CHARACTERIZATION OF SOLUBLE THROMBOMODULIN FRAGMENTS IN HUMAN URINE

The soluble thrombomodulin (TM) subspecies in human urine detected by polyclonal anti-human TM IgG were isolated and characterized. 105, 85, 80, 56, 33, 31 and 28 kDa subspecies under reducing conditions was co mparable to 78, 66, 56, 200, 52, 30 and 25 kDa under non-reducing cond itions, respectively, in the two-dimensional electrophoresis. Each sub species under non-reducing conditions, except the 200 and 52 kDa molec ules, was constituted of single subspecies, whereas the 200 and 52 kDa molecules were constituted of the tetramer of the 56 kDa subspecies o f reducing conditions and a dimer of the 33 kDa subspecies, respective ly. NH2-terminal amino acid sequences of the 105, 85 and 80 kDa subspe cies maintained Ala(1)-Pro(2)-Ala(3)- of intact human TM, however, 56, 33, 31 and 28 kDa subspecies started from Glu(137)-Gln(138)-, Gln(214 )-Gly(215)-, Ser(228)-Val(229)- and Ala(240)-Ile(241)-, respectively. All subspecies obtained under non-reducing conditions exhibited cofact or activity for thrombin-dependent protein C activation ranging from 5 8 to 162 pmol APC/min/nmol TM at 0.4 mM Ca2+ indicating that all of th e subspecies maintained the fourth to sixth repeat of epidermal growth factor-like structure of intact TM. 85, 80, 56, 33, 31 and 28 kDa sub species were suggested to lack both chondroitin sulfate glycosaminogly can (CSGAG), transmembrane and cytoplasmic domains of intact TM, while 105 kDa subspecies lack only CSGAG from the results of kinetic proper ties and the interaction with phospholipid vesicles composed from phos phatidylcholine and phosphatidylethanolamine.