A Significant Fraction of ADAMTS13 Activity Is Associated with Activated Platelets and Their Microparticles (PMP): Implication for Regulating ADAMTS13 Activity.

BACKGROUND: Deficiency of ADAMTS13, vWF cleaving protease, is known to be associated with TTP and some other microangiopathies, but low levels were reported in other diseases such as ITP, DIC, lupus and other thromboses. Although inhibitory autoantibodies were demonstrated in TTP, the mechanisms underlying reduced levels of ADAMTS13 in other disorders remains unclear. We tested the hypothesis that ADAMTS13 is associated with cell membranes and derived microparticles, especially from activated platelet and their microparticles (PMP), which could modulate the enzyme activities of ADAMTS13.

METHODS: PRP was prepared by centrifuging citrated normal blood for 10 min at 160×g, and PPP by further centrifuging for 10 min at 3,000×g, and particle-free plasma (PFP) by further centrifuging for 15 min at 20,000×g. ADAMTS13 activity was assayed by the FRETS-VWF73 method of Kokame et al [Br J Haematol 129:93, 2005] using the Fluoroskan Ascent plate reader. Platelets were activated by ADP (10 μM) or ionophore A23187 (2 μM).

RESULTS: The ADAMTS13 activity (A) of pooled PPP of 10 controls was defined as 100%. (1) In vitro study: (1a) ADAMTS13 activity was not significantly different between PPP and resting PRP. However, if the platelets in PRP were first activated by ADP for 1hr, a significant reduction of activity was observed (A = 85 ±7%, p<0.05). If the activated platelets were removed, the activity of the supernatant fell to 79 ±10% p<0.05) of the control level, and was further reduced by higher centrifugation to remove PMP (A = 66 ±12%, p<0.01). (1b) Activation by A23187, a stronger agonist producing 2–3 fold more PMP than ADP (confirmed by flow cytometry), induced a more dramatic reduction in PRP (A = 78±8%, p<0.01), and after removal of platelets (A = 71 ±11%, p<0.01), and after removal of PMP (48 ±11%, p<0.01). (1c) Interestingly, resuspending the activated platelets did not restore ADAMTS13 activity, although resuspending the PMP did partially restore the activity. (2) In vivo study: PPP from 13 patients (6 ITP, 4 APS, 3 lupus) were analyzed. The majority (11/13) of PPP samples lost activity after removal of PMP (A = 79 ±12% in PPP vs. 64 ±11% in PFP; p <0.02).

CONCLUSION: These data show that a significant but variable fraction of ADAMTS13 activity is associated with activated platelets and PMP. This has several implications. First, distinguishing soluble from membrane-bound ADAMTS13 may lead to better correlation of activities with clinical findings, and may help explain low levels of ADAMTS13 in some disorders associated with platelet activation and high PMP. Second, this interaction may play a role in regulating ADAMTS13 activity. Third, membrane-bound ADAMTS13 may clear more readily from circulation, therefore inhibiting platelet activation or MP formation may have benefits for the management of microangiopathies.