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UDC 615.322 : 615.015.44
HEMOSTATIC BALANCE AT DIFFERENT STAGES OF PHYSIOLOGICAL PREGNANCY AND RISKS OF ITS DISORDER
1 Altai State Medical University, Barnaul
2 Altai branch of National Research Center for Hematology, Barnaul A.P. Momot1,2 , N.I. Fadeyeva1
The present article is devoted to the analysis of the dynamics of a set of leading hemostatic parameters and fibrinolysis studied in 301 women. The research was carried during pregravid period, at different stages of physiological pregnancy and 2-3 days after vaginal delivery.
On the basis of the data obtained it was assumed that hyperproduction of tissue factor and thrombin should play a possible role in the initiation not only of intravascular blood coagulation, but also in the end of pregnancy, which might be important for the understanding of preterm delivery causes.
There was discovered the division of fibrinolytic blood system functions according to the direction of their influence. In particular, the fact of inhibition of parietal fibrinolytic reactions during the late gestation periods was established, which is extremely important for the reduction of blood loss during and after birth injury. Moreover, there was described a previously unknown "shunt" mechanism preventing thrombus formation in late pregnancy, associated with the enhancement of fibrinolytic reactions in blood circulation. It is supposed, that the blocking of the stated mechanism can lead to thrombosis or thromboembolia during pregnancy or early postpartum period.
On the whole, it is observed, that hemostatic balance is maintained during physiological pregnancy, primarily, due to the effective resistance of hemostatic and fibrinolytic reactions.
Keywords: pregnancy; physiological anticoagulants; thrombin generation; blood coagulation markers; fibrinolysis.
Introduction. At the present time the insights into hemostatic balance and mechanisms of its maintenance during pregnancy and delivery are not sufficiently provided. As it is known, this period of woman's life is accompanied by serious changes in activity of organs and systems, happening, however, in terms of adaptation to the changes of the internal and external environment. According to the modern view, pregnancy is associated with hypercoagulation progressing through gestation, the necessity of which is, first of all, important for the reduction of blood loss during delivery and the sustaining of mother's life. Nevertheless, it is beyond controversy, that pregnancy can be accompanied by problems caused by particular disorders of hemostatic reactions.
Thus, the risk of pre-delivery venous thromboembolic complications (VTEC) are 4-5 times higher in pregnant women than in nonpregnant women of the same age. VTEC can occur at any period of pregnancy, but postpartum period is the time of the highest - 20-times risk of the pathology development [1]. No less medically and socially important are massive obstetric bleedings, by which the blood loss exceeds 1,5% of body weight [2]. The main causes of the above stated pathology, being the basis of maternal mortality, are disorders of balance formed by complicated interaction of placental tissue, endotheliocytes and blood cells with a number of proteolytic blood systems. This poses the question - how to maintain this balance and which "breaches" can disturb it, lead to vascular catastrophes at different periods of child-bearing?
The objective of the present work was the
analysis of hemostatic and fibrinolytic blood potentials by pregnancy, favoring its physiological course. The analysis was based on factual data obtained by our center in the period from 2011 to 2015 and presented in some recent publications [39].
Materials and methods
In terms of earlier prospective research approved by the regional ethics committee, there were data obtained from 301 Caucasian women who provided their informed consent. All the women were examined from one to three times during the whole period of examination after the evaluation of their compliance with inclusion and exclusion criteria described earlier [3, 9, 10]. These works also included details and peculiarities of the preanalytical phase of hemostatic reactions research, implied methods, reagents and peculiarities of statistic analysis. The target point of analysis of the hemostatic system were chosen in accordance with the waves of trophoblast invasion and reflected "critical" stages of pregnancy. Women were examined during pregravid period, 6-8 weeks, 12-13 weeks, 22-24 weeks, 34-36 weeks and 2-3 days after spontaneous term delivery.
RESULTS AND DISCUSSION
1. Hemocoagulation activation markers
Despite a large number of scientific publications devoted to the study of blood coagulation activation processes during pregnancy, there are quite few researches related to early stages of blood
coagulation (hemocoagulation initiation) during the stated period of woman's life. The obtained data showed a regular growth of tissue factor (TF) activity in the blood plasma with increase of gestational age (Fig.1, a), thereby the increase of blood coagulation activity is at the peak of intensity in the period close to delivery accompanied by placental expulsion [7].
а
Tissue factor
S '
5
o 300
6
Before pregnancy 6-8
12-13 22-24
5
Peak thrombin
34-36 After delivery
Before pregnancy 6-8
p=0,01
12-13 22-24 34-36 After delivery
B
ETP
3000 2700 2400
S 2100
o
8 1800
Before pregnancy 68 12-13 22-24 34-36 After delivery
Figure 1.
Dynamics of tissue factor conten (a), peak thrombin (b) and endogenous thrombin potential (c) in blood plasma of pregnant and nonpregnant women. Here, and also in figure 2 and 3 the lines stand for medians, rectangles - for upper a nd lower quartiles, bars - for 95% confidence limits, circles - for ejections
As a strong coagulation initiator, TF of primarily placental origin quickly leads to the thrombosis of vessels in order to stop bleeding during delivery. This appears to be quite important, as placental blood flow 700 ml/min-1 can cause heavy bleeding,
if the coagulation system fails to meet its challenge [11]. At the same time, the relevance of significant increase of this indicator and the level of activation factor VII in our work already since 12-13th week of pregnancy in comparison with nonpregnant women is not quite conceivable, because the delivery and, consequently, the reduction of blood loss in the first half of pregnancy are not programmed by nature. However, it is known, that in comparison with the indicators of women with physiological pregnancy, the average concentration of TF in mother's blood plasma is higher by preeclampsia or preterm premature rupture of membranes, which determined the assumption about the connection of redundant expression of TF in the blood flow by preterm delivery [12], which we totally agree with.
The existence of TF in blood leads to the intensification of thrombin formation in blood (Ila factor). In the current work, the mentioned process was studied by means of thrombin generation test suggested by Hemker H. et al. [13]. The results of the implementation of the test in women during pregnancy determined the acceleration of «time to reach peak thrombin» (t-Peak) rate in the blood plasma, and also the changes of two other parameters used for the evaluation of thrombin generation intensity - «Peak thrombin» and «endogenous thrombin potential» (ETP) (Fig.1, b,c). Already since early stages of pregnancy (6-8 weeks) two last indicators had been increasing (in comparison with pregravid period, Peak thrombin
- by 55,1% and ETP - by 39,6%) and complied with each other during the whole period of pregnancy (Spearman rank correlation coefficient constituted 0,80; p <0,001).
As it is known, the thrombin generation test traces the result of a complex interaction of enzymes involved into blood coagulation. According to a number researchers, the measurement of individual capacity of blood plasma for thrombin formation affected by TF might become the most suitable indicator of thrombotic readiness in comparison with the tests aimed at the analysis of fibrin clots formation and indication of potential biomarkers
- prothrombin fragment 1+2, fibrinopeptide A, thrombin-antithrombin complex and D-dimers [14].
The manifestation of blood thrombotic readiness during pregnancy is traditionally conditioned by the increase of D-dimers level, being the result of gradual impact of thrombin on fibrinogen, activation factor XIII and plasmin. The current work determines the progressing growth of D-dimers with the growth of gestation terms (Fig.2, a).
In particular, the D-dimers level, measured with the automatic coagulometer Sysmex CA-1500 with the use of «Auto Red D-dimer 700» reagent on the 6-8th week of pregnancy, corresponded to
а
D-dimers
t
pcftOOl
e pregnancy 6-8 12-13 22-2
б
t-PA, level
34-36 After delivery
I. 6 '
Before pregnancy 6-8
12-13 22-24 34-36 After delivery
PAI-1, level
^ч 40 6
p0,001
p<0,001
Before pregnancy 6-8
34-36 After delivery
Figure 2.
Dynamics of D-dimers level (a), tissue plasminogen activator (b) and inhibitor of plasminogen activatorof type I (c) in blood plasma of pregnant and nonpregnant women
the 10,0-253,3 ng/ml range (median - Me 83,5 ng/ ml), on the 12-13th week - 30,0-311,0 ng/ml (Me 131,0 ng/ml), on the 22-24th week - 152,0-561,1 ng/ ml (Me 271,0 ng/ml) and by the end of pregnancy - 135,4-770,9 ng/ml (Me 330,0 ng/ml) [5].
2. Inhibitors of blood coagulation
Physiological anticoagulants including tissue factor pathway inhibitor (TFPI), antithrombin III (AT III) and protein system C+S resist uncontrolled blood coagulation. During our research there was no marked the growth of TFPI level in the blood plasma of women during the first half of pregnancy, but it grew further with the course of pregnancy [5]. The activity of AT III did not change significantly; however, there were registered
clear changes of anticoagulant system connected with C protein. According to the research results, the activity of C protein had been increasing beginning from the 12-13th week of pregnancy and continuing up to the 22-24th week. At a later stage, before the delivery, the activity of the mentioned anticoagulant decreased.
It can also be oserved, that following the decrease a significant increase of C protein activity was registered during the first days after the delivery. A different dinamics during pregnancy was typical for the cofactor of C protein - S protein, facilitating the anticoagulant and profibrinolytic activity of C protein. According to the research conducted, the activity of a free form of S protein had been decreasing already since the 6-8th week of pregnancy - by 42.1%; in comparison with the women in pregravid period this decrease was stable up to the first days after the delivery. Although the fall of free S protein during pregnancy is a physiological phenomenon, it is not clear, whether it facilitates hypercoagulation during pregnancy and growth of the thromboembolism case number.
3. Fibrinolysis
Fibrinolytic reactions are not less important obstacle on the way of hypercoagulation. The key enzyme of fibrinolysis, as it is known, is plasmin, deriving from inactive proenzyme - plasminogen under the control of activators (tissue plasminogen activator - t-PA, urokinase plasminogen activator -u-PA) and the inhibitors of this process [inhibitors of plasminogen activator of 1 and 2 types - PAI-1 and PAI-2].
A number of researchers suppose that the fibrinolytic activity of blood plasma decreases during pregnancy, keeps low during delivery and return to the normal level after spontaneous delivery. These assumptions are based on the establishment of facts of prolongation of fibrin clot lysis obtained from blood plasma, of the decrease of t-PA concentration and also the decrease of PAI-1 and PAI-2 levels [15]. Some authors believe, that the observed changes of the above mentioned activators and inhibitors of fibrinolysis do not significantly influence the overall fibrinolytic blood activity [16].
However, it contradicts the apparent growth of D-dimers concentration in blood with the increase of gestation terms. Considering the latter and also the growth of «plasmin-a2-AP» complex concentration, other authors state, that fibrinolytic activity by pregnancy is not decreased, but increases in comparison with women in pregravid period [17]. Consequently, this matter lacks unity and it is not clear, why there are no thrombosis by the growth of D-dimers, especially at late stages of pregnancy. In our research the t-PA level was stable during the whole period of pregnancy, though its
в
antagonist for specific influence on plasminogen -PAI-1 showed an "explosive" growth in the second half of pregnancy - by 1.5 and 5.0 times (from the initial level till pregnancy), respectively, on the 2224th and 34-36th weeks (Fig.2, b, c). After delivery the level of this inhibitor returned to the initial value (before pregnancy) [8]. It is noteworthy, that the decrease of t-PA level is connected both with the high risk of thrombosis and the intrauterine growth retardation and preeclampsia [18].
Another important activator of plasminogen - u-PA accounting for 15% of all fibrinolysis activating blood potential and blood vessel walls in normal conditions, grew significantly during pregnancy beginning from the 22-24th week, while the value of this indicator was maximally high at the late stages of pregnancy (Fig.3,a).
The content of thrombin activated fibrinolysis inhibitor in the blood plasma of pregnant women had been already growing since early stages of gestation (Fig. 3, b). Particularly, there was registered the growth of its level on the 6-8th week by 5.9% (in comparison with the data before pregnancy), on the 12-13th week - by 18.8%, on the 22-24th week - by 29.2% and at late stages of pregnancy - by 47.4% [4].
The above-mentioned work also included the study of the dynamics of the content of direct plasmin inhibitor - a2-AP. This the representative of the family of serpins is the basic biological plasmin inhibitor, which accounts for 90% of antiplasmin activity. It is known, that a2-AP connects the plasmin circulating in blood with the formation of inactive complex "plasmin - a2-antiplasmin" [19].
According to the obtained results, the activity of a2-AP had been progressively decreasing with the course of pregnancy (Fig.3, c). Already on the 1213th week of pregnancy the activity of a2-AP was by 17.7% lower in comparison with the data obtained at the pregravid stage. At the further stage, this indicator continued decreasing 40.7% (22-24 week) and 45.6% (34-36 week). Subsequently, on the first 2-3rd days after spontaneous term delivery the a2-AP activity grew, however, there was marked a large spread of data in the selection. It should be noted, that hereditary or acquired insufficiency of a2-AP is associated with heavy bleedings caused by uncontrolled hyperfibrinolysis [20].
Taking the foregoing into consideration, it can be assumed, that the growth of u-PA level on the background of a2-AP activity reduction at late stages of pregnancy creates optimal conditions for the formation and free circulation of plasmin, and, consequently, the increase of fibrinolytic activity of blood in the systemic circulation. It is apparently accompanied by the division of fibrinolytic blood system functions according to the direction of their influence. Parietal (intravascular) fibrinolytic reactions are inhibited, and this is illustrated by
u-PA, activity
1 D
12-13 22-24 34-36 After delivery
5
TAPI, level
Before pregnancy 6-8
12-13 22-24 34-36 After delivery
a2-AP, activity
Before pregnancy 64
After delivery
Figure 3.
Dynamics of urokinase plasminogen activator (a), the level
of thrombin activated fibrinolysis inhibitor (b) and a2-antiplasmin activity ( c) in blood plasma of pregnant and nonpregnant women
the prevalence of PAI-1 growth in comparison with t-PA and the decrease of TAFI level. Because of described processes plasminogen possessing affinity to fibrin considerably loses the ability to turn into a proteolytically active enzyme, which increases the stability of fibrinous masses in the vessels damaged by delivery. The mentioned mechanism can be of critical importance for the reduction of blood loss in the situation, when the considerable amount of "gaping" vessels of the uterus require thrombosing. Evidently, the disorder of this mechanism can lead to the so-called "hyperfibrinolytic" bleedings in terms of fetal distress and early postpartum period.
A separate question arises by the evaluation of the necessity and clinical significance of D-dimers
a
B
determination during pregnancy, normally used for the diagnostics of disseminated intravascular coagulation (DIC syndrome) and exclusion of VTEC. Some authors reflect upon the informational content of the determination of these products of stabilized fibrin lysis in connection with natural growth of their concentration with the pregnancy progress without any pathologies. It is assumed, that the solution of this question requires a fuller insight into the source of D-dimers formation at late stages of pregnancy. Earlier a number of researches had stated, that fibrin can be intensified by covalent links of XIIIa factor at the dissolution stage till its transformation into gel (clot) [21]. Consequently, it is admitted, that the source of D-dimers formation in particular conditions can be represented not only by a fibrin clot, but also its soluble precursors, which is described in detail in the work of Adam S. and Key N. [22].
Taking into consideration the presented data, the hypothesis is made, according to which in the prepartum period, in spite of a considerable growth of hemostatic potential, soluble forms of stabilized fibrin are primarily subject to plasmin proteolysis, which disturbs the classical way of the transformation of fibrinogen into fibrin clot. Upon that, the high level of D-dimers can show the effectiveness of mechanisms protecting from intravascular coagulation and thrombosis before delivery. It can also be assumed, that the reduction of plasmin potential in circulating blood, depending firstly on a2-AP, is able to increase the chances of venous thromboembolic complications in obstetric patients and maternity patients.
Conclusion
The article presents the results of the evaluation of a broad spectrum of participants of hemostatic and fibrinolytic reactions, ring hemostatic balance during pregnancy and delivery.
The obtained data demonstrated a gradual, but explicit growth of hemostatic blood potential with the growth of gestation age, the peak of intensity of which fell at late stages of pregnancy and right after delivery. It is assumed, that preterm intensification of this potential might be an initiator of not only of intravascular blood coagulation, but also of the end of pregnancy and start of delivery, as the possibility of massive obstetric bleedings dangerous for mother's health is minimal in these cases.
There was determined a weak resistance to hypercoagulation during pregnancy on the part of physiological anticoagulants - TFPI, AT III and components of C protein system, the level and activity of which had been changing divergently and unsystematically during pregnancy.
There was discovered the division of fibrinolytic blood system functions according to the direction of their influence. Parietal (intravascular) fibrinolytic
reactions are inhibited at the end of pregnancy, which is extremely important for the reduction of blood loss during delivery. It is obvious, that the disorder of this mechanism ("spontaneous" reduction of PAI-1 and TAFI activity at the 34-36th week) can lead to the so-called "hyperfibrinolytic" during pregnancy and early postpartum period.
There was described a previously unknown mechanism preventing thrombus formation in late pregnancy, associated with the enhancement of fibrinolytic reactions in blood circulation (on the background of multiple growth of a2-AP activity), leading to fibrin dissolution till its transformation into fibrin gel (clot). It is illustrated by the sharp increase of D-dimers level by the lack of thrombosis. The blocking of the stated mechanism can lead to thrombosis during pregnancy or early postpartum period.
Generally, it can be mentioned, that the hemostatic balance is maintained during physiological pregnancy, primarily, due to the effective resistance of hemostatic and fibrinolytic reactions, which the most important adaptation condition of a women during this period of life.
The described "shunt" mechanism of intravascular coagulation, preventing thrombus formation, might have a universal character, the knowledge of which is useful for the deeper insight into the pathogenic mechanism of vascular catastrophes in different spheres of clinical medicine.
The current research was conducted in terms of Government task of the Ministry of Health of the Russian Federation for FSBEI HPE Altai State Medical University of the Ministry of Health of the Russian Federation on the subject of R&D: "Referent values of the indicators of hemostasis and fibrinolysis by physiological pregnancy and clinical importance by preeclampsia.
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