Recombinant human thrombomodulin(csa+): a tool for analyzing Plasmodium falciparum adhesion to chondroitin-4-sulfate

The proteoglycan thrombomodulin has been shown to be involved, via its chon droitin-sulfate moiety, in the cytoadhesion of chondroitin-4-sulfate-bindin g-Plasmodium falciparum-infected erythrocytes to endothelial cells and sync ytiotrophoblasts. We cloned and expressed in CHO and COS-7 cells a gene enc oding soluble human recombinant thrombomodulin, with a chondroitin-4-sulfat e moiety. This system is complementary to the in vitro cell models currentl y used to study the chondroitin-4-sulfate-binding phenotype. It also provid es a means of overcoming the lack of specificity observed in interactions o f infected erythrocytes with modified chondroitin-4-sulfate. This thrombomo dulin displayed normal activity in coagulation, indicating that it was in a functional conformation. The recombinant protein, whether produced in CHO or COS-7 cells, inhibited cytoadhesion to Saimiri brain microvascular endot helial cells 1D infected with Palo-Alto(FUP)1 parasites selected fur chondr oitin-4-sulfate receptor preference. Thus, the recombinant protein was prod uced with a chondroitin-sulfate moiety, identified as a chondroitin-4-sulfa te, in both cell types. In both cases, the recombinant protein bound to the chondroitin-4-sulfate phenotype, but not to CD36- and ICAM-1-binding paras ites. The chondroitin-4-sulfate was 36 kDa in size for CHO and 17.5 kDa for COS-7 cells. There was, however, no difference in the capacities of the re combinant proteins produced by the two cell types to inhibit the cytoadhesi on of infected erythrocytes. Thrombomodulin immobilized on plastic or coupl ed to Dynabeads was used to purify specifically the infected erythrocytes t hat bind to chondroitin-4-sulfate. These infected erythrocytes were culture d to establish parasite lines of this phenotype. We then showed that the th rombomodulin, labeled with FITC, could be used to detect this phenotype in blood samples. Finally, the direct binding of infected erythrocytes to immo bilized thrombomodulin was used to screen for anti-chondroitin-4-sulfate-bi nding antibodies. (C) 2000 Editions scientifiques et medicales Elsevier SAS .