History of Discovery Taking the Thrombin “Fork”

The proverb that probably best exemplifies my career in research is attributable to Yogi Berra (http://www.yogiberra.com/), ie, “when you come to a fork in the road ... take it.” My career is a consequence of chance interactions with great mentors and talented students and the opportunities provided by a succession of ground-breaking improvements in technology. (Arterioscler Thromb Vasc Biol. 2010;30:1293-1299.) As an undergraduate physics major at Manhattan College, I encountered C. W. Batt who hired me as a research technician for his studies involving the enzymology of lipases. I became fascinated with enzyme catalysis and started my slide from “pure” physical science to biology and medicine. After my Biochemistry PhD studies with Carl Vestling at the University of Iowa, I pursued biophysical chemistry with Charles Tanford at Duke as an NIH postdoctoral fellow. I aimed at having a career dealing with the mechanisms of protein folding. In the meantime, Bill Batt’s research program had become involved with the isolation of thrombin from the pharmaceutical “topical thrombin,” and he asked me to characterize the product he had isolated. Curiously, the preparations had constant specific activity toward a synthetic substrate (tosyl arginine methyl ester) but were highly variable in clotting activity. Utilizing newly developed techniques for molecular weight,1 and with the generous support of Charles Tanford, I characterized these preparations and concluded that there were multiple forms of thrombin related by proteolytic cleavage,2 subsequently designated , , and thrombin.3 Prothrombin Activation and Structure I accepted an assistant professorship in Biochemistry at the University of Minnesota in the College of Biological Sciences, where I pursued protein folding as my primary objective. As a side “hobby,” and with the enthusiastic encouragement of Roger Lundblad, I elected to also pursue prothrombin activation using the biophysical techniques that had enabled our studies of thrombin and the new technique of SDS gel electrophoresis.4 At the time, the most prominent laboratories utilizing quantitative protein chemistry to study coagulation reactions were those of Earl Davie,5,6 Russell Doolittle,7,8 Harold Scheraga,9 Donald Hanahan,10 and Stephan Magnusson.11 I sent a research grant to the NIH requesting support for my studies and, with my sense of youthful hubris, I anticipated I would start those studies when the grant money came in. In the meantime, I was befriended by Walter Bowie and Charles Owen at the Mayo Clinic who helped me in interpreting the somewhat confusing coagulation literature. In July 1970, I received a letter from the NIH signed by Fann Harding that indicated that my grant application was not only not to be funded but also it was disapproved based on my inadequate scientific credentials and my naive biophysical approaches to the problem. In those days, before the availability of “pink sheets,” one contacted the NIH study section executive secretary who could provide excerpts from the reviewers’ comments. Dr Harding provided a description from her notes detailing the reviewers citing my lack of appropriate scientific training and the ridiculousness of my approach. My response was determination to show my new colleagues in the Hematology Study Section that they were incorrect. My colleagues in the laboratory at the time were David Fass, who had come to Minnesota as a postdoctoral fellow to work on Neurospora genetics but was abandoned by his preceptor who was more interested in protesting the Vietnam War, and Charles Heldebrandt, a first-year graduate student from Berkeley who overslept his meeting with his primary choice of preceptor and ended up with me. Because the techniques I anticipated using were material-intensive and my budget was limited, at the suggestion of my wife Jeanette, I acquired an antique ($25) cream separator to prepare plasma. We separated bovine citrate blood into plasma and cells using the modified, antique, hand-crank cream separator and developed isolation procedures for prothrombin and thrombin. We then initiated the first SDS4 mass-based analysis for conversion of a zymogen to a protease in studies of prothrombin activation.12 These initial studies were presented at the 1970 Federation of American Societies for Experimental Biology meeting and were sufficiently well-received by the Hematology community that the previously disapproved grant application entitled the “Activation of Prothrombin” was approved and funded, and it has been continuously supported since 1972 as an R01, a Specialized Center of Research, and a Program Project Grant. Our studies and contemporaneous reports from the Jackson13 laboratory led to the evolution of sequences of bond cleavage during prothrombin activation described by pathway 1 to 3 (Figure 1); meizothrombin was not found, and thus pathway 2 to 4 was excluded (but see later). From University of Vermont, College of Medicine, Colchester. Correspondence to Kenneth G. Mann, University of Vermont, College of Medicine, 208 South Park Drive, Room 235, Colchester, VT 05446. E-mail [email protected] © 2010 American Heart Association, Inc. Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org DOI: 10.1161/ATVBAHA.108.179598 1293 by gest on M ay 8, 2017 http://atvajournals.org/ D ow nladed from