Determination for Coagulation Functions and Novel Inhibitory Mechanisms in Acquired Hemophilia A with Type 2 Inhibitors.

Abstract 3483

Poster Board III-420

The development of factor (F)VIII autoantibody inhibitors results in severe hemorrhagic diathesis known as acquired hemophilia A (AHA), although many inhibitors with type 2 behavior incompletely inhibit FVIII activity at saturating concentrations. However, the mechanisms of AHA with type 2 behavior remain to be fully understood. In this study, we evaluated the detailed coagulation function and clarified inhibitory mechanisms for AHA with type 2. Plasma samples of patients were obtained from congenital hemophilia A with severe type {(FVIII:C<0.2 IU/dl; S1-type, n=9) and (0.2-1.0 IU/dl; S2-type, n=6)}, with moderate type (2.1 ± 0.9 IU/dl; M-type, n=10), and AHA-type 2 (2.0 ± 1.9 IU/dl, 201 ± 120 BU/ml; type 2 n=8). Thrombin generation test (TGT) was performed using tissue factor (0.5 pM), phospholipids (PL 4 μM) and ellagic acid (0.3 μM). Although the levels of FVIII:C in M-and S2-types were similar and low compared to those in type 2, TGT parameters in type 2 were significantly decreased than those in M-and S2-types (M/S2/type 2: ETP: 2719±725/1613±684/1352±971 nM*min, peak thrombin: 155±52/110±25/62±35 nM, time to peak: 17.4±2.4/19.3±2.8/31.0±6.8 min). Of note, time to peak in type 2 significantly prolonged than that in S1-type (23.0±4.0 min, p<0.05), suggesting that coagulation function in AHA-type 2 was much worse than that in congenital severe hemophilia A (<0.2 IU/dl). Next, we examined on these inhibitory mechanisms of AHA-type 2 inhibitors. The IgGs from 6 cases were immune-purified from plasmas using protein G-Sepharose. All cases recognized the C2 domain alone of FVIII, whilst any little recognized other coagulation proteins. Competition binding assays showed all anti-C2 cases competed with anti-C2 mAbESH8 (type 2 behavior), whilst little or mildly competed with anti-C2 mAbESH4 (type 1 behavior). FVIII was activated by thrombin, followed by measuring the FVIIIa/FIXa-dependent FX activation in the presence of inhibitors. The addition of purified IgG prior to FVIII activation inhibited the FXa generation by 80∼95% in dose-dependent manners, whilst that of IgG post to FVIII activation little inhibited, supporting these inhibitors affected the cofactor activity for FVIII, but not that for FVIIIa. SDS-PAGE and Western blot revealed that all cases inhibited thrombin cleavage of the FVIII heavy chain at Arg³⁷² by 50∼80% in similar timed- and dose-dependent manners. Furthermore, effects of inhibitors on FVIII/FIXa-dependent FX activation were examined. The presence of all IgGs inhibited the FXa generation by 70∼95% in these circumstances, and these effects were timed- and dose-dependent manners. These also blocked FXa-catalyzed cleavage of the heavy chain at Arg³⁷² by 60∼70%. However, all cases did not inhibit the binding of either von Willebrand factor or PL to FVIII. We concluded that the coagulation functions of AHA-type 2 inhibitors with a C2 epitope like ESH8 were markedly decreased by which inhibitory mechanisms that these inhibitors blocked FVIII activation and cleavage by thrombin and FXa, and these might result in serious hemorrhagic symptoms.