Calix[4]arene methylenebisphosphonic acids as inhibitors of fibrin polymerization.

Calix[4]arenes bearing two or four methylenebisphosphonic acid groups at the macrocyclic upper rim have been studied with respect to their effects on fibrin polymerization. The most potent inhibitor proved to be calix[4]arene tetrakis-methylene-bis-phosphonic acid (C-192), in which case the maximum rate of fibrin polymerization in the fibrinogen + thrombin reaction decreased by 50% at concentrations of 0.52 × 10(-6) M (IC(50)). At this concentration, the molar ratio of the compound to fibrinogen was 1.7 : 1. For the case of desAABB fibrin polymerization, the IC(50) was 1.26 × 10(-6) M at a molar ratio of C-192 to fibrin monomer of 4 : 1. Dipropoxycalix[4]arene bis-methylene-bis-phosphonic acid (C-98) inhibited fibrin desAABB polymerization with an IC(50) = 1.31 × 10(-4) M. We hypothesized that C-192 blocks fibrin formation by combining with polymerization site 'A' (Aα17-19), which ordinarily initiates protofibril formation in a 'knob-hole' manner. This suggestion was confirmed by an HPLC assay, which showed a host-guest inclusion complex of C-192 with the synthetic peptide Gly-Pro-Arg-Pro, an analogue of site 'A'. Further confirmation that the inhibitor was acting at the initial step of the reaction was obtained by electron microscopy, with no evidence of protofibril formation being evident. Calixarene C-192 also doubled both the prothrombin time and the activated partial thromboplastin time in normal human blood plasma at concentrations of 7.13 × 10(-5) M and 1.10 × 10(-5) M, respectively. These experiments demonstrate that C-192 is a specific inhibitor of fibrin polymerization and blood coagulation and can be used for the design of a new class of antithrombotic agents.