Lupus-Derived Antiprothrombin Autoantibodies from a V Gene Phage Display Library Are Specific for the Kringle 2 Domain of Prothrombin

Abstract

Autoantibodies to prothrombin are common in patients with systemic lupus erythematosus. Although their presence is a risk factor for thrombosis, neither their origin nor their precise role in inducing the procoagulant state is known. We have developed a phage-display antibody library from patients with systemic lupus erythematosus with antiprothrombin antibodies, and we have selected two single-chain Fv antibody fragments (ScFvs) by panning on a prothrombin-coated surface. In prothrombin activation assays using purified components, these antibodies promoted prothrombin activation. These ScFvs, termed AN78 and AN129, bound to immobilized prothrombin in a concentration-dependent specific manner but not to other anionic phospholipid binding proteins such as beta2-glycoprotein I or annexin V. Phosphatidylserine-bound prothrombin, but not soluble prothrombin, inhibited the binding suggesting that the epitope is available only on immobilized prothrombin. To localize the epitope, prothrombin was treated with thrombin or factor Xa and various prothrombin activation fragments were subsequently isolated and tested in ELISA with the ScFvs. Both AN78 and AN129 bound to prethrombin I (the fragment lacking the Gla domain and the first kringle domain), to fragment 1.2 (containing Gla and the two kringle domains only) and to fragment 2 but not to thrombin, thus localizing the cognate epitope to the kringle 2 domain in prothrombin. Analysis of the cDNA sequences of these antibodies show clustered mutational patterns in the complementarity determining region, suggesting that variable domains are the products of antigen-driven B cell clonal maturation.

Discovery and characterization of 2 novel subpopulations of aPS/PT antibodies in patients at high risk of thrombosis

“…presence of other and perhaps dominant pathogenic aPL such as aCL and anti-b 2 GPI antibodies could indeed obscure the true contribution of IgG aPS/PT. 39,58 In this regard, the results reported in Figure 5 in the context of current knowledge suggest what could be some of the mechanisms for lupus anticoagulant and prothrombotic state in the presence of type I and type II autoantibodies. By massively recruiting prothrombin onto the membranes, type I Abs could lead to delayed recruitment of other clotting factors, which may slow down the initiation and propagation of the clotting cascade in vitro, similar to anti-b 2 GPI antibodies.…”

“…Specifically, some IgG aPS/PT require a significant structural reorganization of the antigen for proper recognition in solution, whereas IgG aPT-A target epitopes that are constitutively exposed to the solvent in both closed and open forms of prothrombin. [37][38][39] Given that IgG aPS/PT, and not IgG aPT-A, significantly correlate with thrombosis and obstetric complications, 13,14,20 these findings also raise the interesting hypothesis that excessive stabilization of the open form of prothrombin in vivo may contribute to the onset and progression of APS. Second, we discovered that IgG aPS/PT are heterogeneous, and APS patients positive for IgG aPS/PT can be classified into 2 groups (group A and group B) according to their autoantibody profile.…”

“…aPT-A are detected by immobilizing prothrombin alone onto hydrophilic plastic plates, without phospholipids, and are believed to recognize epitopes located primarily in kringle-2 and the protease domain. [37][38][39] In competition experiments, proTWT, proTCC, and proTY93A displayed similar levels of inhibition and comparable IC 50 values against aPT-A (Figure 2E-F; Table 2). The difference between proTCC and proTD154-167, however, was statistically significant, suggesting that the distal portion of Lnk2 may be recognized by aPT-A.…”

Structure of Coagulation Factor II: Molecular Mechanism of Thrombin Generation and Development of Next-Generation Anticoagulants

“…Targeting kringles could, therefore, provide a rational strategy to develop anticoagulants. Interestingly, kringle-2 was found to be the locale of autoantibodies in the antiphospholipid syndrome in patients with Systemic Lupus Erythematosus ( 21 ) and was also found to act as a potential neuroinflammatory factor that stimulates microglial toll-like receptor 4 (TLR4) ( 22 ). Since kringle-2 is part of prothrombin fragment 1.2 which is released from prothrombin upon activation ( 23 ), neutralization or enhanced clearance of this fragment from the circulation may be beneficial to patients with chronic inflammation.…”