Impact of autoantibody-mediated procoagulant platelets and thrombus formation in antiphospholipid syndrome

Introduction Antiphospholipid syndrome (APS) is a prothrombotic autoimmune disease that is associated with recurrent thrombosis and pregnancy loss [1]. The thrombotic risk in patients remains high despite the use of plasmatic anticoagulants [2]. APS is caused by circulating autoantibodies (AAbs) that recognize phospholipids and phospholipid-binding proteins [2]. Anti-β₂-glycoprotein-I APS antibodies (anti-β₂GPI AAbs) are increasingly identified to harbor prothrombotic potential [3] [4] [5]. However, the impact of anti-β₂GPI APS antibody interactions with platelets (PLT) and subsequent relevance on thrombus formation are not well explored.

This study aims to investigate the relevance of anti-β₂GPI antibody interactions with procoagulant platelets and the impact on thrombus formation ex vivo.

Method For the investigation of anti-β₂GPI AAb-mediated PLT alterations, a flow cytometry (FC)-based protocol was employed. To study the impact of anti-β₂GPI AAb-mediated PLT changes on thrombus formation, a microfluidic ex vivo thrombosis model was developed with tetra staining to analyze the multicellular interaction in APS.

Results FC analysis revealed that human anti-β₂GPI AAbs have the potential to induce increased formation of procoagulant (CD62p and PS double positive) PLTs compared to control (p-value 0.0026). Anti-β₂GPI-AAb-induced procoagulant PLT formation was PLT Fc-gamma-RIIA (FcyRIIA)-dependent as specific FcyRIIA blockade resulted in a nearly complete inhibition of AAb-induced procoagulant PLT formation (p=0.0161).

Notably, the prothrombotic PLT changes were not only of phenotypical nature, as the reconstitution of anti-β₂GPI AAb-induced procoagulant PLTs into healthy whole blood resulted in formation of multicellular thrombus ex vivo (p=0.0004). Interestingly thrombus formation was prevented when PLTs were pretreated with therapeutic doses of intravenous immunoglobulin G (IVIG) prior to anti-β₂GPI AAb incubation (p=0.0018) ([Fig. 1]).

Fig. 1 Ex vivo thrombus formation induced by Anti-β2-GPI antibody and inhibited with IVIG; Whole blood from healthy individuals was spiked with plasma that was preincubated with IgG isolates from healthy control, Anti-β₂-GPI antibody (2.5 U/ml or 5 U/ml) or a mixture of antibody and IVIG. Samples were stained with DiOC₆ and perfused through collagen coated microfluidic channels for 15 minutes at an arterial shear rate of 20 dyne. Images were acquired in the green fluorescent channel (upper panel) as well as in the brightfield channel (lower panel). IVIG, Intravenous Immunoglobulin; GPI, Glycoprotein I.

Conclusion Findings from our studies indicate that human anti-β₂GPI AAbs have the potential to induce formation of procoagulant PLT phenotype that harbors dramatic prothrombotic potential. The observation that IVIG treatment could prevent anti-β₂GPI AAb-mediated multicellular thrombus formation directs towards a therapeutic potential of IVIG in the prothrombotic condition often observed in APS patients.

Publication History

Article published online:
26 February 2024

• References

• 1 Ruiz-Irastorza G, Crowther M, Branch W, Khamashta MA.. 'Antiphospholipid syndrome'. Lancet. 2010; 376 (9751) 1498-509 DOI: 10.1016/S0140-6736(10)60709-X.
• 2 Knight JS, Kanthi Y.. 'Mechanisms of immunothrombosis and vasculopathy in antiphospholipid syndrome'. Semin Immunopathol 2022; 44 (03) 347-362 DOI: 10.1007/s00281-022-00916-w.
• 3 Ho Y., Ahuja K., Körner H., Adams M.. 'β2GP1, Anti-β2GP1 Antibodies and Platelets: Key Players in the Antiphospholipid Syndrome'. Antibodies (Basel) 2016; 5 (02) 12 DOI: 10.3390/antib5020012.
• 4 McDonnell T., Wincup C., Buchholz I.. et al. 'The role of beta-2-glycoprotein I in health and disease associating structure with function: More than just APS'. Blood Rev 2020; 39: 100610 DOI: 10.1016/j.blre.2019.100610.
• 5 Baroni G, Banzato A, Bison E, Denas G, Zoppellaro G, Pengo V.. 'The role of platelets in antiphospholipid syndrome'. Platelets 2017; 28 (08) 762-766 DOI: 10.1080/09537104.2017.1280150.