Intrinsic-like blood coagulation activity in fish: Involvement of hepatocyte growth factor activator and interspecific diversity

Most fish lack identifiable orthologs of factor XII and other components of the mammalian contact system; however, their plasma exhibits coagulation responses to negatively charged surfaces. Therefore, hepatocyte growth factor activator (HGFA), an evolutionary relative of factor XII, has been proposed as an initiator of factor XII-independent, contact activation-like coagulation in fish. To examine the distribution of this activity, we compared plasma from multiple fish species using an activated partial thromboplastin time (aPTT)-like assay. Thrombin substrate-cleaving activity was detected under aPTT-like conditions in multiple species lacking identifiable factor XII orthologs, demonstrating that factor XII-independent intrinsic pathway-like activity is broadly distributed among fish. To assess the involvement of factor XII- or HGFA-related proteases, we performed inhibitor assays. The observed inhibitor sensitivities did not consistently support HGFA as the principal initiator of the activity. We next compared the occurrence and intensity of the activity with the genomic presence and domain organization of HGFA orthologs. Neither the presence nor the magnitude of the activity closely corresponded to HGFA occurrence or structure; notably, activity was detected in bichir, in which no clear HGFA ortholog has been identified. Finally, comparative domain analysis showed that fish HGFA generally lacks several structural features associated with the surface-dependent autoactivation of mammalian factor XII. These findings indicate that factor XII-independent, contact activation-like coagulation is broadly present in fish but cannot be explained solely by HGFA. Fish may therefore possess diverse or unidentified proteolytic mechanisms that initiate coagulation under aPTT-like conditions, providing insights into the evolutionary emergence of the vertebrate contact system.