ADAMTS-13 regulates platelet adhesion under flow. A new method for differentiation between inherited and acquired thrombotic thrombocytopenic purpura

ADAMTS-13 cleaves large and ultra-large von Willebrand factor multimers normally secreted by endothelial cells. Severe deficiency of this enzyme leads to thrombotic thrombocytopenic purpura (TTP). We applied the Impact-R system [Cone and plate(let) Analyzer, CPA] to determine optimal conditions for ADAMTS-13 function, to assess it's activity in TTP patients and to distinguish inherited TTP (in TTP) from acquired TTP (ac TTP). The role of ADAMTS-13 in platelet adhesion under different conditions was investigated applying recombinant forms of VWF and ADAMTS-13. rVWF was first treated by rADAMTS-13 either in solution or when immobilized on the surface of the well, under static or flow conditions, in the presence or absence of BaCl₂. The resulting cleaved VWF fragments were then immobilized and served to assess type 3 von Willebrand disease whole blood platelet adhesion under flow. Maximal effect of the rADAMTS-13 (decrease of platelet adhesion in the absence compared to the presence of BaCl₂), was observed when the rVWF was pre-immobilized and the cleavage step took place under flow (85%). Mixing plasma of TTP patients with normal blood (1:3) yielded a 1.6- to 2-fold increase of platelet adhesion under flow compared to mixing normal plasma with normal blood, at shear rate range of 1,800-2,500s^-1. Maximal increase of platelet adhesion was observed under 2,050s^-1. Under these conditions, the extent of adhesion was similarly higher in patients with in TTP and ac TTP versus control [surface coverage (SC) 14.5 ± 2.8% and 14.6 ± 2.5% vs.7.4 ± 1.7%, respectively]. ADAMTS-13 activity measured by collagen-binding test was similarly low (4.2 ± 3.8% and 3.5 ± 2.4% vs. 72.2 ± 8.0%, respectively). An inverse correlation between SC and ADAMTS-13 activity was observed in a patient with in TTP assayed eight times during plasma infusion treatment. Addition of BaCl₂ to the mixture of TTP plasma and normal whole blood yielded a decrease in platelet adhesion in in TTP (by 51%) but not in ac TTP. The lack of reduction of platelet adhesion in ac TTP could presumably be due to the presence of ADAMTS-13 inhibitor in these patients. These results suggest that VWF immobilization and high shear flow yielded optimal conditions for ADAMTS-13 activity, and that introduction of BaCl₂ in the Impact-R (CPA) test may be useful for differentiation between inherited and acquired TTP.