Human Complement Factor H Is a Reductase for Large Soluble von Willebrand Factor Multimers—Brief ReportSignificance
Objective—Ultralarge von Willebrand factor (vWF) strings are secreted by, and anchored to, stimulated human endothelial cells. A disintegrin and metalloprotease with thrombospondin domains-type 13 cleaves the ultralarge vWF strings into large soluble vWF multimers. Normal plasma contains a nonproteolytic reducing activity that subsequently rapidly diminishes the size of the large soluble vWF multimers.
Approach and Results—The vWF reductase activity was isolated from normal cryoprecipitate-poor plasma by chromatography and identified as the complement regulatory protein, factor H (FH), by mass spectroscopy, SDS-PAGE, and monospecific anti-FH antibody. Removal of FH from partially purified vWF reductase by immunoabsorption eliminated the reducing activity, and the activity was recovered in the eluates. Recombinant human FH reduced large soluble vWF multimers in a free thiol-dependent reaction that was not inhibited by a variety of protease inhibitors.
Conclusions—FH contributes to the reduction of large soluble vWF multimers.
Ultralarge von Willebrand factor (ULvWF) multimeric strings, composed of ≈250 kDa subunits connected by disulfide bonds, are secreted by, and anchored to, human endothelial cells (ECs) stimulated by secretagogues (eg, histamine or cytokines).1–3 Exposed A1 domains in vWF monomeric subunits of EC-anchored ULvWF multimeric strings induce platelet adherence, and exposed A2 domains of ULvWF are cleaved at peptide bonds 1605–1606 (in the presence or absence of flow)1,4 by a disintegrin and metalloprotease with thrombospondin domains-type 13 (ADAMTS-13) that is released from ECs.1,2,4,5 This results in the generation of large soluble vWF (LsvWF) multimers.2,5 LsvWF multimers assume a conformation inaccessible to ADAMTS-13 cleavage,5 yet capable of binding to platelet GP Ib (-IX-V) and GP IIb–IIIa receptors and inducing platelet aggregation under conditions of high-fluid shear stress.6,7
LsvWF can be reduced in size by a free thiol-containing molecule in the cryoprecipitate-free fraction of normal plasma (cryosupernatant).5 The LsvWF reducing molecule5,8 is >30 kDa, contains free thiols, and is not albumin, thrombospondin-1,9 cysteine, glutathione, or ADAMTS-13. ADAMTS-13 cleaves EC-secreted/anchored ULvWF strings. ADAMTS-13 can also reduce disulfide bonds that link plasma-type vWF multimers into thicker laterally apposed structures under the influence of shear stress.10 ADAMTS-13 is not capable of reducing the structural disulfide bonds that link vWF monomers into vWF multimers.5
In contrast, as described for the first time in this report, the complement regulatory protein, factor H (FH),11,12 reduces (in the absence of shear stress) the disulfide bonds that link vWF monomers of various chain lengths into LsvWF.
Materials and Methods
Materials and Methods are available in the online-only Supplement.
A partially purified candidate protein with reductase activity for LsvWF, and containing free thiols, was identified as a maleimide–polyethylene glycol (PEG)–biotin–reactive (ie, containing free thiol) band on 7% sodium dodecyl sulfate (SDS) unreduced acrylamide gels (Figure 1). The maleimide–PEG–biotin (and Coomassie stained)–positive band was cut from the gel lanes and identified by mass spectroscopy as compatible with the amino acid composition of FH. The candidate active band reacted with polyclonal antihuman FH antibody (Figure 1).
The LsvWF reducing activity was eliminated from purified preparations by depleting FH, using FH antibody conjugated with protein G or with cyanogen bromide-Sepharose beads (Figure 1). The FH eluted from anti-FH/cyanogen bromide-Sepharose beads was capable of reducing LsvWF (Figure 1).
Control immunoabsorption of C4 and C5, thrombospondin-1, vWF, albumin, or ADAMTS-13 did not remove the reducing activity for LsvWF from cryosupernatant5 or from partially purified LsvWF reductase.
Recombinant FH (rFH; Figure 2) reduced LsvWF in the presence or absence of EDTA, but not in the presence of the free thiol-blocking substance, iodoacetic acid (Figure 2). rFH at concentrations as low as 873 ng/mL (final concentration) were capable of reducing LsvWF. As control, there was no reduction of vWF multimers using the conditioned media from human embryonic kidney (cell line) 293 cells not transfected with the FH gene. (An example of this latter experiment is shown in Figure 3A; top membrane shown.)
In addition to EDTA, several other protease inhibitors did not inhibit the breakdown of the reducing activity for LsvWF contained in normal cryosupernatant, partially purified vWF reductase, or rFH. These results are demonstrated in Figure 4 and the Table. Furthermore, purification of the LsvWF reducing activity (using normal cryosupernatant as starting material) was done in the presence of EDTA.
In some experiments, the lower molecular weight vWF multimers generated by FH reduction of LsvWF are electrically transferred more rapidly than larger vWF multimeric forms from the thin SDS-1% agarose gel into, and through, the underlying membrane. This can be demonstrated by placing a second transfer membrane beneath the first to capture the rapidly migrating lower molecular weight vWF multimers on the second (bottom-most) membrane (Figure 3B). Reduction by rFH of both soluble ULvWF multimers (from EC supernatant; Figure 3A) and soluble recombinant plasma-type large vWF multimers into smaller vWF multimers is demonstrated in the second (bottom-most) transfer membrane in Figure 3B.
We did not detect any ADAMTS-13–mediated proteolytic products of vWF multimers (including 140–176 kDa heterodimers) using unreduced SDS-1% agarose gels, both first and second (bottom) transfer membranes and anti-vWF antibody. However, more rapidly migrating (smaller) vWF antigenic forms were detected from gel lanes that initially contained a relatively large quantity of vWF antigen in normal plasma. (Figure 3A and 3B).
We cannot rule out the possibility that even smaller vWF forms or fragments move through the second (bottom-most) membrane and remain undetected in our transfer system. However, all of our results demonstrate that the FH reducing activity for LsvWF is thiol dependent and not the result of contamination by ADAMTS-13 or other protease.
In contrast to ADAMTS-13, FH reduces LsvWF, but has no effect on ULvWF multimeric strings anchored to ECs, in our in vitro human umbilical vein endothelial cell/microscopy system (Figure 5).
Partially purified FH was isolated in a form with LsvWF reducing activity, perhaps because it was prepared from cryosupernatant that contains a few μmol/L each of cysteine and glutathione.13,14 rFH was also released from transfected cells containing cysteine and glutathione. Equivalent plasma cysteine and glutathione concentrations do not reduce ULvWF (data not shown).
In contrast, a commercial, highly purified preparation of FH (CompTech, Tyler, TX) as supplied (in concentrations ≤500 µg/mL) does not have LsvWF reducing activity. This may be because of reversible inactivation of the FH LsWVF reducing activity during the purification process. Evidence in support of this possibility is provided by the demonstration that commercial FH regains LsvWF reducing activity after incubation at 4°C for 24 hours with either 10 mmol/L EDTA or 1 mol/L urea (Figure 6). These compounds may change the conformation of commercial, purified FH, by altering either the cation environment (EDTA) or the folding pattern (urea) of the highly purified commercial FH molecule, and restore its nascent LsvWF reductase activity. Figure 6 demonstrates that both EDTA-treated and urea-treated commercial, purified FH samples have LsvWF reducing activity associated with a predominant band that is more migrating slowly on unreduced 7% SDS-PAGE than a small quantity of commercial, purified FH.
FH family proteins are present in the plasma in several sizes ranging from 24 to 150 kDa and participate in the regulation of the alternative complement pathway.11,12,15–17 These include FH-like protein (≈40 kDa) and 5 FH-related proteins (≈25–90 kDa), FH monomer (≈150 kDa), and some FH doublets. FH binds to C3b, promotes C3b degradation, and serves as a cofactor for FI-mediated C3b cleavage.
The most abundant FH in normal circulation is the monomeric form. Its estimated size depends on isolation techniques, acrylamide gel/electrophoretic conditions of analysis and, possibly, partial kallikrein proteolysis.18 The full-length FH monomer comprises 20 short consensus repeat (Sushi) glycosylated subunits of 60 amino acids connected by peptide bonds. Each short consensus repeat domain contains 2 internal disulfide bonds.11,12,15–17
We detected free thiols in the FH monomer by maleimide–PEG–biotin, which may have been produced by reduction of ≥1 of the disulfide bonds within short consensus repeat domains. Our experiments using unreduced SDS-1% agarose indicate that FH is likely to decrease the size of LsvWF by reducing some of the disulfide bonds that connect chains of vWF monomers into vWF multimers. The precise region of the multidomain FH molecule responsible for the reduction of intermonomeric disulfide bonds in vWF multimers has not yet been identified. The interaction between FH and LsvWF is likely to be evanescent because we could not detect (using SDS-agarose or SDS-acrylamide electrophoresis) the formation of covalent mixed disulfide bonds between FH and vWF.
FH attaches to cell-anchored ULvWF strings and participates in the regulation of the alternative complement pathway.19 In contrast to its capacity to reduce LsvWF multimers, FH (in the concentrations we have tested) does not reduce EC-secreted/anchored ULvWF multimeric strings. This is the opposite substrate preference from ADAMTS-13 that cleaves EC-anchored ULvWF multimeric strings, but (in the absence of extremely high shear stress) does not cleave either soluble large vWF or soluble ULvWF forms. EC-anchored ULvWF multimeric strings differ considerably in conformation from soluble large vWF and soluble ULvWF forms. These conformational differences are likely to account for the distinct FH (and ADAMTS-13) substrate preferences under our experimental conditions.
The complementary substrate breakdown mechanisms of ADAMTS-13 (for EC-anchored ULvWF multimeric strings) and FH (for soluble large vWF and ULvWF forms) suggest the possibility that ADAMTS-13 and FH may act sequentially to generate the normal vWF multimeric patterns seen in unreduced SDS-agarose gels.
Neither partially purified FH nor rFH at the concentrations tested was capable of reducing EC-secreted/anchored ULvWF multimeric strings in vitro. We do not know, however, whether these results simulate in vivo events because the concentration of FH in normal plasma (≈500 µg/mL) is ≥7-fold greater than we have been able to produce/test on human umbilical vein endothelial cell–secreted/anchored ULvWF strings.
Defective reduction in the size of LsVW and excessive vWF-mediated platelet aggregation in the renal microvasculature20 may contribute to renal dysfunction in some patients with FH deficiency. The clinical effect of a mutation in 1 of the 2 FH genes (as occurs in the usual congenital heterozygous type of atypical hemolytic-uremic syndrome) results in ≈50% of the FH in normal plasma. More severe FH deficiency is sometimes caused by the presence of autoantibody. Insofar as we are aware, vWF multimeric patterns have not yet been studied systematically in either type of FH deficiency. If FH deficiency patients have a shift toward larger vWF multimers in plasma, then they might be more susceptible to shear-induced platelet aggregation mediated by soluble large and soluble ULvWF multimers and, possibly, to accentuated microvascular thrombosis.
We are grateful to the Proteomics Programmatic Core Laboratory at The Methodist Hospital Research Institute, Houston, TX for help with protein identifications.
Sources of Funding
This work was supported by the Mary R. Gibson Foundation, the Mabel and Everett Hinkson Memorial Fund, and a grant from the National Institutes of Health (National Institute of Allergy and Infectious Diseases; V. Afshar-Kharghan).
The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.113.302280/-/DC1.
- Nonstandard Abbreviations and Acronyms
- a disintegrin and metalloprotease with thrombospondin domains-type 13
- endothelial cell
- factor H
- large soluble von Willebrand factor
- recombinant factor H
- ultralarge von Willebrand factor
- Received April 10, 2013.
- Accepted August 16, 2013.
- © 2013 American Heart Association, Inc.
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We demonstrate in this report that factor H, a regulatory protein in the alternative complement pathway, can reduce large soluble von Willebrand factor multimers into smaller von Willebrand factor forms. Defective reduction in the size of large soluble von Willebrand factor multimers may contribute to excessive von Willebrand factor–mediated platelet aggregation in the renal microvasculature, and accentuate renal injury, in patients with factor H deficiency.