Background: We previously identified that apolipoprotein A-IV (apoA-IV) is an endogenous inhibitor of thrombosis through competitive blockade of the platelet αIIbβ3 integrin, specifically from its N-terminal D5/D13 residues (Nat Commun. 2018). Structural analyses indicate that the N-terminal binding site is sterically hindered by its C-terminus. The C-terminus also houses the most common apoA-IV polymorphisms, Q360H and T347S, which are linked with an increased risk of cardiovascular disease as high as 2-fold, however, the underlying mechanism is completely unknown.

Aims: To investigate whether the inhibitory effect of apoA-IV on thrombosis is reduced in its polymorphisms, Q360H and T347S, due to further steric hindrance of the N-terminal platelet binding site.

Methods: We generated recombinant wildtype (WT), Q360H, and T347S apoA-IV using mutagenesis methods. ADP and collagen-induced platelet aggregation were measured using aggregometry. Thrombus formation was measured by flowing human whole blood across collagen-coated microcapillary perfusion chambers under a real-time fluorescence microscope. Platelet spreading on fibrinogen-coated slides was imaged on a confocal microscope. ApoA-IV binding to human platelets was measured using flow cytometry. The flexibility of the C-terminus was assessed through the attachment of a C-terminus-specific fluorophore and measurement of its tumbling and rotation via fluorescence anisotropy.

Results: The inhibitory effect of apoA-IV on platelet aggregation was attenuated in Q360H and even more notably in T347S. This trend was corroborated across platelet-rich plasma and gel-filtered platelets as well as platelet spreading and in vitro thrombus formation. Flow cytometry results demonstrate that Q360H and T347S bind activated platelets less than WT. Interestingly, fluorescence anisotropy results show that the C-terminus is more flexible in Q360H and even further in T347S, which may exacerbate steric hinderance of the N-terminal platelet binding site.

Conclusions: These data identify a novel biomolecular mechanism underlying the elevated cardiovascular risk for individuals with the apoA-IV polymorphisms, Q360H and T347S.

« Back to ISTH 2021 Congress

ISTH Congress Abstracts - https://abstracts.isth.org/abstract/apolipoprotein-a-iv-polymorphisms-q360h-and-t347s-reduce-its-inhibitory-effect-against-thrombosis/