Research Article: Evolution of Primary Hemostasis in Early Vertebrates

Date Published: December 23, 2009

Publisher: Public Library of Science

Author(s): Seongcheol Kim, Maira Carrillo, Vrinda Kulkarni, Pudur Jagadeeswaran, Bruce Riley.

Abstract: Hemostasis is a defense mechanism which protects the organism in the event of injury to stop bleeding. Recently, we established that all the known major mammalian hemostatic factors are conserved in early vertebrates. However, since their highly vascularized gills experience high blood pressure and are exposed to the environment, even very small injuries could be fatal to fish. Since trypsins are forerunners for coagulation proteases and are expressed by many extrapancreatic cells such as endothelial cells and epithelial cells, we hypothesized that trypsin or trypsin-like proteases from gill epithelial cells may protect these animals from gill bleeding following injuries. In this paper we identified the release of three different trypsins from fish gills into water under stress or injury, which have tenfold greater serine protease activity compared to bovine trypsin. We found that these trypsins activate the thrombocytes and protect the fish from gill bleeding. We found 27 protease-activated receptors (PARs) by analyzing zebrafish genome and classified them into five groups, based on tethering peptides, and two families, PAR1 and PAR2, based on homologies. We also found a canonical member of PAR2 family, PAR2-21A which is activated more readily by trypsin, and PAR2-21A tethering peptide stops gill bleeding just as trypsin. This finding provides evidence that trypsin cleaves a PAR2 member on thrombocyte surface. In conclusion, we believe that the gills are evolutionarily selected to produce trypsin to activate PAR2 on thrombocyte surface and protect the gills from bleeding. We also speculate that trypsin may also protect the fish from bleeding from other body injuries due to quick contact with the thrombocytes. Thus, this finding provides evidence for the role of trypsins in primary hemostasis in early vertebrates.

Partial Text: Hemostasis is a defense mechanism that evolved to protect the organism in the event of injury to stop bleeding [1]. In mammals, when a vessel wall is ruptured, the tissue factor on the surface of the cells in the subendothelial matrix binds to the preexisting factor VIIa and initiates coagulation by cleaving factor X to Xa, which then cleaves prothrombin to generate minuscule amounts of thrombin. This thrombin cleaves factor XI to XIa. XIa then cleaves factor IX to IXa, which along with cofactor VIIIa, cleaves larger amounts of factor X to Xa, which then generates explosive amounts of thrombin from prothrombin. Interestingly, these reactions occur on the surface of platelets that are anchored to the subendothelial matrix via collagen and vWF, which themselves activate the platelets along with the newly generated thrombin. These events initiate the secretion of ADP, thromboxane and serotonin, which activate and amplify the aggregation of platelets.

All experiments described below were approved by the Institutional Animal Care and Use Committee.



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