title: Anti-beta-2 glycoprotein 1 antibodies and platelets : their mechanism and effect of interaction
Creators: name: Ho, YC
Publisher: name: University of Tasmania
Year: 2021

description: Anti-beta-2 glycoprotein 1 (β2GP1) antibodies, β2GP1 and platelets are key elements responsible for recurrent vascular diseases in patients with antiphospholipid syndrome (APS) and systemic lupus erythematosus (SLE). The immunocomplexes formed from the binding of anti-β2GP1 antibodies to β2GP1 are believed to interact with the phospholipid membrane and/or receptor(s) of platelets, causing excessive thrombus formation. Despite significant research efforts, the exact mechanism(s) of interaction between anti-β2GP1 antibodies and platelets remain unclear. Therefore, the overall aim of this thesis was to investigate the mechanism(s) and effect(s) of interaction between anti-β2GP1 antibodies and platelets. This investigation is key to understanding the pathophysiology, assisting the development of novel biomarker(s) and treatment(s) for vascular diseases, particularly in APS and SLE. Anti-β2GP1 antibodies bound to the membrane of platelets are hypothesised to affect the collagen receptor, glycoprotein VI (GPVI), which mediates the initial adhesion of platelets to injured blood vessel during clot formation. Three studies were performed to gain further insight into how anti-β2GP1 antibodies, 1) affect collagen-induced in vitro platelet aggregation, 2) induce shedding of the ectodomain of GPVI, and 3) bind/interact with GPVI. These effects of anti-β2GP1 antibodies on platelets were tested with different types of anti-β2GP1 antibodies, including animal-derived anti-β2GP1 antibodies (mono- and polyclonal) and APS/SLE patient-derived IgG fractions (containing anti-β2GP1 antibodies). Light transmission aggregometry, an established model in our laboratory, was initially used to investigate the effects of anti-β2GP1 antibodies on platelet aggregation activated by collagen. The results showed that animal-derived anti-β2GP1 antibodies had a statistically significant inhibitory effect (4-9%, P &lt; 0.01) on collagen induced platelet aggregation compared to baseline control (i.e. no antibody). However, the isotype control antibodies investigated in parallel produced similar inhibitory effect, suggesting this may not be specific to anti-β2GP1 antibodies. Differences in the avidity and specificity of animal compared to human antibodies could explain the non-specific effect of anti-β2GP1 antibodies. Therefore, further aggregometry studies were conducted using APS/SLE patient-derived IgG fractions. Similar to animal antibodies, the APS/SLE patient-derived IgG fractions demonstrated no significant difference in their effect on collagen-induced platelet aggregation compared to baseline and isotype controls. The collective data from both aggregometry studies indicate that anti-β2GP1 antibodies do not affect collagen-induced platelet aggregation. The effects of anti-β2GP1 antibodies may be masked by the strong collagen activation signal and not assessed by in vitro aggregation of platelet-rich-plasma (PRP). Therefore, the direct effects of anti-β2GP1 antibodies on collagen-mediated platelet aggregation were investigated using a platelet activation marker, soluble GPVI (sGPVI). sGPVI is an ectodomain of GPVI shed in response to platelet activation induced by ligands such as collagen and anti-platelet antibodies. Enzyme-linked immunosorbent assay (ELISA) was used to measure the concentrations of sGPVI in 1) platelet-poor-plasma from SLE patients with/without anti-β2GP1 antibodies, and 2) healthy participant-derived PRP treated with SLE-derived IgG fractions or animal-derived anti-β2GP1 antibodies. High concentrations of sGPVI were found to be associated with the presence of anti-β2GP1 antibodies in SLE patients. Yet, SLE-derived IgG fractions and animal-derived anti-β2GP1 antibodies did not increase shedding of the sGPVI in healthy PRP. These results suggest that anti-β2GP1 antibodies do not affect platelets via GPVI. The high concentrations of sGPVI found in this small cohort of patients may be due to other factors, i.e. elevated enzymatic activity of metalloproteinases that increase shedding of sGPVI. Aggregometry and sGPVI studies were designed to test the functional effects of antiβ2GP1 antibodies on platelets, and not the interaction of anti-β2GP1 antibodies with platelets before and after activation by collagen. Therefore, immunocytochemistry (ICC) staining was used to study the binding of anti-β2GP1 antibodies to platelets. Two sets of PRP from healthy individuals were treated with APS/SLE-derived IgG fractions. The first set was incubated for 5, 10 and 20 min without activation, while the other was activated by collagen after 10 min of incubation. ICC stains showed that APS/SLE-derived IgG fractions had significantly higher binding affinity to non-activated compared to collagen-activated platelets. The amount of binding on non-activated platelets further increased with longer incubation time, suggesting that the chronic exposure of resting platelets in patients with APS/SLE to anti-β2GP1 antibodies enhances the binding and effects of anti-β2GP1 antibodies. This increased level of binding may be associated with a higher risk of thrombosis in APS/SLE patients. Though, ICC stains used were not able to identify the exact receptor(s) or pathway(s) targeted by antiβ2GP1 antibodies. Taken together, the results from three studies suggest that anti-β2GP1 antibodies bind and interact with platelets, but have no effect on collagen-induced platelet aggregation. Platelet aggregometry and sGPVI shedding studies demonstrated that animal-derived antiβ2GP1 antibodies and APS/SLE-derived IgG fractions do not affect GPVI receptors and signaling pathways mediated by collagen. ICC staining showed that anti-β2GP1 antibodies may sensitise and affect resting platelets. Since APS/SLE-derived IgG fractions contain a heterogenous group of autoantibodies, efforts were made to purify anti-β2GP1 antibodies with higher specificity. However, although anti-β2GP1 antibodies were isolated from one patient, the yield was insufficient to further explore the effects of anti-β2GP1 antibodies on platelets. Anti-β2GP1 antibodies are speculated not to be the main causative agent, but involved in the two-hit hypothesis for the pathogenesis of APS and SLE. It is proposed that anti-β2GP1 antibodies may sensitise resting platelets asymptomatically after the 1 st injury/hit of a blood vessel. This sensitisation enhances the platelet aggregation and thrombus formation when triggered by the 2 nd injury/hit. The collagen receptor and associated pathways responsible for platelet aggregation are not targeted by anti-β2GP1 antibodies. However, the activation of other receptors and cascade of pathways by the initial collagen activation signal are potentially affected by anti-β2GP1 antibodies that chronically bind to the surface of resting platelets in patients with APS/SLE. Thus, the management of conditions which contribute to the increased binding of anti-β2GP1 antibodies to platelets, or injury of blood vessel, may be an alternative approach to minimise the risk of complications in patients with APS and SLE. In summary, this thesis provides strong evidence that anti-β2GP1 antibodies do not appear to interact, nor affect, the GPVI receptor in platelets. This outcome is based on research conducted using a combination of research methods/techniques and types of antiβ2GP1 antibodies. The results also point towards the need for refining the focus of future work to explore the mechanism of interaction between anti-β2GP1 antibodies and other platelet receptors. The complex pathophysiology of anti-β2GP1 antibodies in APS and SLE may be better resolved using the process of elimination to ultimately assist in the development of novel biomarkers and treatments for vascular diseases in APS and SLE.