|dc.contributor.author||Turner, Nancy A.
Turner, Nancy A. and Moake, Joel. "Assembly and Activation of Alternative Complement Components on Endothelial Cell-Anchored Ultra-Large Von Willebrand Factor Links Complement and Hemostasis-Thrombosis." PLoS One, 8, no. 3 (2013) e59372. http://dx.doi.org/10.1371/journal.pone.0059372.
Background: Vascular endothelial cells (ECs) express and release protein components of the complement pathways, as well
as secreting and anchoring ultra-large von Willebrand factor (ULVWF) multimers in long string-like structures that initiate
platelet adhesion during hemostasis and thrombosis. The alternative complement pathway (AP) is an important nonantibody-
requiring host defense system. Thrombotic microangiopathies can be associated with defective regulation of the
AP (atypical hemolytic-uremic syndrome) or with inadequate cleavage by ADAMTS-13 of ULVWF multimeric strings secreted
by/anchored to ECs (thrombotic thrombocytopenic purpura). Our goal was to determine if EC-anchored ULVWF strings
caused the assembly and activation of AP components, thereby linking two essential defense mechanisms.
Methodology/Principal Findings: We quantified gene expression of these complement components in cultured human
umbilical vein endothelial cells (HUVECs) by real-time PCR: C3 and C5; complement factor (CF) B, CFD, CFP, CFH and CFI of
the AP; and C4 of the classical and lectin (but not alternative) complement pathways. We used fluorescent microscopy,
monospecific antibodies against complement components, fluorescent secondary antibodies, and the analysis of .150
images to quantify the attachment of HUVEC-released complement proteins to ULVWF strings secreted by, and anchored
to, the HUVECs (under conditions of ADAMTS-13 inhibition). We found that HUVEC-released C4 did not attach to ULVWF
strings, ruling out activation of the classical and lectin pathways by the strings. In contrast, C3, FB, FD, FP and C5, FH and FI
attached to ULVWF strings in quantitative patterns consistent with assembly of the AP components into active complexes.
This was verified when non-functional FB blocked the formation of AP C3 convertase complexes (C3bBb) on ULVWF strings.
Conclusions/Significance: AP components are assembled and activated on EC-secreted/anchored ULVWF multimeric
strings. Our findings provide one possible molecular mechanism for clinical linkage between different types of thrombotic
and complement-mediated disorders.
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Assembly and Activation of Alternative Complement Components on Endothelial Cell-Anchored Ultra-Large Von Willebrand Factor Links Complement and Hemostasis-Thrombosis
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