Recombinant ADAMTS13 Processing by FXIa Is Distinct from Thrombin.

ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type1 domain 13) is associated with processing monomeric and multimeric or ultralarge von Willebrand factor (vWF), cleaving between Tyr1605 and Met1606 in the A2 domain. Low levels of ADAMTS13 activity (<5%) are strongly associated with the life threatening hemostatic condition, thrombotic thrombocytopenic purpura (TTP). It is being recognized that not all cases of TTP can be attributed exclusively to the deficiency in ADAMTS13 or presence of ADAMTS13 autoantibodies. Other biological factor(s) or cofactor(s) may play a role in the pathogenesis of TTP.

We initially reported (1) that purified FXIa (1–40 nM) also cleaved purified recombinant A2 domain fragment (100 ug/mL) and FXIa at 200 nM also cleaves purified vWF protein (100 ug/mL) at still unknown sites. We reported (1) that FXIa cleaved purified recombinant (r) ADAMTS13 and that high concentrations of FXIa (>100 nM) resulted in loss of the metalloprotease activity towards VWF73aa FRET (2).

In this study, we found that low concentrations (<20 nM ) of purified FXIa will cleave purified rADAMTS13 which resulted in about 2 fold enhancement of the metalloprotease activity towards the fluorescently labeled VWF73aa peptide substrate using either VWF73aa FRETs (2) or the newly developed Alexa488-VWF86 FRET substrate from American Diagnostica Inc (3). However, at high concentrations (>100nM) of purified FXIa, inactivation of rADAMTS13 metalloprotease activity was observed. The proteolytic patterns at high and low amounts of FXIa will be presented. Since thrombin was reported (4) as possible regulator of ADAMTS13, we used various polyclonal and monoclonal antibodies, recognizing different regions of ADAMTS13 to compare the ADAMTS13 processing by the two serine proteases. The results showed that the proteolytic patterns by FXIa and thrombin are different. In addition, specific monoclonal anti FXI antibodies but not hirudin inhibits the FXIa processing of ADAMTS13. The identification of cleavage sites by FXIa on ADAMTS13 and VWF are in progress. The physiological relevance of FXIa processing of ADAMTS13 and VWF protein and possible link to the etiology of TTP and FXIa related pathophysiology (5) are under investigation.