Establishment of an in vitro assay to detect antibody-mediated procoagulant platelet formation on a single cell level in real time

ABSTRACT

Introduction Procoagulant platelets (PLTs), a subpopulation of PLTs that is characterized by increased externalization of phosphatidylserine (PS), are increasingly identified to promote a prothrombotic environment in different diseases. Recently we observed that procoagulant PLT formation can be induced via engagement of immune receptor Fc-gamma-RIIA by COVID-19, VITT and HIT patient immunoglobulin subclass G (IgG) antibodies (Abs). Here, Fc-gamma- RIIA engagement by patient Abs resulted in significant formation of procoagulant PLTs and loss of mitochondrial potential that was associated with high thrombin formation as well as increased thrombus formation. In the cur- rent study, we aim to establish a PLT adhesion assay that allows investigation of PLT mitochondria during procoagulant PLT formation. Method PLTs were spread on human serum albumin, fibrinogen or collagen precoated glass slides. Adhesion and subsequent shape change of PLTs as well as procoagulant PLT formation were investigated in real time using immune fluorescence microscopy. For the detection of PLT shape change, mitochondrial dynamics and PS externalization, PLTs were double stained with MitoTracker green, a mitochondrial dye that binds to free thiol groups of cysteine residues in the mitochondrial membrane, and Annexin-V, respectively. For the visualization of mitochondrial release from PLTs intracellular compartment, a monoclonal Ab that binds to a subunit of the translocase of the outer membrane (TOM) complex on the mitochondrial membrane, namely TOM22, was used. Results During the observation period, a subgroup of PLTs that was spread on collagen became procoagulant as determined by an increased binding of Annexin- V on the PLT surface. Contrary, these changes were nearly absent in PLTs that adhered to fibrinogen (percentage [ %] of Annexin-V positive cells 19.80 +/- 3.42 % vs. 1.92 +/- 0.62 %, p value 0.0357). Interestingly, procoagulant PLT formation was associated with a significant loss of MitoTracker green signal in PLTs while it remained constant in non-procoagulant PLTs attached on both extracellular matrix coatings. Loss of MitoTracker green signal was associated with translocation of mitochondrial proteins from intracellular to extracellular, as a higher count of TOM22 Ab-positive labelled structures, most likely extracellular mitochondria were detected on collagen but not on fibrinogen coated glass slides. Conclusion Our findings indicate, that the formation of procoagulant PLTs is associated with dramatic changes of the mitochondrial integrity in PLTs. Further attempts, that investigate the potential pathophysiological role of PLT mitochondrial release in Ab-mediated prothrombotic disorders may contribute to a further understanding of the role of PLT mitochondria in these complex diseases.