Distinct and overlapping effects of β2-glycoprotein I conformational variants in ligand interactions and functional assays

J Immunol Methods. 2020 Dec:487:112877. doi: 10.1016/j.jim.2020.112877. Epub 2020 Oct 6.

Abstract

One of the most abundant coagulation proteins is β2-glycoprotein I (β2GPI) that is present in humans at a concentration of around 200 mg/L. Its physiological role is only partially understood, but it adopts several different structural forms the majority of which are the open and closed forms. We isolated native (circular) β2GPI and converted it into an open conformation. The effectiveness of these procedures was assessed by Western blot and negative-staining electron microscopy. We found that in coagulation assays the open form of β2GPI had a significant prolonging effect on fibrin formation in a dilute prothrombin time test (p < 0.001). In the dilute activated partial thromboplastin time test, both conformations had a significant prolonging effect (p < 0.001) but the open conformation was more effective. In a fluorescent thrombin generation assay both conformations slightly delayed thrombin generation with no significant effect on the quantity of formed thrombin. By using surface plasmon resonance assays, the equilibrium dissociation constants of both the open and closed conformations of β2GPI showed a similar and strong affinity to isolated anti-β2GPI autoantibodies (Kd closed β2GPI = 5.17 × 10-8 M, Kd open β2GPI = 5.56 × 10-8 M) and the open form had one order of magnitude stronger affinity to heparin (Kd = 0.30 × 10-6 M) compared to the closed conformation (Kd = 3.50 × 10-6 M). The two different forms of β2GPI have distinct effects in functional tests and in ligand binding, which may considerably affect the intravascular events related to this abundant plasma protein in health and disease.

Keywords: Anti-β(2)-glycoprotein I; Heparin; Surface plasmon resonance; Thrombin generation; β(2)-glycoprotein I conformations.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anticoagulants / pharmacology
  • Autoantibodies / metabolism
  • Binding Sites, Antibody
  • Blood Coagulation* / drug effects
  • Fibrin / metabolism
  • Heparin / pharmacology
  • Humans
  • Ligands
  • Partial Thromboplastin Time
  • Protein Conformation
  • Prothrombin Time
  • Structure-Activity Relationship
  • Thrombin / metabolism
  • beta 2-Glycoprotein I / antagonists & inhibitors
  • beta 2-Glycoprotein I / chemistry
  • beta 2-Glycoprotein I / immunology
  • beta 2-Glycoprotein I / metabolism*

Substances

  • Anticoagulants
  • Autoantibodies
  • Ligands
  • beta 2-Glycoprotein I
  • Fibrin
  • Heparin
  • Thrombin