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Boris Shenkman, Ulrich Budde, Dorothea Angerhaus, Aharon Lubetsky, Naphtali Savion, Uri Seligsohn, David Varon
ADAMTS-13 regulates platelet adhesion under flow. A new method for differentiation between inherited and acquired thrombotic thrombocytopenic purpura
Keywords: ADAMTS-13, TTP, VWF, Impact-R
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 inheritedTTP (inTTP) from acquiredTTP (acTTP). 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 BaCl 2 . The resulting cleavedVWF 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 BaCl 2 ), was observed when the rVWF was pre-immobilized and the cleavage step took place under flow (85%). Mixing plasma ofTTP 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 inTTP and acTTP 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 inTTP assayed eight times during plasma infusion treatment. Addition of BaCl 2 to the mixture of TTP plasma and normal whole blood yielded a decrease in platelet adhesion in inTTP (by 51%) but not in acTTP. The lack of reduction of platelet adhesion in acTTP 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 BaCl 2 in the Impact-R (CPA) test may be useful for differentiation between inherited and acquired TTP.
Thrombosis and Haemostasis, Schattauer
Print ISSN: 0340-6245
Volume: 96, 08/2006
Pages: 160 - 166