Total non-enzymatic fibrinolytic activity in the blood of rats increased three times in response to stress caused by 30 minute immobilization, and the activity of epinephrine-heparin complex increased nine times. In adrenalectomized animals, which showed a weak response to the same stress, intraperitoneal injection of hydrocortisone 30 minutes prior to immobilization normalized the response. Obtained results indicate that adrenalectomy leads to sharp reduction of heparin complexing with thromogenic proteins and epinephrine, while substitution therapy with hydrocortisone restores anticoagulation system function

Kudryashov, B. A.

Lomovskaya, E. G.

Lyapina, L. Y.

Shapiro, F. B.

English

NASA Technical Reports Server

767-/ glalc 
NASA TECHNICAL MEMORANDUM 
 NASA TM-75985
 
THE EFFECTS OF ADRENALECTOMYVAND CORTICOSTEROID INJECTION
 
ON THE FIBRINOLYTIC ACTIVITY OF COMPLEX HEPARIN COMPOUNDS
 
IN THE BLOOD DURING IMMOBILIZATION
 
B. A. Kudryashov, E. G. Lomovskaya,
 
F. B. Shapiro, L. Ya. Lyapina
 
(W11ASA-m-75985j1 T~HE EFFECTS OFR8 -s 5XADENAJLECTOUY AND CORTICSTEROID INJECTION ON
 
THE FIBRINOLYTIC.ACTIVITY OF COfPLEX 
 HEPARIN
COPOUNDS. IN THE BLOOD DURING IMOBIIZATIN Uaclas
(N ational' eronautics and Space G3/51'$7221
 
Translation of "Vliyaniye adrenalektomii i vvedeniya kortikosteroidov na,
fibrinoliticheskuyu aktivnost' kompleksnykh soyedineniygeparinatv kroviw
 
pri immobilizatsii," Fiziologicheskiy zhurnal -SSSR im. I. N. Sechenova,-.
 
Vol. 59, No. 7, 1973, pp. 1108-1113. 
.
 
NATIONAL AERONAUTICS AND SPACE ADMINISTRATION
 
WASHINGTON, D.C. FEBRUARY 19810
 
https://ntrs.nasa.gov/search.jsp?R=19800010409 2020-03-21T20:05:46+00:00Z
STANDARO TITLE PAGE 
1. Report No. 2. Geovrnment Accossion No, 3. Recipient's Catalog No. 
NASA TN-75985;
 
4, Tite ond Subttle 5. Report Dote 
THE EFFECTS OF ADRENALECTOX4Z AND February l98Q
 
CORTICOSTEROID INJECTION ON THE 6. Perforing Orgonizotion Code
 
FIBRrNOLYTIC ACTIVITY OF COMPLEX HEPARIq..
 
7 Autho,(s) 8. Peroarning Org.n saion Report No. 
B. A. Kudryashov, E. G. 'Lomovskaya,
 
F. B. Shapiro, L. Ya. Lyapina, 10. Work Ue, No.
 
Department of Animal Physiology,
 
9. Performing Orgeilsotion Nme NASw,nd Address - 3199
 
Leo Kanner As§ociates 13. Type of Report nd P.,leod coered
 
Redwood City, California 94o65
 
Translation
 
12. Sponsoring 'Agency Nome and Address 
National Aeronautics and Space Admin­
istration, Waahington, D .,C., 2o,546 14. Spon..orIng Agency Code
 
15. Supplementary Noteas 
Tranalation of tt/\lyaniye adrenalektomti i vvedeniya kohtko.
 
ateroidov na fibrinoliticheskuyu aktivnostt kompleknykh___.
 
soyedineniy geparina v krovi pri immobilizatsili,"Fiztolo­
gicheskiy zhurnal SSSR tm. I. N. Sechenovas Vol. 59, Nb. 7, 
1973, pp. 1l08 ll3.
 
16. Abstoct 
Total non-enzymatid flbrinolytc'activity in the blood of ...
 
ratq increesed three times in response to stresas caused by
 
30 minute Immobilization, and the activity of epinephrine­
hepari-n complex increased 9 times. In adrenalectomized­
animals, which showed a weak response to the same stress,
 
intraperitoneal injection of hydrocortisone 30 minutes prior
 
to, immobilization normalized the response. Obtained results
 
indicate that adrenalectomy leads to sharp reduction of
 
heparin complexing with thrombogenic proteind and epineph­
rine, while substitution therapy with hydrocortisone
 
restores anticoagulatipn system function.
 
17. Key Words (Selected by Author{s)) 18. Disunbution Statement 
Unclassified - Unlimited 
M" Sewoy C1 f,I s un'ty Clasi" (of this page) I21. No. of Pages 22 PaceUeNSA 
NASA-HQ 
THE EFFECTS OF ADRENALECTOMY AND CORTICOSTEROID INJECTION
 
ON THE FIBRINOLYIC ACTIVITY OF COMPLEX HEPARIN COMPOUNDS
 
IN THE BLOOD DURING IMMOBILIZATION
 
B. A. Kudryashov, E. G. Lomovskaya,
 
F. B. Shapiro, L. Ya. Lyapina
 
Departent of Animal Physiology, Moscow State University
 
Total non-enzymatic fibrinolytic activity of blood plasma in rats /ll08
 
tripled during stress conditioned by 30-minute immobilization. Epineph­
rine-heparin complex activity concomitantly increased 9 times. A weak
 
response, characterized by a total non-enzymatic fibrinolytic activity
 
approximating that found in intact animals whichlMadtnot btenlsubj(cted to
 
stress, was observed in adrenalectomized animals. Following intraperi­
toneal injection of adrenalectomized rats with hydrocortisone 30 minutes
 
prior to immobilization, stress led to an increase in non-enzymatic
 
fibrinolytic activity to levels present in intact animals which had
 
undergone similar stress (immobilization and intraperitoneal injection
 
of a physiologic salt solution). The obtained results demonstrated that
 
adrenalectomy leads to a sharp reduction in heparin complexing with
 
thrombogenic proteins and epinephrine, while substitution therapy with
 
hydrocortisone ensured complete restoration of the stated system functions.
 
Numerous works have demonstrated the unquestioned participation of 
of adrenal cortex hormones in physiological processes ensuring the 
fluidity of circulating blood. It has been shown that both coagulation 
and anticoagulation systems are disturbed during stress following 
adrenalectomy or blocking of hypophysidl AUT7funtibons,7When "Thetbddy's 
defense mechanisms must be mobilized [7, 8]. 
B. A. Kudryashov and his associates established that complex
 
heparin compounding with specific blood proteins (fibrinogen, thrombin,
 
plasminogen, plasmin) and some hormones (thyroxin, epinephrine,
 
norepinephrine) occurs in the blood during stimulation of the anti­
coagulation system, particularly in stressful situations 1, 3, 41.
 
*Numbers in the margin indicate pagination in the foreign text.
 
1
 
These complex compounds have all of the properties of anticoagulation
 
system agents and, in exerting their non-enzymatic fibrinolytic influence,
 
are of adaptive importance. They inhibit hypercoagulation, lyse unstabil­
ized fibrin clots, and thus, by maintaining the fluidity of the
 
circulating blood, ensure normal functioning of the body in stressful
 
situations.
 
The question naturally arises as to whether or not the process of
 
complexing in adrenalectomized animals during stress is similar to that
 
which occurs with other parameters characterizing the condition of the
 
anticoagulation system. This work was devoted to clarification of
 
this question.
 
Methodology /1109
 
The work was carried out using non-thoroughbred female rats
 
weighing 170-200 grams. The lytic activity of complex heparin compounds
 
was determined 8-12 days after adrenalectomy, when, judging from a
 
sharp decrease in blood corticosteroid content and increase in ACTH
 
secretion, the results of adrenalectomy were sufficiently pronounced [9].
 
Intact rats were used as controls. Stress was induced through 30-minute
 
immobilization (tying to a peg). Blood was taken from the jugular vein
 
and mixed 9:1 with sodium citrate. Total blood fibrinolytic activity
 
was determined using the method of Astrup and MUllertz E103, and
 
non-enzymatlc fibrinolysis caused by the complex heparin compounds was
 
determined using the method of Kudryashov and Lyapina with e-aminocaproic
 
acid (SACA), which suppresses enzymatic fibrinolysis [6]. Fractional
 
separation of the following compex-heparin compounds was carried out:
 
with fibrinogen (FG) -- using the method of Kudryashov, Kalishevsky,
 
and Lyapina [2], epinephrine (ENP) -- using the method of Kudryashov
 
and Lyapina [5], plasminogen (PMG), and plasmin .(PM).
 
Complexes of PMG and PM were separated out after obtaining FG and
 
ENP. To accomplish this, 53.3% ethyl alcohol was added to the
 
supernatant liquid after precipitation of the EN? complex until it
 
constituted 20% in the system and pH=6.5. The precipitate of the 2nd
 
2
 
and 3rd fractions was separated out by Cohn's method [11] using a
 
centrifuge at 00 and dissolved into a physiologic salt solution. Alcohol
 
content in the solution of the 2nd and 3rd fractions was brought to
 
25% and pH=7.2. The 3rd fraction was then separated from the precipitate
 
of the 2nd and 3rd fractions by centrifugation at 00, dissolved into a
 
physiologic salt solution, and the alcohol content of the system was
 
brought to 17% and pH=5.2. The precipitate of the 3rd fraction (PMG,
 
PM + plasminogen) was collected by centrifugation and dissolved in a
 
volume of borate solution equal to the initial plasma volume.
 
Determination of FG, ENP, and PMG complex activity was carried out
 
on plates of fibrin not stabilized by factor XIII. Activity of the PM
 
complex, in distinction-to that of the ether complexes, was determined
 
on plates of stabilized fibrin. The activity of PMG and PM complexes
 
was determined on fibrin plates heated for 30 minutes at 850 [12].
 
To determine activity on the plates, 0.05 ml plasma or complex was
 
.
taken and incubated for 24 hours at 37' The amount of fibrinolytic
 
activity, non-enzymatic fibrinolysis, and FG, ENP, PMG, and PW activity
 
was judged according to the size of the zones of lysis on the plates.
 
of fibrin in mm
 
Results of the Study and Discussion
 
Before studying the lytic activity of complex heparin compounds
 
in adrenalectomized animals during stress caused by 30-minute immobiliz­
ation, it was necessary to establish how this stress affects complexing
 
in intact animals. To do this, comparison was made of total blood
 
non-enzymatic fibrinolytic activity conditioned by complex heparin
 
compounds and the activity of certain complexes (FG, ENP, PMG, and PM)
 
in intact animals subjected to and hot subjected to stress effects.
 
Results of this comparison are shown in tables 1 and 2.
 
As can be seen from the data presented in table 1, the stress we
 
apfflied caused a considerable increase in total non-enzymatic fibr~nolytic
 
activity: its absolute value increased more than three times (32.9 ± 4.0
 
3 
2 
instead of 9.5 - 23 mm2) and its share in overall blood fibrinolytic /1110
 
activity doubled (40.8 ± 4.0 instead of 20.7 ± 5.0%). ENP complex 
activity grew most sharply here -- more than 9 times, thile the activity 
of FG, PMG, and PM increased mbre than 2-5 times (table 2). 
TABLE 1. TOTAL NON-ENZYMATIC FIBRINOLYTIC ACTIVITY
 
ZONE OF LYSIS SIZE (IN MM2 )
 
o W 
P Ow O M1W 
O OH'QM
0 HC 0 HAnimal Group 

Intact, .
 
no immobilization 21 40.0+5.4 9.5 -2.3 5. i 20.7-5.0
 
Intact, _ 
 _.43s
 
with immobilization 29 76.0-6.2 32.094.0 40.S2. 
Adrenalectomized. 
21 54.0-5.5 15.0 3_' 25.S-6.0with immobilization 

TABLE 2. LYTIC ACTIVITY OF COMPLEX HEPARIN COMPOUNDS
 
ZONE OF LYSIS SIZE (IN MM2 )
 
0 W&kJ4 
Animal Group d
 
Intact,
 
no immobilization 23 44.6+10.9 6.7±2.6 17.1+7.0 5.6-'1.2
 
Intact,

with imimobilization 22 93.0+17.9 56.7±10.9 55.6--2.8 26.2-6.1 
AdrenalectomizedI 
with immobilization, 23 27.4 -. 9 19.5 5.5 17.0-M.6 4.8-1.0 
It is thus quite clear that a stressful state caused by 30-minute
 
immobilization was accompanied in intact rats by a sharp increase in
 
blood non-enzymatic fibrinolytic activity conditioned by the lytic
 
properties of complex heparin compounds.
 
When adrenalectomized animals were subjected to such stress,
 
complexing actually occurred no more intensely in them than in intact
 
animals not subjected to stress, Despite some increase in total
 
'4
 
non-enzymatic fibrinolytic activity noted in adrenalectomized animals
 
during stress (15.0 ± 3.4 instead of 9.5 ± 2.3 m 2), there was no
 
statistically significant difference between them and intact animals
 
not subjected to stress (t = 1.36). At the same time, non-enzymatic
 
fibrinolytic activity in adrenalectomized animals duting stress
 
differed significantly from fibrinolytic activity in intact animals 
which had been subjected to this stress (15.0 ± 3.4 versus 32.9 ± 4.0 
mm2; t = 3.4) (table 1). 
The same picture may be seen in reference to the activity of
 
certain other complexes (table 2). If, as has been indicated above,
 
the activity of all complexes sharply increases in intact animals
 
during stress, the activity of PMG and PM complexes in adrenalectomized
 
rats in the same situation remains the same as in intact ones free of
 
stress, ENPRicomplex activity is three times higher (in intact animals
 
more than 9 times higher), and the activity of the FG complex even
 
decreases 1.5 times.
 
These data are evidence of the fact that disturbance of the body's
 
adaptive capabilities after adrenalectomy also includes disturbance ¢t>
 
of the process of heparin complex formation and, consequently, the
 
defense reaction of the anticoagulation system in response to stress
 
cannot (in this case) be fully implemented.
 
Such a reduction in adaptive respunces after adrenalectomy, as we
 
know, is conditioned by inadequate body corticost-zoids, primarily
 
glucocorticoids. Hence, there is every reason to believe that injection
 
of the corresponding hormone preparations can normalize the process
 
of complexing in response to'stress.
 
To test this, hydrocortisone (1 mg/100g of weight: 0.5 ml volume)
 
was injected intraperitoneally into adrenalectomized rats 30 minutes
 
before tethering, thereby compensating for their lack of natural
 
cobttcetef£Mtd output in response to the stimulation of stress g8J.
 
5 
Adrenalectomized and intact animals which had received Inraperitoneally/l1l
 
equal volumes of physiologic salt solution served as controls. The data&
 
obtained are shown in tables 3 and 4.
 
TABLE 3. TOTAL NON-ENZYMATIC FIBRINOLYTIC ACTIVITY
 
ZONE OF LYSIS SIZE (IRifMM2 )
 
Number 
Animal Group of 
animals 
Intact, intraperitoneal 
salt solution, with 
titobilization. .. ........ ,3
4dfenalegtomizdl . 
with immob zation ........... 7 
Adrenalectomized2intraperi neal Aydrocortsn- 24 

with immobilization ......... 24 

Lysis 
zone 
(A) 
Lysis 
zone w/ 
EACA(B) 
B X 100 
A 
12.8-4.0 S2.6-26 
50.5±5.9 93..5 
9.+.27.00e 
09.5 73.0-0. 
653-2,1 
0+. 
63.3-1.1 
001.I.5 
TABLE A- LYTIC ACTIVITY OF COMPLEX HEPARIN COMPOUNDS
 
ZONE OF LYSIS SIZE (IN MM 2 )
 
Number
 
Animal Group of 

animals 

Intact intraperjtoneal

salt solu ion, with 
immobilization ................ 25 

Adrenalectomized,
 
intraperito~eal.ialt sol'n
 
with iZatmmooi 1on ......... 

Adrenalectomizedo
i~ra~erioealt yrocortsn._
W3 mmo Iilza Ion ......... 24 

PG ENP PMG PM 
- , 
---
1o0.52S.6 68.0±9.0 8J.5-5.6 49.39 
27.3'5.S 18.7-2.7 8.3-4.1 3.9-1.7 
112.5-8.9 64.5±7.3 83.2- 5._ -18.7±5.2 
While reviewing the data cited in these tables, it is most
 
important to bear in mind the following fact. Intact and adrenalectom­
ized animals reacted to additional stress, such as intraperitoneal
 
injection of the physiologic salt solution, with a more significant
 
increase in non-enzymatic fibrinolytic activity and, consequently,
 
a more intensive process of complexing, than they did to stress
 
caused only by immobilization. In the intact animals, the absolute
 
value of non-enzymatic fibrinolytic activity and its share in overall
 
6 
ORIGINAL PAGE IS
 
'F -PCCP n,,AIUTy
 
blood fibrinelytic activity rose significantly (t = 10.30 and t = 5.30)
 
in comparison tdt he corresponding indices in intact animals which were
 
subjected to immobilization stress alone. ENP and PMG complex activity
 
was especially elevated here. The proportion of non-enzymatic fibrin­
olytic activity in overall fibrinolytic activity for adrenalectomized
 
animals also increased with some degree of significance in comparison
 
to that which occurred in them after immobilization alone. This
 
increase of complexing after additional stress, however, proceeded at /1112
 
a markedly lower level in them than in the intact animals. In fact,
 
only in the presence of additional stress did this level approach that
 
whichtioccurred in intact animals which had been subjected only to
 
immobilization (cf. tables 1 and 3).
 
It is interesting to note that the activity of FG, ENP, PMG, and
 
PG complexes did nbt change with addtitbndlsktress (table 4).
 
The picture changes completely when adrenalectomized animals were
 
injected intraperitoneally with hydrocortisone rather than a physiologic
 
salt solution. After this all indices were comparable with th6se from 
intact animals which had been subjected to similar combined stresses. 
- . Bothethbnab'solteva1Ue-of total 
Snon-enzymatic fibrinolytic activity
 
;Lo] &ftd its share in overall blood
 
- .fibrinolytic activity increase to 
[t 'levels in intact animals, and the 
! activity of FG, ENP, PMG, and PM 
_i complexes reach control values.
 
-;In other words, when blood cortico-

Fig. 1. Total non-enzymatic steroid levelsaare raised, the
 
fibrinolytic activity adrenalectomized animals begin to
 
l-- intact, no immobilization; react to stress with as intensive a
 
2 -- intact, immobilization; 
3 .-. intgct, intraperitonealprocess of heparin complex formation 
physibigc salt solution; as intact animals do. This is 
ith iliza&Uen&Lectomizea, with particularly graphically illustrated 
immobilization; 5 - adrenalec- by figures 1 and 2, in which changes 
tomized, intraperitoneal in nonenzymatic fibrinolytlc activity
 
hydrocortisone, immobilized;
 
6 -- adrenalectomized, intraperi­
toneal salt solution, immobilized.
 
A -- absolute value; B -- % of 7
 
overall activity.
 
and the activity of certain heparin complexes in all groups of animals
 
are presented in comparison with corresponding indices in intact
 
animals which had not been subjected to stress, (in fig, 2 these indices.
 
are taken as the initial level),.
 
- M, "L, 

F// 2 Mprocess 
EM 

Aevidence 
 Hl !KI 

o-

FG ENP P4G PM 

Fig. 2. Activity of complex 
heparin compounds . 
Normalization of the complexing
 
during stress in adrenalec­
tomized animals with substitution
 
therapy using hydrocortisone is
 
oftthe re-establishment
 
of the functioning of the anti­
coagulation system. This c6&ndides
 
with data from works in which it 
was shown that disturbance of the
 
coagulation and anticoagulation
 
1 -- intact., with immobilization; potentials'of the blood, acutely 
2 -- intact, intraperitoneal manifested during stress in 
physiologic salt solution, with 
immobilization;,3-- adrenalec- adrenalectomized animals and animals 
tom zed, with immobilization; with impeded hypophysial ACT function, 
4 -- adrenalectomized, intra­
peritoneal hydrocortisone, with returns to normal under the influence 
immobilization; 5 -- adrenalec­
tomized, intraperitoneal physio­
logic salt solution, with corticosterMi6daddACA2H T->81.
 
immobilization.
 
Horizontal line -- activity level
 
for complexes in intact rats In conclusion, we should also
 
without stress, note that the formation of heparin
 
complexes, which are active agents in the anticoagulation system, is a
 
process which is sensitive,to stress; intensification of stress immed­
iately entails intensification of complexing. In a body which has been
 
deprived of the capability of mobilizing proper defensive mechanisms
 
in response to stress because of adrenalectomy, the process of heparin
 
complea formation is not only significantly weakendd, but quite disrupted.
 
Increasing the level of corticosteroids through exogenous hormone intro­
duction into adrenalectomized animals,normalizes the process of
 
heparin complex formation.
 
0frt;S.8 
REFERENCES
 
El] Kudryashov, B. A., T. M. Kalishevskaya, L, A. Polyakova,'Vopr.
 
midit-i$rfsk. khnii, 12, 114, 1966.
 
[2] 	 Kudryashov, B. A., T. M. Kalishevskaya, L. A. Lyapina, Vopr.
 
meditsinsk. khimii, 14, 217, 1968.
 
[3] Kudryashov, B. A., Usp. fiziolog. nauk, 1, 17, 1970.
 
[4] Kudryashov, B. A., Arkhiv patologii, 33, 3, 1971.
 
95] Kudryashov, B. A., L. A. Lyapina, Vopr. meditsinsk. khimii,
 
17, 46, 1971.
 
[6] Kudryashov, B. A., L. A. Lyapina, Labor. delo, 6, 326, 1971.
 
[7] Lomovskaya, E. G., F. B. Shapiro, Biolog. nauki, 6, 38, 1969.
 
[8] Shapiro, F. B., E. G. Lomovskaya, Biolog. nauki, 5, 42, 1972.
 
[9] Shchedrina, R. N., Problemy endokrinolog., 17, 58, 1971.
 
[10] Astrup, T., S. MNlle'tz, Archiv. Bicch. a. Biophys., 40, 346, 1952.
 
fill Cohn, W., L. Strong, N. Hughes, J. Amer. Chem. Soc.,' 68, 459, 1946.
 
[12] Lassen, M., Acta Physiol. Scand., 27, 371, 1952.
 
9 


The effects of adrenalectomy and corticsteroid injection on the fibrinolytic activity of complex heparin compounds in the blood during immobilization

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