title: Assembly and activation of alternative complement components on endothelial cell-anchored ultra-large von Willebrand factor links complement and hemostasis-thrombosis.
abstract: 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 non-antibody-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.
  
  
  RESULTS
  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
  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.
authors.affiliation: Department of Bioengineering, Rice University, Houston, TX, USA. nturner@rice.edu
authors.forename: Nancy A
authors.initials: NA
authors.lastname: Turner
authors.forename: Joel
authors.initials: J
authors.lastname: Moake
mesh_terms: D051722:ADAM Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology; D000071120:ADAMTS13 Protein; D002478:Cells, Cultured; D003170:Complement Pathway, Alternative; D003171:Complement Pathway, Classical; D037582:Complement Pathway, Mannose-Binding Lectin; D003165:Complement System Proteins / Q000235:genetics / Q000276:immunology*; D015870:Gene Expression; D006487:Hemostasis / Q000276:immunology*; D006358:Hot Temperature; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding; D055503:Protein Multimerization; D013927:Thrombosis / Q000276:immunology* / Q000473:pathology; D014841:von Willebrand Factor / Q000235:genetics / Q000276:immunology* / Q000378:metabolism
chemical_list: D014841:von Willebrand Factor; D003165:Complement System Proteins; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human
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