Thrombomodulin mutations in atypical hemolytic-uremic syndrome

Abstract

Background: The hemolytic-uremic syndrome consists of the triad of microangiopathic hemolytic anemia, thrombocytopenia, and renal failure. The common form of the syndrome is triggered by infection with Shiga toxin-producing bacteria and has a favorable outcome. The less common form of the syndrome, called atypical hemolytic-uremic syndrome, accounts for about 10% of cases, and patients with this form of the syndrome have a poor prognosis. Approximately half of the patients with atypical hemolytic-uremic syndrome have mutations in genes that regulate the complement system. Genetic factors in the remaining cases are unknown. We studied the role of thrombomodulin, an endothelial glycoprotein with anticoagulant, antiinflammatory, and cytoprotective properties, in atypical hemolytic-uremic syndrome.

Methods: We sequenced the entire thrombomodulin gene (THBD) in 152 patients with atypical hemolytic-uremic syndrome and in 380 controls. Using purified proteins and cell-expression systems, we investigated whether thrombomodulin regulates the complement system, and we characterized the mechanisms. We evaluated the effects of thrombomodulin missense mutations associated with atypical hemolytic-uremic syndrome on complement activation by expressing thrombomodulin variants in cultured cells.

Results: Of 152 patients with atypical hemolytic-uremic syndrome, 7 unrelated patients had six different heterozygous missense THBD mutations. In vitro, thrombomodulin binds to C3b and factor H (CFH) and negatively regulates complement by accelerating factor I-mediated inactivation of C3b in the presence of cofactors, CFH or C4b binding protein. By promoting activation of the plasma procarboxypeptidase B, thrombomodulin also accelerates the inactivation of anaphylatoxins C3a and C5a. Cultured cells expressing thrombomodulin variants associated with atypical hemolytic-uremic syndrome had diminished capacity to inactivate C3b and to activate procarboxypeptidase B and were thus less protected from activated complement.

Conclusions: Mutations that impair the function of thrombomodulin occur in about 5% of patients with atypical hemolytic-uremic syndrome.

Figure 1. Alternative Pathway of Complement Activation and Regulation

In this schematic representation, the alternative pathway cascade on a complement-activating surface is shown on the right side, and the proposed mechanisms of complement regulation by thrombomodulin on host cells are shown on the left side. C3 spontaneously undergoes cleavage at a slow rate, amplified by bacterial and viral products. C3 releases the anaphylatoxin C3a and the fragment C3b, which is deposited on almost all cell surfaces that are in contact with plasma. C3b deposited on bacterial surfaces that lack complement regulators binds to CFB to form the C3 convertase of the alternative pathway, an enzyme complex (C3bBb) that cleaves additional C3 molecules. C3b also participates in the formation of the C5 convertase (C3b2Bb), which by cleaving C5, releases C5a, an anaphylatoxin, and C5b, which initiates assembly of the membrane attack complex (MAC), a pore-like structure that inserts into the cell membranes, causing cell activation or lysis. In host cells, several membrane-anchored and fluid-phase regulators control this cascade. CFH and C4bBP in the fluid phase bind to cell-surface glycosaminoglycans and to C3b and act as cofactors for CFI-mediated cleavage of C3b to iC3b. This reduces downstream activation of C3 and C5, thereby protecting the cell membrane. Thrombomodulin, an integral membrane protein on all endothelial cells, provides additional protection of the membrane by enhancing CFI-mediated inactivation of C3b in the presence of either CFH or C4bBP; by binding to thrombin, thereby preventing it from activating C5; and by promoting the generation of carboxypeptidase B (TAFIa), which inactivates C3a and C5a. See Glossary for explanation of complement components.

Figure 2. Pedigrees of Patients with Familial Atypical Hemolytic–Uremic Syndrome

Solid symbols (squares for male family members and circles for female family members) indicate affected persons, and slashes deceased persons. The patient number and age are shown below each symbol.

Figure 3. Effect of Mutations Associated with Atypical Hemolytic–Uremic Syndrome on the Ability of Thrombomodulin to Enhance Complement Inactivation

CHO-K1 cells were stably transfected for equal cell-surface expression of wild-type and mutated forms of thrombomodulin (Panel A). The control column represents cells stably transfected with empty vector. The amount of iC3b relative to (C3b+iC3b) deposited on the CHO cells after complement activation in serum was measured with the use of flow cytometry (see the Methods section of the Supplementary Appendix). Cells expressing wild-type thrombomodulin provided significant protection, as compared with control cells. Variants had a significantly lower percentage of iC3b on the cell surface than wild-type cells. The P value for wild type is for the comparison of wild-type thrombomodulin with control cells. Other P values are for the comparison of variants with wild-type thrombomodulin. The results shown are the mean values from three independent experiments. In Panels B and C, the direct interaction of CFH and C3b with thrombomodulin is shown by coprecipitations followed by immunoblotting, with the use of immobilized thrombin (IIa)–sepharose to pull down thrombomodulin in the presence of purified proteins (1 μg each). C3b interacts with thrombomodulin, and this interaction is increased in the presence of CFH (Panel B). CFH directly interacts with thrombomodulin (Panel C). HEK293 cells were stably transfected for equal expression of wild-type and thrombomodulin variants, and membrane preparations were immobilized in 96-well plates (Panel D). Binding of biotinylated C3b or CFH was quantified with an enzyme-linked immunosorbent assay, as described in the Methods section in the Supplementary Appendix. As compared with binding to wild-type thrombomodulin, there was a significant increase in specific binding of C3b or CFH to all thrombomodulin variants (P<0.001) except for D486Y (P = 0.73 and P = 0.80, for binding of C3b and CFH to D486Y, respectively). Nonspecific signals, determined with non–thrombomodulin-expressing cells, were subtracted from the results. Assays were performed in triplicate. See the Glossary for an explanation of complement components.

Figure 4. Inactivation of Complement Factor C3b by Complement Factor I, as Facilitated by Thrombomodulin

Panel A is a diagrammatic representation of cleavage and conversion of C3b to its inactivated form (iC3b) by CFI in the presence of cofactors (CFH, C4bBP). On the right is a Western blot of reduced purified C3b and iC3b, revealing component fragments. Reactions were performed with purified proteins (Panels B and C). After 90 minutes of reaction in solution with the purified proteins, reactions were separated by means of sodium dodecyl sulfate–polyacryl-amide-gel electrophoresis (SDS–PAGE) followed by Western blotting with anti-C3 antibodies. Increasing the concentrations of thrombomodulin yielded more inactivation of C3b (less α′ and more α′2), as quantified by densitometry of three blots (Panel B). With the use of the same approach, thrombomodulin also significantly enhanced the cofactor activity of C4bBP in CFI-mediated inactivation of C3b (Panel C). T bars indicate standard errors. See the Glossary for an explanation of complement components.

Figure 5. Effect of Thrombomodulin Mutations Associated with Atypical Hemolytic–Uremic Syndrome on Inactivation of Complement Factor C3b and on Activation of Plasma Procarboxypeptidase B (TAFI)

HEK293 cells were stably transfected for equal expression of wild-type and thrombomodulin variants. CFI-mediated inactivation of C3b in the presence of CFH (Panel A) or C4bBP (Panel B) was assessed by Western blotting, and densitometry was performed to measure the generation of inactive cleavage fragment (iC3b) α′2, relative to control cells transfected with empty vector. Mutant forms of thrombomodulin were significantly less effective than wild-type thrombomodulin in facilitating CFI-mediated inactivation of C3b in the presence of CFH. Only the P501L variant exhibited defects in C4bBP cofactor activity. Thrombin–thrombomodulin–dependent generation of TAFIa was determined by incubating TAFI and thrombin on HEK293 cells expressing thrombomodulin (Panel C). Significantly less TAFIa was generated with thrombomodulin variants. The results shown are the mean values for three independent experiments. T bars indicate standard errors. See the Glossary for an explanation of complement components.