Therapeutic Anticoagulation with Argatroban and Heparins Reduces Granulocyte Migration: Possible Impact on ECLS-Therapy?

Introduction Anticoagulants such as argatroban and heparins (low-molecular-weight and unfractionated) play an immense role in preventing thromboembolic complications in clinical practice. Nevertheless, they can also have a negative effect on the immune system. This study is aimed at investigating the influence of these substances on polymorphonuclear neutrophils (PMNs), whose nonspecific defense mechanisms can promote thrombogenesis. Methods Blood samples from 30 healthy volunteers were investigated, whereby PMNs were isolated by density gradient centrifugation and incubated with 0.8 μg/mL of argatroban, 1.0 U/mL of low-molecular-weight heparin (LMWH), 1.0 U/mL of unfractionated heparin (UFH), or without drug (control). A collagen-cell mixture was prepared and filled into 3D μ-slide chemotaxis chambers (IBIDI® GmbH, Germany). Stimulation was initiated by using a chemokine gradient of n-formyl-methionine-leucyl-phenylalanine (fMLP), and microscopic observation was conducted for 4.5 hours. The cells' track length and track straightness, as well as the number of attracted granulocytes, level of ROS (reactive oxygen species) production, and NET (neutrophil extracellular traps) formation, were analyzed and categorized into migration distances and time periods. Results All three anticoagulants led to significantly reduced PMN track lengths, with UFH having the biggest impact. The number of tracks observed in the UFH group were significantly reduced compared to the control group. Additionally, the UFH group demonstrated a significantly lower track straightness compared to the control. ROS production and NET formation were unaffected. Conclusion Our data provide evidence that anticoagulants have an inhibitory effect on the extent of PMN migration and chemotactic migration efficiency, thus indicating their potential immune-modulatory and prothrombotic effects.


Introduction
Critically ill patients frequently require anticoagulation in order to prevent deep vein thrombosis (DVT) and pulmonary embolism (PE). Patients suffering from organ failure and in need of organ replacement therapy, i.e., receiving treatment with Extra Corporeal Life Support (ECLS) for respiratory or cardiocirculatory distress or hemodialysis for kidney failure, are-even under anticoagulation therapy-at high risk for thrombotic events due to activation of the coagulation system as mediated by the artificial surfaces involved [1][2][3]. Anticoagulation optimization is, therefore, mandatory in these patients [4,5]. Clot formation within the membrane lung or the extracorporeal circuit is the most common technical complication, with an incidence of up to 22% [6] during ECLS therapy resulting in high mortality rates despite technical improvements and more sophisticated anticoagulation regimens [7].
In recent years, several studies have provided evidence for the interaction between polymorphonuclear granulocytes (PMNs) and the coagulation system. While forming the first line of innate immunity, imbalanced granulocyte activation can result in autoimmune diseases and thrombotic complications [8][9][10]. Furthermore, leukocyte deposits can be found in thrombi formed on gas exchange membranes of membrane lungs [11,12].
Unfractionated heparin (UFH) or low-molecular-weight heparins (LMWH) are usually used for anticoagulation [7]. The selective factor-IIa (thrombin)-inhibitor argatroban is an upcoming alternative which is used for anticoagulation in patients suffering from heparin-induced thrombocytopenia [13]. Although studies have repeatedly shown suppression of granulocyte function in a dose-dependent manner for UFH and LMWH [14][15][16][17], far less is known about the impact of argatroban on granulocyte migration [18,19].
The aim of this study was to investigate the impact and time-dependent viability of UFH, LMWH, and argatroban on the function of isolated granulocytes by performing in vitro assays for the comparative analysis of granulocyte migration capacity, chemotaxis, reactive oxygen species (ROS) production, and neutrophil extracellular trap formation (NETosis).

Materials and Methods
2.1. Granulocyte Preparation. The experimental setup was based on our previous studies [20,21]. Briefly, whole blood drawn from 30 healthy blood donors (Table 1) with their informed consent (as approved by the local ethics committee, Vote No.: 15-101-0043) was anticoagulated using ethylenediaminetetraacetic acid (EDTA) in a final concentration of 1.6 mg/mL blood. The blood samples were diluted with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and Tyrode solution (modified to 1 mM CaCl 2 and 1 mM MgCl 2 ). PMNs were isolated by density gradient centrifugation (756 g) at an ambient temperature of 21°C for 20 minutes with Lympho Spin Medium on top of Leuko Spin Medium (pluriSelect Life Science, Leipzig, Germany) according to the manufacturer's instructions. After two washing steps, PMNs were resuspended in RPMI 1640 (Pan-Biotech GmbH, Aidenbach, Germany) made up with 10% fetal calf serum (FCS, Sigma-Aldrich GmbH, Steinheim, Germany).
2.2. Microscopy and Live Cell Imaging. To visualize the production of ROS 1 μM di-hydro-rhodamine (DHR, Invitrogen D632, Sigma-Aldrich GmbH, Steinheim, Germany) and, accordingly, its fluorescence, active oxidized product rhodamine 123 was used. NET formation was detected by staining released extracellular DNA with 0.5 μg/mL of 4 ′ ,6-diamidin-2-phenylindol (DAPI, D9542, Sigma-Aldrich GmbH, Steinheim, Germany). Light and fluorescence microscopy was utilized to examine the cells of all four groups (argatroban, LMWH, UFH, and control), which were suspended in type I collagen gel (1.5 mg/mL of PureCol with 1.67% FCS, Advanced BioMatrix Inc., San Diego, CA, USA) and filled into μ-Slide Chemotaxis chambers (IBIDI GmbH, Martinsried, Germany) in accordance with the manufacturer's protocol. There is a reservoir on both sides of the chamber's gel channels; one was filled with n-formyl-methionine-leucyl-phenylalanine (fMLP 10 nM; Sigma-Aldrich GmbH, Steinheim, Germany) with RPMI/10% FCS and the other with RPMI with 10% FCS to establish a linear gradient of the chemoattractant. Each channel was filled with cell-gel suspensions preincubated with either argatroban, LMWH, UFH, or none of the aforementioned (control) in order to exclude interdonor variability.
The PMN observation was carried out with a Leica DMi8 (Leica GmbH, Wetzlar, Germany) with an automatically adjustable microscope stage and camera (DFC9000, Leica GmbH, Wetzlar, Germany). To ensure stable test conditions (37°C, 5% CO 2 ), a stage top incubator (IBIDI) was used. During the observation period of 4.5 hours, fluorescence and phase contrast images were taken automatically every 30 seconds by the Leica Application Suite X (LAS X 3.0.4.16529, Leica GmbH, Wetzlar, Germany).
2.3. Image Data Analysis. Image data analysis was based on previous experiments [20,21]. Briefly, a total of 540 images per chamber were analyzed using Imaris software (version 9.0.0, Bitplane, Zurich, Switzerland). To evaluate cell migration, the software recognized and tracked migrating cells semiautomatically over a predetermined time period. After monitoring the migration over the entire observation time, the 4.5-hour period was divided into blocks of 120 images per hour and the calculated spots and tracks within these blocks were analyzed. Data was then exported to Excel (Microsoft Corp., Redmond, WA, USA), including the total track length (μm) (length of the migration route of each individual cell) and the track straightness (fraction of Euclid track length and total track length showing the cell's tendency to migrate directly; higher factors refer to straighter lines) of each track. A track straightness above 0.2 and track lengths between 11 μm and 200 μm were required to identify and track granulocytes and exclude artefacts. Furthermore, the number of tracked PMNs was analyzed in categorized migration distances (the blocks of total track Relationships were examined through contingency analyses, and crosstabs were created comparing the observed and expected normal distribution values.
Pearson's chi-square test was used to test the significance of the differences between the variables. In terms of the track straightness comparison, a track length above 50 μm was required.
To investigate the time point of maximum ROS production (Tmax), images were taken every 2.5 minutes during the 4.5-hour observation period and the sum of the surface areas with DHR/rhodamine 123 staining was calculated. These surface areas were also recognized semiautomatically by Imaris and exported to Excel files. A third degree polynomial trendline was fitted to the parabolic course (time vs. sum of voxels) to determine the Tmax of ROS (min). Tmax values above 120 min were defined as implausible.
The response of DAPI contact with extracellular DNA (NET formation) was also analyzed by summing up single surface areas per time point, which resulted in sigmoidal curves. The time points at which the half-maximal effects (Et 50 ) were reached were calculated by population analysis with Phoenix 64 software (sigmoidal baseline E max -model; Certara Inc., Princeton, NJ, USA). ET 50 values beyond the observational period were defined as implausible. The materials and methods for quantifying ROS production have been described in detail in previous papers [22,23]. Briefly, cells were preincubated in 500 μL of HEPES buffer and Tyrode solution (modified to 1 mM CaCl 2 and 1 mM MgCl 2 ), 5 μL of DHR (10 μM), and 5 μL of seminaphtharhodafluor (SNARF, 10 μM, Invitrogen). Stimulation was induced by adding either 5 μL of fMLP (10 μM) and 5 μL of human tumor necrosis factor alpha (TNFα, 1 μg/mL, Pepro-Tech Inc., Rocky Hill, NJ, USA) or 5 μL of phorbol-12-myristate-13-acetate (PMA, 10 μM, Sigma-Aldrich GmbH). Finally, 5 μL of PI (33671, 1.5 mM, Serva Electrophoresis GmbH, Heidelberg, Germany) was added to detect dead cells.

Statistical Analysis. All Excel data (Microsoft Excel 2016)
were subsequently transferred to SPSS Statistics (Version 25, IBM Corp., Armonk, NY, USA), and the Kolmogorov-Smirnov test was used to confirm normal distribution for each group. Where there was a normal distribution, the mean values (MV) were compared, specifying the standard deviation (±SD). Raw data were compared using the Kruskal-Wallis one-way analysis of variance (ANOVA). Where there was variance homogeneity (Levene), post hoc analysis was carried out using the Bonferroni correction or, alternatively, the Dunnett-T3 test. In nonparametric comparison of groups, medians were given with the interquartile range (IQR) and plotted as box plots and lower and upper quartiles, as well as minima and maxima that had been calculated. Contingency analyses, crosstabs, and Pearson's chi-square test were used to detect significant contexts. p values below or equal to 0.05 were considered statistically significant.
The crosstab containing the observed track counts in defined length categories, and the expected number of tracks which were calculated based on an assumed normal distribution, revealed a significant correlation between drug and 3 Cardiovascular Therapeutics number of tracks (chi-square ð15Þ = 165, p < 0:001, n = 13552 tracks). The comparison showed no significant deviations in the control and the argatroban groups, for which the relative deviations were in the range of -1.82 and -4.32%. For all distance categories above 151 μm (-7.77 and -22.1%), deviations tended to be of higher magnitude in the LMWH group but did not reach significance. The highest discrepancies between the observed and expected counts were found in the UFH group. Here, deviations ranged from -23.0 to -72.9% in the categories beyond 51 μm total track length, indicating that the relative number of nonmigrating granulocytes (total TL < 50 μm; mean + 35:4%) is elevated and the relative number of active granulocytes (total TL > 51 μm; mean -44.3%) is reduced (Table 2).
Consequently, only the mean granulocyte counts of the UFH group (n = 869 tracks) showed a significant deviation of -31:1 ± 36:9% between the tracked and expected cells per track length categories, compared to the control group  , the LMWH group (n = 10), and the UFH group (n = 12), respectively. Thus, all medians of fluorescence intensity caused by ROS production were found to be indistinguishable (Figure 4).
After the 30-minute preincubation period with the drugs, cells of the argatroban group (n = 23) showed a median of 98.6% (IQR = 1:50%) vital granulocytes. In the LMWH group (n = 11) with a median of 98.7% (IQR = 2:15%), vital cells could be found, which was similar to that of the UFH group (n = 12) with a median of 98.8% (IQR = 2:30%). Therefore, compared to the control (n = 23) with a median

Discussion
This study investigated the impact of the clinically used anticoagulants UFH, the LMWH Enoxaparin, and the direct thrombin-inhibitor argatroban on isolated granulocytes in terms of granulocyte migration, time dependency of ROS production and NET formation, and vitality.
PMNs were obtained from healthy volunteers of both sexes without prior mobilization and were then incubated in parallel with UFH, LMWH, and argatroban after two washing steps in order to exclude any external influences on baseline granulocyte function, e.g., infections or humoral and hormonal factors. Therefore, measurements of humoral factors such as clotting factors, chemokines, or cytokines were not performed. All three anticoagulants are known to reduce inflammatory mediators produced by blood cells and other tissues (e.g., IL-6, IL-8, TNFα, or nuclear factor kappa B (NFκ-B)) after longer incubation times, ranging from three hours to four weeks [18,24,25].
UFH is used in clinical routine since 1971. It activates antithrombin and exerts its anticoagulatory effects by facilitating the interaction between antithrombin (AT) and thrombin, as well as the interaction with coagulation factor X [26]. LMWH directly inhibits coagulation factor X, creating a dose-dependent anticoagulant effect. It has also been 6 Cardiovascular Therapeutics used in clinical routine for two decades, and its use has been proven safe in medical and surgical patients [27,28]. Argatroban is a direct thrombin inhibitor used to manage anticoagulation during HIT II [13]; its feasibility for anticoagulation during ECLS and hemodialysis has already been proven [29,30]. The dosages of the three anticoagulants under investigation were chosen to match clinical routine. According to the manufacturer, an activated prothrombin time (aPTT, baseline 27-36 seconds) prolonged by a factor of 1.5 to 3 is required for therapeutic anticoagulation with argatroban [31]. Plasma levels of 0.8 μg/mL of argatroban result in an aPTT of 62-65 seconds [32]. The dosage for the LMWH Enoxaparin was chosen according to the manufacturer's prescription information for therapeutic anticoagulation. After a subcutaneous application of 1 mg/kg bodyweight twice daily, plasma levels of anti-Xa-activity of 1.1 IU/mL can be measured in the steady state [33]. An aPTT prolonged by a factor of 1.5 to 2.5 is required for therapeutic anticoagulation with UFH [34]. The concentration of UFH was chosen based on the same parameters as for Enoxaparin, with-due to ethical considerations-an added 1 IU/mL of anti-Xa-activity to avoid having to administer an UFH infusion to healthy volunteers for several hours in order to achieve stable aPTT values. Furthermore, adjustment of UFH dosage based on anti-Xa assays has been proven more reliable than aPTTbased assays in terms of achieving a faster rate of therapeutic anticoagulation with less variance [35].
For the migration assay, neutrophil activation was initiated by using the chemoattractant fMLP. Stimulation with fMLP has been proven in several studies to be a practicable way to induce NET formation [36]. It enabled the observation of the cellular events in our live cell imaging setting, which was a prerequisite for the precise evaluation of migration. We decided against activation with PMA for the live cell imaging because of the agent's already known substantial impact on granulocyte function [37]. In our flow cytometric assay, granulocyte activation for ROS production was achieved using fMLP, with the addition of PMA to serve as a positive control [38]. Unpublished data from our laboratory reveal an approximate 50% ET 50 reduction with a PMA concentration above 10 nM.
Migration assays were performed using a collagen I matrix to mimic the physiological extracellular environment. Our collagen I-based experimental set-up enabled live cell imaging and parallel testing of migration, ROS production, and NETosis [20,21]. In addition, a well-established method of FACS analysis was used for respiratory burst measurement [22,23].
All three anticoagulants under investigation significantly reduced neutrophil migration compared to the control group. While argatroban already began to suppress PMN migration within the first observation period, LMWH took longer to exert its effect. The biggest impact was measured in the UFH group during the first 180 minutes of the experiment. Furthermore, UFH preincubated granulocytes showed significantly lower relative track numbers of migrating cells compared to the control. Additionally, track straightness was reduced significantly, resulting in less effi-cient migration in the UFH group. Heparin and LMWH are known to reduce granulocyte migration in various clinical settings. In other studies, researchers found reduced granulocyte infiltration after traumatic brain injury when using the same dosing regimen for UFH and LMWH [39,40]. Our observations match the findings of these studies, despite our study having used a different matrix. These inhibitory effects have been attributed to stimuli unrelated to the anticoagulant capacities of UFH and LMWH, such as inhibition of thrombin, Mac-1, interaction with heparin-binding proteins, NFκ-B, growth factors, and cytokines [39,40]. After the administration of argatroban, a lower infiltration of granulocytes into the brain tissue of rats suffering traumatic brain injury was observed; another study showed reduced granulocyte infiltration into the liver tissue of rats suffering from fatty liver disease [18,19]. Both studies postulate that this effect is due to thrombin inhibition, which leads to increased plasma levels in both the brain and liver tissue and serves as a potent activator of granulocytes. After the administration of argatroban in rats with sepsis, Fuchs et al. observed improved intestinal microcirculation with reduced leucocyte adhesion and improved capillary perfusion [41]. These effects were also attributed to thrombin inhibition and subsequent reduction of microthrombi, as well as reduced endothelial activation with less leukocyte adherence. Thrombin also has direct proinflammatory effects due to its receptor (protease-activated receptor (PAR)), resulting in increased levels of AKT, NFκ-B, and caspase-3 [42]. In a recent study by Bulani et al., a significant improvement in diabetic cardiomyopathy could be detected [43]. The administration of argatroban reduced granulocyte migration in the first 120 minutes of both experiments. Surprisingly, in the present work, neutrophils incubated with argatroban showed greater migration distances after 120 minutes compared to the control, though migration during this time period was significantly reduced compared to baseline values for both groups.
In our study, no significant differences in ROS production between the groups investigated could be observed, whether it concerned fluorescence intensity or its chronological sequence. This result is in line with the findings of a study by Xu et al.; the researchers could not observe reduced ROS production following a daily subcutaneous administration of 1.5 mg of Enoxaparin in a rat model of liver fibrosis and splenectomy [44]. Xu et al. speculated that Enoxaparin had an inhibitory effect on thrombin and platelets. In contrast, Li and coworkers showed reduced ROS production in cell cultures of the bronchial epithelial cell line HBE 16 after incubation with 50 to 450 IU/mL of UFH [45]. Li et al.'s study postulated that the inhibitory effects of UFH on intracellular signaling pathways are unrelated to the anticoagulant effects responsible for this observation. Hence, the dosages used to achieve reduced ROS production are far beyond the plasma levels of UFH used in our study and far beyond clinical routine.
Concerning the applied dosages of the three substances investigated in our experiment, no significant NET formation differences between the groups could be found. Contrary findings regarding the absolute amounts, but not the chronological sequence, were made by other research groups in 7 Cardiovascular Therapeutics studies on UFH and LMWH. Fuchs et al. observed significantly reduced levels of NETs after administration of high-dose UFH at 100 μg/mL, which is equivalent to approximately 10 IU/mL [15]. Reduced NET formation was observed by Manfredi et al. for an identical dose of 1 IU/mL of Enoxaparin [17]. This contrary effect observed in the latter study may be due to different handling regimens concerning how the PMNs were obtained and isolated. In Manfredi et al.'s study, granulocytes were incubated for 30 minutes at 4°C with Enoxaparin, which may have had an inhibitory effect due to hypothermia. To the best of our knowledge, no literature on the interaction of argatroban and NET formation is currently available.
Finally, in our experimental setting, none of the three substances under investigation affected the study's viability. Although UFH is known to be able to induce apoptosis in granulocytes in a dose-dependent manner via the CD61b pathway, plasma levels above 20-50 IU/mL are required to have a significant effect [14,16,45]. As previously mentioned, this dosage is far beyond a rational anticoagulation regime.
Further studies are needed to elucidate the different or additional effects due to PMNs coming into contact with artificial surfaces or the shear force effects induced by extracorporeal systems.

Conclusion
In conclusion, our in vitro study with a collagen-I-matrix provides evidence that the PMN activity sequence is initialized with migration before the cells start to change their shape and produce ROS followed by NET formation. Our data show that anticoagulants in therapeutic dosage, especially UFH, have an inhibitory effect on the ability of granulocytes to migrate, as well as the extent of migration and the ability to migrate efficiently. Anticoagulants might, therefore, have immune modulatory effects. It could be hypothesized that PMNs are less able to migrate in anticoagulated patients. Furthermore, it could be speculated that PMNs of anticoagulated patients could stick to artificial surfaces due to their reduced migration capacity, resulting in a prothrombotic state that might affect therapy with extracorporeal devices. Other granulocyte functions were not affected, and the available literature suggests that higher doses of anticoagulants are required for the inhibition of these functions.

Data Availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Disclosure
Parts of this work was presented as an abstract at the Euro-ELSO Congress 2018.

Conflicts of Interest
The authors declare that no conflict of interests exists.

Authors' Contributions
AB and AR were responsible for the study design, statistical analyses, and drafting the manuscript. MK, MG, DB, and SMP performed the experiments and critically revised the manuscript. KL, WP, and TS were responsible for statistical analysis. All authors read and approved the final manuscript. Andre Bredthauer and Manuel Kopfmueller contributed equally to this article and share first-authorship.