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399 VISUALIZING THE MOLECULAR BASIS OF BLOOD COAGULATION.
  1. M. H. Johnson1
  1. 1University of Washington School of Medicine, Seattle, WA

Abstract

Purpose of Study Cardiovascular disease remains the #1 cause of mortality in the United States, affecting 71 million Americans in 2006 and costing $403.1 billion in direct and indirect costs. An initiator of hemostasis/thrombosis is tissue factor (TF), a constitutively expressed protein on the plasma membrane of cells that do not usually encounter blood. In the event of vascular injury, blood is exposed to TF-expressing tissues, which initiate the coagulation cascade and ultimately lead to the deposition of fibrin. Interestingly, TF antigens have been detected in whole blood in the absence of endothelial injury. These findings challenge the conventional mechanism of TF as the coagulation initiator. However, this is resolved by the hypothesis that TF exists in two enzymatic states based on the biochemical environment.

Methods Investigating the purported dual state of TF, as well as blood coagulation kinetics, has relied on the development of novel technologies. Intravital microscopy, which combines confocal and bright-field microscopy, allows real-time visualization of thrombogenesis in vivo. Following laser-induced endothelial injury, circulating fluorescent antibodies allow for quantifiable detection and interaction of multiple coagulant molecules. Another novel technique aimed at identifying the activity state of TF is also being actively developed. The proposed redox-based, disulfide switch on TF's surface can be detected by covalently attaching fluorescent maleimide derivatives to superficial sulfhydryl groups. This technique has been demonstrated in vitro and, once mature, will provide a fluorescence or mass spectrometry-based method for determining the enzymatic state of TF in vivo.

Results Intravital microscopy has allowed real-time, in vivo imaging of thrombogenesis. Many of the major proteins traditionally involved in blood coagulation have been fluorescently labeled and imaged during blood clot formation. As a result, a library of quantitative measurements is being created, cataloging the biomolecular roles of coagulant proteins. Methods to determine the cryptic versus coagulant conformation of TF in vivo are ongoing but show promise in vitro with an albumin standard protein.

Conclusion Understanding the molecular basis of hemostasis and thrombosis is a goal that is both achievable and medically relevant. New technologies to visualize and quantify thrombogenesis are providing valuable methods for biomedical discovery.

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