelines[13] in combination of LA sensitive APTT (Cephascreen, Stago UK LTD) and diluted Russell's viper venom time (dRVVT) using dRVVT STA-Staclot DRVV Screen and STA-Staclot DRVV Confirm assays (Stago UK LTD, Sulhamstead United Kingdom).

Data was analyzed using a combination of descriptive statistics and non-parametric tests following assessment for normality. Descriptive statistics are presented as percentages for categorical data, and median and interquartile ranges for continuous data. Correlation between patient characteristics and laboratory variables with detection of anti-PS/PT was analyzed using univariate and multivariate logistic regression models. Fisher's exact test was used to compare differences between groups where appropriate. All statistical analysis was performed using standard statistical software package (R studio). p-Values ≤0.05 were considered statistically significant.

A total of 65 patients with confirmed APS were included in this study. Patient characteristics are summarized in [Table 1]. The median age was 56 years (IQR 40) and 69.2% were female. Of the 65 patients, 15.4% were triple positive (presence of LA, aCL, and anti-β2GPI). The laboratory parameters, other than aPLs, tested at routine clinic visits are summarized in [Supplementary Table S2] (available in the online version only). Lymphopenia was the most common hematological finding, present in 30.7%, of whom 65% (13/20) had an additional autoimmune diagnosis.

Table 1 Baseline characteristics and current treatment regiments of the patients included in the study

Characteristics	% of 65 patients
Median age	56 (IQR = 40)
Male	30.8 (n = 20)
Female	69.2 (n = 45)
History of any thrombotic event	84.6 (n = 55)
History of arterial thrombotic event	72.3 (n = 47)
History of venous thrombotic event	30.8 (n = 20)
History of combined arterial and venous thrombosis	10.8 (n = 7)
History of obstetric complications	16.9 (n = 11)
History of both thrombotic and obstetric complications	13.9 (n = 9)
Cardiovascular risk factors	58.5 (n = 38)
Diagnosis of additional autoimmune condition	46.2 (n = 30)
Triple aPL +	15.4 (n = 10)
Dual aPL +	47.7 (n = 31)
Single aPL +	36.9 (n = 24)
Other autoantibodies	26.2 (n = 17)
Current treatment
Warfarin	61.5 (n = 40)
DOAC	12.7 (n = 7)
Heparin	5.5 (n = 3)
Anti-platelet + anti-coagulation	34.6 (n = 19)
Hydroxychloroquine	45.5 (n = 25)
Statin	52.7 (n = 29)

Abbreviations: aPL, anti-phospholipid antibody; DOAC, direct oral anticoagulant.

Of the 65 patients, 84.6% had a history of at least one thrombotic event, with arterial being the most common (85.5%, 47/55); 31% (18/55) of these patients had a history of recurrent thrombotic complications prior to attending to our center. Furthermore, of the 45 female patients, 24.4% had prior obstetric complications, with 82% (9/11) of these patients also having a history of thrombosis.

The overall incidence of IgG and/or IgM anti-PS/PT antibodies was 18%, with 16.9 and 10.8% having IgG anti-PS/PT and IgM anti-PS/PT antibodies, respectively. Within the cohort, 6% were tetra-positive defined as presence of all three traditional diagnostic aPL plus anti-PS/PT and 9.2% were triple positive (traditional diagnostic aPL) ([Fig. 1]). [Supplementary Table S3] (available in the online version only) summaries the thrombotic complications in tetra-positive APS patients in this cohort.

Fig. 1 Antiphospholipid profile of the study population. The Venn diagram illustrates that 12 patients had detectable levels of anti-PS/PT, with four of these patients having a tetra-positive antiphospholipid antibody profile. The most frequently observed aPL detected in the study cohort was anti-β2GPI (41/65). anti-B2GPI, anti-β2-glycoprotein I; aCL, anti-cardiolipin; anti-PS/PT, anti-phosphatidylserine/prothrombin; LA, lupus anticoagulant.

Anti-PS/PT antibodies were more specific for LA than aCL. The presence of anti-PS/PT antibodies (either IgG or IgM) was strongly linked with the presence of LA (p = 0.006) ([Table 2]) such that patients with positive LA were 5.28-fold more likely to be positive for anti-PS/PT antibodies (95% CI 1.65–17.9), compared with patients without LA. Patients with IgG aCL specifically, were 6.04-fold more likely to be positive for anti-PS/PT antibodies (95% CI 1.42–41.83, p = 0.04) ([Table 2]). Furthermore, patients triple positive for standard laboratory criteria for APS (LA, anti-β2GPI, and aCL) were 13.7-fold (95% CI 2.02–101, p = 0.007) more likely to have IgG anti-PS/PT antibodies than those with non-triple positive APS ([Supplementary Table S4], available in the online version only). There was a trend for patients with triple positive APS to be more likely to have IgM anti-PS/PT antibodies, but this was not significant (OR: 6.42, 95% CI 0.91–26.1, p = 0.064).

Table 2 Association of anti-PS/PT with the three diagnostic aPLs, and their respective isotypes (multiple logistic regression analysis)

Laboratory marker	OR	95% CI	p-Value
LA	5.28	(1.65–17.9)	0.006
aCL (IgM and/or IgG)	1.97	(0.63–6.94)	0.3
Anti-B2GPI (IgM and/or IgG)	0.44	(0.14–1.29)	0.14
aCL IgG	6.04	(1.42–41.83)	0.04
aCL IgM	1.39	0.33, 5.19	0.7
aCL IgA	2.45	0.31, 14.48	0.6
Anti-b2GPI IgG	1.51	0.40, 5.45	0.6
Anti-b2GPI IgM	0.97	0.23, 3.54	0.9
Anti-b2GPI IgA	0.39	0.02, 2.39	0.6

Abbreviations: aCL, anti-cardiolipin; anti-b2GPI, anti-β2-glycoprotein I; DOAC, direct acting oral anticoagulant; IgG, immunoglobulin G; LA, lupus anticoagulant.

Sensitivity and specificity of detection of anti-PS/PT antibodies for the presence of LA and aCL were 75, 81.5% and 83.3 and 43.4%, respectively. However, there was no association between the presence of IgM or IgA aCL, and IgM, IgG, or IgA anti-β2GPI antibodies with the detection of anti-PS/PT antibodies.

All 12 patients positive for anti-PS/PT had previous thrombotic complications. Eight patients (66.7%) had a history of arterial thrombosis, with stroke being the most common arterial event observed. Ten patients with positive anti-PS/PT antibodies (83.3%) had a history of recurrent thrombotic complications, defined as ≥2 incidences of arterial or venous thrombotic events. Patients with positive anti-PS/PT antibodies were at 6.3-fold (95% CI 1.68–30.81, p = 0.02) greater risk of developing recurrent thrombosis compared with those without anti-PS/PT antibodies ([Supplementary Table S5], available in the online version only). With regard to the obstetric complications, there was no statistically significant association seen between the detection of anti-PS/PT and obstetric complications on their own [OR 1.12 (95% CI 0.05–13.50, p >0.9)] or in combination with thrombotic complications [OR 2.25 (95% CI 0.35–14.70, p = 0.6)]. There was no difference in recurrent thrombosis between patients with tetra positive aPL or triple positive aPL majority of patients (75%) with tetra positive aPL had recurrent thrombosis while one-third of patients with traditional triple positive aPL had recurrent thrombosis (33.3%) (2/6); p = 0.56. Of the 12 patients with positive anti-PS/PT antibodies, seven patients were female and 42.8% (3/7) had a previous history of obstetric complications. Presence of anti-PS/PT antibodies was therefore more strongly linked with thrombosis than obstetric complications.

The strong correlation between the presence of LA and the presence of anti-PS/PT has been noted in previous studies[14] [15] including a multicenter study conducted by Sciascia et al.[16] [17] This has a major clinical significance, as anti-PS/PT can be used in situations where detection of LA is problematic: the most significant being patients on anti-coagulation therapy (DOACs and warfarin), which can lead to false positive LA results.[6] Currently, either LA testing is postponed until anticoagulation is completed, the patient is switched to alternative anticoagulant such as low molecular weight heparin or alternative tests such as Taipan venom test/Ecarin clotting time are employed.[18] [19] Additionally, some studies have suggested that absorption methods to remove the DOACs are effective.[20] [21] However, the challenge around the detection of LA in the presence of DOACs poses a dilemma for clinicians due to the well-reported importance of LA testing, both as a diagnostic and prognostic markers of APS.[22] The addition of anti-PS/PT to current laboratory diagnostic tests for APS may therefore help to remove the ambiguity around potential false positives, allowing for clinical management to be altered and ultimately lead to more accurate diagnosis and monitoring of APS.

Another significant finding in this observational study was that patients with anti-PS/PT were shown to have a significantly increased risk (OR = 6.30, 95% CI 1.68–30.81, p = 0.02) of recurrent thrombosis, which is not previously reported.[4] [23] A systematic review, conducted by Sciascia et al using retrospective data collated using Medline-published reports between 1988 and 2013, encompassing more than 7,000 patients and controls from 38 studies,[4] found that patients positive for anti-PS/PT were over five times more likely to develop either arterial or venous thrombotic complications.[4]

This finding that anti-PS/PT antibodies are strongly linked with thrombosis has important clinical implications because thrombotic events are the most frequently observed complication of APS, in comparison to either obstetric or non-criteria events. The strong correlation with recurrent thrombosis observed in this observational study suggests the potential for anti-PS/PT, both as an adjunct to traditional diagnostic aPL markers and independently, to predict thrombotic events earlier, aiding risk stratification and tailoring management appropriately. It is well known that patients with triple positive aPL are at higher risk of recurrent thrombosis.[24] Further studies are needed to determine whether patients with tetra-positive aPL have an even higher rate of recurrent thrombosis and whether it should be used in standard risk stratification of patients with APS.

The pathogenic mechanism(s) by which anti-PS/PT antibodies promote thrombosis is not well understood but appears to be distinct from the effect on endothelial nitric oxide synthase proposed for anti-β2GPI antibodies. One proposed mechanism hypothesizes that anti-PS/PT mediate activation of the mitogen-activated protein kinase pathway, inducing increased expression of tissue factor (TF).[25] It is important to consider the development of thrombosis is multifactorial and predicting thrombotic risk in APS patients encompasses investigating laboratory findings, alongside patient demographic data, and presence of cardiovascular risk factors. For instance, in this observational study, 58.46% (n = 38) of the cohort had one or more cardiovascular risk factors, of which hypercholesterolemia was the most common.

The main limitation of this study is the relatively small sample size, especially of patients with obstetric APS. Only 16.7% (n = 11), had obstetric complications, of whom 27.2% (n = 3) were positive for anti-PS/PT. Hence, the study was not able to investigate the previously observed correlation between obstetric complications and anti-PS/PT.[7] [8]

In conclusion, in this observational study of patients with APS, the frequency of anti-PS/PT antibodies was 18% and there was a significant association of anti-PS/PT with LA, aCL IgG, and specifically IgG anti-PS/PT with triple positive aPL. The study supports the reported potential for IgG and IgM anti-PS/PT to act as a surrogate for LA. This has major implications in clinical practice, as the inclusion of anti-PS/PT with traditional aPL, could lead to earlier identification of patients at the highest risk for recurrent thrombosis in APS. Additionally, anti-PS/PT antibodies may be used in patients on oral anticoagulants where detection of LA is not practical. However, larger population-based studies are required to assess the true positive and negative predictive values for the presence of anti-PS/PT to have LA.

Authors' Contributions

D.J.A. conceptualized the study and led the methodology including study plan, data analysis plan, interpretation of the data, manuscript design, and reviewing the manuscript. H.N. and C.C.-T. were involved in sample processing, performing some of ELISA and writing the first draft of the manuscript. Z.O. contributed to data analysis. N.K. and K.L. were involved in sample collection and processing. C.P. and M.L. contributed to data interpretation, review, and editing the manuscript. All authors reviewed and approved the final version of the study.

^* These authors contributed equally to this work.

Publication History

Article published online:
01 November 2022

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