Deficiency of ADAMTS13 is implicated in the pathogenesis of congenital and some acquired idiopathic thrombotic thrombocytopenic purpura (TTP). However, injury to endothelial cells (EC) is also believed to play a major role. Endothelial microparticles (EMP), markers of EC injury, are elevated in acute TTP, and subsets of EMP have been shown to carry ULvWF. The aim of this study was to further characterize the role of EMP in TTP by assessing their effect on ADAMTS13 activity.
METHODS. EMP derived from activated renal microvascular EC (RMVEC) and coronary artery EC (CAEC) were depleted of phenotypic subsets by negative panning with immunomagnetic beads against CD62E, CD31, or CD54. For clinical studies, platelet-poor plasmas (PPP) were selectively depleted of PMP and LMP by beads against CD41 and CD45, respectively. Antigenic phenotypes of EMP were measured by flow cytometry. vWF multimer size was analyzed by Western blotting after 0.8% argarose gel electrophoresis. Proaggregatory activity of vWF (free in plasma or on EMP) was assessed by incubation of washed platelets with EMP (some are vWF+) or soluble plasma vWF, and ristocetin for 10min, then measuring platelet aggregation by flow cytometry. To measure interaction of ADAMTS13 with EMP, PPP was incubated with EMP (1x105 – 1x108 EMP/mL) for 15min – 3hr, then centrifuged to remove EMP (15,000xg, 1hr), and ADAMTS13 activity remaining in the plasma was assayed by the method of Furlan et al [Blood, 1997, 89:3097].
RESULTS. (1) RMVEC released >2-fold more EMP than CAEC and the proportion of vWF+ EMP was 2-fold higher with RMVEC (65 ±17% of total) than with CAEC (30±12%), p<0.01. (2) We next selectively depleted total EMP of CD31+, CD62E+ or CD54+ subsets, then incubated with normal plasma (defined as 100% ADAMTS13 activity) and measured activity remaining after centrifugation. Maximal reduction of activity was seen with non-depleted (total) EMP, which was nearly the same as with the CD54-depleted preparations (all experiments at 1x108 EMP/mL for 3hr), resulting in a 71 ±23% reduction after centrifugation (p<0.01). However, depletion of EMP positive for CD31 or CD62E resulted in significantly less removal or inactivation of ADAMTS13 (55 ±17%, 35 ±8% inhibition, respectively. Similar results were observed with EMP from CAEC except that the effect was about 25% less. (3) To determine whether EMP from TTP patients can also negatively regulate ADAMTS13 activity, we isolated total MP by centrifugation from plasma of 3 acute TTP patients, and removed MP of platelet and leukocyte origin by immunomagnetic beads. The resulting EMP were incubated with normal plasma. As observed with in vitro-generated EMP, similar concentrations of patient EMP removed or inactivated ADAMTS13 activity in plasma, by 66 ±18% in these experiments (p<0.02). (4) To further test the ability of EMP to adsorb ADAMTS13, we found that preincubation of plasma with EMP, resulted in 57 ±10% increased platelet aggregate formation relative to absence of EMP preincubation, (p<0.01).
CONCLUSION. EMP released from EC culture and EMP isolated from TTP plasma inactivated ADAMTS13 activity and enhanced formation of platelet aggregates. These findings further support the hypothesis that EMP play an important role in TTP by downregulating ADAMTS13 and promoting the formation of platelet rich microthrombi in microvasculature.