Journal Article

Assembly and Activation of Alternative Complement Components on Endothelial Cell-Anchored Ultra-Large Von Willebrand Factor Links Complement and Hemostasis-Thrombosis

Turner, Nancy A., Moake, Joel

2013

Overview

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. 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. 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.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: ADAM Proteins - antagonists & inhibitors / ADAM Proteins - genetics / ADAM Proteins - immunology / ADAMTS13 Protein / Anchoring / Antibodies / Assembly / Bioengineering / Biology / Cells, Cultured / Complement / Complement activation / Complement component C3 / Complement component C4 / Complement component C5 / Complement Pathway, Alternative / Complement Pathway, Classical / Complement Pathway, Mannose-Binding Lectin / Complement system / Complement System Proteins - genetics / Complement System Proteins - immunology / Endothelial cells / Endothelium / Fluorescence / Gene Expression / Hemostasis / Hemostasis - immunology / Hemostatics / Hot Temperature / Human Umbilical Vein Endothelial Cells - cytology / Human Umbilical Vein Endothelial Cells - immunology / Human Umbilical Vein Endothelial Cells - metabolism / Humans / Lectins / Medicine / Microscopy / Mutation / Pathways / Protein Binding / Protein Multimerization / Proteins / Purpura / Strings / Thrombocytopenic purpura / Thromboembolism / Thrombosis / Thrombosis - immunology / Thrombosis - pathology / Thrombotic thrombocytopenic purpura / Umbilical vein / Veins & arteries / Von Willebrand factor / von Willebrand Factor - genetics / von Willebrand Factor - immunology / von Willebrand Factor - secretion