Interaction between Antiphospholipid Antibodies and Protein C Anticoagulant Pathway: A Narrative Review

Thrombotic antiphospholipid syndrome (APS) is a condition in which thrombosis in venous, arterial, and/or small vessels is ascribed to the presence of antiphospholipid antibodies (aPL). Among the various proposed pathogenic theories to explain thrombotic APS, those involving the interaction between aPL and the protein C system have gained much consensus. Indeed, robust data show an acquired activated protein C resistance (APC-R) in these patients. The role of aPL in this impairment is clear, but the mechanism of action is uncertain, as the type of aPL and to what extent aPL are involved remains a gray area. Lupus anticoagulant (LA) is often associated with APC-R, but antibodies generating LA comprise those directed to β2-glycoprotein I and antiphosphatidylserine/prothrombin. Moreover, the induction of APC-R by aPL requires the presence of phospholipids and is suppressed by the presence of an excess of phospholipids. How phospholipids exposed on the cell membranes work in the system in vivo is unknown. Interestingly, acquired APC-R due to aPL might explain the clinical phenotypes of thrombotic APS. Indeed, the literature reports cases of both venous and arterial thromboembolism as well as skin necrosis, the latter observed in the severe form of protein C deficiency and in catastrophic APS.

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 β2 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.

Pathophysiology of the Antiphospholipid Antibody Syndrome

The antiphospholipid syndrome is characterized by antibodies directed against phospholipid-binding proteins and phospholipids attached to cell membrane receptors, mitochondria, oxidized lipoproteins, and activated complement components. When antibodies bind to these complex antigens, cells are activated and the coagulation and complement cascades are triggered, culminating in thrombotic events and pregnancy morbidity that further define the syndrome. The phospholipid-binding proteins most often involved are annexins II and V, β2-glycoprotein I, prothrombin, and cardiolipin. A distinguishing feature of the antiphospholipid syndrome is the “lupus anticoagulant”. This is not a single entity but rather a family of antibodies directed against complex antigens consisting of β2-glycoprotein I and/or prothrombin bound to an anionic phospholipid. Although these antibodies prolong in vitro clotting times by competing with clotting factors for phospholipid binding sites, they are not associated with clinical bleeding. Rather, they are thrombogenic because they augment thrombin production in vivo by concentrating prothrombin on phospholipid surfaces. Other antiphospholipid antibodies decrease the clot-inhibitory properties of the endothelium and enhance platelet adherence and aggregation. Some are atherogenic because they increase lipid peroxidation by reducing paraoxonase activity, and others impair fetal nutrition by diminishing placental antithrombotic and fibrinolytic activity. This plethora of destructive autoantibodies is currently managed with immunomodulatory agents, but new approaches to treatment might include vaccines against specific autoantigens, blocking the antibodies generated by exposure to cytoplasmic DNA, and selective targeting of aberrant B-cells to reduce or eliminate autoantibody production.

Prevalence of Anti-Nuclear Antibodies and Anti-Phospholipid Antibodies in an Egyptian Cohort with Schizophrenia: A Case-Control Study

Background: Psychiatric disorders, including schizophrenia could herald other manifestation(s) of systemic lupus erythematosus (SLE) potentially hindering timely and optimal management. Moreover, schizophrenia is among the described ‘extra-criteria’ manifestations of anti-phospholipid syndrome (APS). Hence, screening schizophrenia patients for SLE and APS may pose diagnostic and therapeutic implications. Objectives: Examine schizophrenia patients with no overt connective tissue disease(s) manifestation(s) for clinical and/or serologic evidence of SLE and/or APS. Methods: The study included 92 schizophrenia patients [61 (66.3%) males] and 100 age- and gender-matched healthy controls. Both groups were tested for anti-nuclear antibodies (ANAs), anti-double stranded deoxyribonucleic acid (anti-dsDNA) antibodies, complement 3 (C3) and C4, and criteria anti-phospholipid antibodies (aPL) [anticardiolipin Immunoglobulin (Ig) G and IgM, anti-beta-2-glycoprotein I IgG and IgM, and lupus anticoagulant (LAC)]. Results: The patients’ mean age and disease duration were 28.8 ± 8.1 and 5.7 ± 2.2 years, respectively. The prevalence of ANA positivity, height of titre, and pattern was comparable between patients and controls (p = 0.9, p = 0.8 and p = 0.1, respectively). Anti-dsDNA antibodies and hypocomplementemia were absent in both groups. A significantly higher frequency of positive LAC was observed among patients compared with controls (7.6 % vs. 1 %, p = 0.02), whereas other aPL were comparable between both groups. None of the patients or controls demonstrated clinically meaningful (medium or high) aPL titres. Conclusion: In our study, schizophrenia was solely associated with LAC. Thus, in the absence of findings suggestive of SLE or APS, routine screening for both diseases is questionable.

Abundance of B Cell Receptors Harboring Elongated Polytyrosine and Polyserine Rich Motifs within Their Heavy Chain CDR3 Distinguishes Catastrophic and Antiphospholipid Syndrome

ACKGROUND: Antiphospholipid syndrome (APS) is defined by arterial and/or venous thrombosis and/or pregnancy morbidity along with persistent circulating antiphospholipid antibodies (aPL). Central to APS pathogenesis are B cells that generate autoreactive antibodies, including anticardiolipin antibodies (aCL), and anti-β2-glycoprotein-I antibodies (anti-B2GPI). Several studies have implicated specific germline and/or antigen-driven somatic hypermutations within the complementarity determining regions (CDRs) of pathogenic antibodies in APS, as well as other thrombotic disorders. Nevertheless, the B cell repertoire in thrombotic APS remains poorly characterized. Recently, autoreactive and platelet activating pathogenic antibodies harboring elongated tyrosine rich CDR3 motifs were reported in patients with heparin-induced thrombocytopenia (HIT) (Zhu et.al, Blood 2019, 2020). Whether such antibodies are present in APS and their potential role, if so, has not been determined. APPROACH : We leveraged single cell immunoprofiling to characterize the presence of antibodies harboring elongated tyrosine rich CDR3 motifs in the circulating B cell repertoire obtained from peripheral blood mononuclear cells (PBMC) from patients with thrombotic (n=4) and catastrophic APS (n=3) as well as healthy individuals (n=3). We also correlated the abundance of these antibodies with criteria aPL and inflammatory cytokines in plasma. The modified Ham (mHam) test was used to assess functional complement activation in plasma in plasma from all of the subjects studied (Chaturvedi et al. Blood 2020). RESULTS : B cell clones containing elongated polytyrosine rich CDR3 motifs were most abundant in patients with a diagnosis of catastrophic APS (n=3) , and were markedly elevated in patients with APS (n=4) compared to healthy counterparts (n=3) (Table 1). Elongated polytyrosine rich CDR3 motifs from CAPS patients contained penta-tyrosine (Y5), hexa-tyrosine (Y6), hepta-tyrosine (Y7), and octa-tyrosine (Y8) motifs (table 2) and contained more tyrosine residues than the penta-tyrosines previously reported in HIT. In addition, all of the polytyrosine containing motifs CDR3 motifs were present on the heavy chain and was followed by MDVW motif of IGHJ6. The presence of B cell clones containing elongated polytyrosine rich CDR3 motifs exhibited significant positive correlations with complement activation measured by mHAM test (r=0.728, p=0.0032), criteria aPLs including anticardiolipin antibodies (aCL) and anti-β2-glycoprotein-I antibodies (anti-B2GPI), as well as plasma inflammatory cytokines; TNFα (r=0.901, p=1.5x10 -5), IL-23 (r=0.780, p=0.0016), IL-17C cytokines (r=0.729, p=0.0033), sICAM1 (r=0.740, p=0.0025), sVCAM1 (r=0.764, p=0.0015), (Figure 1). The presence of elongated polytyrosine rich CDR3 motifs was also associated with the increase abundance of polyserine rich CDR3 motifs that were present predominantly in patients with CAPS. CONCLUSION : These preliminary studies provide the first characterization of the prevalence of B cell clones containing elongated polytyrosine rich CDR3 motifs, previously reported in HIT, in the B cell repertoire of APS patients. Notably, the abundance of these B cell clones is markedly elevated in patients with CAPS and is associated with elevated levels of inflammatory cytokines TNFa, IL-23, IL-17C, sICAM1, and sVCAM1. Validation studies in larger cohorts from APS patients and other thrombotic disorders are ongoing. Figure 1 Figure 1. Disclosures Chaturvedi: Alexion: Other: Advisory board member; Dova: Other: Advisory board member; Sanofi Genzyme: Other: Advisory board member; Argenx: Other: Advisory board member; UCB: Other: Advisory board participation. Khorana: Bristol Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Anthos: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Halozyme: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Bayer: Consultancy, Honoraria. McCrae: Sanofi, Novartis, Alexion, and Johnson & Johnson: Consultancy, Honoraria; Dova, Novartis, Rigel, and Sanofi Genzyme: Consultancy.

Acquired and Inherited Thrombophilia Testing in Patients with Chronic Thromboembolic Pulmonary Hypertension: Value of Testing in an Academic Health Center

Introduction Chronic thromboembolic pulmonary hypertension (CTEPH) is classed as group 4 in the present classification of pulmonary hypertension. The pathophysiology of CTEPH is complex, mainly is a consequence of prior acute pulmonary embolism with failure of thrombi to resolve and the recent recognition of added small vessel changes which impacts long-term outcomes even after surgical management. The role of thrombophilia testing in this condition has been debated. Hence, we here analyzed the utility of thrombophilia testing in CTEPH from a center in a developing country. Methods This is a single institution (Narayana Health City, Bangalore); retrospective study including patients ≥ 18 years of age who underwent thrombophilia workup in a diagnosis of CTEPH from January 2019 till July 2021. Tests done to evaluate thrombophilia included factor V Leiden; prothrombin F20210A mutation; MTHFR gene mutation; Protein C, S, and antithrombin deficiency; lupus anticoagulant, anti-beta2 glycoprotein I (IgM and IgG) and anticardiolipin antibody (IgM and IgG); hyperhomocysteinemia and anti-nuclear antibody testing (ANA-IF). The study was approved by the ethics committee of the institute and was carried out in accordance with the principles of the declaration of Helsinki. Results and discussion The study included 56 patients with a median age of 37 years (range 23-50), and 36 (64%) were males. Patients with recurrent venous thrombosis included 37 (66%), with the majority having thrombosis at 2 sites (53%; 22 patients with associated deep vein thrombosis). A family history of thrombosis was present in 4 patients. The majority of patients received vitamin K antagonists (76%), with the rest receiving direct oral anticoagulants (DOAC). Among the tests sent for acquired thrombophilia, ANA-IF and antiphospholipid antibody (APLA) were most frequently evaluated (94%). ANA-IF and APLA tests were positive in 5.6% and 30.1%, respectively. Among the APLA tests, Anti-beta2 glycoprotein I (IgM or IgG) was the most commonly detected antibody (13/46), followed by anticardiolipin antibody (IgG or IgM) (9/43) and lupus anticoagulant (7/40). Double and triple positive APLA were present in 3 and 4 patients, respectively. Homocysteine levels were high in 93.7% though only 16 patients were tested in this cohort. Among the tests for inherited thrombophilia, genetic tests (factor V Leiden, prothrombin F20210A mutation, and MTHFR gene mutation) were tested in only ~50%. Twenty-three percent were positive for heterozygous MTHFR followed by MTHFR compound heterozygous (10%) and heterozygous factor V Leiden heterozygous (10%). Antithrombin III, protein C, and S were tested in ~30% of patients. Antithrombin III was low in only 1 patient, with protein C and S assays being normal in all the patients. The cost analysis was calculated, showed a median of $364 (₹ 27,055) was spent per person on thrombophilia workup. The median cost incurred per patient for inherited thrombophilia workup was $232 (₹ 17,300) and for acquired thrombophilia was $132 (₹ 9814), respectively. Conclusion This single-institution study on thrombophilia workup in CTEPH patients reveals that APLA was the most commonly performed test with high positivity rates of 30.1%. Among the inherited thrombophilia, the positivity rate of MTHFR mutation was highest (33.3%), with other tests having a low positivity rate (0-10%). Hence, we would recommend APLA testing in all patients with CTEPH considering its high positivity and clinical utility. Testing for other thrombophilias should be pursued judiciously especially in economically restrictive settings. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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 (β2GPI) 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.

Evaluation of Frequency, Clinical Correlation, and Antibodies Confirmation Profile in Patients with Suspected Antiphospholipid Syndrome

Antiphospholipid syndrome (APS) is a systemic autoimmune disease characterized by arterial and venous thrombotic manifestations and/or pregnancy-related complications in patients with persistent antiphospholipid (aPL) antibodies. The introduction of Sapporo's classification criteria allowed uniformity in the classification of this pathology, representing a considerable advance in its diagnosis. However, currently some doubts about the application of these criteria still persist. The aim of this study was to contribute to the better understanding of APS by the assessment of aPL prevalence, the association between clinical and laboratory tests, and evaluation of the aPL confirmatory profile.In this study, 1,179 samples from patients with suspected APS of both genders, without age restrictions, who were advised to test for complete aPL's profile were analyzed. The samples were tested for lupus anticoagulant (LAC), anticardiolipin immunoglobulin (Ig) G/IgM and anti-β-2-glycoprotein I IgG/IgM antibodies. Patient samples with isolated test requests for analysis and samples from patients under the influence of anticoagulants or in an infectious process were excluded.The overall positivity found was 17.9% and the most frequent aPL was LAC. The antibodies were determined in isolation and in association. The prevalence of triple positivity was 0.8% and double positivity was 1.8%. Positivity was higher in inpatient/emergency services compared with outpatient services. There was a higher positivity in individuals over 41 years, males, patients with systemic lupus erythematosus, kidney complications, and deep vein thrombosis/thrombophlebitis. The positivity confirmation with second sample was 39.5% and the confirmation profile shows that 50.6% of samples confirmed with same positivity profile; 17.3% with a different profile and regarding to these, 2.5% of the samples confirmed positivity with a different antibody from the previously detected.This study suggests that the aPL's positivity tends to increase with age, showing that the aPL's testing should be avoided during an acute event and reinforces the need for complete aPL laboratory profile in the second sample and subsequent determinations.

Antiphospholipid Antibody Assays in 2021: Looking for a Predictive Value in Addition to a Diagnostic One

Antiphospholipid antibodies (aPL) are mandatory for the diagnosis but are also a risk factor for the antiphospholipid syndrome (APS) clinical manifestations. Lupus anticoagulant (LA), anticardiolipin (aCL), and anti-beta2 glycoprotein I (β2GPI) assays are the formal laboratory classification/diagnostic criteria. Additional nonclassification assays have been suggested; among them, antiphosphatidylserine-prothrombin (aPS/PT) and antidomain 1 β2GPI antibodies are the most promising ones although not yet formally accepted. aPL represent the example of a laboratory test that moved from dichotomous to quantitative results consistent with the idea that reporting quantitative data offers more diagnostic/prognostic information for both vascular and obstetric manifestations. Although the general rule is that the higher the aPL titer, the higher the test likelihood ratio, there is growing evidence that this is not the case for persistent low titers and obstetric events. LA displays the highest diagnostic/prognostic power, although some isolated LAs are apparently not associated with APS manifestations. Moreover, isotype characterization is also critical since IgG aPL are more diagnostic/prognostic than IgA or IgM. aPL are directed against two main autoantigens: β2GPI and PT. However, anti-β2GPI antibodies are more associated with the APS clinical spectrum. In addition, there is evidence that anti-β2GPI domain 1 antibodies display a stronger diagnostic/prognostic value. This finding supports the view that antigen and even epitope characterization represents a further step for improving the assay value. The strategy to improve aPL laboratory characterization is a lesson that can be translated to other autoantibody assays in order to improve our diagnostic and prognostic power.