Activation of Prothrombin to Thrombin in 25% Trisodium Citrate and Other Concentrated Salt Solutions

               The conversion of prothrombin to thrombin in concentrated solutions of trisodium citrate was first reported in detail by Seegers in 1949 (1).   The rate and extent of activation of prothrombin was dependent on the concentration of trisodium citrate with most reactions being performed in 25% trisodium citrate (approximately 0.85 M assuming the dihydrate, M.W. 294.099; unadjusted pH 8.0 to 8.5).  As a note, do not confuse the trisodium salt with the disodium salt (sodium citrate, acid) as there will be no activation The activation of prothrombin in sodium citrate is pH dependent with optimal activation at alkaline pH.2  As part of their comprehensive study, Schultze and Schwick (2) showed that the activation of their prothrombin (which likely contained factor X and factor VII), proceeded best at pH 7.10-9.95 with a distinct decrease at 6.65 and more so at 5.85.   They also showed that the reaction proceeded best at 37oC and 45oC with a distinct drop at 28oC; the difference was in rate (extent of lag phase) and not yield.   The concentration of prothrombin in the reaction mixture was also critical. The effect of citrate was noted by Seegers as a result of adding the salt to prevent premature activation of his prothrombin preparations (3,4). Activation of some prothrombin preparation was also observed with other polyvalent inorganic anions such as ammonium sulfate, potassium oxalate, and potassium hydrogen phosphate (5).  As noted above, the rate and extent of thrombin formation from prothrombin in trisodium citrate is dependent on the concentration of sodium citrate with little, if any, activation at 5% sodium citrate (5). Seegers also observed that diphenyl sulfones had a profound effect on the rate and extent of prothrombin activation in 25% trisodium citrate; both acceleration (2-methyl,4-amino, 4’.6’-diamino diphenyl sulfone and inhibition (4-amino, 4’,6’diamino diphenyl sulfone) were observed.   As with citrate activation, this was a fortuitous finding by Seegers as as he had added the diphenyl sulfones to inhibit possible bacterial growth (4). It is reasonable to suggest the inhibitory effect of diphenyl sulfones would be directed at factor Xa (autoprothrombin C)(4).  Rationalization of the enhancement of rate of formation of and yield of thrombin is more difficult.  This would be an interesting project but unlikely to gain support in the current age which is dominated by cell biology (I do recall one of the prominent figures of my youth commenting that he was a cell biologist, a spectrophometer cell).    There is one paper (that I could find) on the effect of diphenyl sulfones on proteolytic enzymes which suggest a modest inhibition of ‘leucoprotease.” (6) Diphenyl sulfone has a log P value of 2.4 suggesting that the derivatives would be poorly soluble in water and indeed Seegers commented that the solubility of the various diphenyl sulfones limited the use to the addition of only several milligrams (1).  Inhibition of citrate activation of prothrombin is also inhibited by DFP and p-toluenesulfonyl-L-arginine methyl ester (TAMe)(7,8). The citrate activation of prothrombin is also inhibited by the addition of diluted serum (9) likely due to antithrombin.

                    The majority of work on the citrate activation of prothrombin was performed by Professor Seegers or his associates.  He interpreted the results as the autocatalytic activation of prothrombin in trisodium citrate (7).  Although this was later shown to be an incorrect interpretation of the data, it was not unreasonable at the time considering the state of the art in protein purification and analysis.  It is clear now that the early prothrombin preparations contained other coagulation factors as it was possible for other investigators to obtain prothrombin preparations which did not form thrombin in 25% trisodium citrate (10,11).  Lechner and Deutsch (10) had been able to demonstrate the presence of factors VII and X in the preparations of prothrombin obtained by the method of Seegers and associates.  Somewhat earlier, Seegers and Landaburu (12) had obtained prothrombin which did not undergo citrate activation but attributed the difference to changes in the structure of the prothrombin.  Subsequently, Marciniak and Seegers described autoprothrombin C (now factor Xa, Milstone’s thrombokinase) a as second enzyme derived from bovine prothrombin in 25% trisodium citrate (13). Autoprothrombin C was derived from a precursor, autoprothrombin III (now factor X, Stuart-Prower factor) , and could activate prothrombin and prethrombin (likely prethrombin 2) to thrombin (14).  Somewhat later, Teng and Seegers (15) described the activation of factor X in 25% trisodium citrate and ascribed the activation to thrombin which they described as reciprocal proenzyme activation where factor Xa activated prothrombin in 25% trisodium citrate and thrombin activated factor X in trisodium citrate.  I don’t know of any other work describing the activation of factor X by thrombin, but I don’t think anyone has tried to perform the reaction in 25% trisodium citrate. I should add that Seegers in what might have been his final opus, maintained that thrombin did activate factor X in 25% trisodium citrate (4).   Inhibition of thrombin formation from prothrombin in 25% trisodium citrate  by soybean trypsin inhibitor (16) provided support for the role factor Xa in the process of the  citrate activation of prothrombin.   Factor Xa is one of the  few activated coagulation factors, all of which can be considered to be trypsin-like serine proteases, that is inhibited by soybean trypsin inhibitor (17).  Although there was no thrombin formation, the chemical changes in prothrombin occurring during activation (18) (electrophoretic mobility in Tiselius apparatus (19) and material soluble in 7% trichloroacetic acid) were unchanged when compared to a control activation without the inhibitor.   A similar effect is observed by inclusion of 3,4,4’-triaminodiphenyl sulfone, the inhibitor described above.  Additional work from Dave Aronson showed that factor VII was also required for the citrate activation of prothrombin (20).  Current work on the citrate activation of prothrombin either use partially purified prothrombin (21) such as a barium sulfate eluate or a Cohn Fraction III/IV or add a source of factors VII and X such as defibrinated plasma (22).   Phyllis Roberts, at MCV in Richmond observed that the rate of prothrombin activation was slower in potassium citrate than in sodium citrate (23) and suggested that the decreased rate of thrombin formation was due to the inhibition of factor Xa activity by potassium ions and enhancement by sodium ions.  While most of the work cited above suggests that purified prothrombin will not activate in concentrated trisodium citrate, a patent (24) described the activation of recombinant prothrombin to thrombin in 35% trisodium citrate (added as a solid) for 32 hours at 37oC.  The activation of purified plasma prothrombin is also claimed with 35% trisodium citrate (24-28 hours, 37o C).    The patent also claims pH 6.5-7.5 which is slightly more acid than the more alkaline pH of most unadjusted solutions of trisodium citrate as noted above. Schultze and Schwick (2) note that citrate activation is more rapid at alkaline pH than at pH 6.6.   I would note that there has been renewed interest in the autoactivation of prothrombin (25-27) and it is possible that high concentrations of citrate may favor the open conformation which is suggested to be susceptible to autoactivation (27).  Citrate activation has been useful for preparing multiple forms of thrombin (e.g. β-thrombin, γ-thrombin) (21,22,28-30).  The citrate activation of prothrombin has been used to prepare human thrombin for therapeutic purposes (31).

1.  Seegers, W.H., Activation of purified prothrombin, Proc.Soc.Exptl.Biol.Med. 77, 677-680, 1949.
2.  Schultze, H.E. and Schwick, G., Uber den mechanisimus der Thrombinbildung im isölieren System, Hoppe-Seyler Zeitsch.fur Phyiol.Chem. 289, 26-   , 1951.
3.  Seegers, W.H., Prothrombin in Enzymology, Thrombosis, and Hemophilia. Chapter 4, Development of thrombin activity in concentrated salt solutions, pps. 88-98, C.C. Thomas, Springfield, Illinois, USA, 1967.
4.  Seegers, W.H., IV. Activation of prothrombin and factor X in 25% sodium citrate, Sem.Thromb.Hemost. 17, 213-220, 1981.
5.  Seegers, W.H., McClaughry, R.I., and Fahey, J.L.., Some properties of purified prothrombin and its activation with sodium citrate, Blood 5, 421-433, 1950.
6. Grob, D., Proteolytic enzymes. I. Control of their activity, J.Gen.Physiol. 29, 219-247, 1946
7.  Seegers, W.H., Marciniak, E. and Cole, E.R., Autocatalysis in prothrombin activation, Am.J.Physiol. 203, 347-400, 1962.
8. Miller, K.D. and Van Vunakis, H., The effect of diisopropyl fluorophosphate on the proteinase and esterase activities of thrombin and on prothrombin and on its activation, J.Biol.Chem. 223, 227-237, 1956.
9. Seegers, W.H. and Landaburu, R.H., Inhibition of prothrombin activation in sodium citrate solutions, Proc.Soc.Exptl.Biol.Med. 95, 710-713, 1957.
10.  Lechner, K. and Deutsch, E., Activation of factor X, Thromb.Diath.Haemorrh. 13, 314-329, 1965.
11.  Esnouf, M.P., Prothrombin, in Human Blood Coagulation, Haemostasis and Thrombosis, ed. R.Biggs, Chapter 3, pps. 42-45, Blackwell Scientific, Oxford, United Kingdom, 1972.
12.   Seegers, W.H. and Landaburu, R.H., Purification of prothrombin and thrombin by chromatography on cellulose, Canad.J.Biochem.Physiol. 38, 1405-1416, 1960.
13. Marciniak, E. and Seegers, W.H., Autoprothombin C:  A second enzyme from prothrombin, Canad.J.Biochem.Physiol. 40, 597-603, 1962.
14.  Seegers. W.H. and Marciniak, E., Some activation characteristics of the prethrombin subunit of prothrombin, Life Sci. 4, 1721-1726, 1965.
15. Teng, C.-M. and Seegers, W.H., Activation of factor X and thrombin zymogens in 25% sodium citrate solution, Thromb.Res. 22, 203-212, 1981.
16.  Alkjaersig, N., Deutsch, E., and Seegers, W.H., Prothrombin derivatives and the inhibition of thrombin formation with soy bean trypsin inhibitor, Amer.J.Physiol. 180, 367-370, 1955.
17.  Milstone, J.H., Effect of blood thrombokinase, as influenced by soy bean  trypsin inhibitor, ultracentrifugation, and accessory factors, J.Gen.Physiol. 38, 757-769, 1955.
18.  Lorand, L., Alkjaersig, N., and Seegers, W.H., Carbohydrate and nitrogen distribution during the activation of purified prothrombin in sodium citrate solution, Arch.Biochem.Biophys. 45, 312-318, 1953.
19.  Tiselius, A., Electrophoresis past, present, and future, Clin.Chim.Acta 3, 1-9, 1958.
20.  Aronson, D.L. and Mustafa, A.J., The activation of human factor X in sodium citrate: the role of factor VII, Thromb.Haemost. 36, 104-113, 1976.
21.  Lundblad, R.L., Nesheim, M.E., Straight, D.L. et al., Bovine α- and β-thrombin. Reduced fibrinogen-clotting activity of β-thrombin is not a consequence of reduced affinity for fibrinogen, J.Biol.Chem. 259, 6991-6995, 1984.
22. Mann, K.G., Heldebrant, C.M., and Fass, D.N., Multiple active forms of thrombin II. Mechanism of production from prothrombin, J.Biol.Chem. 246, 6106-6114, 1971.
23. Roberts, P.S., The effects of Na+ and K+ on the citrate activation of prothrombin and on the esterase activities of thrombokinase, Biochim.Biophys.Acta 201, 340-349, 1970.
24.  Ralston, A.H. and Drohan, W.N., Activation of pure prothrombin to thrombin with about 30% to 40% sodium citrate, US Patent 6,245,548, 2001.
25.  Pozzi, N., Chen, Z., Zapata, F., et al., Autoactivation of thrombin precursors, J.Biol.Chem. 288, 11601-11610, 2013.
26.  Pozzi, N., Chen, Z., Gohara, D.W., et al., Crystal structure of prothrombin reveals conformational flexibility and mechanism of activation, J.Biol.Chem. 288, 22734-22744, 2013.
27.  Pozzi,  N., Bystranowska, D., Zhu, X., and Di Cera, E., Structural architecture of prothrombin in solution revealed by single molecule spectroscopy, J.Biol.Chem. 291, 18107-18116, 2016.
28. Lanchantin, G.F., Friedmann, J.A., and Hart, D.W., Esterase and clotting activities derived from citrate activation of human prothrombin, J.Biol.Chem. 242, 2491-2501, 1967.
29. Mann, K.G., Heldebrant, C.M., and Fass, D.N., Multiple active forms of thrombin I. Partial resolution, differential reactivities, and sequential formation,  J.Biol.Chem. 246, 5994-6001, 1971.
30.  Fass,D.N., and Mann, K.G., Activation of fluorescent-labeled prothrombin, J.Biol.Chem. 248, 3280-3287, 1973.
31. Alzawa, P., Winge, S., and Karlsson, G., Large-scale preparation of thrombin from human plasma, Thromb.Res. 122, 560-567, 2008.

Roger L. Lundblad
December, 2019
Chapel Hill, North Carolina USA