Progestational agents and blood coagulation

Progestational agents and blood coagulation V. Changes

IRVING

induced

B.

by sequential

MINK, G.

COUREY,

KENNETH

R.

NISWANDER,

H.

MADELINE

MOORE, A.

JULIAN

L.

Springuille,

Buffalo,

and

therapy

M.D. M.D. M.S.

LILLIE,

A M

contraceptive

M.S.

NORMAN

ROBERT

oral

M.S.

B R U

S,

M.D.,

Niagara

Falls,

New

PH.D. York

A sequential oral contraceptive therapy regimen was monitored in a group of women over a two-year period. Blood samples were drawn at intervals during the study, and levels of coagulation and jibrinolytic factors were measured and cornFared with those oj a control group of women. Significant increases toward hypercoagulability were observed in the treated groufi accompanied by possible compensatory changes toward hypocoagulability.

1N A papels,”

s E R I E s o F previously published 5. G we reported the results of our studies on the effects of various types and dosages of oral contraceptive medications

upon the blood coagulation and fibrinolysin systems and reviewed the findings of 0thers.l In continuation of this research program, we have also evaluated the effects of an oral sequential-type hormone combination upon the two systems. The present report deals with these findings.

From the Roswell Park Memorial Institute, Department of Health, State of New York, The Deaconess and Children’s Hospitals of Buffalo, The Planned Parenthood Clinic of Niagara Falls, and the State University of New York at Buffalo.

Subjects,

for

publication

Accepted

February

February

and

methods

One hundred and sixty-four women were entered into the study for a projected twoyear interval. They were all healthy volunteers of childbearing age who desired to practice some form of birth control and were enrolled, in most cases, shortly post partum. After initial examination and blood sampling, at the next menstrual cycle, 112 of the volunteers started on a regimen of sequential oral contraceptive therapy (progestin plus

Supported by Grants 2-MOl-RR-0026208 from the National Institutes of Health, United States Public Health Service, and 9333-047900 from the American Heart Association. Fge;;ived

material,

I,

26, 1974.

Reprint requests: Dr. Julian L. Ambrus, Roswell Park Memorial Institute. Springville Laboratories, Spring~ille, New York 14141. 401

402

Mink et al.

Am.

Table I. Number interval,

and per cent of Patients in each treatment to blood groups and smoking history

according

Bleeding interval

Treatment

Base line

P

3 Months

9 Months

24 Months

group

+ E* sequential

I /

Total

112

Control

52

P t E sequential

93

Control

49

P + E sequential

77

Control

47

P + E sequential

51

Control

32

0

/

group

Blood

groufl

A

/

June 1, 1974 J Obstet. Gynecol.

at each bleeding

B

(

AB

Smoker,

45 (40% ) “4 (46%) 35 (38 % )

40 (36%)

, ‘) (iti%)

(&,

73 (65%)

(&I, 35 ( 1-i8 % )

jlk) 18 ( 19 <,: )

(4%) 5 ( .57%)

28 (54%)

(& 31) (39%) ‘9 (47%)

(&, 29 (38%) 15 (2%)

(li%)

1.5 (51%)

(ifs,)

(I%, 4 (5%) 2 (4%)

,:;,;j

,:i,,

i:d%)

(P%,

30 (59%)

(ii%)

59 (63%)

47 (61%~) 24 (51%)

14 (44%) *Progeatin

plus estrogen.

estrogen). From Days 5 through 14, they received one 50 pg tablet of ethinyl estradiol (an estrogen alone) daily. On Days 15 through 25, the tablets contained 50 pg of ethinyl estradiol plus 1 mg. of norethindrone acetate (a progestin) .* The 52 remaining women served as nonmedicated controls ; most used intrauterine contraceptive devices. Preprandial titrated blood and nonanticoagulated blood samples were drawn by the plastic 2 syringe technique at time of enrollment (base-line premeditation levels) and at 3, 9, and 24 month intervals thereafter. Platelet counts and thrombelastograms were obtained. The titrated samples M‘ere centrifuged at 1,500 x g from 35 minutes at 4’ C., and the platelet-poor plasma samples w-ere separated, frozen, and stored in plastic tubes at -20’ C. until tested. The nonanticoagulated tubes were incubated at 37’ C. until completely clotted, centrifuged, and separated; the sera were frozen. Plasma assays were performed with the same lots of reagents and factor-deficient subtrates throughout the study. Tests included the Hicks and Pitney thromboplastin generation time (TGT) -screening test with calculation of “Norlestrin. Joseph Campau

Parke. Davis & Co.. P. 0. Box 118 GPO, Aw. At the River. Detroit, Michigan 48232

generation factor (GF) units, the Quick one-stage prothrombin time, determination of factors I, II (prothrombin), V, VII, VIII, IX, and X, plasminogen, and antiplasmins. We employed the factor VII assay of Lechner and Deutsch,” and the other techniques were as described previously.‘, ’ Alkaline phosphatase and glutamic oxalacetic transaminase ( SCOT) determinations were performed on the serum samples. Normal activity curves for all the clotting and fibrinolytic determinations were established with pools of base-line plasmas. Whenever assays \vere done, a standard reference control was obtained along with each set of determinations, all performed concurrently on both treated and control patient samples. When the last patient enrolled had finished two years in the study, the data were tabulated and analyzed statistically with paired t tests. (Each patients’ values were compared with her own base-line value for each parameter, and paired differences were obtained. These were then grouped according to medication category.) We started with 164 patients but unfortunately were not able to analyze changes from baseline Lvith that number in all parameters. Table I indicates the number of patient samples available for testing at each time

Volume

119

Number

I?

Progestational

agents

and

blood

coagulation.

V

403

mm3

0-l ‘hi $

5

(bITEgram,

rtk 1 (a)FACTOR

min

V

I

5

(n)ANTIPLASMIN

150 100 % 50 0.5 1 (alALK.

PHOSPH.

1

0

200 150 100 50 0 -50 3

9

24

BLOOD

3

SAMPLE

9

COLLECTION

24

INTERVALS

(MONTHS)

Fig. 1. interval after base-line sampling. Over the two-year study period, many women dropped out of the study for various reasons. Consequently, only half the patients (51 treated, 32 control) finished the entire two-year program. In addition, some assay results were considered unacceptable because of technical problems associated with long-term storage and therefore were not tabulated (e.g., TGT screen, GF, and factors VIII and IX of the 24 month samples). The relative percentages of women in each blood group remained fairly constant at each bleeding interval (Table I), so that blood group influences (if there were any) remained constant within each treatment group. This applied as well to smoking habits within each treatment group. Results Fig. 1 delineates the changes from base line which occurred with each measured param-

eter during each bleeding interval in both treated and control patients. These are the means of paired differences with 95 per cent confidence intervals for each. If the confidence interval lines do not overlap the base line or each other, the compared mean differences are statistically significantly different from base line and/or one another at least at the 95 per cent level (and in some cases at the 99 per cent level) . The sequential progestin plus estrogentreated group exhibited highly significant increases from base line and difierences from control values in the following parameters and bleeding intervals. Results of Quick one-stage prothrombin determination at 3 and 9 months [Fig. 1 (e)] (a composite of activities of prothrombin, factor V, factor VII, factor X, and fibrinogen) were extremely variable at 24 months in both treated and control groups, as a probable reflection of storage loss of

404

Mink et al.

labile factor V (g). Also significantly different were prothrombin (factor II) (f), all intervals; factor V (g), 3 months; factor VII (lx), all intervals; factor IX (i), all intervals; factor X (k), all intervals; and plasminogen (m) , all intervals The treated group also demonstrated significant increases from base line in activities or levels of: TGT screen generation factor (d), 3 and 9 months; factor V, (g), 9 months; factor VIII (;), 3 and 9 months, and fibrinogen (factor I) (1)) 3 months. These increases were greater in magnitude than those of the control group, but the differences between the groups were not statistically significant. The treated group showed significant decreases from base line and differences from controls in: antiplasmins (n) , 24 months, and alkaline phosphatase (0) , all intervals. Both groups had similarly significant increases in platelets (a) at 24 months and decreases in thrombelastogram r + k times (6) at 9 and 24 months. Both groups showed elevations in thrombelastogram maximal amplitude values (c) in all intervals, but here the control values were higher each time. There were no significant differences between groups in SGOT levels (p) , although both increased above base line at 9 months. Comment Our previous studies?. 5. ’ and those of others’ have associated oral contraceptive medications (estrogen plus progestin compounds in various dose combinations) with hyperproduction of clotting and fibrinolytic system factors. The present report demonstrates the same pattern of findings already evident at a three-month observation period, with a sequential-type (reduced total progestin) medication regimen. This is not surprising, since our group6 and others7 have demonstrated that progestins alone do not cause such changes, at least in short-term studies, thereby indirectly implicating the estrogen component in the effected rises. At three months, the vitamin K-dependent factor activities (prothrombin and factors VII,

Am.

June 1, 1974 J. Obstet. Gynecol.

IX, and X) rose dramatically in the treated patients, as did factor V and plasminogen (Results) . These levels remained significantly elevated, as compared with control values, throughout the two-year treatment period (except for factor V in the two-year samples, which probably deteriorated in storage). The other activities and levels which rose (Results) also indicated a tendency toward hyperproduction or hyperactivation attributable to estrogen-progestin therapy. An unusual finding (Results) were the significant decreases in serum antiplasmins at 24 months in the treated group. These reflected either decreased synthesis or increased catabolism (possibly the complexing with activated plasminogen) and possibly the eventual delayed development of a compensatory response in the fibrinolytic system to the hyperactivity of the coagulation system. Also unusual were the significantly decreased levels of alkaline phosphatase in the treated patients at all three time intervals. These may represent hormonally induced increases in leukocyte uptake or retention, since serum alkaline phosphatase levels are ordinarily equilibrated with intraleukocyte levels. We have no evidence for this, however, and the serum decreases remain unexplained. The changes which occurred over a period of time in thrombelastogram r + k and ma readings (Results) were of interest from a technical standpoint. The values for r + k decreased in both groups at the same rate, and since these evaluations were obtained from fresh blood, storage problems were nonexistent. Measurements were made with disposable plastic cups but with the same washable stainless steel pins over the entire study period. It is probable that the pins accumulated minute scratches and residues over the time of the study, thereby gradually producing shorter clotting times in all samples tested because of altered surface contact. The concomitant increases in maximum amplitude could also have been attributed to this technical flaw. However, the treated patients had consistently lower ma values than the control subjects, control indicating possible steroid-induced decreases in clot ten-

Volume Number

119 3

Progestational

sile strength, again an unusual finding and a possible indicator of a compensatory response to other changes toward hypercoagulability. During the two-year study period, none of the patients in either group demonstrated any clinical evidence of thrombosis. The sequential oral contraceptive therapy regimen we monitored in volunteer women was associated with significantly increased levels of clotting factors. These changes paralleled the previously reported increases found to accompany the use of combination steroid preparations which contained monthly dosages of at least twice as much progestin but the same levels of estrogen.

agents

and

blood

coagulation.

V

405

These findings confirm those of others and conclusively implicate the estrogen component of these preparations in the induced rises of coagulation factors observed in the laboratory. We wish to acknowledge the dedicated assistance of our research nurses, technologists, and student fellows: Mrs. Patricia Armstrong, Mrs. Anne Eddy, R.N., Mrs. Elizabeth Guay, A.A.S., Mr. Philip Niswander, and Mrs. Bernadette Striegel, R.N. We are also grateful for the advice and assistance of Mrs. Alice Chamberlain, Department of Biostatistics, Roswell Park Memorial Institute, Stanford Copley, M.D., and Ronald J. Foote, M.D.

REFERENCES

1. Ambrus, C. M., G., and Mink, 1970. 2. Ambrus, J. L., G., Wamsteker,

Niswander, K. R., Courey, N. 1. B.: Hematol. Rev. 2: 163,

Niswander, K. R., Courey, N. E. F., and Mink, I. B.: AM. J. OBSTET. GYNECOL. 103: 994, 1969. 3. Denson, K. W.: Acta Haematol. 25: 105, 1961. 4. Lechner, K., and Deutsch, E.: Thromb. Diath. Haemorrh. 18: 739, 1972.

5. Margulis, R. R., Ambrus, J. L., Mink, I. B., and Stryker, J. C.: AM. J. OBSTET. GYNECOL 93: 161, 1965. 6. Mink, I. B., Courey, N. G., Moore, R. H., Ambrus, C. M., and Ambrus, J. L.: AM. J. OBSTET. GYSECOL. 113: 739, 1972. 7. Poller, L., Thomson, J. M., Thomas, W., and Wray, C.: Br. Med. J. 1: 705, 1971.