Insight from studies with recombinant fibrinogens.
Using a two-step cloning strategy, we have synthesized more than 20 variant human fibrinogens for biochemical studies. In preliminary experiments we showed that normal fibrinogen produced in CHO cells serves as an accurate model for plasma fibrinogen. We focus here on those variants whose characterization has provided insight into the mechanism of thrombin-catalyzed polymerization. Analysis of N-terminal variants showed that thrombin specificity dictates the ordered release of fibrinopeptides. Nevertheless, analysis of C-terminal variants indicated that fibrinopeptide B (FpB) release is dependent on polymerization. Changes in the a polymerization site and the high-affinity calcium-binding site were associated with a complete loss of polymerization. These experiments showed that alterations in the calcium-binding site influenced function of the a site; in contrast, alterations in the a site did not alter calcium binding. Analysis of variants in the N-terminus of the B beta chain provided the first direct evidence that this region impacts predominantly on lateral aggregation, as has long been presumed. These experiments also suggested that lateral aggregation facilitated by this region proceeds without the release of FpB. From these studies we learned that individual sites within fibrinogen do not function in isolation. We conclude that thrombin-catalyzed polymerization is mediated by a continuum of concerted interactions.
Lord
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Departments of Pathology and Laboratory Medicine, and Chemistry, Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA. stl@med.unc.edu
Gorkun
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D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D006224:Cricetinae; D005340:Fibrinogen / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism
D011994:Recombinant Proteins; D005340:Fibrinogen; D013917:Thrombin; D002118:Calcium