Monoclonal whole IgG impairs both fibrin and thrombin formation: hemostasis and surface plasmon resonance studies.

OBJECTIVES: Monoclonal gammopathies frequently associate with hemostatic alterations. Thrombotic events occur with high incidence particularly upon treatment, while in rarer cases hemorrhagic diathesis can be observed. The pathology of these tendencies could be caused by thrombocytopenia or hyperviscosity burden of circulating monoclonal antibodies. Studies also suggest interference of monoclonal antibodies with primary hemostasis. We isolated monoclonal whole IgG paraproteins from two myeloma patients to observe their effect on thrombin formation and fibrin polymerization. METHODS: Monoclonal whole IgG was prepared from sera of two newly diagnosed untreated multiple myeloma patients and control normal plasma samples. Fibrin formation was measured using thrombin time and dilute prothrombin time tests and thrombin formation was detected with a fluorimetric thrombin generation assay. In addition, molecular interactions were investigated by surface plasmon resonance (SPR). RESULTS: Thrombin time was prolonged upon addition of monoclonal IgG even at 30 g/L by 12 %, increasing up to 36 % at 60 g/L concentration. Dilute prothrombin time was prolonged by 20 % even at 30 g/L. Thrombin generation assay indicated an impairment in thrombin formation at the presence of monoclonal IgG compared to polyclonal at equivalent concentration. By an SPR assay we determined that both clonality IgG preparations interacted with fibrinogen, however interaction with human thrombin was only detected with monoclonal immunoglobulins (KD=1.03 × 10-7 M). CONCLUSIONS: Here we provide evidence that isolated monoclonal whole IgG from myeloma patients can impair both fibrin and thrombin formation and we demonstrate by SPR assay that it interacts with components of the final phase of the coagulation system.

Distinct and overlapping effects of ß2-glycoprotein I conformational variants in ligand interactions and functional assays.

One of the most abundant coagulation proteins is ß2-glycoprotein I (ß2GPI) 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) ß2GPI 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 ß2GPI 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 ß2GPI showed a similar and strong affinity to isolated anti-ß2GPI autoantibodies (Kd closed ß2GPI = 5.17 × 10-8 M, Kd open ß2GPI = 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 ß2GPI 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.