Experimental warfarin poisoning in the dog

J. COMP.

PATH,

1973. VOL. 83.

EXPERIMENTAL PLATELET

173

WARFARIN FUNCTION,

POISONING

COAGULATION

IN THE

AND

DOG

FIBRINOLYSIS

BY C.

D. FORBES, University

C.

THOMSON, C. R. M, G. P. MCNICOL*

Department

of Medicine, Glasgow Rgml

PRENTICE

and

In@nary

A. D. MCEWAN Department

of Veterinary

Medicine,

Universi[v

of Glasgow

INTRODUCTION

Poisoning of animals with anticoagulant compounds is a common clinical problem in veterinary practice. The depression of the synthesis of the vitamin K dependent factors in the liver is well documented, but this accounts only for a part of the profound haemostatic defect. In some experimental animals the bleeding time is also prolonged (Zucker, 1947 ; Newland and Nordoy, 1967), but it has not been established whether these changes are due to a specific platelet defect. Following warfarin ingestion there have been reports of decreased platelet adhesiveness (Spooner and Meyer, 1944 ; Murphy and Mustard, 1960) and increased adhesiveness (Horlick, 1961) in various animal species whilst O’Brien (1961) found no change in platelet adhesiveness in man after anticoagulant therapy. The object of this paper was to investigate haemostasis in dogs experimentally poisoned with warfarin. MATERIALS

AND

METHODS

Animals. The dogs used were 3 female and 3 male adult greyhounds with an average weight of 25 kg. ; they had no history of abnormal bleeding. On the first day blood samples were taken for baseline assays and then a standard dose of warfarin (10 mg.1 kg.) was given with their food. This method of administration was chosen as it more closely approximated to accidental poisoning. By day 4 or 5 of administration the dogs had stopped eating and so no further warfarin was ingested. Collection of blood. Blood samples were collected by clean venepuncture using plastic syringes. Nine vol. of blood from the foreleg or jugular vein were added to 1 vol. of 3.8 per cent. sodium citrate. Samples for clotting factor and fibrinolytic assay were kept on ice at 4 “C. and samples for platelet function tests were kept at room temperature. Platelet rich plasma. This was prepared from titrated whole blood by centrifugation at 6009 for 5 min. at room temperature. Adenosine diphosphate (ADP) .t A stock solution of 100 pg. ADP per ml. was prepared and kept at -20 “C. Dilutions referred to in the text were made from this. Collagen. This was prepared from human tendo Achilles. * Present address: Department of Medicine, The Martin Wing, Leeds General Infirmary, Leeds. t Sigma Chemical Co., St Louis, Missouri.

174

c.

D.

FORBES

et al.

Platelet aggregation. This was measured by the turbidimetric method of Born (1962). Platelet adhesiveness was measured by the glass bead column method using the principles described by Hellem (1960). The apparatus used in the study gave a mean contact time between blood and glass of 30 & 1 s. Euglobulin lysis time. This was carried out by the method described by Nilsson and Olow (1962). Results are presented as suggested by Sherry, Lindmeyer, Fletcher and Alkjaersig (1959) in arbitrary units of activity assigning to a lysis time of 300 min., one unit of activity. Urokinase sensitivity test. This was carried out as described by McNicol, Gale and Douglas (1963). Results are presented in arbitrary units, based on a double logarithm plot of lysis times against units of activity, and assigning one unit of activity to a lysis time of 10 min. Plasminogen assay. The method has been described by Remmert and Cohen (1949) and modified by Alkjaersig, Fletcher and Sherry (1959). Fibrinogen USXZJS.The method has been described by Ratnoff and Menzie (1951) as modified by Alkjaersig (1960). One-stage “pothrombin” time. The method has been described by Douglas (1962). Thrombin clotting time. The method has been described by Fletcher, Alkjaersig and Sherry (1959). Platelet count. This was performed by the method of Dacie (1956) using form01 citrate as the diluting fluid. Kaolin-cephalin clotting time. This has been described by Biggs and Macfarlane (1966). The bleeding-time. This was performed by the method of Ratnoff (personal communication). A No. 11 scalpel blade was used to make a stab wound 3 mm. deep on the dorsum of the nose of the dog. The time taken for complete cessation of bleeding was taken as the bleeding time. This was repeated before and after anticoagulant therapy in each animal. Assays. Warfarin assay was performed as described by O’Reilly, Aggeler, Hoag and Leong (1962). Assays of the specific coagulation factors were carried out as described by Breckenridge and Ratnoff (1965). Blood urea, bilirubin, alkaline phosphatase and serum oxaloacetic and pyruvic transaminases (SGOT, SGPT) were measured using standard autoanalyzer techniques. RESULTS

Clinical

Course

The clinical course was similar in all the animals; by day 4 or 5 they became apathetic and stopped eating and drinking. By day 7 or 8 their condition had deteriorated with the onset of anorexia and vomiting. At this time it was noted that excess bleeding had occurred at venepuncture sites. Two of the dogs developed severe respiratory distress, but had no radiological evidence of intrapulmonary bleeding; 2 died suddenly from intraperitoneal bleeding. The rest of the dogs were given parenteral vitamin K, (“Konakion”) and made a rapid clinical and biochemical recovery. Fig. 1 shows the rapid return of the clotting mechanism to normal after 100 mg. vitamin K,, given intravenously. One dog was given a trial of plasma infusion without effect. Assays Platelet count. No significant differences were found in the platelet before and after warfarin. The mean count before dosing was 164&31 161&40 x 10 platelets per mm.3. platelets per mm3 and after poisoning

counts x lo3

EXPERIMENTAL ma

y

WARFARIN

POISONING

K,lOOmg

IN Vltomin

Plasma I

175

DOG K, IOOmg

’ (b)

Warfarin

Fig.

1. Coagulation response to vitamin K,.

factor levels the infusion

in dog No. 1 at the time of maximum poisoning of 300 ml. of fresh dog plasma, (b) rapid response

treated

showing (a) to intravenous

no

Bleeding time. As seen in Fig. 2 warfarin treatment caused significant prolongation of the bleeding time. One of the dogs died of intraperitoneal bleeding before the test could be carried out. The mean value before therapy was 240 s. & 32 s. and after treatment 630 s. &270 s. Two animals had bleeding times in excess of 15 min. at which point the test was terminated. The differences are highly significant (P < O-001). Platelet adhesiveness. The mean adhesiveness before therapy was 41 per cent. f7 and at the time of maximum poisoning 37 per cent. &19. The results are not significantly different.

000

z

600

J

0 Fig.

2. Bleeding persisted

times performed from the wound

I

Pre

I

Past

on the dog nose before and after for 24 h. in the 2 dogs with bleeding

warfarin poisoning. Bleeding times in excess of 15 minutes.

176

et

C. D. FORBES

al.

Platelet aggregation. No significant difference was obtained before and after warfarin therapy. The mean optical density 2 min. after addition of ADP in the pre-treatment group was 0*45&0.09 compared with 0*46&0.10 after therapy. In 3 dogs platelet aggregation by collagen was tested and no significant differences were found. Euglobulin I_si.s. The time was significantly prolonged after warfarin treatment (Fig. 3). There was no significant difference in the urokinase sensitivity test before and after warfarin.

-.

..A .+%.i \\\\

.

Pre

Fig.

3. Euglobulin

lysis times

l

before

and after

Post

poisoning

with

warfarin.

Thrombin clotting time, prothrombin and kaolin-cephalin clotting time (KCCT) . The results are shown in Table 1. There was no significant change in the thrombin clotting time after warfarin administration. As expected, there were significant changes in the prothrombin time and the KCCT (P < 0.001).

Thrombin

clotting time

One-stage prothrombin time

(s.1

Dog

BtTfOW

After

Before

Kaolin-cephalin

(s.j

time

After

Before

c1ottin.g (s.)

24fter

1 3 4

ti 11 8

11 10.5 12 9

180 180 180 180

27 ;:

180 68

12 IO.5

19

1:;

;

1:

12 13

1:

180

19 19.5

108 87

EXPERIMENTAL

WARFARIN

POISONING

IN DOG

177

Factors 11, IX and X. The levels of these factors at the time of maximum poisoning showed the predictable highly significant fall. Factor II level was 7 per cent. 16 per cent., factor IX was 3.5 per cent. 13 per cent., factor X was 3 per cent. &2 per cent. Factor VII was not assayed in all animals. Fibrinogen and plasminogen levels. No significant differences were found in these levels after warfarin poisoning. Warfarin levels in plasma. These are shown in Table 2. The mean plasma levels on day 4 were 32.6 pg./ml. and on day 7 were 11.9 pg./ml. TABLE 2 WARFARIN

LEVELS

Level &Lg./ml.) Day 4

Day 7

1 2 3 4

32.6 30.6 38.0 18.6

10.2 19.5

ii Mean

43.2 32.6

13.8 7.8 11.9

Dog

8<

Plasma urea and liverfunction tests. The blood urea rose in all 6 dogs, the level after poisoning being significantly different from the pre-treatment level (Fig. 4). There was no correlation with the degree of illness of the dog and none of

Fig.4.

The

rise in the blood

urea after

warfarin

poisoning.

178

C. D.

FORBES

et

d.

the animals had evidence of gastro-intestinal bleeding. Bilirubin, alkaline phosphatase, serum glutamic oxaloacetic transaminase (SGOT) and glutamic pyruvic transaminase (SGPT) showed no significant change. DISCUSSION

The effect of coumarin drugs on the hepatic enzyme system responsible for the synthesis of factors II, VII and X is well documented. When warfarin is administered there is a delay of up to 48 h. before reduction in the level of factor VII is seen. This is followed by a fall in the levels of factors IX, X and II, in sequential order (Douglas, 1962). The rate of onset of this effect is the same whether a large loading dose is used or not, up to a certain critical dose level (0.7 mg. warfarinlkg. body weight). This was confirmed in our experiments; despite high plasma levels of warfarin, on day 4 (mean 32.6 pg/ml.) the maximum reduction in the levels of the plasma coagulation factors did not occur until the 7th day by which time the plasma level of warfarin had fallen (mean 11.9 pg./ml.). The fall in plasma-warfarin level was probably due to the inability of the sick dogs to ingest the warfarin tablets after the fourth day of administration. Depression of factors II, IX and X followed the predicted pattern with factor II being less severely depressed than IX or X. As seen in Fig. 1 the administration of vitamin K, (Konakion) intravenously rapidly reverses the action of warfarin and results in a fall in the one-stage prothrombin time to normal and a rise in the levels of factors II, VII, IX and X. In the example shown there was no significant benefit from the infusion of 300 ml. of fresh dog plasma. In all the animals there was a significant increase in the bleeding time. This was most marked on the 7th day when the animals were most severely poisoned and this coincided with maximal depression of the coagulation factors. In 2 of the animals the bleeding time was greater than 15 min. and oozing continued intermittently from the wounds for the next 24 h. despite vitamin K, therapy. Dog 4 showed only slight prolongation of the bleeding time and this corresponded to less marked depression of the levels of coagulation factors than in the other dogs. Excessive coumarin administration in experimental animals has been previously shown to prolong the bleeding time (Zucker, 1947) and Newland and Nordoy (1967), using the tail bleeding time in rats given toxic doses of warfarin, found that many of the animals died of exsanguination from this wound. Histology of the vessels in the tails of the warfarin-treated animals showed unstable loosely aggregated platelets, whereas in the control animals the vessels were plugged with densely packed platelets with fibrin strands at the periphery of the platelet mass both inside and outside the vessel. Similarly, it is possible that in dogs a degree of fibrin formation is necessary for primary haemostasis and that in our animals the prolonged bleeding time was due solely to this defect. Coumarin anticoagulants have also been shown to have a direct action on the walls of capillaries causing loss of plasma proteins from the blood stream (Nelson, 1960) vasodilatation (Bingham, Meyer and Pohle, 1941) and increased vascular fragility (F&on, Akers and Lutz, 1953).

EXPERIMENTAL

WARFARIN

POISONING

IN

DOG

179

In these studies we detected no abnormalities in the platelet function tests of the dogs despite the high warfarin level achieved. There have been numerous reports in other species of platelet abnormalities after anticoagulants both in therapeutic and toxic doses (Cunningham, McNicol and Douglas, 1965; Murphy and Mustard, 1960). This may be due to the marked interspecies variations in the reactions of platelets to aggregating agents (Le Roy, Mason and Brinkhous, 1960). The only significant change in the fibrinolytic enzyme system was a decrease in the euglobulin lysis time. This could be consistent with stress as the dogs became well. As the urokinase sensitivity test was unchanged, this probably represented increased amounts of circulating plasminogen activator. In therapeutic doses, however, oral anticoagulants have been shown to produce no change in fibrinolysis (Lackner and Merskey, 1960) or in fibrinogen level (Sherry et al., 1959). No changes were seen in various biochemical tests of liver function (bilirubin, alkaline phosphatase, SGOT and SGPT). All the animals had a rise in blood urea which was not due to intestinal blood loss. It probably represents dehydration as at the point of maximum poisoning they stopped eating and drinking. No specific changes attributable to a toxic action of warfarin were found in the histology of the kidneys in the dogs that died. SUMMARY

Experimental warfarin poisoning in dogs produced a profound haemostatic defect with depression in the levels of factors II, VII, IX and X and gross prolongation of the bleeding time. Surprisingly platelet function was unaltered suggesting that in the dog the intrinsic coagulation pathway plays an important part in the normal bleeding time. There was slight shortening of the euglobulin lysis time, but no other changes in the components of the fibrinolytic enzyme system. Rapid reversal of the clotting abnormality was produced by the intravenous injection of vitamin K,. REFERENCES

Alkjaersig, N., Fletcher, A. P., and Sherry, S. (1959). J. clin. Invest., 38, 1086. Alkjaersig, N. (1960). In Conference on Thrombolytic Agents, Eds. H. R. Roberts and J. D. Geraty, p. 316, University of North Carolina Press; Chapel Hill, N.C. Biggs, R., and Macfarlane, R. G. (1966). In Treatment of Haemophilia and Other Coagulation Disorders, Blackwell Scientific Publications; Oxford. Bingham, J. B., Meyer, 0. O., and Pohle, J. F. (1941). Amer. 3. med. Sci., 202, 563. Born, G. V. R. (1962). Nature, Lond., 194, 927. Breckenridge, R. T., and Ratnoff, 0. D. (1965). 3. clin. Invest., 44, 302. Cunningham, G. M., McNicol, G. P., and Douglas, A. S. (1965). Lancet, i, 729. Dacie, J. V. (1956). In Practical Haematology, 2nd edit., Churchill; London. Douglas, A. S. (1962). In Anticoagulant Therapy, Blackwell Scientific Publications; Oxford. Fletcher, A. P., Alkjaersig, N., and Sherry, S. (1959). J. clin. Innvest., 38, 109. Fulton, G. P., Akers, R. P., and Lutz, B. R. (1953). Blood, 8, 140. Hellem, A. J. (1960). &and. 3. clin. Lab. Invest., 12, Suppl., p. 51. Horlick, L. (1961). Amer. 3. Cardiol., 8, 459. Lackner, H., and Merskey, C. (1960). Brit. 3. Haemat., 6, 402.

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Le Roy, E. C., Mason, R. C., and Brinkhous, K. M. (1960). Amer. J. Physiol., 199, 183. McNicol, G. P., Gale, S. B., and Douglas, A. S. (1963). Brit. med. J., i, 909. Murphy, E. A., and Mustard, J. F. (1960). Circulat. Res., 8, 1187. Nelson, T. E. (1960). Ibid., 8, 889. Newland, H., and Nordoy, A. (1967). Cardiovasc. Res., 1, 362. Nilsson, I. M., and Olow, B. (1967). Acta chir. stand., 123, 247. O’Brien, J. R. (1961). 3. clin. Path., 14, 140. O’Reilly, R. A., Aggeler, P. M., Hoag, M. S., and Leong, L. S. (1962). Thromb. Diathes. Haemorrh., 8, 82. Ratnoff, 0. D., and Menzie, C. (1951). 3. Lab. clin. Med., 37, 3 16. Remmert, L. F., and Cohen, P. P. (1949). 3. biol. Chem., 181: 431. Sherry, S., Lindmeyer, R. I., Fletcher, A. P., and Alkjaersig, N. (1959). J. clin Invest., 38, 810. Spooner, M., and Meyer, 0. 0. (1944). Amer. J. Physiol., 142, 279. Zucker; M. B. (1947). Ibid., 148, 275. [Received for publication,

March

6th, 19721