VWF cleavage by endothelial cell lysates depicted by immunoblotting.

<p>A. Recombinant ADAMTS13 cleaves the A2 domain of VWF whereby 176 kDa and 140 kDa bands appear (lane 1). Full-length VWF is depicted by an arrow. Cell buffer, incubated with VWF and used as the negative control, did not exhibit the cleavage fragments (lane 2). CiGEnC cell lysate exhibited VWF cleaving activity as shown by the presence of similar cleavage fragments (lane 3) which were inhibited by 20 mM EDTA (lane 4). HMVEC lysate also showed the two breakdown products (lane 5) which were partially inhibited by EDTA (lane 6). B. Immunoadsorption of ADAMTS13 inhibited VWF cleaving activity. Recombinant ADAMTS13 showed the two cleavage fragments (lane 1) and the cleaving activity was abrogated by removal of ADAMTS13 (lane 2). CiGEnC cell lysate also induced VWF cleavage (lane 3) which was inhibited by immunoadsorption of ADAMTS13 from the lysate (lane 4). Experiments were carried out three times with reproducible results. C. VWF cleavage products were derived from exogenous VWF. Exogenous VWF was added to rADAMTS13 resulting in two cleavage products (lane 1). CiGEnC lysate, without added exogenous VWF, did not show cleavage bands but exhibited a weak band corresponding to full-length VWF (lane 2). When purified exogenous VWF was added to CiGEnC lysates VWF cleavage was demonstrated (lane 3). D. Recombinant ADAMTS13 cleaves exogenously added VWF in cell medium (that was not in contact with endothelial cells, lane 1). Cell medium (that had not been exposed to cells) without added ADAMTS13 exhibited full-length VWF and a very weak band at 176 kDa (lane 2). Cell medium derived from unstimulated CiGEnC exhibited a stronger band at 176 kDa and a weak band at 140 kDa (lane 3). Cell medium derived from CiGEnC stimulated with estradiol exhibited a band at 176 kDa and a weak band at 140 kDa (lane 4). All lanes were run on the same gel. Experiments were carried out twice with reproducible results.