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UDC 612.115:612.273.2:612.223.11:616-005.2-092.4
UNIVERSAL MECHANISMS OF REACTION OF THE HEMOSTATIC SYSTEM ON THE ACTION OF VARIOUS STRESSORS
1 Altai State Medical University, Barnaul
2 Research institute of physiology and fundamental medicine
I.I. Shakhmatov 12, V.I. Kiselev 12
The analysis of hemostasis system was carried out on rats during short-term exposure to stressors of different nature. Physical exertion, immobilisation, hypercapnic hypoxia, hypothermia and hyperthermia were simulated as stress influences. One-type reaction independent of irritant nature was observed during the investigation (the combined activation of aggregative platelet function, contact phase of blood coagulation as well as anticoagulative and fibrinolytic system is registered).
As the exposure was prolonged successive involvement of all the hemostasis system components into reciprocal response was registered. By the end of a single peak term exposure the totality of hemostasis features characteristic of the initial stage of DIC (disseminated intravascular coagulation) - syndrome was markedly registered in experimental animals.
Key words: stress, hemostasis, DIC-syndrome.
Introduction. One of the topical issues of both pathologic physiology and normal physiology is the evaluation of acute and long-term adaptive reaction of the organism, occurring by various stress stimulations. Upon that a particular stressor can cause either adaptive or maldaptive response reactions on the part of organism depending on its parameters [1,2]. By the present time the reactions to different stress activities of the blood circulation system and a number of other vital systems have been studied in detail [3,4]. At the same time, there are data, that in the development of complications on the part of systemic hemodynamics by extreme stress stimulations an important role is played by the disorders of the hemostatic system [5, 6].
The discovery of mechanisms of hemostatic system reaction to various stressors is significantly important for the elaboration of methods of effective prevention and treatment of various manifestations of intravascular blood coagulation (thrombosis, DIC syndrome) [7, 8]. All the above stated determined the objective of the current work.
Research objective: to determine the mechanisms of hemostatic system reaction to the influence of stressors of various nature by one-time effect, and also to evaluate the state of hemocoagulation by consistent increase of duration of stressor effect.
Materials and methods
Wistar type of rats was used as the object of the research. The involvement of the rats in the experiment was conducted according to the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes and Directives 86/609/ EEC [9], and also international regulations on the animal care and their involvement in the experiment.
In terms of the research there was investigated the state of hemostatic system by one-time effect of such stressors as physical activity (running and walking on the moving platform with given speed), immobilization, influence of hypercapnic hypoxia and also thermal (hypo- and hyperthermal) effect.
Physical activity in rats was modelled in the form of imposed running (30 min with 28-30m/min speed) and walking of various duration (during 0,5; 2; 4 and 8 hours with 6-8m/min speed) [10].
The effect of moderate immobilization was studied by one-time placement of rats into freely ventilated transparent chambers for 20 minutes [11]. The influence of hypercapnic hypoxia was studied by means of placing the rats into the chambers for 20 minutes with the following (according to the gas analysis indicator «Spirolyt-2» (Germany)) gas environment: 9,0-11,0 % 02, 7-8 % C02 [12].
Hypothermal effect was modelled by placing the animals, being in the individual cages, into the tanks with water at the depth of 4,5 sm at the water temperature 5^ and air temperature - 7° С [13].
Hyperthermal effect was modelled by placing the animals, being in the individual cages, into the air bath at the temperature of +45^ for 30 min [14].
The control group included intact animals with free ration in large cages (n=70).
The material of the research was the blood drawn right after the end of stressor influence. The plasm samples were gathered according to the recommendations of Z.S. Barkagan and A.P. Momot [15].
In all animals there were examined the parameters of thrombocytic and coagulatory hemostasis, and also anticoagulant and fibrinolytic plasm activity by means of the kits of "Technologiya-Standart" firm (Russia). The analysis of the parameters of peripheral blood was carried by means of hematological analyzer "Drew-3" (USA).
Data processing
The comparison of obtained results was made by the calculation of mean values and error of mean (M ± m). The statistical analysis was carried by nonparametric methods (Mann-Whitney U test) on the personal computer using application statistical software pack Statistica 6.0 (StatSoft, USA). The accepted critical level of significance by the validation of statistical hypothesis in the current research was 0,05.
Results and discussion
Influence of one-time effect of various stressors on the hemostatic system of rats
It is well known, that during lifetime an organism is exposed to a constant influence of stress factors of various nature. Adaptive reactions on the part of separate systems of the organism at early stages of stressor influence form a total generalized effect, which is determined as the first stress stage - "anxiety stage".
The experiments showed, that by one-time short-term effect of all stressors there was registered a standard one-type reaction on the side of plasmic hemostasis , characterized by combined activation of the aggregative function of thrombocytes, blood coagulation (mainly of its contact phase), fibrinolytic system by the increase of anticoagulant activity and blood fibrinogen (Table 1). Such changes in the hemostatic system in response to one-time effect of stressors can be evaluated as one of the chains of the reaction of acute adaptation of the organism, increasing the advance preparation of the system to the termination of bleeding and can be characterized as "eustress".
Influence of various duration of one-time stressor effects on the reaction of the hemostatic system of rats
The aim of the next series of experiments was to study the influence of stressor effects of various duration on the hemostatic system.
In our article, the influence of the increase of duration of stressor effect on hemostasis is presented on the example of physical activity, being the most common stressor. Moreover, physical activity is quite a convenient modelled influence from the point of view of extrapolation of the experimental data.
The influence of one-time physical activity of various duration in rats was modelled in the form of imposed walking on a moving platform with the speed 6-8 m/min during 30 min, 2, 4 and 8 hours respectively.
It is shown, that 30-minute imposed walking insignificantly activated the contact phase of coagulation and more significantly - anticoagulant and fibrinolytic plasm activity, which is a stereotypic response reaction of the hemostatic
system on one-time threshold effects (Table 2).
Two-hour physical activity was accompanied by the appearance of unfavorable shifts in separate chains of hemostasis. It was reflected in expressed activation of thrombocytic and in a lesser degree plasmatic hemostasis on the background of a significant suppression of fibrinolytic activity of the blood plasma.
The registered discrepancy between the changes in coagulation and fibrinolytic systems in response to two-hour physical activity can be evaluated as the first stage of formation of pathologic reaction of the hemostatic system (or "distress") on the stressor with increasing duration of influence.
One-time 4-hour physical activity was accompanied by significant shifts in the systems of thrombocytic and coagulatory hemostasis. The aggregation of thrombocytes retained increased. From the side of coagulation system, together with the activation by intrinsic coagulation pathway, registered by 2-hour influence, there was also registered the activation of the extrinsic coagulation pathway for the first time. There was also revealed the disorder of fibrin polymerization at the final stage of hemocoagulation. There was firstly registered the increase of level of soluble fibrin complexes. The registered decrease of the main anticoagulant level - antithrombin III and suppression of fibrinolytic activity of the blood plasma considerably worsened the hemostasiological picture. The combination of a number of characteristics of thrombogenesis (activation of coagulation, increase of SFC, decrease of antithrombin III and fibrinolysis) allows to state the existence of first symptoms of DIC syndrome.
Upon the expiry of eight hours of the influence, there was clearly registered the combination of hemostasiological symptoms typical for thrombinemia (activation of coagulation accompanied by increased SFC and active fibrinogen consumption, decrease of fibrinolytic and anticoagulant activity of the blood plasma, accompanied by the reduction of antithrombin level in blood), which corresponds to the early stage of DIC syndrome. The obtained data fit into the concept of the development of stage of exhaustion, forming by overdosing of activity [16].
Thus, it can be assumed, that stress factors by a relatively short-term effect irrespective of the nature of stressor effect kept within the eustress, causing balanced changes in the state of coagulatory and the confronting anticoagulant and fibrinolytic systems. Further, the overall favorable picture of acute adaptation in terms of eustress becomes more and more threatening with the growth of duration of one-time effect. This fact can be considered as the manifestation of a gradual shift of the response reaction from the side of hemostatic system from eustress into distress.
Table 1
Coagulogram of rats in response to one-time short-term stressor effects of various nature (M±m)
Research methods Control (n=70) Physical activity (n=12) Immobilization (n=ll) Hypercapnic hypoxia (n=15) Hypothermia (n=12) Hyperthermia (n=17)
Induced thrombocyte aggregation, c 21,7±0,5 15,5±1,1 p<0,001 12,8±0,4 p<0,001 14,3±2,9 p<0,02 13,3±1,2 p<0,05 16,5±2,3 p<0,05
Coagulation time, c 220,4±5,7 191,9 ±7,5 p<0,05 188,9±10,5 p<0,05 152,0±11,4 p<0,001 198,5±5,6 p<0,05 185±13,2 p<0,05
APTT, c 21,8±0,4 20,6±1,1 p>0,05 22,9±0,6 p>0,05 21,9±0,7 p>0,05 22,5±2,0 p>0,05 23,5±26 p>0,05
Prothrombin time, c 13,9±0,2 13,0±0,2 p<0,05 12,5±0,3 p<0,01 12,7±0,3 p<0,01 12,8±2,8 p<0,05 12,8±3,8 p<0,05
SFC, mg/100 ml 3,3±0,1 3,5±0,3 p>0,05 3,5±0,4 p>0,05 3,7±0,3 p>0,05 3,0±0,3 p>0,05 3,5±1,5 p>0,05
Fibrinogen content, g/1 1,77±0,07 2,48±0,23 p<0,01 2,55±0,20 p<0,001 2,29±0,19 p<0,02 2,1±0,1 p<0,05 2,8±0,4 p<0,05
Antithrombin III, % 97,3±1,4 88,8±1,2 p<0,001 89,6±7,9 p>0,05 80,6±1,4 p<0,001 81,2±4,5 p<0,05 80 ±7,7 p<0,05
Fibrinolytic activity, min. 332,1±14,0 205,3±18,1 p<0,001 216,8±20,6 p<0,001 169,0±24,4 p<0,001 215,5±15,8 p<0,05 286,4±13,2 p<0,05
Note: n - number of observations; p - level of statistical significance of differences of the compared parameters in relation to the control.
Table 2
Dynamics of changes of the hemostatic system registered during one-time physical activities
with increasing duration (M±m)
Research methods Control (n=70) Experiment 1 (30 min) (n=10) Experiment 2 (2 hours) (n=10) Experiment 3 (4 hours) (n=18) Experiment 4 (8 hours) (n=8*)
Thrombocytes, 109M 772,1±23,9 470,0±62,5 701,7±22,0 707,6±32,0 850,0±27,4
<0,001 <0,05 >0,05 <0,01
Induced thrombocyte aggregation, c 21,7±0,5 22,2±1,8 10,5±0,8 13,9±0,9 9,0±0,6
>0,05 <0,001 <0,001 <0,001
Coagulation time, c 220,4±5,7 249,0±18,4 237,0±12,9 202,1±15,5 162,1±16,0
>0,05 >0,05 >0,05 <0,01
APTT, c 21,8±0,4 24,2±0,8 16,7±0,3 18,9±0,6 20,0±0,4
<0,01 <0,001 <0,001 <0,01
Prothrombin time, c 13,9±0,2 14,4±0,3 13,2±0,3 12,7±0,5 12,4±0,4
>0,05 >0,05 <0,05 <0,01
Thrombin time, c 28,1±0,7 23,8±0,3 30,3±1,4 37,3±1,8 19,0±0,5
<0,001 >0,05 <0,001 <0,001
SFC, mg/100 ml 3,3±0,1 3,2±0,2 3,2±0,2 5,8±1,5 7,8±1,8
>0,05 >0,05 <0,05 <0,001
Fibrinogen content, g/1 1,77±0,07 1,88±0,12 1,90±0,10 1,69±0,08 0,65±0,13
>0,05 >0,05 >0,05 <0,001
Antithrombin III, % 97,3±1,4 107,7±1,3 82,9±5,1 79,5±3,3 64,6±3,3
<0,001 <0,01 <0,001 <0,001
Fibrinolytic activity, min. 332,1±14,0 251,5±23,4 458,0±21,3 479,0±36,5 775,0±71,6
<0,05 <0,001 <0,001 <0,001
Note: * - out of 10 rats, exposed to one-time 8-hour physical activity, 2 animals died during the experiment; p - level of statistical significance of differences of characteristics in control and experimental groups; n - number of rats in the group.
A consistently developing picture of distress leads to the increase of the degree of blood coagulation activation, both initial and final stages of hemocoagulation are involved into the process, external mechanism also joins the activation of by intrinsic coagulation pathway. The decrease of anticoagulant and fibrinolytic activity aggravates the picture even more, which finally leads to the appearance of SFC in the blood flow and active consumption of fibrinogen. All these data allow to suggest the fact of thrombinemia and threat of DIC syndrome development.
This fact was confirmed by the death of 20% of animals with detected pathomorphological signs of intravital inopexia during maximally positive influence. Similar results were obtained also by other researchers, who showed, that by sufficient intensity of stressor even the reaction of anxiety can lead to the death of the organism [17; 18].
The development of the clinical picture of thrombinemia and symptoms of disseminated intravascular coagulation can be the indicator of distress development in experimental animals and is a specific manifestation of the disorders of hemostatic system.
Conclusion
1. In response to one-time threshold effect of various stressors the hemostatic system reacts in combined activation of the aggregative function of thrombocytes, contact phase of blood coagulation and also anticoagulant and fibrinolytic blood systems. The described complex of changes does not depend on the nature of stressor and is a stereotypic response reaction of the hemostatic system in response to one-time threshold stressor effect.
2. The increase of duration of the stressor effect leads to the growth of activation of blood coagulation. Both initial and final stages of hemocoagulation are consistently involved into the process, there is registered hypercoagulation by intrinsic and further extrinsic coagulation pathway. Anticoagulatory and fibrinolytic activity decreases gradually. By most durative effect in the animals there is registered thrombinemia and risk of DIC syndrome development.
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