Inhibition of calcium oxalate crystal growth and aggregation by prothrombin and its fragments in vitro - Relationship between protein structure and inhibitory activity

During blood coagulation, prothrombin (PT) is ultimately degraded to three fragments, thrombin, fragment 1 (F1) and fragment 2 (F2), which, collective ly, contain all of the structural features of PT. One of these fragments, F 1, is excreted in human urine and is the principal protein occluded into ca lcium oxalate (CaOx) crystals precipitated from it. This urinary form of F1 , which we have named urinary prothrombin fragment 1 is present in calcium stones and is a potent inhibitor of CaOx crystallization in urine in vitro. The aim of this study was to determine whether PT itself and its other act ivation products, namely, thrombin, F1 and F2 also inhibit CaOx crystalliza tion, by comparing their effects in a seeded, inorganic crystallization sys tem. A secondary objective was to assess the relationship between the struc tures of the proteins and their inhibitory activities. PT was isolated from a human blood concentrate rich in vitamin K-dependent proteins. Following initial cleavage by thrombin, the resulting fragments. F1 and F2, were puri fied by a combination of reversed phase HPLC and low pressure column chroma tography. The purity of the proteins was confirmed by SDS/PAGE and their in dividual effects on CaOx crystallization were determined at the same concen tration (16.13 nM) in a seeded, metastable solution of CaOx using a Coulter Counter. [C-14]Oxalate was used to assess deposition of CaOx and crystals were visualized using scanning electron microscopy. The Coulter Counter dat a revealed that the proteins reduced the size of precipitated crystals in t he order F1 > PT > F2 > thrombin. These findings were confirmed by scanning electron microscopy which showed that the reduction in particle size resul ted from a decrease in the degree of crystal aggregation [C-14]Oxalate anal ysis demonstrated that all proteins inhibited mineral deposition, in the or der F1 (44%) > PT (27.4%) > thrombin (10.2%) > F2 (6.59). It was concluded that the gamma-carboxyglutamic acid domain of PT and F1, which is absent fr om thrombin and F2, is the region of the molecules which determines their p otent inhibitory effects. The superior potency of F1, in comparison with PT , probably results from the molecule's greater charge to mass ratio.