Original Papers Haemostasis 7: 189-201 (1978)

Rat Coagulation Factors V, VIII, XI, and XII: Vitamin K Dependent Charles A. Owen, jr. andE.J. Walter Bowie Department of Biochemistry and Section of Hematology Research, Mayo Clinic and Mayo Foundation, Rochester, Minn.

Key Words. Vitamin K-dependent factors • Vitamin K deficiency • Warfarin • Biliary fistula • Perfused rat liver Abstract. When rats were given single or multiple doses of warfarin, the levels of prothrombin and factors VII, IX, and X were depressed, as expected. However, modest reductions of factors V, VIII, XI, and XII, but not of fibrinogen, also occurred. The levels of all eight factors promptly returned to normal when vitamin K, was given. Warfarin-resistant rats had no depression of any of the eight factors. When vitamin K deficiency was induced by internal or external biliary fistula, factors II-VII-IX-X decreased sharply and factors V-VIII-XI-XII decreased modestly. Again, all depressions were promptly reversed by vitamin K,. Isolated livers from warfarinized rats did not generate the classic vitamin K-dependent factors during 5 h of perfusion but did generate small amounts of factors V, XI, and XII, although less than normal. The isolated rat liver apparently does not generate factor VIII.

Introduction Vitamin K deficiency or the use of oral anticoagulants depresses four plasmatic coagulation factor activities, namely factors II (prothrombin) and factors VII, IX, and X. Recently, another factor, of unknown function, has been observed by Stenflo and Ganrot (30). The literature, as reviewed by Kazmier

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Received: October 18, 1977; in revised form: December 2, 1977; accepted by editor P. Brakman: December 12, 1977.

Owen, jr. /Bowie

190

(10), has had very few reports of the effect of vitamin K or oral anticoagulants on other plasmatic clotting factors. In 1949, Olwin (14) observed a decrease of 40% in the levels of factor V of his patients receiving dicumarol. He and his associates (4) found comparable depressions in dogs given dicumarol. They further noted that changes in factor V of patients given anticoagulants tended to be self-correcting after a month or so of continuous anticoagulant treatment. Greater depressions of factor V were observed by Perlick (22) to between 30 and 40% of prephenindione levels —at a time when factor VII levels were between 10 and 20% of normal. Despite these claims, Quick and Stefanini (25), Owren and Aas (20), and Douglas (2) could not confirm the findings. In 1965, Jaques ( 8) noted, Other components of the coagulation system are also affected by the indirect anti­ coagulant drugs when given in larger doses or on prolonged dosage fibrinogen (I), antihemophilic globulin (VIII), Christmas factor (IX).

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No other suggestion that oral anticoagulants depress factor VIII levels could be found. In fact, levels of factor VIII have been observed to increase moder­ ately in patients given coumarin drugs (2, 3, 23, 24, 29). Graham (5) dismissed any relationship between dicumarol and factor VIII. The evidence for a link between vitamin K and the contact activation factors XI and XII is also obscure. Naeye (13) noted reductions of factor XI levels in patients with nontropical sprue (9% of normal), obstructive jaundice (40%), and fibrocystic disease (7%). He also found factor XI levels of 22 and 24% in 2 patients receiving dicumarol. In all 5 patients, the factor XI level, as well as the levels of other factors, promptly increased when vitamin K was administered. By contrast, the lowest levels of factor XI detected by Rapaport (26) in 14 patients receiving dicumarol, phenindione, or warfarin were between 68 and 84% of normal. Egeberg (3) found no trend in patients receiving phenindione. Boyles and Nichol (1) found depressions of factor XII in 300 patients receiving various anticoagulants. But Jim and Goidfein (9) did not observe any change in a newborn infant or in a patient receiving dicumarol. Further, contact activation was normal, apparently reflecting adequate levels of both factors XI and XII, in patients given oral anticoagulation (28,33). The effect of vitamin K is apparently not limited to the coagulation mechanism - reviewed by Owen (15) and the malformations induced in fetuses early in pregnancy by oral anticoagulants are preventable by the use of

Rat Coagulation Factors V, VIII, XI, and XII

191

vitamin K, (11). That a primary or secondary deficiency of vitamin K might have a broader influence than on just the four classic vitamin K-dependent factors might not be too surprising, and is, in fact, what we report here.

Materials and Methods

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Adult male Sprague-Dawley rats, weighing between 300 and 400 g, were used. Warfarin-resistant rats were from our colony (obtained originally through the kindness of Dr. John W. Suttie, Madison, Wise.). Vitamin K deficiency was induced in normal rats by two biliary fistula techniques: one external and the other choledochocystostomy. In the latter procedure, a No. 24 soft plastic tube of 5 -6 cm length was doubly ligated in the bile duct, and the other end (after heating to produce a flange) was put in the urinary bladder and held in place by a single stitch around the hole and tube. Choledochocystostomy was the more satisfactory, and between 2 and 4 weeks, a severe vitamin K deficiency had regularly occurred. Isolated livers of rats were perfused for 5 h with washed, frozen rat erythrocytes suspended in Tyrode’s solution containing 6% albumin (16, 17). Although the perfusates were assayed every 1 -2 h, only the 5-hour values are reported. Warfarin sodium was administered intravenously in single doses of 0.15-2 mg or about 0.5-6 mg/kg body weight. The usual priming dose in man is about 0.5 1 mg/kg. When rats received daily warfarin sodium for 4 -1 2 days, doses ranged from 0.025 to 0.15 mg or about 0.075 0.45 mg/kg. The preparations (Coumadin, Endo Laboratories, Garden City, N.Y.) were freshly made by diluting the original solution (75 mg/ampule) in saline such that the volumes of the injections varied from 0.5 to 2.0 ml, usually 1.0 ml. Vitamin K, (Aquamephyton, Merck Sharp & Dohme, West Point, Pa.) was always administered once in a 1.0-mgdose (0.1 ml) intraperitoneally. Fibrinogen was measured by a biuret assay of thrombin-clottable protein (18, pp. 134 135). Prothrombin was assayed by the two-stage test (19) using rat brain, acetonedried thromboplastin. Factors V, VII, and X were measured by a prothrombin time assay. One volume of serially diluted, normal, citrated rat plasma was added to 9 vol of human plasma lacking factor V, VII, or X, and a prothrombin time was performed. The unknown rat plasmas, in the same proportions, were similarly mixed, and the concentration of each factor was assessed from the normal rat plasma curve plotted on a double-logarithmic scale (18, pp. 139 140). Factors VIII, IX, and XII were measured by the activated partial thromboplastin time assay technique, again using human plasmas congenitally deficient in a single factor (18, pp. 140 -141). One might question the use of human plasmas as sub­ strates. However, all percentages were calculated by comparisons of the clotting times of the unknown rat plasmas with serially diluted normal rat plasma, not with normal human plasma. Because all six of these substrate plasmas contained normal concentrations of 5 of the 6 factors being studied V, VII, VIII, IX, X, and XII and because the substrate plasmas constituted 90% of the final plasma mixtures, any variation in the concentration in these factors in the unknown plasmas could influence the total concentrations of those factors not being assayed by only 10% at most. Neither the prothrombin time test nor the

Table I. Responses of coagulation factors to single intravenous injections of warfarin sodium' VII

IX

X

(8) (6) (4) (3) (2)

93(6) 69 (6) 76(2) 71 (3) 200 (2)

100(2) 64 (4) 59 (4) 90(2)

82(4) 68 (6) 75 (2) 71 (3) 100 (2)

47 (4) 5 (4) 24 (6) 83(4)

245 283 287 315

97 (4) 49 (9) 76(6) 100(2)

100(2) 54(7) 64 (6) 88 (2)

— 58 (5) 51 (4) 76(2)

100 (2) 57(7) 70 (6) 100 (2)

69 (4) 12(16) 8(6) 28 (5)

14(4) 5 (16) 16(8) 56 (5)

260 (4) 263 (2) -

82(8) 44 (18) 52(8) 67 (5)

91 (4) 58 (18) 54 (6) 56(5)

70 (8) 23(9) 6(5) 16(6) 40 (5)

34(8) 5(9) 1 (7) 35(3) 79 (5)

_

74 (12) 50(17) 48 (7) 50(7) 88 (5)

75 66 47 40 83

89(8) 73(4) 80(2) 96 (4) 110 (2)

32(8) 5 (6) 54 (4) 71 (4) 93(2)

69 (8) 23(6) 33 (2) 56 (4) 91 (2)

29(8) 11 (4) 51 (4) 86 (4) 100 (2)

0.50

8 24 48 72

74 (4) 21 (2) 36 (4) 107 (2)

32 (4) 1 (9) 19(6) 87 (4)

88 (4) 27 (4) 16(6) 62 (4)

1.0

8 24 48 72

8 (6) 26(6) 50 (5)

17 4 8 45

-

23(12) 2(17) 1 (7) 11 (8) 77 (5)

2.0

8 24 48 72 96

XII

70 55 65 81 105

8 24 48 72 96

(8) (18) (8) (5)

XI

280 (4) 232 (2)

0.15

_

VIII

V

I

(2) (2) (4) (2)

290(2) 312 (2) 295 (2) -

(10) (16) (5) (7) (5)

88 46 56 56

(2) (18) (6) (5)

100 (2) 60(10) 25 (4) 47 (3) 76 (5)

81 59 56 59

(2) (18) (6) (5)

96 (7) 65 (15) 59(5) 58 (10) 79 (5)

192

' Levels were measured 8 96 h after injections. Results are means; the numbers of rats are indicated in parentheses. Factor I (fibrinogen) is expressed in mg/dl plasma; all other factors are expressed as percent of normal rat plasma.

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II

Owen, jr./Bowie

Dose, mg Hours

Rat Coagulation Factors V, VIII, XI, and XII

193

activated partial thromboplastin time test can distinguish between concentrations of 90 and 100% of any factor. Factor XI was assayed w'ith bovine factor Xl-deficient plasma generous­ ly donated by Dr. Gary Kociba of Cleveland, Ohio. In all instances, normal rat plasma was used for control.

Results Single Doses o f Warfarin to Normal Rats (table I) No change in fibrinogen was evident; the normal rat fibrinogen value in our laboratory is between 247 and 331 mg/dl (± 2 SD). As expected, levels of factors II, VII, IX, and X decreased with increasing doses of warfarin, but prothrombin and factor IX levels decreased somewhat less than did the other two factors. If the reduction of factor VII level, with all doses and at all intervals of time, was arbitrarily called ‘100%’, the average depression of factor X was 91%, of factor IX 83%, and of prothrombin 60%. Factors V, VIII, XI, and XII also decreased but more moderately. Again, if the average factor VII depression is 100%, factor XI decreased on the average 49%, factor V 45%, factor VIII 41%, and factor XII 37%. Multiple Doses o f Warfarin to Normal Rats (table II) Eight factors were regularly measured, but fibrinogen was measured only occasionally. All the eight factors were depressed by the warfarin in a dosedependent fashion, with factors VII, IX, and X again being the most sensitive. Least sensitive was factor VIII, which was virtually unaffected by daily doses of 0.06 mg or less. When the daily dose reached 0.10 mg of warfarin, the levels of factors II, VII, IX, and X had decreased more than 80%, but levels of the other four factors had decreased less than half (factors VIII and XII 47%, factor V 40%, and Factor XI 28%). Responses o f Warfarinized Rats to Vitamin K , (table III) When vitamin K] was injected in 1-mg doses 24 h after rats received 1.0 mg of warfarin intravenously, and blood was drawn for assay 24 h after the injection of vitamin K, the larger reductions of factors VII, IX, and X and the more modest reductions of factors II, V, VIII, XI, XII were abolished.

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Warfarin-Resistant Rats In our rat colony, doses of warfarin of 1 mg or less have no effect on the clotting factors of warfarin-resistant rats and doses of 2 mg have little or no

Table II. Responses of coagulation factors to multiple intravenous injections of warfarin sodium'

0.025 0.05 0.06

0.10 0.15

4 4 4 8 12 4 11 4

II

VII

IX

X

I

V

VIII

XI

XII

86 (2) 64 (6)

75 (4) 41 (6) 25 (2) 15 (2) 44 (4) 5 (4) 20 (2) 2(4)

64 (4) 16(6) 17(2) 11 (2) 19 (4) 4(4) 7 (2) 1 (4)

72(4) 29 (6) 39 (2) 20 (2) 46 (4) 11 (4) 30(2) 3(4)

_

99 (4) 64 (6) 93 (2) 84(2) 72(4) 52 (4) 18(2) 36 (4)

87(4) 93(6) 100(2) 100 (2) 100 (4) 53(4) 42(2) 67(4)

76 (4) 66 (6) 88 (2) 46(2) 50(4) 65 (2) 33(4)

89 88 76 86 61 45 69

-

16(2) 11 (2)

Rat coagulation factors V, VIII, XI, and XII: vitamin K dependent.

Original Papers Haemostasis 7: 189-201 (1978) Rat Coagulation Factors V, VIII, XI, and XII: Vitamin K Dependent Charles A. Owen, jr. andE.J. Walter B...
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