Von Willebrand factor (VWF) is unable to interact spontaneously with platelets because this interaction requires a conversion of the VWF A1 domain into a glycoprotein Ib␣ (GpIb␣) binding conformation. Here, we discuss a llama-derived antibody fragment (AU/VWFa-11) that specifically recognizes the GpIb␣-binding conformation. AU/VWFa-11 is unable to bind VWF in solution, but efficiently interacts with ristocetin-or botrocetin-activated VWF, VWF comprising type 2B mutation R1306Q, or immobilized VWF. These unique properties allowed us to use AU/ VWFa-11 for the detection of activated VWF in plasma of patients characterized by spontaneous VWF-platelet interactions: von Willebrand disease (VWD) type 2B and thrombotic thrombocytopenic purpura (TTP). For VWD type 2B, levels of activated VWF were increased 12-fold (P < .001) compared to levels in healthy volunteers. An inverse correlation between activated VWF levels and platelet count was observed (R 2 ؍ 0.74; P < .003). With regard to TTP, a 2-fold (P < .001) increase in activated VWF levels was found in plasma of patients with acquired TTP, whereas an 8-fold increase (P < .003) was found in congenital TTP. No overlap in levels of activated VWF could be detected between acquired and congenital TTP, suggesting that AU/VWFa-11 could be used to distinguish between both disorders. Furthermore, it could provide a tool to investigate the role of VWF in the development of thrombocytopenia in various diseases. (Blood. 2005;106: 3035-3042)
Thrombotic thrombocytopenic purpura (TTP) is a microangiopathy often associated with a severely decreased activity of ADAMTS13. In plasma of the majority of patients with TTP, antibodies are present that inhibit the von Willebrand factor (VWF) processing activity of ADAMTS13. We describe a sensitive assay that monitors binding of recombinant ADAMTS13 to immobilized IgG derived from patient plasma. Analysis of fifteen patients with TTP and severely reduced ADAMTS13 activity revealed that in all patients antibodies directed to ADAMTS13 were present. Levels of anti-ADAMTS13 antibodies varied considerably among patients, specific antibody levels in plasma range from less than 100 ng/ml to over 1 microg/ml. Longitudinal analysis in three patients revealed that anti-ADAMTS13 antibody levels declined with different kinetics. For further characterization of anti-ADAMTS13 antibodies, we prepared a series of recombinant fragments corresponding to the various ADAMTS13 domains. All seven TTP plasma samples tested, showed reactivity of antibodies towards a fragment consisting of the disintegrin/TSR1/cysteine-rich/spacer domains. In one patient, we also observed reactivity towards the TSR2-8 repeats. No binding of antibodies to propeptide, metalloprotease and CUB domains was detected. To further delineate the binding site in the disintegrin/TSR1/cysteine-rich/spacer region, we prepared additional ADAMTS13 fragments. Antibodies directed towards the cysteine-rich/spacer fragment were found in all plasma samples analyzed. No antibodies reacting with the disintegrin/TSR1 domains were detected. A recombinant fragment comprising the spacer domain was recognized by all patients samples analyzed, suggesting that the 130-amino-acid spacer domain harbors a major binding site for anti-ADAMTS-13 antibodies.
Patients with antiphospholipid syndrome are characterized by the association of thrombosis or pregnancy morbidity and the presence of antiphospholipid autoantibodies. Particularly, anti- 2 -glycoprotein ( 2 GPI) autoantibodies correlate with thrombosis, suggesting an antibodyinduced gain of prothrombotic function and/or an antibody-induced loss of antithrombotic function of  2 GPI. In the search for potential antithrombotic properties of  2 GPI, we found that  2 GPI inhibits von Willebrand factor (VWF)-induced platelet aggregation. In addition, platelet adhesion to a VWF-coated surface was decreased by 50% in the presence of  2 GPI (P < .03).  2 GPI binds to the A1 domain of VWF but preferably when the A1 domain is in its active glycoprotein Ib␣-binding conformation. Anti- 2 GPI antibodies isolated from a subset of antiphospholipid syndrome patients neutralized the  2 GPI-VWF interactions and thus the inhibitory activity of  2 GPI. In comparison to healthy individuals, the amounts of active VWF in circulation were increased 1.5-fold (P < .001) in patients positive for lupus anticoagulant (LAC) due to anti- 2 GPI antibodies. Thus,  2 GPI is a biologically relevant inhibitor of VWF function by interfering with VWFdependent platelet adhesion. Anti- 2 GPI autoantibodies neutralize this inhibitory function and are associated with increased levels of active VWF. This mode of action could contribute to the thrombosis and consumptive thrombocytopenia observed in patients with anti- 2 GPI antibodies. (Blood.
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