The initiation phase--a review of old (clotting-) times.

Thromb Haemost 2007; 98: 20–23 To browse through old work is a pleasure in the indian summer of a career, not devoid of nostalgia.As the large majority of our early work appeared inThrombosis and Diathesis Haemorrhagica, the present issue is an appropriate occasion to do so and to review old results, holding them against the light of our present day knowledge – and apologising beforehand for the shameless self-citation that it automatically involves. Even in the mild light of hindsight this soon turns out to be primarily an exercise in modesty and an occasion to recognise old errors. Our early attempts to extract a maximum of information from clotting times now may look a bit naïve.Yet some of the conclusions and insights still seem to hold and still may contribute to the understanding of what we now call the “initiation phase” of blood coagulation. My first contribution to Thrombosis and Diathesis Haemorrhagica was in the supplement that reported the proceedings of the Gleneagles conference of the International Committee for the Nomenclature of Blood Clotting Factors in 1963. It was on PIVKA, protein induced by vitamin K absence(or antagonists) and reports my first appearance before the big personalities of coagulation of those days, such as Biggs, Brinkhous, Deutsch, Koller, Macfarlane, Seegers and Soulier. From sheer anxiety my right patella kept trembling during the full seven minutes that my talk lasted. And nobody paid more than a polite interest – with the notable exception of Peter Esnouf and François Josso. My own initiation phase had begun some eight months before, when Fredi Loeliger had confronted me with a riddle in the control of oral anticoagulation, viz. the discrepancy between “prothrombin times” and single factor determinations. For those who are so modern as to know clotting times only from figures that come out of laboratory automatons, I will give some background information: As everybody knows, the “prothrombin” defect in oral anticoagulation was, and still is, routinely assessed by the “prothrombin time”, introduced by Armand Quick, often in a variant introduced by Owren, i.e. with the addition of BaSO4 adsorbed cow-plasma, so as to make it dependent upon factors II, VII and X only. (For a review see e.g. [1]). We used a variant in which we added 0.1 ml of the plasma to be tested, 0.1 ml of BaSO4 adsorbed cow plasma that contained human brain thromboplastin and recalcified at zero time with 0.1 ml of a 100 mM CaCl2 solution in buffer. The “prothrombin” of Quick had turned out, in the meantime, to be a set of proteins but the “prothrombin time” remained, although “thromboplastin time” is more exact. This clotting time was recognised to be dependent upon the factors II, VII and X (factor IX standing offside at the high thromboplastin concentrations used). Each of these factors can also be determined separately. In that case one takes 0.1 ml of plasma deficient in the factor that you want to determine; 0.1 ml of 1:10 diluted sample of the plasma to be tested and 0.1 ml thromboplastin solution in 100 mM CaCl2. The 1:10 dilution was necessary to prevent that the other clotting factors present in the sample would influence the clotting time and in order to cover a useful range of clotting times.To relate clotting times to clotting factor concentrations, reference lines were made: the clotting time (tc) at dilutions of normal plasma, by definition containing 100% of each factor, were plotted, on logarithmic paper, against the clotting factor concentration and the clotting time obtained with an unknown sample was related to a concentration using this graph. Loeliger had found that in stable oral anticoagulation the four vitamin-K-dependent clotting factors were decreased to about the same level when tested individually (2). But in a “prothrombin time” type of experiment consistently about half of that concentration was found. Why? Was there an elusive, rate limiting fifth vitamin-K-dependent clotting factor? In 1962 none of the clotting factors had been isolated to purity. It was not even certain that all of them were proteins. Walter Seegers and his school maintained that factors II, VII, IX and X were different aspects of one and the same prothrombin (1).Antibodies were not available, monoclonals did not exist. Clotting factors were defined as an activity required to make blood clot normally and that was congenitally absent in certain patients. Factor X e.g. was the entity lacking in the plasma of Mr. Stuart and obviously in every plasma that would not correct the defect, like that of Mrs. Prower (1). Therefore the possibilities to approach the problem were in practice limited to juggling with clotting times. While doing this, I recalled the Lineweaver-Burke plot of enzyme kinetics: inverse reaction velocity against inverse sub50 th A n n iv er sa ry (1 95 7– 20 07 )