Association of Complement with Microvascular Endothelial Cell Injury in Thrombotic Microangiopathy In Vivo and in In Vitro Model Systems.

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We have previously shown that plasma from patients with idiopathic as well as HIV and thienopyridine-linked TTP induces apoptosis of microvascular endothelial cells (MVEC) in vitro, with specific sparing of large vessel and pulmonary MVEC, consistent with the clinical syndrome. This correlates with in vivo measures of apoptosis in splenic tissue of TTP patients. We found that osteoprotegrin (OPG), an inhibitor of multiple inducers of MVEC apoptosis, could block TTP plasma-mediated MVEC injury. Utilizing OPG affinity-binding columns and MS-TOFI analysis of column eluates, we subsequently identified complement (C) components and C-regulatory factors as potentially involved in TTP plasma-mediated EC injury. Building on the observation that familial TTP syndromes have been linked to genetic deficiencies of the C-regulatory factors Factor H and MCP, and three cases of sporadic HUS in children have been accompanied by autoantibodies to factor H, we hypothesized that the C-system plays an important role in TTP-induced MVEC injury, facilitated by autoantibody-mediated suppression of C-regulatory proteins. To evaluate the role of the complement system we looked at:IFA staining of splenic tissue from TTP patients versus controls for C-components in conjunction with CD34+ indentification of MVEC.ELISA-based assays of complement activation/deposition on MVEC exposed to TTP vs. control plasma.Ability of anti-C agents to block TTP plasma-mediated MVEC apoptosis.DNA microarray analysis of TTP plasma MVEC exposed to TTP vs. control plasma.ELISA-based search for autoantibodies against C-regulatory proteins in TTP plasma.IFA staining demonstrated increased deposition of C-components on MVEC in vivo on splenic tissue of TTP vs. control patients (4/5 TTP, 0/3 control). Increased C-activation was demonstrated on MVEC exposed to plasmas of TTP patients in vitro as evidenced by decreased C3b (p=0.002) and C4d (p=0.0002) deposition. Microarray data revealed increased expression of C-regulatory factors in resistant vs. susceptible MVEC exposed to TTP plasma. C-inhibtion resulted in prevention of TTP-plasma induced MVEC apoptosis by annexin-V staining (P<0.02). The search for autoantibodies against five C-regulatory components: CD46 (MCP), CD55 (DAF), CD59, Factor H, and Factor I by ELISA assay is currently ongoing. In conclusion, the clinical expression of thrombotic microangiopathy likely requires a double-hit of endothelial cell injury and autoantibodies against proteins vital to hemostasis and/or MVEC survival. In some patients this may involve autoantibodies to ADAMTS13; in others, C-system activation and inhibition of C-regulation by C-regulatory protein autoantibodies may be involved. Inhibition of the C-system may be a feasible clinical approach in some patients with thrombotic microangiopathies.