PROTHROMBIN CLEAVAGE BY HUMAN VASCULAR SMOOTH-MUSCLE CELLS - A POTENTIAL ALTERNATIVE PATHWAY TO THE COAGULATION CASCADE

Thrombin is a potent mitogen for human vascular smooth muscle cells (H VSMC) and its enzymatic activity is required for this function. The pr esent study demonstrates that prothrombin is also mitogenic for HVSMC due to the generation of enzymatically active thrombin which occurs up on incubation of prothrombin with the cells. Analysis by SDS-PAGE, imm unoblotting, and amino acid sequencing revealed that prothrombin incub ated with HVSMC undergoes limited proteolysis. Prethrombin 1 was forme d through cleavage at R(155)-S-156. Cleavage at R(271)-T-272 generated fragment 1.2 and prethrombin 2 whilst cleavage at R(284)-T-285 yielde d truncated prothrombin 2 (prethrombin 2'). However, cleavage at R(320 -)I(321) which, during prothrombin activation produces two-chain alpha -thrombin, was not detectable. Studies on HVSMC-conditioned medium rev ealed that a similar pattern of prothrombin cleavage occurred by a cel l-secreted factor(s). Amidolytic activity analysis indicated that 1-3% catalytically active thrombin-like activity was generated upon incuba tion of prothrombin with HVSMC-conditioned medium. By treating conditi oned medium with various classes of proteinase inhibitors or hirudin, it was determined that prothrombin is cleaved by a cell-derived serine proteinase-like factor(s) at R(271)-S-172 and by alpha-thrombin at R( 155)-S-156 and R(284)-T-285. Antibodies neutralising the activity of e ither urokinase, tissue plasminogen activator, or factor Xa failed to alter the prothrombin cleaving activity of conditioned medium. This ac tivity which may catalyse an alternative pathway for the generation of thrombin, was eluted from a gel filtration column as a single peak wi th apparent molecular mass of 30-40 kDa. (C) 1995 Wiley-Liss, Inc.