PLATELET FUNCTION IN VENOUS THROMBOSIS AND LOW-DOSAGE HEPARIN

1302

PLATELET FUNCTION IN VENOUS individual uraemic patients. We do not know whether an of indicator this will prove to be total-body magTHROMBOSIS AND LOW-DOSAGE nesium excess. However, the findings of Wallach et al. HEPARIN of a rapid rise in red-blood-cell magnesium in a M. ETHERINGTON J. R. O’BRIEN patient with acute renal failure and the high redM. R. KLABER SANDRA JAMIESON blood-cell magnesium seen in hypothyroidism suggest Portsmouth and Isle of Wight Area Pathology Service that factors other than total-body magnesium may influence erythrocyte-magnesium levels.7 We and others ó,6 have found muscle-magnesium Compared with preoperative levels, Summary content to be normal or raised in patients with chronic patients immediately after a major renal insufficiency. This finding is at variance with operation showed a decrease in the platelet-aggregation the studies of Lim and co-workers.3,44 However, response to adenosine diphosphate (A.D.P.) and thrombin, but the percentage of platelets retained in a glassmuscle-magnesium content may not reflect total-body bead column was increased. By the next day the results magnesium. Muscle magnesium varies with potassium of the aggregation tests and of the glass-bead-column balance, being low with potassium deficiency, and is test had all exceeded the preoperative levels. Heparin low in protein-calorie malnutrition syndromes.14,15 Our findings suggest that muscle-magnesium content (5000 U. subcutaneously) prevented the immediate is not a good indicator of magnesium excess. postoperative decrease in aggregation. It is tentatively Little is known about the significance of soft-tissue suggested that a decreased responsiveness to A.D.P. and is related to the cause of postoperative thrombin excess. The increased magnesium content magnesium found in myocardium, lung, and skin samples in deep-vein thrombosis. uraemic patients has been shown to be related to the Introduction excess calcium content of these tissues.l6 Also magnesium has been found in a constant molar ratio with SINCE platelets contribute to a thrombus they might calcium and phosphorus in visceral calcification in be expected to demonstrate increased responsiveness uraemic patients, and is felt to be an integral part of when thrombosis occurs. There are, however, only a this material. 17 Whittier and Freeman 18 showed that few reports of such an increase. 1-We have reported a magnesium excess potentiates the degree of soft-tissue statistically significant decrease in response to A.D.P. in calcification occurring in vitamin-D-intoxicated rats. patients who later developed venous thrombosis after They also found significant correlation between caloperations compared with those with no thrombosis.5 cium and magnesium content in soft tissue and suggesImmediately after major operations the plateletted that a (Ca)x(Mg)y(P04)z compound was formed. aggregation response to A.D.P. and to collagen when Bone-magnesium content may be a better indicator tested in vitro was decreased: a temporary refractory of magnesium stores than other tissue. Neuman and state had developed. In-vitro evidence suggested Mulryan have shown that apatitic magnesium is a that this decrease might be due to exposure of the surface-limited ion and is readily exchangeable. 19 Siniiplatelets to A.D.P. in the body.6 These findings larly, Blaxter showed that up to 75% of skeletal magmight be related to postoperative venous thrombosis. nesium was lost in calves with severe magnesium depletion. 20 Conversely, Clark and Belanger 21 and DR. CONTIGUGLIA AND OTHERS: REFERENCES Cunningham 22 showed that bone content of magne1. MacIntyre, I., Hanna, S., Booth, C., Read, A. E. Clin. Sci. 1961, sium parallels the serum-level and that the skeleton 20, 297. 2. Lim, P., Jacob, E., Dong, S., Khoo, O. T. J. clin. Path. 1969, 22, can sequester large amounts of absorbed magnesium. 417. Thus the increased bone magnesium found in uraemic 3. Lim, P., Chir, B., Dong, S., Khoo, O. T. New Engl. J. Med. 1969, 280, 981. patients may be a consequence of raised plasma 4. Lim, P., Khoo, O. T. Br. med. J. 1969, i, 414. magnesium levels. 5. Maschio, G., Campanacei, L., Mioni, G., Bazzato, G., Bruschi, E., In 1933 Cunningham suggested that magnesium Riz, G., Gianfranceschi, G. Minerva Nefrol. 1969, 16, 41. 6. Bergström, J., Hultman, E. ibid. p. 33. excess had an adverse effect on bone formation in 7. Wallach, S., Cahill, L. N., Rogan, F. H., Jones, H. L. J. Lab. clin. rats. Clark and Belanger have shown that magnesium Med. 1962, 59, 195. 8. Mahler, D., Walsh, J., Heynie, G. Am. J. clin. Path. 1970, 56, 17. excess will produce a demineralised matrix in rats.21 9. Schmidt, P., Katzaurek, J., Zazgornik, J., Hysek, H. Clin. Sci. 1971, Magnesium has also been shown to stabilise amorphous 41, 131. 10. Mansouri, K. J., Halstead, A., Gombos, E. Archs intern. Med. secondary calcium phosphate and inhibit hydroxy1970, 125, 88. apatite formation.23, 24 11. Shils, M. E. Am. J. clin. Nutr. 1964, 15, 133. 12. Walser, M. Ergeben. Physiol. 1967, 59, 185. Pletka et al. 25 have suggested that high-magnesium 13. Peliligrino, E. D., Biltz, R. Medicine, Baltimore, 1965, 44, 397. dialysis may suppress hyperparathyroidism in dialysed 14. Baldwin, D., Robinson, P., Zierler, K., Lilienthal, J. L. J. clin. Invest. urasmic patients and thus may be effective in reducing 1952, 31, 850. 15. Wacker, W. E., Parisi, A. New Engl. J. Med. 1968, 278, 712. the incidence of bone disease. However, our findings 16. Contiguglia, S., Alfrey, A., Miller, N., Hammond, W., Ogden, D. suggest that high-magnesium dialysis would further Clin. Res. 1972, 20, 245. 17. Contiguglia, S., Alfrey, A., Miller, N., Runnels, D. ibid. p. 590. increase bone and total-body magnesium content. 18. Whittier, F., Freeman, R. Am. J. Physiol. 1971, 220, 209. Until more is known about magnesium excess and its 19. Neuman, W. F., Mulryan, B. J. Calc. Tissue Res. 1971, 7, 133. 20. Blaxter, K. L. in Bone Structure and Metabolism (edited by G. E. W. relation to metastatic calcification and osteomalacia Wolstenholme and C. M. O’Connor); p. 117. London, 1956. this form of therapy is unwarranted. 21. Clark, I., Belanger, L. Calc. Tissue Res. 1967, 1, 204. 22. Cunningham, I. N.Z. Jl Sci. Tech. 1933, 15, 191. Requests for reprints should be addressed to A. C. A., Veteran! 23. Posner, A. Physiol. Rev. 1969, 49, 760. Administration Hospital, 1055 Clermont, Denver, Coloradc 24. Bachra, B., Trautz, O., Simon, S. Archs oral Biol. 1965, 10, 731. T A 25. Pletka, P., Bernstein, D., Hampers, C., L. M. Lancet, 1971, ii, 462.

Merrill, J. P., Sherwood,

1303

There have been

two reports 7,8that subcutaneous in small doses greatly decreases the incidence heparin of postoperative thrombosis as detected by the 1251tagged-fibrinogen method. The extent of the platelet contribution to the fibrin deposited in venous thrombosis is unknown, nor is it known whether the decreased platelet responsiveness has any influence on thrombosis; nevertheless, it seemed worth while to study the effect of heparin on the postoperative change in platelet aggregation.

Patients and Methods

Patients The thirty-six patients in the main study all had thoracotomies. Twenty-one of these-the controls-were studied twice before the operation, immediately after operation and on the first postoperative day. Eight patients were similarly studied but were given 5000 u. heparin subcutaneously 2 hours preoperatively and 12-hourly thereafter. The treatment of these patients was not revealed while the tests were carried out. Seven patients were studied only during the preoperative period. They were studied twice before heparin was given and twice after the

12-hourly injections began. Methods Blood was collected slowly by syringe; some was citrated (032%) and some was used at once to determine the percentage retention of platelets when whole blood was passed rapidly through a column of glass beads.9 On each occasion two counts were made from each of two glass-bead columns and the four results were averaged. Platelet-rich plasma (P.R.P.) was prepared and used at once to determine the amount of platelet aggregation 10 induced by a small dose of A.D.P. (M x 10-7 or 2M x 10-7) as appropriate. Aggregation was also induced by thrombin 0’1 u. per ml. (final concentration). The aggregation results were adjusted according to the platelet-count. Platelet-counts were performed on sedimented ethylenediaminetetraacetic acid (E.D.T.A.) blood using a Coulter counter (model B) and a

Fig. 2-Mean percentage change relative

immediately after

preoperative results operation. heparin, tested with A.D.P. to

and 24 hours after

0- • 21 patients not on •- - - 8 8 patients on heparin, tested with A.D.P. 0- 0 17 patients not on heparin, tested with thrombin. 0- - - 0 8 patients on heparin, tested with thrombin. p-- [] 7 patients not on heparin, glass-bead-column retention test. 0- - - [] 8 patients on heparin, glass-bead-column retention test.

70 µ aperture. The mean platelet size was determined on the Coulter mean-cell-volume computer. The thrombin clotting-time was determined by adding 0-1 ml. of thrombin (1 u. per ml.) to 0-1 ml. P.R.P. The heparin/thrombin clotting-time was determined by adding 0.1 ml. heparin (0.4 u. per ml.) to 0-1 ml. citrated whole blood, and twenty seconds later 0.1 ml. of thrombin (10 u. per ml.). These tests were carried out at least in duplicate in siliconed glass tubes at 37 °C.

Results

The Effect of Operation The results from the twenty-one control patients studied before and after operation, who received no heparin, confirmed and amplified our previous findings.The A.D.P.-adjusted slopes for the two preoperative tests were averaged and the difference between this and the postoperative result was expressed as a percent-

Fig. 1—Individual

results for the adjusted slope of A.D.P.

aggregation and percentage retention in glass-bead column expressed as percentage change relative to mean preoperative results.

Patients - - - - -

on

heparin compared with controls (no heparin),

indicates

mean

value.

age of the preoperative value. Eighteen out of the twenty-one patients had a decrease in response to A.D.P. immediately after the operation; the average decrease was 56% (fig. 1). The following day the aggregation responses had returned to their preoperative level, and indeed often showed increased responsiveness; the average result was then +41% (fig. 2). The effect of operation on the glass-bead-column retention test was completely different from that on A.D.P.-induced aggregation. Immediately after operation there was a pronounced rise in retention ( ,’-86%), and as previously found5 this was still greatly increased the next day (-r63%) (fig. 2). The Effect of Heparin and Operation Of the eight patients on heparin studied fully, seven demonstrated a relative increase in platelet response to A.D.P. immediately after operation; one showed a decrease in response and the mean figure was -21% (fig. 1). By the next day (fig. 2) the mean response had increased to +31 % and was then similar to the results

1304 MEAN REACTION-TIME

(SECONDS)

AND

ADJUSTED AGGREGATION SLOPB

Number of observations in parentheses.

of the control patients not on heparin (+41 %). The difference in the percentage change immediately after the operation between those on heparin (mean +21 %) and the controls not on heparin (mean -56%) is

significant (p<0.05). The platelet-retention test value rose immediately after the operation-a mean increase of 89%, which was similar to the figure for the controls (+86%). Next day (fig. 2) the results were still greatly increased relative to the preoperative figure and again similar to the controls.

The Effect of Heparin Alone Theoretically, since heparin reversed the effect of operation on platelet aggregation it is possible that the observed small increase in aggregation might be due to a balance between moderate inhibition caused by the operation and pronounced potentiation caused by the heparin. Accordingly, seven patients were studied preoperatively twice before and twice after heparin therapy was begun. There was no change in the aggregation tests; the mean values for A.D.P.-induced platelet aggregation before heparin and on heparin were 1-00 and 0-91, respectively, and for thrombin 0-77 and 0-77. (These figures are the observed slope squared, divided by the platelet-count.)

by Thrombin All these patients had undergone a thoracotomy and so were comparable, but we have not carried out enough thrombin-aggregation tests on thoracotomy patients. Nevertheless, results are available from seventeen patients who had various operations and no heparin and the eight thoracotomy patients on heparin. These groups are not strictly comparable and no formal analysis was carried out. Nevertheless, the means are reported in fig. 2. They indicate that the thrombinaggregation response decreases immediately after operation (mean value -280/’) in the controls and that heparin prevents this decrease (11 %). These findings then are similar to those for A.D.P. In the untreated controls the thrombin-aggregation response increased after 24 hours relative to the preoperative level (+12%). This result is similar to the 1-day mean results for the patients on heparin (+8%). Thus the effect of heparin on the postoperative aggregation response to a challenge with thrombin is the

Aggregation

WITH

*

A.D.P., THROMBIN, AND ADRENALINE AS AGGREGATING AGENTS’*

Data derived from O’Brien

et

al., 1971.6

per c.mm., but this was not significant. The of those patients treated with heparin did not differ from those without heparin. The platelet size of the three treatment groups of patients showed no difference between groups and no change after

208,000 counts

operation. Clotting-tests The thrombin clotting-time of P.R.P. was not significantly changed by thoracotomy; the patients given heparin and those given none had similar results, and this agrees with the published figures.’,’ However, all seven patients on heparin studied only before operation had prolonged thrombin clotting-times (i.e., 27 seconds before heparin and 31 seconds on heparin). Similarly, the heparin/thrombin clotting-times were prolonged from 40 to 46 seconds. Thus heparin in this dosage without operation does produce a slight but detectable extension of two sensitive clotting-times. In both the controls and those patients on heparin the heparin/thrombin clotting-time of whole blood was on average shorter after operation (36 seconds) than before operation (46 seconds). This difference was not significant but it agrees with two other reports.5,11 Thus apparently more heparin is inactivated after operation, indicating an increase in anti-heparin activity or platelet factor 4.

Induced

that with A.D.P. The whole-blood platelet-count

same as

immediately after the operation (average 248,000 per c.mm.) showed no change from the preoperative level (259,000 per c.mm.). By the first day the count had fallen on average to

Discussion to thrombosis and transient reduction in platelet aggregation induced by A.D.P., and probably by thrombin, which occurs for only a few hours after

Operations predispose patients

it is established that there is

a

major operations. Heparin in small subcutaneous doses noticeably decreases the incidence of thrombosis as detected by the 125I-fibrinogen method.’,’ Heparin also prevents the postoperative reduction in platelet-aggregation response. Heparin alone without operation has no on these tests. Platelet aggregation induced by A.D.P., thrombin, and adrenaline is less preoperatively in those patients who subsequently get a thrombus than in those patients who do not get a thrombus. This difference

effect

after operation. The trend in all the tests consistent and a number of these comparisons

persists was were

highly significant

5

(see accompanying table).

The response to A.D.P. and probably to thrombin is decreased in those patients who subsequently get postoperative venous thrombosis and in patients undergoing operation whether they get a thrombus or not, provided they are not on heparin. Patients under-

1305

going operation and receiving small doses of heparin apparently reduces the incidence of throm

which

bosis,7,8do

not

show this decrease.

This decrease*

response may reflect the existence of a past or presen thrombotic or pre-thrombotic state, or be causally related to thrombosis, or be coincidental and un

related to the There are

development of thrombosis.

other reports of decreased platele responsiveness in heart-valve replacement and myo cardial infarction 12-14—both situations in whicl thrombosis is liable to occur. However, we usec different tests and it is incorrect to extrapolate fron If decrease( one set of observations to another. platelet responsiveness to A.D.P. and thrombin i: associated with thrombosis, then the mechanisn involved must be considered. We have suggested tha exposure to A.D.P. or thrombin in the wound, o: indeed elsewhere, could induce a refractory state. However, the addition of A.D.P. or thrombin to citratec blood or P.R.P. in vitro produces immediate aggrega. tion and high platelet retention in the glass-beac column. After some minutes a decreased response t( both tests follows-the refractory state. Thus our finding immediately after operation of decreased aggregation but increased retention is difficult to reconcil( with the in-vitro studies on the refractory state. Another suggestion is that the more active platelets presumably large young ones,15 are utilised in thrombosis, but the observations do not support this hypothesis. Postoperatively, at least in the glass-columr. test, the platelets are more, not less, reactive. The platelet-count and size did not change postoperatively, suggesting there was no change in the platelet population. Fletcher et al. 16reported that an excess of largemolecular-weight complexes of fibrinogen (polymers; occurs in some patients before operation, suggesting that some thrombin formation may occur in these patients. This could lead to a refractory state in platelet aggregation. This explanation also seems unlikely, because in an unpublished study in collaboration with Dr. A. P. Fletcher and Dr. N. Alkjaersig we found that patients who had an excess of polymer preoperatively did not have decreased platelet aggre-

gation.

Heparin added in vitro has little or no effect on platelets (although some workers report changes) and we have shown that, in the absence of operation, heparin given in vivo had no effect on our platelet tests. What postoperative change affecting platelet function is influenced by heparin ? There have been reports of fatty acids affecting platelet aggregation," and these doses of heparin certainly activate lipoprotein lipasethe heparin-clearing factor-but there is no evidence that this activation influences platelet function, except possibly in extreme situations. 18 Yin et al. 19 report that small doses of heparin potentiate the activity of a natural inhibitor of activated clotting-factor X (Xa), and Kakkar et al. suggest that the activation of this inhibitor may be responsible for the prevention of thrombosis. However, there is no evidence that factor Xa depresses platelet aggregation. Factor-Xa inhibitor may be identical to anti-thrombin III, and both inhibitors are potentiated by heparin. 19

Our finding of an increase after operation of plateletfactor-4 activity may also be relevant. Thus again there is indirect evidence that thrombin or perhaps factor Xa may be implicated. There does not seem to be sufficient evidence to indicate what mechanism is involved in the postoperative decrease in aggregation or in the prevention of this decrease by heparin; nevertheless, these findings, if they are related, must help towards an ultimate understanding of the processes involved. J. R. O’B. gratefully acknowledges grants from the Wellcome Trust and the National Institute of Health, U.S. Department of Health, Education, and Welfare, contract no. 71-2302. We thank Mr. M. Bradmore for permission to study his patients. Requests for reprints should be addressed to J. R. O’B., Central Laboratory, St. Mary’s General Hospital, Portsmouth P03 6A9. REFERENCES 1. Emmons, P. R., Mitchell, J. R. A. Lancet, 1965, i, 71. 2. O’Brien, J. R., Heywood, J. B., Heady, J. A. Thromb. Diath. hœmorrh. 1966, 16, 752. 3. Bennett, P. N. J. clin. Path. 1967, 20, 708. 4. Zahavi, J., Dreyfuss, F. Thromb. Diath. hœmorrh. 1969, 21, 76. 5. O’Brien, J. R., Tulevski, V., Etherington, M. in Proceedings of XVI Scientific Conference of the Faculty of General Medicine, Charles University, Prague; p. 46. 1971. Blood, 1971, 38, 781 6. 7.

8. 9. 10. 11. 12.

13. 14. 15. 16.

17. 18. 19.

(abstract). O’Brien, J. R., Etherington, M., Jamieson, S. Lancet, 1971, ii, 741. Kakkar, V. V., Field, E. S., Nicolaides, A. N., Flute, P. T., Wessler, S., Yin, E. T. ibid. p. 669. Williams, H. T. ibid. p. 950. O’Brien, J. R., Heywood, J. B. J. clin. Path. 1967, 20, 56. O’Brien, J. R., Heywood, J. B., Heady, J. A. Thromb. Diath. hœmorrh. 1966, 16, 752. Godal, H. C., Fichera, C. Acta med. scand. 1961, 170, 417. Fitzgerald, D. E., Butterfield, W. J. H., Smink, D., Kruisheer, H. E. J. Atherosclerosis, 1971, 13, 217. Stormorken, H. Fedn Proc. 1971, 30, 1551. McKenzie, F. N., Dhall, D. P., Arfors, K. E., Nordlund, S., Matheson, N. A. Br. med. J. 1969, ii, 795. Karpatkin, S. A. Rev. Med. (in the press). Fletcher, A. P., Alkjaersig, N., O’Brien, J. R., Tulevski, V. G. Trans Ass. Am. Physns, 1970, 83, 159. Shore, P. A., Alpers, H. S. Fedn Proc. 1963, 22, 504. Hoak, J. C., Connor, W. E., Warner, E. D. ibid. 1962, 21, 60. Yin, E. T., Wessler, S., Stoll, P. J. J. biol. Chem. 1971, 246, 3703, 3712.

VENOUS THROMBOSIS FOLLOWING STROKES CHARLES WARLOW D. OGSTON A. S. DOUGLAS Department of Medicine, University of Aberdeen

Thirty patients with a recent cerebrovascular accident resulting in a " stroke " were studied for evidence of leg-vein thrombosis using the 125I-labelled fibrinogen technique. 60% developed a deep-venous thrombosis, as measured by this technique, in their paralysed leg; 7% had, in addition, a thrombus in the non-paralysed leg. Summary

Introduction

thrombosis of the legs is a recognised of cerebrovascular accidents with a complication but its frequency has not been stroke ",1 resulting " established. This is a preliminary study on the incidence of venous thrombosis in the lower limbs of patients following a stroke " using the 125I-labelled DEEP

venous

"

fibrinogen technique.2,3