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• W2010004644 abstract "This story will discuss how Whyte Owen and I discovered thrombomodulin. As this story unfolds, it is clear that many people contributed significant insights that led to the identification of thrombomodulin. Perhaps the most interesting aspect is that Whyte and I had worked together on prothrombin activation. We then separated to work on different parts of the coagulation process but were able to combine our separate research experiences into the experiments that revealed the existence of thrombomodulin. In the mid 1970s, I was working as a postdoctoral fellow in John Suttie's laboratory at the University of Wisconsin. John was interested primarily in how vitamin K functioned. We had the great fortune to have Johan Stenflo join the laboratory. Johan had just described the presence of gamma carboxyglutamic acid, so it was natural to work together on how the vitamin might function. Both Johan and I also had a real interest in the control of blood coagulation. Johan had found a ‘new’ vitamin K-dependent protein, named protein C because it was the third major peak from a barium eluate of bovine plasma to elute from a DEAE ion exchange column. As an aside, had this been human plasma, the protein C pathway would now be termed the protein B pathway due to the differences in chromatographic behavior. There were already four vitamin K-dependent proteins identified, but this seemed to be a new one. We set out to test our idea that this was not just an improved yield of factor (F) VII. The N-terminal sequence was slightly different, but we reasoned that this could be sequencing error. However, we could absorb protein C on antibodies without removing the FVII activity. We were unsure of the function of the protein, but because of the presence of Gla residues, we tested whether protein C would bind to phospholipids and whether it could be converted to a protease, both of which proved to be true. Trypsin activated protein C fairly effectively, but at the concentrations we employed, thrombin was ineffective [1]. Walter Kisiel subsequently showed that more thrombin for longer times would activate protein C. The thrombin concentrations and time required for protein C to be activated made it unlikely that thrombin alone was the physiological activator. Furthermore, we found that removing protein C from plasma had no effect on the clotting time and Walter Kisiel found that almost all of the protein C could be recovered in serum [2, 3]. At this point we only knew that it was a protease and would probably need phospholipids to function. As it turns out, the ‘new’ protein C was shown by the Seegers laboratory to be equivalent to a coagulation inhibitor that they had described in the early 1960s and named autoprothrombin IIa [4]. At that time, autoprothrombin IIa was thought to be a competitive inhibitor of prothrombin activation. The time then came for Johan to return to Malmo and join the department of Clinical Chemistry and for me to move to Oklahoma City to join the Pathology department where Fletcher Taylor was setting up a new ‘thrombosis’ division. When I moved to Oklahoma, my major project was to isolate FV, figure out how it was activated and determine why it was so sensitive to chelating agents. Fortunately, I was able to isolate FV and show that it was converted from a high molecular weight, apparently single chain protein into two required subunits held together in a calcium ion dependent fashion [5]. Of course, as luck would have it, Mike Nesheim and Ken Mann also isolated FV at almost the same time. A second project was to determine how activated protein C functioned as an anti-coagulant. To put it mildly, it was a stroke of fortune that these two projects dovetailed so well. We were able to show that activated protein C inactivated FVa primarily by cleaving the heavy chain and this process was partially blocked by FXa [6], probably explaining the apparent competitive inhibition seen earlier by the Seegers group. Walt Kisiel showed at a similar time that activated protein C could inactivate a partially purified FV preparation [2]. At about this time, I put in a grant for support to study the protein C system. The grant was funded but the budget was reduced by more than 3-fold. The major critique was that there is no known physiological activator and so you might be studying the ‘appendix’ of the coagulation system. Similar comments were flying in from my collaborator Phil Comp who advised me, probably wisely, to focus on the FV project, because we all know it is important and who knows about protein C. Whyte Owen visited our research program at about this time. Whyte had been a postdoctoral fellow in Craig Jackson's laboratory while I was a graduate student there. During that time we worked together on prothrombin activation. When Whyte went to the University of Iowa, he became interested in the role of the endothelial interactions with thrombin. In a seminar Whyte presented in our program, he told us about how he and his colleagues were developing the idea of the endothelium as a regulatory organ. In the early 1970s, John (‘Jack’) Hoak became possessed of the idea that endothelium should bear thrombin receptors. Jack had been one of the early players in endothelium culture, primarily via his interest in thrombosis. With no evidence, just an intuition, he set medical student Brian Awbrey on the problem, and drew Whyte in as a skeptical, if not unwilling, collaborator. The resulting paper [7] had some problems in design and interpretation, but it spawned follow-up work by Haematology fellow Pete Lollar (whom Jack had directed to Whyte's laboratory) that clarified the physical nature of thrombin binding to endothelium [8], including resolving a reversible site from a covalent component identified by others as protease nexin. Pete's work also led the Iowa group to consider the surface area of endothelium and ultimately to study the association of thrombin with endothelium in vivo[9] and in isolated Langendorff heart preparations [10]. At this point, they knew that thrombin bound reversibly to sites on endothelium, that these sites did not distinguish active from inactive (diisopropyl phosphorofluoridate [DFP]-treated) thrombin, and that the active thrombin bound there reacted rapidly with circulating antithrombin. Whyte's hypothesis was that the binding site comprised a sulfated glycosaminoglycan and functioned with antithrombin to detoxify traces of circulating thrombin. To get at the issue, Pete began using perfused rodent hearts, and Christer Busch, on sabbatical from the University of Uppsala, set up columns of bovine endothelium cultured on beads so that he could flow thrombin and related reagents over a large endothelial surface [11]. They interpreted their results to indicate that thrombin bound to endothelium drove the thrombin antithrombin reaction catalytically, though it may have been the consequence of mass action. At that time, my lab was not in a position to exploit this important concept. We had considered the possibility that endothelial cells might carry the critical protein C activation capability, but no one on campus at that time had cultured these cells nor were there any tissue culture facilities in our area. Realistically, the chances of getting funding for these facilities without preliminary data seemed remote at best. Fritz Markwardt, working in East Germany, then published a paper showing that he could detect plasminogen activator activity from perfused pigs' ears. I figured that what we lacked in tissue culture facilities could be made up easily by simply collecting a lot of pigs' ears from one of the many local packing houses that existed at that time. Initially Phil Comp and I used these perfused ears to look for activated protein C-mediated release of plasminogen activators, but without any consistent success. These pigs' ears were kept in a walk-in warm room. The perfusion lines were running in and out of about eight ears in a typical experiment. It really did look like something from a Frankenstein movie. It is noteworthy that the warm room had a glass door and was located across from the offices so as secretaries and visitors came in, the scene elicited quite a response – no faints, but a lot of screams. Using this perfused pigs' ear approach, I decided to test the hypothesis that thrombin might become an activator of protein C when bound to the vessels in the ear. We ran the perfusion and I asked a new technician to do the assays to see if it worked. The technician grumbled a fair bit because he did not like clotting assays and came back happily saying the experiment failed. I looked at the data. I was not convinced it had failed. On average, the clotting times of the perfusate were increased about 2 s compared to buffer control but the assay precision was about ± 5 s. After some discussion as to whether the experiment worked or not, I decided to simply redo the assays myself. When each point was run in quadruplicate, it was clear that there was a real prolongation of the clotting time, but it was only about 1–2 s. Given that we had no idea how much thrombin or protein C to add and no idea as to an appropriate flow rate, I thought optimization was needed badly. I asked several technicians and a student whether they would be willing to pursue this, but no one was interested or willing. The most common response reflected the following sentiment: ‘if you want this done, do it yourself’. There was a major reason that was not possible. Specifically, I had a 3-year grant to work on FV, much of my salary came from the grant, and renewal was approaching. I was really the only person in my laboratory at that time that could do the key experiments on FV so this observation was laid to rest for the time being. At about this time, I applied for and received an Established Investigator Award from the American Heart Association. As this covered my salary, it allowed me to broaden my scope of interests. The old problem of the FVa subunit dissociation/reassociation was still of interest. We had not been able to follow this process biophysically and I thought that CD/ORD methods might be helpful. At that time, we did not have an instrument available on campus but I knew that Whyte did have access to one at Iowa. I called him and we thought this would be worth doing. After Whyte confirmed the instrument was up and running, I got on a plane to Iowa, FVa subunits in my pocket and looking forward to a fun collaborative interaction. Fortunately, between take off and landing the CD/ORD instrument died. This was very fortunate because also in my pocket was a tube of protein C. I had taken this with me in case we finished the spectroscopy experiments and had time to exploit the rabbit heart method for testing the hypothesis that thrombin in the microcirculation was indeed the protein C activator. As I recall, this was a Friday afternoon, but we figured ‘why not try it.’ The experiments worked beyond our wildest expectations (1, 2). When thrombin and protein C were perfused together and the perfusate was added to a plasma clotting test, the clotting time was approximately doubled. No change in clotting time was observed when either thrombin or protein C was perfused alone. We repeated that experiment, got surprisingly similar results, and left to plan the rest of the experiments. The experiments for the Proceedings of the National Academy of Science USA paper we knew we would write were mapped out to generate a tentative figure by figure outline. We were now running low on protein C, so I called my wife Naomi to ask her to send up what ever we had left as soon as possible. Even though we did not work together then, I am told she went bouncing up and down the hall on the way to the laboratory, secure in the knowledge that my future employment was secure if the FV research fizzled. Chuck Esmon and Whyte Owen pointing to the Langendorff Heart at the University of Iowa. Chuck Esmon and Pete Lollar at the University of Iowa. The old Zeiss spectrophotometer to Pete Lollar's right is still in frequent use in Whyte's lab at the Mayo Clinic. Although we were confident of our results, the next day we figured we would repeat the perfusion study one more time. It was about 10 in the morning before we had the heart mounted and ready to go. To my astonishment, this time absolutely nothing happened. No anticoagulant response could be detected at all. This sent me from giddy excitement to near depression. Whyte, on the other hand, took it in stride and suggested we go to lunch. I remember a distinct lack of appetite which nausea can certainly bring on. Whyte had a nice lunch. When we returned to the laboratory, Whyte went back to check the freezer and get some more thrombin. Upon his return, he proclaimed that he figured it out – we had inadvertently perfused platelet F4 (which he kept next to the thrombin) with the protein C. When we actually repeated the experiments, all was well again. It is ironic that PF4 has now been shown to augment substantially the activation of protein C by the thrombin–thrombomodulin complex [12]. There were at least two different models compatible with our results. In the first, thrombin bound to a cofactor to form a functional protein C activation complex. In the second, thrombin activated a zymogen on the vessel surface that would then become a direct protein C activator. If the first model were true, we reasoned that active site-blocked thrombin would be a competitive inhibitor of thrombin catalysis, whereas if the second model were true, there should be little if any effect. The first model turned out to be correct. Some rough calculations indicated that the rate enhancement we were seeing in the microcirculation was about 20,000-fold. Therefore, even though the surface-to-volume ratios are horribly unfavorable over cultured cells, we felt it was worthwhile seeing if cultured endothelium would work also. Fortunately for us, Jack Hoak was still at Iowa and had a very productive tissue culture facility. Jack was extremely generous and Whyte and I took all of his available endothelial cells to study the phenomenon further (Fig. 3). Probably because thrombin is such a very poor protein C activator on its own, especially in physiological buffers (the effect of Ca2+), we could clearly see endothelial cell catalysis of thrombin-dependent protein C activation, even though less than 1% of the thrombin was bound to the cells. Whyte Owen negotiating for Jack Hoak's total supply of endothelial cells. The suggestion derived from the kinetics that the cofactor should be able to bind to active site-blocked thrombin suggested that immobilized active site blocked thrombin was a strategy to isolate the cofactor. Whyte came down to Oklahoma and by this time, Naomi had joined my laboratory so the three of us set out to isolate the cofactor. Attempts to elute the cofactor activity with high salt and chelating agents yielded nothing, but detergent extraction resulted in activity in the soluble extract. Passing the extract over the thrombin columns resulted in isolation of a protein with cofactor activity that we subsequently called thrombomodulin (TM) [13]. Naomi, Whyte and I decided to use rabbit lung homogenate as the source of thrombomodulin because it was a very highly vascularized organ, was simple to perfuse and could be obtained fresh. As it turns out, this was a very good choice since rabbit thrombomodulin has a very high specific activity and is expressed at high levels in the rabbit lung. Had we chosen human, bovine or porcine lung, the procedure would have been much more difficult. The isolation of thrombomodulin allowed us to look at other physiologically relevant things. If thrombomodulin functioned by binding thrombin and thereby concentrating it in the microcirculation, this could lead to microvascular thrombosis unless the binding event blocked procoagulant activities as well as accelerate protein C activation. Indeed, thrombomodulin did block thrombin-dependent platelet activation [14] and fibrinogen clotting activity [15]. Thus the cofactor that we had found was really a thrombomodulin – switching the physiological function of thrombin from clotting to anticoagulation. How did the system shut off? Both on cells and in purified systems, thrombin bound to thrombomodulin was readily inhibited by plasma inhibitors, particularly antithrombin and protein C inhibitor. Interestingly, the chondroitin sulfate that Lindahl's group described on thrombomodulin actually accelerated thrombin inactivation [16]. Later studies designed to look at the impact of thrombomodulin on thrombin specificity revealed that thrombomodulin dramatically enhanced the ability of protein C inhibitor to inactivate thrombin [17]. Kinetic evidence suggested that the thrombin-inhibitor complex would dissociate rapidly from thrombomodulin and we know now, largely from the work of James Huntington and colleagues, that the likely mechanism for this rapid dissociation involves ‘smashing’ thrombin by the sprung inhibitor and distorting the thrombomodulin binding region. The process seemed reasonably understood until we began searching for receptors that could modulate activated protein C functions. This quest was initiated because we and many others (reviewed in [18]) had observed that activated protein C could dampen inflammatory cytokine production in animals challenged with endotoxin/bacteria. In quest of the responsible receptor, Kenji Fukudome and I set out to expression clone candidate receptors [19]. We identified the endothelial cell protein C receptor (EPCR). To my surprise this receptor plays not only a major role in orchestrating the anti-inflammatory activities of APC, but increases activated protein C generation in response to thrombin about 10- to 20-fold. The existence of this receptor had been predicted from earlier studies. In these studies, we found that some rabbit-derived endothelial cell lines activated protein C much more rapidly than Gla-domainless protein C, while others did not discriminate [20]. This has been an amazing journey filled with good luck, timely funding, fortuitous and key interactions with critically important people, and did I say, unbelievably good luck. Johan Stenflo might not have visited John Suttie's laboratory and therefore, I might not have become interested in this problem. The CD/ORD machine in Iowa might have worked and Whyte and I might not have tested the rabbit heart perfusion experiments. (What would have happened if the hearts had retained a bit of thrombin and the first thing through was PF4?) Perhaps more amazing was the fortuitous change in Whyte's direction from the protein biochemist I met at Washington University into a physiologically oriented investigator. I might not have paid attention to sepsis studies that were emerging with Taylor and Hinshaw. Had that been the case we might not have looked for EPCR. We wish to thank Naomi L. Esmon for many helpful suggestions and historical recollections." @default.
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• W2010004644 title "The discovery of thrombomodulin" @default.
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