We identified six missense mutations in the gene encoding thrombomodulin in seven unrelated patients with atypical hemolytic–uremic syndrome, accounting for 4.6% of the 152 cases. We also found that the resultant thrombomodulin variants did not protect cultured cells against complement activation. SNPs in the coding region of the THBD
gene are rare,38–40
and only eight missense changes are reported in the dbSNP database (www.ncbi.nlm.nih.gov/projects/SNP
). One insertion frameshift mutation has been described in a kindred with myocardial infarction,41
whereas no homozygous mutations have been reported. All the THBD
gene mutations that we identified were heterozygous.
In one carrier of a mutation, the disease was first manifested when the person was 15 years of age, and in four carriers, the hemolytic–uremic syndrome has not developed. These data suggest that mutation of a single THBD
allele is not sufficient by itself to cause atypical hemolytic–uremic syndrome. Rather, additional factors — environmental, genetic, or epigenetic — are probably required. Indeed, in two patients, episodes of the hemolytic–uremic syndrome were triggered by viruslike illnesses. This finding is relevant to thrombomodulin, since the expression of thrombomodulin is down-regulated during inflammation and infection.42–45
Overall, the findings are similar to those observed with mutations in CFH, CFI, C3, C4bBP, and CFB in the hemolytic–uremic syndrome, which are often missense mutations (in the case of CFH, CFI and C3)17,27
and are usually heterozygous4,9–13,15–17
and associated with disease after an infection.27
Thrombomodulin is a 557-amino-acid endothelial glycoprotein that is anchored to the cell by a short cytoplasmic tail and a single transmembrane domain.46
A series of six epidermal growth-factor–like repeats are required for thrombin-mediated generation of activated protein C, which has anticoagulant and cytoprotective properties, and the generation of activated TAFI, which has C3a-degrading and C5a-degrading properties.23,47,48
Farthest from the transmembrane domain is the lectinlike domain, which confers resistance to proinflammatory stimuli, including endotoxin and ischemia–reperfusion.25,49
It is notable that three of the missense mutations that we found to be associated with atypical hemolytic–uremic syndrome are in the lectinlike domain of thrombomodulin.
The A43T mutation is a rare variant that has been associated with venous thrombosis,50
atherosclerosis, and myocardial infarction.51
A computer-generated model of the lectinlike domain of thrombomodulin predicts that A43 is positioned on the surface of the molecule, where it could potentially bind to proteins in the circulation.52
This observation is in line with our findings that thrombomodulin variants associated with the atypical hemolytic–uremic syndrome that involve the lectinlike domain alter CFH and C3b binding and, in turn, the regulation of complement activation.
The other three mutations in sporadic cases of atypical hemolytic–uremic syndrome — D486Y, P495S, and P501L — reside in the serine–threonine-rich region of thrombomodulin. D486Y and P501L have been reported rarely in patients with venous thrombosis.53
These mutations are near the consensus sequence for attachment of chondroitin sulfate at serine 49254
; in vitro, P495S and P501L moderately reduce expression of thrombomodulin on the cell surface, and P495S decreases the affinity of thrombomodulin for thrombin.55
All three of these variants exhibited defects in suppressing activation of the alternative complement pathway through CFI-mediated C3b inactivation in vitro, implicating them in the pathogenesis of atypical hemolytic–uremic syndrome. The additional impairment in TAFIa generation probably aggravates the disease.36
Thrombomodulin has a binding site for C3b and CFH, thereby accelerating CFI-mediated inactivation of C3b. Beyond these activities, which negatively regulate the alternative pathway, thrombomodulin plays a wider role in suppressing complement-mediated cell injury. Thrombomodulin interferes with thrombin-mediated activation of C556
and is necessary for thrombin-mediated generation of TAFIa. Thus, in addition to its role in coagulation, thrombomodulin interacts with several complement pathways ().
Despite advances in delineating the pathogenesis of atypical hemolytic–uremic syndrome, effective therapies are lacking, and in most patients, end-stage renal failure requiring dialysis develops. Since thrombomodulin simultaneously suppresses the complement and coagulation systems, its administration may have therapeutic value for some patients with the atypical hemolytic–uremic syndrome. The efficacy and safety of recombinant thrombomodulin for disseminated intravascular coagulation have been shown in a phase 3 clinical trial.57
In conclusion, we have shown that thrombomodulin is a negative regulator of the complement system and that mutant variants of thrombomodulin may contribute to the development of atypical hemolytic–uremic syndrome.