THE PERSONALISED ASPECTS OF DEVELOPMENT OF VENOUS TROMBOEMBOLIC COMPLICATIONS IN POLYTRAUMA
Chita State Medical Academy,
Chita, Russia
The recent years are associated with the increasing rates of injuries. So, 13.28 million cases of injuries, poisoning and other consequences of external factors have been registered in the medical facilities of Russian Health Ministry in 2014 [1]. Among the men at the age of 20-24, accidents are the causes of death in more than 80 % [2].
Polytrauma is characterized with specific severity of clinical manifestations, is accompanied by significant disorder of vital functions, difficulties for diagnostics and treatment, high rate of complications, long period of hospital stay and disability [3, 4].
Thromboembolic complications are in 40-77 % of cases with polytrauma and are characterized with latent clinical course, difficult treatment and high mortality [5].
Currently, thrombophilia has been recognized as the cause of complications and burdening course of various diseases. It was found that the greatest part of surgical pathology proceeds at the background of disorders in hemostasis and immune system. The condition of hemostasis and immune systems significantly influences on the course of diseases, efficiency of treatment and outcomes. Unfortunately, the transbaikalian population is poorly studied in this regard. For the current moment some population studies of frequency of prothrombotic mutations FV (Leiden G1691A), mutations in the prothrombin II gene and gene MTGFR (C677T) among healthy individuals and patients with cerebral vascular diseases, infectious diseases in children, pulmonary tumors, traumatic brain injury in ethnic and age aspects have been performed [6]. However, such studies do not include patients with polytrauma. Trauma is a trigger for enzyme cascade of hemostasis system. Breakdown of spare capacity of the body happens in patients with thrombophilia, and it inevitably causes some rude pathophysiological changes [7, 8].
Therefore, search of genetic markers determining the features of individual responses of protective systems of pathogenesis of complications is an actual task for prediction of the course, prevention of hemocoagulation disorders and outcomes of injuries.
The objective of the study - to identify the personalised prognostic criteria of development of venous thromboembolic complications (VTEC) in patients with polytrauma.
MATERIALS AND METHODS
The study was conducted according to World Medical Association Declaration of Helsinki (the amendments 1964, 2011) and the Rules for Clinical Practice in Russian Federation confirmed by the Order of Health Ministry of the Russian Federation, June 19, 2003, No.266.
The examination included 114 patients at the age of 20-40 with polytrauma. They received the treatment in the City Clinical Hospital No.1, Chita. The men were 71.9 % (82 persons), the women – 28.1 % (32 patients). The inclusion criteria were polytrauma with ISS > 9. The exclusion criteria were the patients with malignant tumors, acute inflammatory diseases and chronic diseases at the inflammation stage, the women with pregnancy or menstrual flux, the individuals with previous anticoagulant therapy.
The patients were distributed into the following groups: the group 1 – 73 patients with uncomplicated course of polytrauma, the group 2 – 41 patients with venous thromboembolic complications of polytrauma (thrombosis in the vessels of the lower extremities, pulmonary embolism including the small branches of the artery). The control group included 100 almost healthy men and women at the age of 20-40.
The mechanisms of the injuries were as indicated below: the main part included the victims of road traffic accidents – 70 patients (61.5 %), falling from height – 23 persons (20.2 %), injuries inflicted by other persons – 12 persons (10.5 %), injuries as result of contact with various mechanisms and devices – 6 persons (5.2 %), other types of injuries – 3 persons (2.6 %).
The diagnosis was made on the basis of the clinical examination, the data of X-ray examination, computer tomography, ultrasonic scanning, the laboratory investigations. The severity of the injuries was estimated with Abbreviated Injury Scale (AIS) and Injury Severity Score (ISS). Thromboembolic complications were confirmed by the clinical, instrumental (electrocardiography, plain chest radiography, computer tomography, vascular ultrasonic doppler) and laboratory data (quantitative estimation of D-dimer, APTT, INR, SFMC) [4].
The sets of the reagents from Vector-Best Ltd. (Novosibirsk, Russia) were used for estimation of the levels of cytokines in the serum (IL-1b, IL-2, IL-8, IL-10, TNFα and TGFβ). The level of cytokines was measured with the sandwich type of enzyme-linked immunosorbent assay (ELISA). The coagulation hemostasis was estimated with the following tests: prothrombin time, INR, APTT, fibrinogen level, resistance of Va factor to APC with coagulometry techniques, D-dimer – with the turbidity method. LTA was measured with the technique by Yu.A. Vitkovsky et al. (1999) [8].
The genes SNP – IL-2 (T330G), IL-10 (G1082A, C819T), FV (Leiden – G1691A), FII (prothrombin G20210A), MTHFR (C677T) – were measured with polymerase chain reaction with use of the primers (Litekh, Moscow, Russia). The analysis included genome DNA separated from leukocytes of whole blood with use of the reagent DNA-express-blood. Then the response of amplification with two pairs of allel-specific primers was conducted [9].
The received data was analyzed with Microsoft Office 2003 for Windows XP Professional and STATISTICA 6.1 (StatSoft, USA). The non-parametrical methods were used for comparison of the clinical and laboratory values. The results were presented as median (Me) and the interquartile (25th and 75th percentiles) range. Mann-Whitney test was used for estimation of the statistically significant differences between the groups. Fisher’s exact test (c2 test) was used for comparison of the groups according to the qualitative binary feature. Hardy-Weinberg's law was used for estimation of frequencies of the allelic types of the genes. The relative probability of an event and odds ratio (OR) were calculated. Multiple regression analysis was used for prediction of the values of the range of dependent variables with use of the known values of other variables.
RESULTS AND DISCUSSION
The occurrence of T SPN allele of the gene (T330G) was higher in the group of the patients with complicated polytrauma as compared with the group with uncomplicated course. The homozygote T/T is more often in presence of complications (the table 1).
| The table 1 | |||
| The frequency of polymorphism of IL-2 gene (T330G), IL-10 gene (G1082A) and IL-10 gene (C819T) in patients with uncomplicated course of polytrauma and development of thromboembolic complications | |||
| A value | Uncomplicated course | Complicated course | c2 |
| (n = 73) | (n = 41) | ||
| IL-2 (Ò330G) | |||
| Genotype | |||
| T/T | 48 (0.66) | 36 (0.88) | Ò/Ò ê Ò/G T/T to T/G |
| 7.47 (ð = 0.0063) | |||
| T/G | 24 (0.32) | 4 (0.10) | T/G ê G/G T/G to G/G |
| 1.71 (ð = 0.19) | |||
| G/G | 1 (0.014) | 1 (0.02) | T/T ê G/G |
| 0.04 (ð = 0.84) | |||
| Allel | |||
| T | 120 (0.82) | 76 (0.93) | 4.79 (ð = 0.028) |
| G | 26 (0.18) | 6 (0.07) | |
| IL-10 (G1082A) | |||
| Genotype | |||
| G/G | 33 (0.45) | 29 (0.71) | Ñ/Ñ ê Ñ/Ò C/C to C/T |
| 4.44 (ð = 0.035) | |||
| G/A | 32 (0.44) | 11 (0.27) | Ñ/Ò ê Ò/Ò C/T to T/T |
| 0.14 (ð = 0.71) | |||
| A/A | 8 (0.11) | 1 (0.02) | Ñ/Ñ ê Ò/Ò C/C to T/T |
| 4.10 (ð = 0.043) | |||
| Alleles | |||
| G | 98 (0.67) | 69 (0.84) | 7.76 (ð = 0.0053) |
| A | 48 (0.33) | 13 (0.16) | |
| IL-10 (Ñ819Ò) | |||
| Genotype | |||
| Ñ/Ñ | 34 (0.47) | 26 (0.76) | Ñ/Ñ ê Ñ/Ò C/C to C/T |
| 3.85 (ð=0.049) | |||
| Ñ/Ò | 38 (0.52) | 13 (0.19) | Ñ/Ò ê Ò/Ò C/T to T/T |
| 2.39 (ð = 0.12) | |||
| Ò/Ò | 1 (0.01) | 2 (0.05) | Ñ/Ñ ê Ò/Ò C/C to T/T |
| 0.63 (ð = 0.43) | |||
| Alleles | |||
| Ñ | 106 (0.73) | 65 (0.793) | 1.24 (ð = 0.26) |
| Ò | 40 (0.27) | 17 (0.207) | |
| Note: p - statistical significance of differences with p < 0.05. | |||
It was found that polytrauma was accompanied by increasing levels of IL-2 regardless of a genotype and presence of complications as compared with the control group (p < 0.001). The highest level of IL-2 was identified in the carriers with the homozygote variant of T/T. If the course of polytrauma was without complications, the level of IL-2 was 9.5 (7.3; 10.9) pg/ml, whereas homozygotes T/T showed the level of 6.3 (4.2; 7.0) pg/ml (p < 0.001).
The homozygote variant of the carrier state of SNP IL-10 (G1082A) G/G was more common in the group with thromboembolic complications. The homozygote mutation A/A was identified in 8 patients without complications and in 1 patient with a complication. Allele G was more common in the patients with complications, whereas allele A was more frequently identified in the patients with uncomplicated course of polytrauma (the table 1).
After investigating the level of IL-10 in the patients with polytrauma with dependence on SNP genotype of IL-10 gene (G1082A) and presence of thromboembolic complications, it was found that polytrauma was accompanied by the increasing levels of IL-10 regardless of the genotype and presence of complications in comparison with the control group (p < 0.001). The highest level of IL-10 was identified in the carriers with the homozygote variant of A/A. Thus, the level of IL-10 was 12.2 (6.3; 13) pg/ml in the uncomplicated course of polytrauma as compared with 5.6 (3.8; 6.1) pg/ml (p < 0.001) for the homozygotes G/G.
It was found that the normal homozygotes (C/C) of the polymorphism of IL-10 gene (C819T) were more frequently registered in the group of the patients with thromboembolic complications. There were not any statistically significant intergroup differences in the occurrence of the heterozygotes C/T, homozygotes T/T and the alleles C and T (the table 1).
It was found that polytrauma was accompanied by the increasing level of IL-10 regardless of the genotype of SNP of IL-10 gene (C819T) and presence of complications as compared with the control group (p < 0.001). The highest level of IL-10 was identified in the carriers of the homozygote variant of T/T. So, the level of IL-10 was 11.2 (8.8; 12.6) pg/ml in the uncomplicated course of polytrauma, whereas it was 4.9 (3.6; 5.7) pg/ml for the homozygotes C/C (p < 0.001).
Therefore, presence of the allele variants of IL-2 genes (-330G), IL-10 gene (-819T, -1082A) significantly reduces the risk of thromboembolic complications in patients with polytrauma. The odds ratio and relative risk were lower than 1.0 in the carriers of the above-mentioned alleles.
Both clinical groups demonstrated the relative and absolute values of LTA functioning in the patients with polytrauma in dependence on presence of hemocoagulation complications and the genotypes of the examined polymorphisms of IL-2 and IL-10 genes on the 3rd day after trauma. The maximal values were noted in the patients with hemocoagulation disorders and 2.8-fold increase in the absolute value as compared with the control group (p < 0.001). It was found that the highest activity of lymphocyte-platelet interaction was registered among the carriers of T/T genotype of IL-2 gene (T330G). The absolute level of LTA was 0.56 (0.43; 0.61) × 109/l in the group without complications and 0.75 (0.64; 0.86) × 109/l in the group with complications, whereas the healthy individuals demonstrated the value of 0.26 (0.23; 0.35) (p < 0.001). The absolute value of LTA was lower in the patients with heterozygote variant of T/G of polymorphism of IL-2 gene (T330G) as compared with the homozygote carriers of G/G both in the groups without complications and with them – 0.43 (0.38; 0.56) and 0.58 (0.42; 0.76) correspondingly. The degree of LTA and the level of lymphocytes did not differ from the homozygotes T/T for this genotype. The lowest values of LTA were identified in the carriers of G/G polymorphism of the IL-2 gene (T330G). The absolute value was 0.34 × 109/l in the group without complications and it differed from the control values and the homozygote variant of T/T of the studied polymorphism. The high levels of LTA were registered in the carriers of G/G genotype during the analysis of LTA activity in the patients depending on the polymorphism of IL-10 gene (G1082A). The amount of coaggregates was 0.51 × 109/l in the group without complications and 0.65 × 109/l in the group with complications, with differences from the control group – 0.25 × 109/l (p < 0.001). For the carriers of G/A genotype of polymorphism of IL-10 gene (G1082A) the values of LTA differed from the control values and the carriers with G/G genotype. The absolute value was 0.38 (0.26; 0.52) in the group without complications and 0.53 (0.36; 0.64) in the group with complications.
The following stage was the incidence of the prothrombotic mutations: V factor Leiden 1691A, prothrombin G20210A, MTHFR C677T. In the patients with the uncomplicated course the homozygote carrier state of G/G SNP gene of V Leiden factor (G 1691A) was 96 %, in the control group – 99 %. The genotype G/A did not differ from the values in the healthy persons. The homozygote carriers of A/A were absent in both groups. The genotype G/A and the allele A were more frequent in the patients with thromboembolic complications that were higher than in the control group (the table 2).
| Table 2 | |||
| The frequency of polymorphism of gene of factor V (Leiden) (G1691A), prothrombin (G20210A), MTHFR (C677T) in patients with uncomplicated course of polytrauma and development of thromboembolic complications | |||
| A value | Controls | Uncomplicated course | Complicated course |
| (n = 100) | (n = 73) | ||
| (n = 41) | |||
| V (Leiden) (G1691A) | |||
| Genotype | |||
| G/G | 99 (0.99) | 70 (0.96) | 33 (0.80) */** |
| G/A | 1 (0.01) | 3 (0.04) | 7 (0.18) |
| A/A | - | - | 1 (0.02) |
| Allel | |||
| G | 199 (0.995) | 143 (0.979) | 73 (0.90) */** |
| A | 1 (0.005) | 3 (0.021) | 9 (0.10) */** |
| Prothrombine (G20210A) | |||
| Genotype | |||
| G/G | 98 (0.98) | 72 (0.99) | 36 (0.85) */** |
| G/A | 2 (0.02) | 1 (0.01) | 5 (0.15) */** |
| À/À | - | - | - |
| Allel | |||
| G | 198 (0.99) | 145 (0.99) | 77 (0.93) */** |
| A | 2 (0.01) | 1 (0.01) | 5 (0.07) */** |
| MTHFR (Ñ677Ò) | |||
| Genotype | |||
| Ñ/Ñ | 44 (0.44) | 52 (0.71) * | 12 (0.29) ** |
| Ñ/Ò | 45 (0.45) | 17 (0.23) | 12 (0.29) */** |
| Ò/Ò | 11 (0.11) | 4 (0.06) * | 17 (0.42) */** |
| Allel | |||
| Ñ | 133 (0.67) | 121 (0.83) * | 36 (0.44) */** |
| Ò | 67 (0.33) | 25 (0.17) * | 46 (0.56) */** |
| Note: * - statistical significance of differences with the controls; ** - statistical significance of differences between the clinical groups (reliable for p < 0.05). | |||
G/G homozygote was more frequently identified in the patients with the uncomplicated course of polytrauma as compared with the complicated course. The incidence of the genotypes G/A and A/A was higher in the patients with hemocoagulation complications. The allele A was identified in 10 % of the patients with polytrauma with complications and in 2 % in the patients without complications (the table 2).
The incidence of SNP genotypes of the prothrombin gene (G20210A) was similar in the healthy individuals and in the patients with the uncomplicated course of polytrauma. No carriers with the homozygote variant of A/A were found in all examined persons. The homozygote carriers with G/G were more frequent in absence of complications. The frequency of the allele A of the indicated polymorphism is higher for polytrauma with complications (the table 2).
The rate of SNP of MTHFR gene (C677T) showed the significant differences in the patients with the complicated course of trauma as compared with the uncomplicated course (the table 2). So, the allele T is 3 times higher in the patients with thromboembolic complications as compared with the group without complications. The groups of the individuals with complicated polytrauma demonstrated the proportion of the carriers with the homozygote variant of C/C in the group of the uncomplicated course, and the increasing proportion of T/T homozygotes (the table 2).
The simultaneous presence of the allele G of the polymorphism of FII gene (G20210A) and the allele T of the polymorphism of MTHFR gene (C677T) was in 3 patients, but the combination of polymorphisms of the gene V of Leiden factor1691A and the gene MTHFR C677T was in 2 cases; in these patients the course of polytrauma was accompanied by development of pulmonary embolism, whereas such combinations were not found in the group with the uncomplicated course of polytrauma.
It is known the mutant homozygote FVL is accompanied by the decreasing sensitivity of the plasma factor to activated protein C and, as result, to hypercoagulation. The carriers of the heterozygote and homozygote polymorphism of the prothrombin G20210A demonstrated the increase in biosynthesis of the proenzyme that caused the significant thrombinemia during activation of FII resulting in increasing blood clotting. The same high rate of the carrier state of the allele T of the polymorphism of MTHFR gene was identified in both groups. The abnormal molecule MTHFR is characterized with thermolability and influences on the processes of the folate cycle. Herewith, the blood level of homocysteine increases and the atrombogenic function of the vascular wall decreases. The above-mentioned mutations create the genetic readiness to hypercoagulation – primary thrombophilia. Trauma and long term immobilization are the triggering factors for appearance of coagulation disorders and for development of thrombotic complications [10].
The table 3 shows the parameters of coagulation hemostasis in the patients with polytrauma.
| Table 3 | |||
| The values of coagulation hemostasis in patients with polytrauma | |||
| Values | Controls | Patients with polytrauma | |
| (n = 100) | Uncomplicated course | Complicated course | |
| (n = 73) | (n = 41) | ||
| INR, units | 1 | 0.91 | 0.88 |
| [0.97 – 1.03] | [0.88 – 0.97] | [0.82 – 0.92] | |
| ð1 < 0.05 | ð1 < 0.001 | ||
| ð2 > 0.05 | |||
| APTT, sec. | 28.2 | 26.9 | 26.3 |
| [26.4 –29.3] | [26.1 –28.2] | [26.0 –28.8] | |
| ð1 > 0.05 | ð1 > 0.01 | ||
| ð2 > 0.05 | |||
| Fibrinogen, g/l | 3.1 | 4.8 | 5.3 |
| [2.8 – 3.3] | [3.7 – 5.2] | [4.1 – 5.6] | |
| ð1 < 0.01 | ð1 < 0.01 | ||
| ð2 > 0.05 | |||
| D-dimer, mg/l | 0.26 | 0.38 | 0.48 |
| [0.21 – 0.29] | [0.32 – 0.43] | [0.39 – 0.55] | |
| ð1 < 0.001 | ð1 < 0.001 | ||
| ð2 > 0.05 | |||
| SFMC, mg/l | 28.2 | 37.6 | 44.5 |
| [22.4 – 31.6 ] | [29.3 – 45.4] | [35.8 – 51.2 ] | |
| ð1 < 0.05 | ð1 < 0.001 | ||
| ð2 > 0.05 | |||
| Note: ð1 – level of statistical significance of differences in comparison with the control group (Mann-Whitney test); | |||
| ð2 – level of statistical significance of differences between the clinical groups (Mann-Whitney test). | |||
The level of INR decreased and APTT, fibrinogen, D-dimer and SFMC increased on the 3rd day as compared with the control group (p < 0.01). The decrease in INR characterized the state of hypercoagulation and is estimated as the risk of clot formation. The levels of APTT, fibrinogen, D-dimer and SFMC significantly increase during the situations relating to the increase in formation in fibrin in blood flow and development of thromboembolic complications [11].
We investigated the association between the anamnesis data, the clinical values, the results of the laboratory and instrumental examinations. We used the regression model including the examined values in the patients with the uncomplicated course and development of thromboembolic complications in polytrauma.
The results of this multifactorial step by step regression analysis showed that identification of LTA was closely associated with thromboembolic complications in the patients with polytrauma (the step 1). The accuracy of prediction increased after addition of the data about the level of IL-2 (the step 2), the level of D-dimer (the step 3), the polymorphism of MTHFR gene (-677T) (the step 4), the polymorphism of IL-10 (-1082G) (the step 5), APTT (the step 6), the polymorphism of IL-2 gene (-330G) (the step 7) and the polymorphism of FV (Leiden) (-1691A) (the step 8). The addition of other values to the selected ones did not give any predictive power (the table 4).
| Table 4 | |||||
| Predictive significance of the values in the multivariable model of development of thromboembolic complications in polytrauma | |||||
| N = 114 | *ÁÅÒÀ *BETA | Ñò. Îø. ÁÅÒÀ St. OR BETA | Â | Ñò. Îø. St. OR | **ð-óðîâ. **p-level |
| Â | |||||
| ËÒÀ / LPA | 0.34 | 0.06 | 0.03 | 0.01 | 0.0001 |
| IL-2 | 0.26 | 0.07 | 0.04 | 0.01 | 0.0004 |
| D-äèìåð / D-dimer | 0.17 | 0.06 | 1.05 | 0.39 | 0.009 |
| MTHFR-677Ñ>Ò | 0.37 | 0.08 | 0.23 | 0.05 | 0.0001 |
| IL10-1082G>A | -0.22 | 0.05 | -0.16 | 0.04 | 0.0001 |
| À×Ò / APTT | -0.17 | 0.05 | -0.04 | 0.01 | 0.0008 |
| IL2-303Ò>G | -0.28 | 0.1 | -0.24 | 0.08 | 0.004 |
| FV(Leiden)-1691G>A | 0.18 | 0.06 | 0.25 | 0.09 | 0.007 |
| Note: *beta – the regression coefficient; **ð – level of statistical significance (reliable for p ≤ 0.05). | |||||
After the analysis of the predictive model, which was developed as result of the conducted multifactorial regression analysis, the value of multiple coefficient of correlation was 0.855. It supposes the presence of strong lineal relationship between the factors of influence and the response (disordered union). The determination coefficient (R-square) – 0.78. This fact shows the high degree of correspondence of the regression model to the empirical data. The level of significance of the regression model was p < 0.0001, i.e. high sensitivity and reliability [12].
Therefore, the highest predictive value is associated with estimation of LTA, IL-2, D-dimer, the polymorphism of MTHFR-677C>T gene, the polymorphism of IL10-1082G>A gene, APTT, the polymorphism of IL2-303T>G gene and the polymorphism of FV(Leiden)-1691G>A gene. It can be used for the diagnostic process with the aim of prediction of development of hemocoagulation disorders and it will allow preventing the development of thromboembolic complications.
Considering the results of our investigations, we offer the scheme of pathogenesis of the hemocoagulation complications in the patients with polytrauma (Fig.).
Figure
The scheme of pathogenesis of hemocoagulative complications in the patients with polytrauma
The results indicate the increased primary risk of thrombosis in the patients with the separate allele variants of the prothrombotic genes and the modulating influence on thrombogenesis of translated IL-2 and IL-10, their levels depend on SNP of the corresponding genes. We think that the studied polymorphisms of the genes and the translated proteins (the clotting factors, cytokines, the enzyme) are directly or indirectly involved in the pathogenesis of clot formation and thromboembolism which often complicates polytrauma.
Therefore, the investigation of the polymorphisms of the cytokine genes, the prothrombotic genes and lymphocyte-platelet adhesion are the demonstrative example of close relationship between immunity and hemostasis which are the components of the whole integral cellular and humoral system of body protection, where cytokines take the role of the link [8, 11, 13].
CONCLUSION
Estimation of LTA, IL-2, D-dimer, APTT, the polymorphism of MTHFR-677Ñ>Ò gene, the polymorphism of IL10-1082G>A gene, the polymorphism of IL2-303Ò>G gene and the polymorphism of FV(Leiden)-1691G>A gene in polytrauma can be used during the diagnostic process for prediction of development of hemocoagulation disorders and will allow performing the individual preventive measures for thromboembolic complications.