Biological and nonbiological activation of normal and dicoumarol-treated prothrombin

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BIOLOGICAL AND NONHIOLOGICAL ACTIVATION OF NORMAL AND DICOUMAROL-TREATED PROTHROMB~i Om P. Malhotra and John A. Carter Veterans Administration Hospital and Iastitate of Pathology, Case Western Reserve University, Cleveland, Ohio 44106

ateceived 9 Febrwtry 1978; in ~ tca~m 80 March 1978) 9ummaryand Çonclusion Similar to normal, prothrombin obtained from dicoumaroltreated steers required factor X for activation in addition to factor V, thromboplastin and Cat+ . The shortest clotting time, however, persisted for approszmately an hour as compared to 10 mimitgs for normal prothrombin. Factor X from dicoumaroltreated plasma also corrected the clotting (activation) detect . Poly-L-lysin, protamine sulfate and 25°J6 sodium citrate solution activated both normal and dicoumarol-treated preparations containing factor X within s few hours . Thrombdn yield for normal was half that of its potential but 50°J6 greater than the potential for dicoumarol-treated preparations . This greater yield from dicoumsrol-treated prothromMn represented the (nonbiologicsl) activation of "abnormal" molecules . In the absence of factor X, poly-L-lysin and protamine sulfate, 'on the other hand, did not activate the two prothrombins . In 25°}6 sodium citrate, such preparations activated very slowly taking days or weeks rather than hours . These results indicate the requirement of factor X even for nonbiological activation of prothrombin. Correlation of structure with function of proteins and eaeymes has been the subject of intensive i~estigation and speculation. Aside from the accepted congenital and familial inborn errors of metabolism causing disease and resulting from genetic-controlled alteration of molecular structure, e. g. hemoglobin in sickle cell anemia, virtually all molecular alteration studies have been made by inducing such changes in vitro by a variety of techniques 3npported by YA funds and in part by NIH grant HE-10571 and a grant from Heart Association of Northeastern Ohio . ~B

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or by observation of those alterations which have occurred si untaneously. The induction of reversible or irrevb .aible alterations in vivo remains speculative, however.

For example, in 1948, Lein and Lein (1) hypothesised the

synthesis of abnormal prothrombin by dicoumarol administration . Since then, particularly in recent years, several in vivo and in vitro studies (utilising isolated liver perfusion, liver slices etc . from normal and vitamin Kdeficient animals) have been undertaken to determine the effects of vitamin K and dicoumarol on the biosynthesis of prothrofnbin in the presence or absence of puromycin, cycloheaimide or actinomycin-inhibitors of protein bio synthesis .

The results have been conflicting (2-9) .

To prove or disprove the acquired molecular alteration hypothesis, a singular approach has been to characterize the highly purified preparations of prothrombin obtained from normal and dicoumarol-treated steers .

Except for

low specific activity of dicoumarol-treated prothrombin, the two preparations (normal and dicoumarol-treated) were similar (10) .

The low specific activity

was determined to be the result of the presence of so-called "abnormal" (prothrombin) molecules. Strong salt sôlutione such ae 25% sodium citrate, 0 . 2 M (NH4)2504, polyamino acids (poly-L-lysin, poly-L-ornithine) and protamine sulfate have been reported to convert prothrombin to thrombin (autocatalysis) (11-13) . The mechanism by which they activate prothrombin is disputed, however. For example, there are those who believe that they do so through factor X which is present as a contaminant (14-16) .

Others, on the contrary, believe

that these activating agents activate prothrombin directly, and the preparations which did not activate were either altered during purification or contained inhibitors) (17, 18) .

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Since our highly purified preparations of prothramMn (normal and dicoumarol-treated) were free from factor X and showed s single component by several rigorous physicochemical and immunological criteria of purity (10, 19), their activation in the presence or absence of factor X by the above activation agents seemed as ideal system to employ to solve the controversy . Further, since the biological and autocatalytic activation kinetics are different, it seemed possible that the low specific activity of dicoumaroltreated prothrombin, measured by biological assay, could be obviated sutocatalytically by conversion of "abnormal" prothrombin molecules to thrombin, and therefore document the thrombin potential of "abnormal" prothrombin. The objectives of these studies were to determine whether 1) the "abnormal" molecules present in dicoumarol-treated preparations could yield thrombin by activating agents ; 2) these agents activate prothrombin directly or indirectly through factor X; 3) biological activation characteristics of the two prothrombins were similar . Materials and Methods Prothrombin, normal and dicoumarol-treated, was isolated and purified by the methods described previously (10, 19).

Prothrombin prepared from

plasma which had been treated previously with adsorbants

LH .

BaS04,

bentonite and kaolin) was highly purified and was free from factor X. inch preparations are designated as "adsorbed", versus "nonadsorbed", i.e . purified from plasma without prior treatment with these adsorbents . nonadsorbed preparations contained factor X .

The

Factor X was isolated a~ puri-

Sed from the slants (596 sodium citrate, pH 7 . 8) of the BsS04 employed for the adsorption of factor X from plasma by ammoni~mn sulfate fractionation, isoelectric precipitation and polyscrylamide gel electrophoresis . Factor X

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so obtained was essentially free from prothrombin .

Vol. 11, No. 9 In some cases, plasma

deficient in prothrombin or in prothrombin and factor VII (Sigma ; personal communication) prepared by a modification of the procedure of Koller et al . (20) and Hjort et al . (21), respectively, was used as a source of factor X.

Thrombin was measured essentially by the same technique se that

used for prothrombin (22), ezcept that accelerators (serum) were not added to the activation mizture, and the assays were performed at 0 time . Factor X-deficient plasma was prepared from normal citrated bovine plasma (sodium citrate, 2 .85% ; 1:B dilution) by preferential adsorption with Ha504 (J . T . Baker Co . , Phillipsburg, N. J . , analytical grade) .

The plasma

was stirred twice with BaS04 at 4oC for 30 min. in concentrations first of 70 mg/ml followed by 20 mg/ml. by centrifugation .

After each adsorption, BaS0 4 was removed

Plasma dsHcient in factor X made by filtration through

charcoal, was obtained from Diagnostic Reagent Ltd; Thame (Oaon), England . The plasmas were essentially free from factor X (~1%) as assayed by the method of Denson (23) . Natural factor X-deficient (Stuart) plasma was obtained through the courtesy of Dr . John Graham of the University of North Carolina . Biological activation of normal and dicoumarol-treated prothrambin or plasma prothrombin in the absence of factor X was performed as described previously (10, 19). The techniques used for nonbiological activations were essentially those of Seegers (11, 12) and Miller et al . (13) . Results and Discussion In addition to thromboplasHn, factor V and Cat+, normal adsorbed prothromMn (or nonsdsorbed from which factor X had been removed by disc

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electrophoreei~) required factor X for rapid and complete activation .

In the

absence of factor X, s minimum of 18 instead of 6 minutes way required, and the yield of thrombin was approximately 25°j6 lea (Fig . 1) .

It ws~ immaterial

whether the factor X used was isolated from normal or dicoumsrol-treated pis~ms .

Similar to normal, dicoumsrol-treated prothrombin sl~o required

factor X; however, its shortest clotting time persisted for approzimately an hour as compared to 10 minutes (Fig . 1) . Since the specific activity of

FIG. 1

Activation of dicoumsrol-treated prothrambin in presence or sb~ence of factor X. ~ activation mixture contained thromboplsetin, factor V and Ca + . dicoumarol-treated prothrombin way about half that of normal (10), and data glow activation of the englobin fraction of~dicoamsrol-treated pL~ma hs~ been reported (2~), .it ie conceivable, that the pereietancs of the ehorteet clotting

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time could be the result of delayed activation of "abnormal" molecules preyumably present in the dicoumarol-treated preparations .

To accelerate the

rate of activation of "abnormal" molecules and thereby improve thrombin yield, serum (factory V, VII and X) concentration was increased 2 to 3 fold . However, neither the rate nor the yield increased. In 25°go sodium citrate solution, nonadsorbed normal prothrombin preparations activated within a few hours (Fig . 2) .

The yield of thrombin was

about half that of the total potential, confirming the results .of others (11, 12). In the case of dicoumarol-treated prothrombin, however, the yield was 50q'o greater than the potential (Fig . 2) . In other words, dicoumarol-treated

FIG . 2 ActivaHot< of nonadsorbed normal and dicoumirol-treated prothrombin in 256 sodium citrate solution . prothrombin yielded 3z ay much thrombin a did normal preparations -

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suggesting activation ctf "abnormal" molecules which presumably were not activated by Mological procedures .

These results, therefore, explain the

Iow specific activity and persistence of shortest clotting times of dicoumarol-treated preparations . Contrary to nonadsorbed, adsorbed preparations (normal and dicoumaroltreated) activated very sloavly taking days or even weeks rather than hours . For normal prothrombin, the thrombin yield was comparable to nonadsorbed preparations . For dicoumarol-treated (adsorbed) prothrombin, however, it was equivalent to its (thrombin) potential but not greater .

UnacHvated pro-

thrombin accounted for lower yield than that obtained from nonadsorbed dicoumarol-treated preparations (Fig . 3) .

In say event, the results do

FIG. 3 Activation of adsorbed prothrombin (normal and dicoumsrol-treated) in protamiae sulfate solution and in 25°J6 sodium citrate solution.

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substantiate the thrombin potential of "abnormal" prothrombin molecules the molecules responsible for the low specific activity of dicoumarol-treated prothrombin. The slow rate of activation of adsorbed preparations in strong sodium citrate solution suggests the necessity for factor X.

That the preparation,

essentially free from factor X, could be activated by 25% sodium citrate was conürmed by the formation of thrombin from DEAE-cellulose chromstographed prothrombin, contrary to other reports (16) .

This discrepancy could

be ezplained by the fact that either activation was not followed for a sufficient period of time and/or the prothrombin concentration used at 0 time was low (less than 3000 U/ml) . It is our e~erience that the activation rate not only depends upon the presence of factor X but also on prothrombin concentration. Whether the latter influenced the activation by increasing factor X which may be present in trace amounts cannot be ezplained by these ezperiments. The requirement of factor X for nonbiological activation of prothrombin is further documented by the fact that poly-L-lysin and protamine sulfate activated both notmal and dicoumarol-treated preparations only in the presence of factor X (Fig . 3, 4) .

Evidently, the preparations used by other

investigators for autocatalytic activations contained factor X (11-13) . Since polypeptide chain of "abnormal" molecules appears to be similar (or almost similar) to that of normal prothrombin (25) and since vitamin K is implicated for the attachment of carbohydrates to prothrombin (7, 26) the differences in activation characteristics of the two preparations (normal and dicoumarol-treated) therefore, could be due to varied carbohydrate moiety of "abnormal" molecules .

To this effect, experiments are in progress to

separate the "abnormal" molecules from normal and to compare their

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carbohydrate moieties . Fnrthsr, whether or not dicoumsrol Mnd~ with "abnormal" molecnle~ and thereby alters its activsNon characteristics needs to be ezplored .

FIG. 4 Activation of nonad~orbed and adsorbed prothrombin (normal and dicoumarol-treated) in poly-L-lysin . Reference~ J. LEIN and P. S. LEIN, Am . J. Phy~iol.

155, 394 (1948) .

G. F. ANDERSON and M. I. BARIfiHART, Am . J. Ph siol. _206, 929 (1964) . 3.

L. F. LI, R. K. KIFFER and R. E. OLSON, Arch. Hiochem . Biophys. 137, 494 (1970) .

4.

J . W. SUTTIE, Fed . Proceedings 28, 1696 (1969) .

5.

R. B. HILL, S. GASTANI, A. M. PAOLUCCI, P. B. RAMA RAO, R. ALDEN, G. S. RANHOTRA, D. V. SHAH, V. K. SHAH and B. C. JOHNSON, J. Hiol . Chem . 243, 3930 (1968) .

6.

G. S. RANHOTRA and B. C . JOHNSON, Proc . Soc . Ezp. Biol . Med. 132, 509 (1970) .

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7. M. PEREIRA and D. COURI, Biothun. Biophye. Acta. 237, 348 (1971) . 8. R . J. BERNACKI and H. B. BOSMANN, Biochem . Biophye . Res . Comm. 41, 498 (1970). 9 . D. V. SHAH and J. W. SUTTIE, Proc. Nat . Acad . Sci . USA 68, 1653 (1971) . 10. O. P. MALHOTRA and J. R. CARTER, J . Biol. Chem. 246, 2665 (1971) . 11 . W. H. SEEGERS, Proc . Soc . Exp . Biol. Med . 72, 677 (1949) . 12 . R. H. LANDA BURU and W. H. SEEGERS, Am. J . Physiol . 193, 169 (1958) . 13 . K. D. MILLER, W. H. COPELAND and J. F . McGARRAHAN, Proc . Soc . Exp. Biol. Med . 108, 117 (1961) . 14. B. ALEXANDER In: E. Deutsch ed. , Blood Clottin~Factors, proceedings of the Fourth International Congress of Biochemistry, p. 37, Pergamon Press, Inc ., New York (1959) . 15 . F. STREULI, Thrombos . Diathes . Haemorrh . 3, 194 (1959). 16 . K. LECHNER and E. DEUTSCH, Thrombos . Diathes . Haemorrh. _13, 314 (1965) . 17. W. H . SEEGERS and R. H. LANDA BURU, Proc. Soc . Exp . Biol. Med . 95, 710 (1957) . 18. W. R. THOMAS and W. H. SEEGERS, Biochim . Biophye . Acta 42, 556 (1960) . 19 . O. P. MALHOTRA and J. R. CARTER, Thrombos . Diathee . Haemorrh. 19, 178 (1968) . 20. F. KOLLER, A. LOELIGER and F. DUCKERT, Acta Haematol . 6, 1 (1951) . 21 . p. HJORT, S. I. RAPAPORT and P. A. OWREN, J . Lab . Clin. Med . 46, 89 (1955). 22. O. P. MALHOTRA and J . R . CARTER, Fed . Proceedings 24, 154 (1965). 23 . K. W. DENSON, Acta Haematol. 25, 105 (1961) . 24. H. C . HEMKER, A. D. MULLER and E. A. LOELIGER, Thrombos . Diathes . Haemorrh. 23, 633 (1970) . 25. O. P. MALHOTRA, Life Sciences, In Press . 26. H. V. JOHNSON J. MARTINOVIC and B. C . JOHNSON, Biochem . Biophye . Res . komm . 43, 1040 (1971) .