The combined presence of anti-phospholipid (PL) Ab, including lupus anticoagulants (LAC) and/or anticardiolipin Ab (aCL), and thrombosis is recognized as the antiphospholipid syndrome (APS). LAC are detected as an inhibitory effect on PL-restricted in vitro blood coagulation tests, and are comprised mainly of Ab against β2 glycoprotein I and prothrombin (PT). Recently, anti-PT Ab (aPT) were found to be associated with thrombosis by some investigators, although this is not confirmed by others. Considering that aPT are heterogeneous in patients and that PT is converted into thrombin, we hypothesize that certain aPT in patients may bind to thrombin, and that some of such anti-thrombin Ab may interfere with thrombin-antithrombin (AT) interaction and thus reduce the AT inactivation of thrombin. To test this hypothesis, we searched for anti-thrombin Ab in APS patients and then studied those found for their effects on the AT inactivation of thrombin. The results revealed that most, but not all, aPT-positive patient plasma samples contained anti-thrombin Ab. To study the functional significance of these Ab, we identified six patient-derived mAb that bound to both PT and thrombin. Of these mAb, three could reduce the AT inactivation of thrombin, whereas others had minimal effect. These findings indicate that some aPT in patients react with thrombin, and that some of such anti-thrombin Ab could inhibit feedback regulation of thrombin. Because the latter anti-thrombin Ab are likely to promote clotting, it will be important to develop specific assays for such Ab and study their roles in thrombosis in APS patients.

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Previously we reported that the variable heavy chain region (V H ) of a human beta 2 glycoprotein I-dependent monoclonal antiphospholipid antibody (IS4) was dominant in conferring the ability to bind cardiolipin (CL). In contrast, the identity of the paired variable light chain region (V L ) determined the strength of CL binding. In the present study, we examine the importance of specific arginine residues in IS4V H and paired V L in CL binding. The distribution of arginine residues in complementarity determining regions (CDRs) of V H and V L sequences was altered by site-directed mutagenesis or by CDR exchange. Ten different 2a2 germline gene-derived V L sequences were expressed with IS4V H and the V H of an anti-dsDNA antibody, B3. Six variants of IS4V H , containing different patterns of arginine residues in CDR3, were paired with B3V L and IS4V L . The ability of the 32 expressed heavy chain/light chain combinations to bind CL was determined by ELISA. Of four arginine residues in IS4V H CDR3 substituted to serines, two residues at positions 100 and 100 g had a major influence on the strength of CL binding while the two residues at positions 96 and 97 had no effect. In CDR exchange studies, V L containing B3V L CDR1 were associated with elevated CL binding, which was reduced significantly by substitution of a CDR1 arginine residue at position 27a with serine. In contrast, arginine residues in V L CDR2 or V L CDR3 did not enhance CL binding, and in one case may have contributed to inhibition of this binding. Subsets of arginine residues at specific locations in the CDRs of heavy chains and light chains of pathogenic antiphospholipid antibodies are important in determining their ability to bind CL.

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