The present study revealed significantly higher concentrations of investigated parameters (t-PA Ag, PAI-1 Ag, D-dimer, fibrinogen) and insignificantly elevated PLT count in LEAD patients when contrasted with healthy subjects. Moreover, the observations indicated significantly increasing values of fibrinogen and PLT, and an insignificant (due to the number of subjects) but clear trend of increasing concentrations of D-dimer at the successive stages of LEAD (in accordance with the Fontaine classification). This was also reflected in the obtained negative correlation of IC distance, fibrinogen concentrations and PLT count. Besides, the ABI negatively correlated with D-dimer concentrations and PLT count. No influence of gender on the concentrations of the studied parameters was found, yet, the age of patients with LEAD positively correlated with D-dimer concentrations and the subgroup of patients ≥ 65 years of age revealed significantly elevated values of this parameter when compared to the subjects in the younger age group.
The world literature refers to LEAD patients as PAD or PAOD. Zalewska-Rydzkowska et al. measured t-PA Ag and PAI-Ag levels in 40 patients with PAOD classified as stage II of Fontaine’s classification. They found significantly elevated concentrations of t-PA Ag and PAI-1 Ag in the blood patients against healthy subjects [14]. Similar results – high values of t-PA Ag and PAI-1 Ag – in the study of 103 patients (also stage II of Fontaine’s classification) were obtained by Strano et al. [15]. A comparative analysis conducted by Killewich et al. on two groups of patients: with PAD and mild ischemia (stage IIa of Fontaine’s classification, n = 18) and subjects with PAD and severe ischemia (stage IIb, n = 51) found significantly higher values of t-PA Ag in patients at stage IIb of Fontaine’s classification, in comparison to IIa subjects and the control group, as well as a negative correlation between t-PA Ag levels and the claudication-free distance [16]. The studies carried out by Rość et al. on patients at stage II (n = 13), III (n = 10) and IV (n = 2) of Fontaine’s classification reported increasing values of t-PA Ag with the disease progress [17]. Elevated concentrations of PAI-1 in patients with lower extremity artery disease against healthy subjects were also observed by Trifiletti et al. [18]. No distinctive differences were obtained in this research in terms of concentrations of t-PA Ag and PAI-1 Ag in patients with division to LEAD patients.
One of significant cardiovascular risk factors is the reduced fibrinolytic activity of plasma depending on t-PA synthesis and/or the increased activity of PAI-1 [19,20]. A considerable part of plasma t-PA circulates as complexes with PAI-1, therefore, the concentration of t-PA is believed to be primarily dependent on PAI-1 levels [21]. t-PA-PAI-1 complexes do not demonstrate any catalytic activity to plasminogen and their active form is free t-PA. The observations made in the present study, just like the examples provided by the scientific literature about high concentrations of t-PA in patients with LEAD/PAD indirectly indicate that patients with atherosclerosis have damaged endothelium which is the main source of t-PA. However, given the high concentration of PAI-1 Ag in PAD patients, t-PA forms inactive fibrinolytic complexes with this inhibitor.
The research studies conducted by van der Bom et al. reported elevated D-dimer levels in the blood of patients with PAD [22]. Similar, significantly higher values of D-dimer were observed by Trifiletti et al. in patients with lower extremity artery disease in comparison to healthy subjects [18]. Likewise, significantly higher D-dimer levels were obtained by Strano et al. in the blood of 103 patients with PAD (stage II of Fontaine’s classification) in comparison to the control group [15]. High FDP levels were reported by Zalewska-Rydzkowska et al. in 40 patients at stage II of Fontaine’s classification [14].
Our studies on D-dimer are consistent with the findings of the authors referred to above. They indicate increased dissolution of stabilized fibrin that is excessively formed during blood coagulation activated by the tissue factor (TF). The research of our center presented in another publication demonstrates increased thrombogenesis and excessive formation of TF in the blood of PAD patients. The present study, despite the observed tendency to increasing D-dimer values at successive Fontaine stages (IIa < IIb < III < IV), did not find statistical significance which may have been associated with a relatively small number of subjects, especially in the group of stage III patients.
This research study observed a significant increase in the concentrations of fibrinogen in the course of successive disease stages in accordance with the Fontaine classification (IIa, IIb, III and IV) in the test group, and the values in patients classified as IIb, III and IV were significantly higher when compared to healthy subjects. The above-mentioned study by Zalewska-Rydzkowska et al. found significantly higher fibrinogen levels in the blood of patients with PAOD classified as II against healthy subjects [14]. Increased fibrinogen concentrations are an independent thrombosis factor in patients with atherosclerosis. A high fibrinogen concentration changes the rheological properties of the blood through increased aggregation of platelets and erythrocytes and intense adhesion of granulocytes. It also contributes to increase the content of fibrinogen in thrombus and to change the thrombus structure.
Available publications do not analyze the absolute number of PLT at subsequent Fontaine stages. Various platelet parameters including platelet count in patients with PAD were investigated by Zeiger et al. who did not find any significant differences between patients and healthy subjects in terms of PLT count. Yet, they demonstrated a negative correlation between platelet count and volume [23]. The role of platelet count, in addition to other platelet parameters, was also mentioned by Bigalke et al. [24]. Still, Braun et al. in their pilot study reported elevated PLT count in patients with PAD against the control group [25]. The study by Demirtas et al. analyzed 82 PAD patients divided in accordance with the Fontaine classification and determined mean platelet volume (MPV) obtaining the increasing values of MPV respectively in class I, II and III of Fontaine’s classification [26]. The importance of MPV as a significant PAD risk factor was highlighted by Berger et al. in the NHANES (National Health and Nutrition Examination Survey) that included as many as 6354 subjects [27]. The analysis of subgroups of PAD patients classified as IIa, IIb and IV of Fontaine’s classification – in the present publication – found a significant increase in PLT count at successive stages of disease. In the present study, lack of statistical significance between these subgroups of patients and class III of Fontaine’s classification was probably affected by a small sample size of patients in this class (n = 4). It should be also added that the values in class IIa, IIb and III were not significantly higher in comparison to the control group, whereas a significant difference was observed between class IV and healthy subjects. Regardless of the aforementioned limitations, it appears that determination of PLT count, due to the fact that this test is readily available in everyday medical practice, should be more appreciated. The findings require further studies on a larger number of LEAD patients.
The ABI is an indispensable parameter in the diagnostics and assessment of LEAD/PAD stage. Van der Bom et al. analyzed t-PA and PAI-1 concentrations depending on the severity of disease classified in accordance with the decreasing value of ABI in 325 patients with PAD selected from a large cohort study group - Rotterdam Study (including nearly 8 thousand people). They demonstrated higher values of t-PA Ag and PAI-1 Ag in patients against healthy subjects, and a significant increase in t-PA Ag with the decreasing ABI [22]. The publication of Mota et al. indicated elevated levels of PAI-1 in PAD patients as compared to healthy subjects and a negative correlation between PAI-1 concentrations and ABI values [28]. However, in the study conducted by McDermott at al. on a large number of patients with PAD (n = 370), significant correlations were not observed between t-PA Ag and ABI concentrations and the levels of PAI-1 Ag and ABI [29]. This study also did not report any statistically significant correlations between t-PA Ag, PAI-1 Ag and IC distance or rest ABI.
Findings reported by Mota et al. highlighted high concentrations of D-dimer in patients with PAD which insignificantly increased with the decreasing ABI [28]. In the above-mentioned research, however, McDermott et al. obtained a significant negative correlation of D-dimer concentrations and the ABI [29]. The severity of lower extremity artery disease assessed as the decreasing ABI, correlated – in the study by Reich et al. – with the increasing values of D-dimer [30], which is consistent with the statistically significant negative correlations between D-dimer levels and the ABI observed in our patients. In their publication, McDermott et al. also described a significant negative correlation between fibrinogen concentrations and the ABI [29]. A similar relationship between increasing values of fibrinogen with the decreasing ankle-brachial index was evidenced by Reich et al. [30]. Such an interdependence was not reported in the present study, but a significant negative correlation was observed between the values of fibrinogen levels and IC distance in the walk test.
Important observations were made in this study in terms of statistically significant positive correlations of D-dimer levels and age in the LEAD group, in addition to significantly higher concentrations of D-dimer in the subgroups of patients over the age of 65 in comparison to LEAD patients under 65. It is evident that the process of blood coagulation increases with age due to atherosclerosis progression and endothelial dysfunction [31]. Since the concentration of D-dimer reflects secondary-to-coagulation fibrinolysis, high D-dimer levels in patients over 65 years of age manifest intensified coagulation and an increase in secondary fibrinolysis. Even though the present study did not find a significant difference in PLT count between LEAD patients and healthy subjects, the comparison of patients and healthy subjects under the age of 65, allowed to determine significantly higher PLT count in patients as compared to healthy subjects from the same age group. Elevated PLT count in the blood of patients with atherosclerosis always increases the risk of thrombosis.
Limitations of the study
Based on the study results it is hard to explain the apparent contradiction of high concentrations of t-PA Ag, PAI-1 Ag and D-dimer in PAD patients. A considerable limitation of the present study was the use of methods for quantitative detection of antigen proteins only (t-PA Ag, PAI-1 Ag) without measuring the activity of t-PA and PAI-1 at the same time. The measurement of t-PA activity allows to determine the size of t-PA active fraction responsible for transforming plasminogen into plasmin within the entire antigen range of t-PA protein. The research methodology of the activity demonstrated by these parameters is complicated and incredibly hard to conduct considering the approved research protocol of the present study. Another limitation of the present study is also a small number (n = 4) of LEAD patients classified as III of Fontaine’s classification.
Conclusion
1. High t-PA Ag concentrations in LEAD patients reflect damaged endothelium which comprises the main source of this factor. With high PAI-1 Ag levels, inactive fibrinolytic t-PA-PAI-1 complexes are formed.
2. Increasing concentrations of fibrinogen and rising PLT count at the successive stages of Fontaine’s classification, confirmed by negative correlations with IC distance (and with the ABI value in case of PLT count) suggest increasing ischemia (hypoxia) and inflammatory condition which is associated with progression of atherosclerosis.
3. Elevated values of D-dimer reflect the aggregation of secondary fibrinolysis activation as the LEAD patient ages and along with impaired extremity vascularization manifested by the decreasing ABI at rest.