In the present study we are first to demonstrate that anticoagulant-naïve patients with AF and elevated serum lipopolysaccharide have the prothrombotic fibrin clot phenotype characterized mainly by decreased susceptibility of fibrin clots to tPA-mediated lysis. High LPS levels were associated not only with impaired fibrinolysis, as reflected by a global fibrinolysis assessment, i.e. prolonged CLT in association with increased PAI-1, but also endothelial injury, reflected by increased vWF, along with oxidative stress and inflammation, reflected by elevated GDF-15. Importantly, elevated LPS was the independent predictor of prolonged CLT in AF. Our findings shed new light on prothrombotic effects of low-grade endotoxemia in humans, indicating that in AF and possibly in other cardiovascular diseases [21], elevated LPS impairs fibrinolysis largely via increased PAI-1 concentrations in circulating blood, which might at least in part explain an increased risk of ischemic cardiovascular events in nonseptic patients with elevated LPS. We also observed CLT in association with NT-proBNP and cTnI, which is in line with the previous findings and supports their utility in the multi-marker assessment of the prothrombotic state in patients with AF [22,23,24].

The conversion of plasminogen into plasmin by t-PA is under control of PAI-1 released from platelets, endothelium, hepatocytes, adipocytes, and, to some extent, from fibroblasts [14, 25]. Several antifibrinolytic proteins (i.e. PAI-2, α2-antiplasmin, TAFI) further contribute to fibrinolysis efficiency in vivo [14]. Enhanced PAI-1 release in response to inflammation has been demonstrated to be associated with structural and electrical remodeling of the atria in patients with AF [26]. In experimental models overproduction of PAI-1 by endothelial cells via TLR4-related inflammatory response to LPS addition has been observed [2]. As PAI-1 acts as an acute phase protein, it is secreted by a number of cells in response to inflammatory cytokines [25, 27]. It is postulated that the LPS-induced PAI-1 expression is modulated through NF-κB and MAP kinases activation [2, 25]. Consequently, LPS induces PAI-1 oversecretion and thus, initiates an antifibrinolytic response [28, 29]. These molecular pathways might contribute to the antifibrinolytic effects in the setting of low-grade LPS-related endotoxemia. Our findings suggest that LPS is potent enough to modulate fibrinolysis in human circulation, however the underlying mechanisms remain unclear and require further research.

Prothrombotic clot phenotype and its role in the prediction of thrombotic and bleeding complications have been reported in AF patients regardless of the CHA2DS2-VASc score [15, 17, 22, 30]. Impaired fibrinolysis in AF patients reflected by elevated plasma PAI-1 is associated with thromboembolic events [31]. However, little is known about fibrin clot properties modulation by enhanced LPS levels. To the best of our knowledge, the only study on this topic was published by Nunes et al. [32], who investigated the effects of LPS on clot formation and architecture in plasma from healthy individuals and in purified fibrinogen models. They found the LPS-dependent formation of denser fibers and altered clot mechanics, but they did not evaluate the clot permeability, which is a well-established indirect measure of clot density, and clot lysability [32]. Although Ks has been demonstrated to be lower in cardiovascular disorders associated with increased risk of thromboembolism, including AF [14], in our patients LPS showed no significant impact on this variable. Taken together, higher LPS modulates efficiency of fibrinolysis, which increases the current knowledge on the complex regulation of this process in AF and possibly in other cardiovascular diseases.

We also found that LPS is weakly positively associated with vWF antigen. In the study by Carnevale et al. LPS strongly correlated with vWF in liver cirrhosis patients, which was attributed to the LPS-related endothelial release of factor VIII and vWF [20]. Despite vWF is being primarily considered a marker of endothelial cells injury, it is also synthetized and stored in platelets. It is known that vWF is increased in AF [33]. It might be speculated that LPS exerts the native impact on prognosis in part by vWF mediated effects.

Menichelli et al. reported unfavorable antioxidant status in AF patients with elevated circulating LPS [34]. Hu et al. linked the thrombus formation with elevated GDF-15 in anticoagulant-naïve AF patients [35]. In our study, a novel findings is the association of LPS and GDF-15, which may reflect inflammatory actions of LPS in concordance with other findings [2, 25, 27]. It might be hypothesized that enhanced GDF-15 level, currently known as an integrative disease severity marker [36], might in part contribute to prothrombotic effects of LPS in AF [24].

Altered fibrin clot properties in AF patients can predict cerebrovascular outcomes [30, 37].

Impaired fibrinolysis in AF patients reflected by elevated PAI-1 is associated with thromboembolic events [31]. cTnI and vWF, markers of myocardial and endothelial injury, have also been reported to predict clinical outcomes in patients with AF [38,39,40]. The biomarker substudy in the ARISTOTLE trial with apixaban has demonstrated that GDF-15 alone and in addition to cTnI are predictive of major bleeding, mortality, and stroke in AF patients [36]. We found a significant association between elevated LPS and cTnI, vWF, and GDF-15. It would be interesting to investigate cerebrovascular outcomes in relation to the low-grade LPS-related endotoxemia, however this issue was beyond the scope of our study.

A potential impact of concomitant medications deserves a comment. Since there is evidence that aspirin, statins, antihypertensive medication, and antidiabetic therapy, may improve both Ks and CLT [14, 41], in our study such effects were not related to LPS. In the context of anticoagulation which potently affects fibrin clot properties [14], it should be highlighted that our study group involved solely AF patients who were not treated with any anticoagulant at the enrollment. To our knowledge, there have been no reports showing that anticoagulation affects low-grade endotoxemia, therefore it might be speculated that benefits from anticoagulation, including less prothrombotic fibrin clot phenotype, are not mediated by decrease in circulating LPS concentrations. We consider this the strength of our study, as the current OAC prescription rates reach 87% at one year from the therapy initiation [42].

Although the sample size was relatively small, it was sufficiently powered to show intergroup differences. The study has inherent limitations of a cross-sectional design. We did not assess the dietary intake. No other markers of dysbiosis including short-chain fatty acids, bile acids, and trimethylamine N-oxide were measured. The results cannot be extrapolated to the patient subsets defined in the exclusion criteria and those treated with OAC [43]. All laboratory findings were assessed once therefore some changes with time (i.e. the management strategy including medication) cannot be excluded given the inclusion period over a time span of five years. The fibrin clot-related measurements are feasible, however further work on the standardization is warranted [13], and now CLT cannot be used in everyday practice. Finally, mechanistic studies to explore precise mechanisms behind PAI-1-associated hypofibrinolysis in subjects with elevated LPS were beyond scope of the present research, and the study should be considered hypothesis-generating.

In conclusion, elevated serum LPS was the independent predictor of prolonged CLT in anticoagulant-naïve patients with AF. The antifibrinolytic effect was largely driven by enhanced PAI-1 release. Further studies are needed to elucidate the impact of low-grade endotoxemia on a prothrombotic state in AF patients and its potential modulation by agents affecting LPS in this clinical context.