Thrombotic and cardiac disease in the antiphospholipid syndrome

The antiphospholipid syndrome (APS) is a highly heterogeneous disease that presents with obstetric complications and/or thromboembolic events that can hit any side of the vascular tree in an unpredictable way. The common trait among all patients affected by the syndrome is the persistent presence of the antiphospholipid antibodies (aPL) and/or a prolonged coagulation time in vitro, the lupus anticoagulant (LA) test. The pathogenesis of APS has not yet been clearly defined. Many molecules are involved in the maintenance of the equilibrium called hemostasis, interacting and cross-reacting in an incredibly perfect orchestrated balance that somehow, in APS, is broken. The aim of this thesis has been to try to shed some light on the complex mechanisms behind APS, by focusing on some of the many pieces of the puzzle that characterize the disease. The complement, coagulation and fibrinolytic systems have been the subjects of this fascinating journey, and two proteins in particular have been on focus: C4b-binding protein (C4BP) and Thrombin activatable fibrinolysis inhibitor (TAFI). These are both regulators of complement activation that at the same time play a role in coagulation. Moreover, the link between myocardial infarction (MI) and aPL/LA has been explored.

Paper I: TAFI and its activated form, TAFIa, were studied in patients affected by mainly primary APS (i.e. without other autoimmune diseases) and compared with a healthy control group. Moreover, C5a, a marker of complement activation, and markers of fibrinolysis were investigated and correlated with TAFI and TAFIa. Both TAFI and TAFIa are significantly higher in APS compared to controls. TAFIa is increased in APS patients affected by arterial thrombosis compared to other clinical manifestations, independently of traditional cardiovascular risk factors. TAFI is positively correlated with C5a, confirming its increase upon inflammation. TAFIa positively correlates with thrombomodulin, marker of endothelial damage/activation. The values of the clot lysis time (CLT) and of the permeability coefficient confirm impaired fibrinolysis in APS, with clots more resistant to lysis.

Paper II: we investigated the prevalence of aPL in a large and well-characterized cohort of patients after 6-10 weeks from a first MI, and compared it with age, gender and region matched controls. We demonstrated ten times higher prevalence of aPL of the IgG isotype in patients versus controls, independently of traditional cardiovascular risk factors, suggesting that IgG aPL positivity may be considered a potential risk factor for MI in the general population. No significant differences were observed for IgM and IgA isotypes.

Paper III: C4BP was investigated in a large cohort of patients affected by systemic lupus erythematosus (SLE), in primary APS and in controls. C4BP is lower in patients persistently positive for aPL and in patients treated with warfarin. C4BP correlates with markers of complement activation. Both persistent aPL positivity and warfarin are associated with C4BP reduction, and by means of a mediation analysis we were able to assess the relative contribution of these two variables: aPL have a direct reducing effect on C4BP of 11%, while warfarin contributes to 9% of the observed reduction.

Paper IV: After the results of paper III, we decided to study the effect of warfarin on complement and C4BP in the general population, comparing it with the direct oral anticoagulants (DOACs), during and after treatment discontinuation. Warfarin, as opposed to DOACs, is associated with increased markers of complement activation, which persist for at least three weeks after withdrawal. Higher C4BP levels characterize the patients after warfarin discontinuation, as a rebound effect. DOACs have no effect on complement, but, in contrast, we demonstrate that warfarin is associated with complement activation, partly but probably not only through inhibition of C4BP. This study is of relevance in the context of APS, since different anticoagulant mechanisms have been subjects of debate in recent years.

In conclusion, as also Ames stated regarding paper I , this thesis has tried to insert other linking pieces in the puzzle of APS. We confirm the presence of impaired fibrinolysis and complement activation in APS, and we have opened the path for a new era of research in the syndrome, where also the treatment with anticoagulants has to be considered for its potential impact on complement activation. Moreover, although causality could not be proven, we demonstrate that the prevalence of aPL after myocardial infarction in the general population is considerable and higher than generally anticipated.

List of scientific papers

I. Thrombin activatable fibrinolysis inhibitor (TAFI) - A possible link between coagulation and complement activation in the antiphospholipid syndrome (APS). GIORGIA GROSSO, Anna Vikerfors, Barry Woodhams, Mariette Adam, Katarina Bremme, Margareta Holmström, Anna Ågren, Anna Eelde, Maria Bruzelius, Elisabet Svenungsson, Aleksandra Antovic. Thrombosis Research. 2017, vol. 158:168-173.
https://doi.org/10.1016/j.thromres.2017.06.028

II. Antiphospholipid antibodies in patients with Myocardial Infarction. GIORGIA GROSSO, Natalie Sippl, Barbro Kjellström, Khaled Amara, Ulf de Faire, Kerstin Elvin, Bertil Lindahl, Per Näsman, Lars Rydén, Anna Norhammar, Elisabet Svenungsson. Annals of Internal Medicine. 2019, vol. 170(4):277-280.
https://doi.org/10.7326/M18-2130

III. The complex relationship between C4b-Binding Protein, warfarin and antiphospholipid antibodies. GIORGIA GROSSO, Kerstin Sandholm, Aleksandra Antovic, Iva Gunnarsson, Agneta Zickert, Anna Vikerfors, Lennart Truedsson, Maria Bruzelius, Bo Nilsson, Kristina Nilsson-Ekdahl, Elisabet Svenungsson. Thrombosis and Haemostasis. 2021 Jan 7.
https://doi.org/10.1055/a-1347-5655

IV. Anticoagulants and complement. Kerstin Sandholm*, Manal Ibrahim-Kosta*, GIORGIA GROSSO, Aleksandra Antovic, Maria Hårdstedt, Camilla Mohlin, Oskar Eriksson, Elisabet Svenungsson, Bo Nilsson, Pierre-Emmanuel Morange, Maria Bruzelius, Kristina Nilsson-Ekdahl. *Equal contribution. [Manuscript]