Ion-Exchange Resin Treatment of Blood instead of Anticoagulant With a Preface on Serum


I have previously written about the fact that serum is not plasma (1,2) and there is an earlier post on this site.  Lost in antiquity (or at least not available electronically) are some early studies on the purification of factor IX from serum for research (3) and a therapeutic product (4). White and coworkers (3) had shown that the infusion of serum did correct the defect in hemophilia B patients.   While prothrombin is "consumed" during blood clotting, most likely by removal of fragment 1, factor IX is only slightly "consumed" during the formation of serum.   The greater extent of prothrombin consumption, the more efficient coagulation process.   The patient described by White and coworkers (3) showed 10-20 % prothrombin consumption which was increased on the infusion of serum or plasma.  A therapeutic factor IX concentrate has been prepared from human serum which demonstrated higher in vivo recovery than therapeutic concentrates prepared from c plasmaa (4,5).   It is also noted that serum can be the source of therapeutic immunoglobulins(convalescent serum)(6,7) although isolated immunoglobulin from post-exposure serum (convalescent serum) is more effective (7).    There was early work from Netherlands which showed that a fraction could be prepared from human serum which was therapeutically effective in hemophilia patients (8).  To assure transparency in this note, the author's introduction to the purification of proteins was the use of the technique developed in Paul Ageller's laboratory in San Francisco (3).    While, this technique seems primitive by today's standards, in all fairness to Dr. Ageller who made major contributions to hemostasis and thrombosis, this work was performed three years prior to the development of ion-exchange celluloses for protein fractionation (9,10).  


Some thirty years ago my laboratory in Chapel Hill was involved in the study of the chemistry of factor IX and factor IXa (11).  We converted factor IX to factor IXa by the action of factor XIa.   It was necessary to prepare factor XIa from serum and this involved early morning trips from Chapel Hill to Wilson, North Carolina to an abattoir which processed cattle.  For reasons that have never been completely (or for that matter even partially) understood, the blood had to be collected in one-pint glass canning  jars and allowed to stand for 24 hours  to form serum to maximize the production of factor XIa.  We also collected bovine blood in citrate to prepare blood plasma for the preparation of factor IX, factor X, and prothrombin.   It occurred to us that the process could be much cleaner if we could prepare the serum from the citrated plasma after recalcification in the presence of glass.   This would not save us from the early morning trips to Wilson but would be a lot cleaner,  I should also say that the trips to Wilson were rewarded with a stop on the way back in Middlesex for the best country ham biscuits (there is a reason for elevated cholesterol in Eastern North Carolina).   Unfortunately, the recalcification of the plasma obtained from bovine blood collected in citrate did not yield factor XIa nor did recalcification of citrated bovine whole blood.   We then embarked on a study using human blood to study factors influencing the formation of factor XIa.  We were able to duplicate the findings from the bovine blood in being unable to prepare factor Xia from recalcified citrated  human blood.   We were also unable to prepared factor XIa from recalcified EDTA blood.   We were finally able to prepare factor XIa from serum prepared from human plasma obtained by passing blood through a resin (12) which removed calcium ions and other divalent cations as well as monovalent cations.  We were indebted to the late John Borden Graham of the Department of Pathology at the University of North Carolina at Chapel Hill for the suggestion of the resin which was obtained from the Renal Division of Baxter Healthcare.   I should also note that Ken Mann and associates have reported that calcium citrate inhibits the process of blood coagulation possibly by inhibiting Factor Xase (13).  I recognize that citrate and other chelating agents such as EDTA will chelate other divalent cations, such as magnesium and manganese; magnesium has been suggested to be important in the function of factor IX (14,15) and in the tissue factor pathway (16).  In looking back at our work from 1977 (12), we should have used a mixture of divalent cations such as Hank's balanced salt solution (instead of only calcium chloride).   I could find one study from Raymond Machovich's laboratory which used a physiological solution (Hanks) in the study of the effect of fibrin on inactivation of plasmin and neutrophil elastase by plasma proteinase inhibitors (17).  I could not find any other studies on blood coagulation which compared a physiological solution with only calcium chloride or other single metal ion.   While I think that it is unlikely, it is possible that we would have obtained different results had we used Hanks or another physiological solution instead of 0.025 M CaCl2.


The use of ion-exchange resins for the chelation of calcium ions in blood has a longer history than its use in renal dialysis.  Steinberg (18) introduced the use of resin (Amberlite IR-100b) for obtaining and preserving blood in the fluid state.   This work was part of the effort in World War II to support field blood transfusion services by identifying an anticoagulant process not requiring a fluid (19).   Amberlite IR-100 also binds magnesium ions somewhat more effectively than calcium ions (20) and magnesium has been observed to be important in blood coagulation.   There is one very interesting observation on the use of an ion-exchange resin in blood coagulation.  Breen and Tullis (21) reported 100% recovery of factor IX (4a) from a factor IX concentrate prepared from resin blood  by DEAE-cellulose chromatography(22).    I have been unable to find any additional work on the use of resin blood for the preparation of single-factor IX concentrates or prothrombin complex concentrates, I have no reason to doubt the work from James Tullis and his associates.   Tullis was a distinguished investigator responsible for significant contributions to hematology research during his career at Harvard Medical School (New England Deaconess)(23).   There is some evidence to suggest that the recovery of plasma-derived factor IX is greater than recombinant factor IX (24); it is not clear on how this data related to the apparent effect of citrate anticoagulant.


Footnotes

a Dr. Ernest Briet's thesis contains a Table (Table 1, p.47) listing the in vivo recoveries of various therapeutic concentrates.  The recovery of the serum concentrate was 76% while that repaired from resin blood was 100%.   While likely not significant, recovery from concentrates prepared from EDTA plasma (44%,48%) was slightly higher than that observed with concentrates prepared from citrated plasma (20%-48% with majority in the range of 20% to 33%).  The low recovery of factor IX reflects an expanded volume of distribution (Kessler, C.M. and Maniani, G., Clinical Manifestations and therapy of the hemophilias, in Thrombosis and Hemostasis. Basic Principles and Clinical Practice, ed. R.W. Colman, V.J. Marder, A.W.Clowes, J.N.George, and  S.Z. Goldhaber, Chapter 59, pps. 887-904, Lippincott, Philadelphia, Pennsylvania, USA, 2006) possible due to factor IX binding to collagen IV in the basement membrane (Cheung, W.F., van den Born, J., Kühn, K., et al., Identification of the endothelial cell binding site for factor IX, Proc.Natl.Acad.Sci. USA 93, 11068-11973, 1996).

b Amberlite IR-100 is a cation-exchange resin possessing sulfate, carboxylate and hydroxyl functional groups used for the removal of inorganic cations from water and other polar solvents.  Amberlite IR-100 was used for the removal of copper ion from tyrosinase (Dressler, H. and Dawson, C.R., Nature and mode of action of the copper protein, tyrosinase. I. Exchange experiments with radioactive copper and the resting enzyme, Biochim.Biophys.Acta 45, 408-414, 1960) and for ascorbic acid oxidase (Magee, R.J. and Dawson, R.J., Exchangeability of copper at the active site in ascorbic acid oxidase, Arch.Biochem.Biophys. 99, 338-347, 1962).

 
References              
1.  Sapan, C.V. and Lundblad, R.L., Considerations regarding the use of blood samples in the proteomic identification of biomarkers cancer diagnosis, Cancer Genomics and Proteomics 33, 227-231, 2006
2.  Burgess, C. and Lundblad, R.L., Plasma or serum in proteomic analysis, Clinical Laboratory International 31, 10-11, 2007.
3.  White, A.G., Aggeler, P.M., and Glendening, M.B., Plasma thromboplastin component (PTC), a hitherto unrecognized blood coagulation factor; case report of PTC deficiency, Blood 8, 101-124, 1953.
4.  Ménaché, D., Josso, F., and Dufour, L., Durée, de vie des facteurs VIII and IX (facteurs anti-hémophilique A et B) injectés à des hémophiles, Hémostase 3, 139-148, 1963.
5.  Briet, E., Three Problems of Hemophilia B, Thesis, University of Leiden, Netherlands, 1977.
6.   Rambar, A.C., Mumps; the use of convalescent serum in the prevention of mumps in adults, Am.J.Nursing 48, 86-87, 1948.
7.  Young, M.K., Nimmo,G.R., Cripps, A.W., and Jones, M.A., Post-exposure passive immunisation for preventing measles, Cochrane Database Syst.Rev.April 1; 4:CD010056, 2014.
8.  Benden, W.M. and van Crevald, S., Investigations on hemophilia, Amer.J.Dis.Children 54, 713-725, 1937,.
9.  Peterson, E.A. and Sober, H.A., Chromatography of proteins. I.  Cellulose ion-exchange adsorbents, J.Am.Chem.Soc. 78, 751-758, 1956.
10. Sober, H.A., Gutter, F.J. Wychoff, M.M., and Peterson, E.A., Chromatography of proteins. II. Fractionation of serum proteins on anion-exchange cellulose, J.Am.Chem.Soc. 7, 756-763, 1956.
11. Lundblad R.L. and Kingdon H.S.,  Biochemistry of the Interaction of Bovine Factors XIa and IX.  Thromb Diath Haemorrh (Stuttg.), Suppl 57:315-333, 1974
12.  Kingdon, H.S., Lundblad , R.L., and Dingman, G., Factors affecting the evolution of factor XIa during blood coagulation, J.Lab.Clin.Med. 85, 826-831, 1975.
13.  Mann, K.G., Whelihan, M.F., Butenas, S., and Orfeo, T., Citrate anticoagulation and the dynamics of thrombin generation, J.Thromb.Haemost. 5, 2055-2061, 2007.
14.  Sekiya, F., Yoshida, M., Yamashita, T., and Morita, T., Magnesium (II) is a crucial constituent of the blood coagulation cascade.  Potentiation of coagulant activity of factor IX by Mg2+ ions, J.Biol.Chem. 271, 8541-8544, 1996.
15.  Kim, W.H., Kim, J.S., Yoon, Y., and Lee, G.M., Effect of Ca2+ AND Mg2+ on the activation of recombinant factor IX produced in Chinese hamster ovary cells, J.Biotechnol. 142, 275-278, 2009.
16. van den Besselaar, A.M., Magnesium and manganese ions accelerate tissue factor-induced coagulation independently of factor IX, Blood Coagul.Fibrinolysis  13, 19-23, 2002.
17.  Kolev, K., Léránt, I, Tenekejiev, K., and Machovich, R., Regulation of fibrinolytic activity of neutrophil leukocyte elastase, plasmin, and miniplasmin by plasma proteinase inhibitors, J.Biol.Chem. 269, 17030-17034, 1994.
18. Steinberg, A., A new method of preventing blood coagulation, Proc.Soc.Exptl.Biol.Med. 56, 124-127, 1944.
19. Kendrick, D.B., Blood Programs in World War II, Chapter XX, Pt. II, Section VI, p. 769, Office of the Surgeon General, Superintendent of Documents, Washington, DC, 1964.
20.  Heymann, E. and O'Donnell, I.J., Physicochemical investigation of a cation-exchange resin. II. Resin conductance, J.Colloid Sci. 4, 405-416, 1949.
21.   Breen, F.A. and Tullis, J.L., Prothrombin concentrates in the treatment of Christmas disease and allied disorders, J.Amer.Med.Assoc. 208, 1848-1852, 1969.
22. Tullis, J.L., Melin, M., and Jungian, J. Clinical use of human prothrombin complex, New Engl.J.Med. 273, 667-674, 1965.
23. http://www.nytimes.com/1996/04/04/us/james-l-tullis-81-leading-researcher-on-blood.html
24.  Alamelu, J., Bevan, D., Sorensen, B. and Rangarajan, S., Pharmacokinetic and pharmacodynamic properties of plasma-derived vs. recombinant factor IX in patients with hemophilia B: a prospective crossover study, J.Thromb.Haemost. 12, 2044-2048, 2014.

Copyright Roger L. Lundblad Chapel Hill, NC February 2016