{"paper_id":"10cd3edc-c84f-47ba-99c1-34ddd729ca24","body_text":"1 The Potential Effect of Vitamin D Supplement on Selected \n2 Coagulability Predictors in Vape-Exposed Female Rats\n3\n4 Aman M. Hammad1, Mahmoud Abu Samak1*, Rana Abu Farha1, Lujain F. Alzaghari 2, \n5 Abdelrahim Alqudah3, Diana Malaeb4, Khaldoun Rasem Shnewer5, Souheil Hallit6, Muna \n6 Barakat1*\n7 1 Department of Clinical Pharmacy and Therapeutics, Faculty of Pharmacy, Applied Science Private \n8 University, Amman 11937, Jordan, aman20.hammad@gmail.com, r_abufarha@asu.edu.jo, \n9 m_abusamak@asu.edu.jo, m_barakat@asu.edu.jo \n10 2 Medab Pharmacy, Madaba, Jordan. lujainfalzaghari@gmail.com \n11 3 Department of Clinical Pharmacy and Pharmacy Practice, Faculty of Pharmaceutical Sciences, The \n12 Hashemite University, Zarqa 13133, Jordan. Abdelrahim@hu.edu.jo\n13 4 College of Pharmacy, Gulf Medical University, Ajman, UAE. dr.diana@gmu.ac.ae  \n14 5 Smart Medical Lab, Amman, Jordan. Khaldoun@clemjo.com \n15 6 School of Medicine and Medical Sciences, Holy Spirit University of Kaslik, Jounieh, Lebanon, \n16 souheilhallit@hotmail.com \n17 *Correspondence: \n18 Muna Barakat, Department of clinical pharmacy and therapeutics, Faculty of Pharmacy, Applied Science \n19 Private University, Amman, 11937, Jordan, Email m_barakat@asu.edu.jo \n20 Mahmoud Abu Samak, Department of clinical pharmacy and therapeutics, Faculty of Pharmacy, \n21 Applied Science Private University, Amman, 11937, Jordan, Email: m_abusamak@asu.edu.jo\n22\n23\n24\n25\n26\n27\n28\n29\n30\n31\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint \n\n32 Abstract\n33 Background: Vaping and vitamin D deficiency impact blood coagulation and health. This study \n34 aimed to investigate the effects of vitamin D supplementation on coagulation predictors in female \n35 rats exposed to E-cigarette vaping.\n36 Objective: To examine the effect of vaping alone and vaping with different VD doses on some \n37 coagulation predictors, lungs, liver, and kidney functions \n38 Methods: Forty-two female Wistar rats were divided into six groups, including vaping and non-\n39 vaping with high (50,000 IU) and low (1000 IU) vitamin D doses. Blood samples and \n40 histopathological analyses were conducted after one and three months. Nicotine, cotinine, \n41 Interleukin-6 (IL-6), D-dimer, coagulation factor X (FX), thrombomodulin (TM), Alanine \n42 Transaminase (ALT), and Creatinine levels were analyzed. Additionally, histopathological \n43 analyses were conducted on the rats' liver, kidney, and lung. \n44 Results: Exposing rats to vaping for one month caused a significant acute increase in D-dimer, \n45 FX, and TM levels to 4402.05 ng/mL ± 785.15, 1.8687 μg/mL ± 0.3132, and 34.71 ng/mL ± 8.42, \n46 respectively. However, after three months of exposure, those levels decreased significantly \n47 compared to the one-month levels. Supplementation of the vape-exposed rats with a high vitamin \n48 D dose reduced levels of IL-6, D-dimer, FX, and TM levels to become 93.285 pg/mL ± 12.715, \n49 439.95 ng/mL ± 294.05, 0.647 μg/mL, and 17.375 ng/mL ± 3.895, respectively, at the end of the \n50 three months. Moreover, vaping rats supplemented with the low and high doses of vitamin D had \n51 significantly lower nicotine and cotinine levels than the EC group, with a p-value of <0.0001. The \n52 histopathological examination revealed that the rat’s lung had necrotic pneumonia when exposed \n53 to vaping without vitamin D treatment. Moreover, all vaping groups had an alveolar hemorrhage. \n54 Bacterial pneumonia was seen in the high-dose vitamin D vape-exposed group. However, the \n55 histopathological examination of the liver indicated no major differences between the groups. \n56 One month of vaping raised D-dimer, FX, and TM levels, which decreased after three months. \n57 High-dose vitamin D supplementation reduced IL-6, D-dimer, and FX levels while increasing TM \n58 levels after three months. Vaping rats receiving vitamin D had lower nicotine and cotinine levels. \n59 Histopathological findings showed necrotic pneumonia and alveolar hemorrhage in vaping rats, \n60 with bacterial pneumonia in the high-dose group. \n61 Conclusion: Vaping activates inflammatory and coagulation pathways, while high-dose vitamin \n62 D appears to mitigate inflammation and blood coagulation issues associated with vaping, \n63 potentially aiding in reducing nicotine dependence.\n64 Keywords: Vitamin D, E-cigarette, D-dimer, Coagulation factor X, Creatinine.\n65\n66\n67\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint \n\n68 Introduction \n69 Smoking significantly contributes to early mortality and public health issues globally. E-cigarettes \n70 (E-cigs), emerging around 2006-2009, have gained popularity, particularly among young adults, \n71 with over 683,300 users in Jordan as of 2020 [1-4]. \n72 E-cigs are devices that heat a solution usually composed of propylene glycol or glycerin, nicotine, \n73 and flavoring ingredients to produce an aerosol, also known as vapor [5]. While traditional \n74 smokers are switching to vaping as a quitting method, concerns arise regarding their role in \n75 cardiovascular diseases. Yet, some recent studies concluded that e-cigs are not blameless for \n76 causing cardiovascular diseases, including blood coagulation problems. A recent study has shown \n77 that the thermal decomposition of vape components generates harmful compounds, which are \n78 associated with a significant enhancement in platelet aggregation in vape-exposed mice, \n79 suggesting an elevated risk of thrombosis-related cardiovascular disorders[6-8].  Concurrently, \n80 vitamin D (VD) is highlighted for its health-modulating effects, including its anticoagulant \n81 properties and association with reduced thrombosis risk [9]. Supporting this, a five-year cross-\n82 sectional study reported that higher serum levels of VD were significantly associated with a \n83 reduced risk of deep vein thrombosis and other serious health outcomes [10].\n84 Based on published literature, this study will focus on female rats due to the higher reported risk \n85 of thrombosis in females and the increasing use of e-cigs among women, especially during \n86 pregnancy [11]. This research aims to investigate the effects of vape exposure on blood coagulation \n87 and inflammation, as previous studies have reported conflicting results. In addition, the study will \n88 evaluate the potential protective effect of vitamin D against vaping-induced changes in coagulation \n89 and inflammatory markers.\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint \n\n90 Materials and methods \n91 Study design \n92 VOOPOO DRAG M100S vape device with PnP-TM2 0.8 Ohms coil resistance, VGOD berry \n93 bomb (sour strawberry belt) flavor, 50% propylene glycol, 50% vegetable glycerin, and 18mg/ml \n94 nicotine. Hi Dee® (2000 IU VD/5 drops) and tera D ® (400 IU VD/ml) were used. The study was \n95 conducted in two phases as follows:\n96 Phase one:\n97 Three groups of female rats were exposed to vaping for one month, and the other three groups \n98 were not exposed to vaping nor received any treatment. \n99 Phase two:\n100 After the first month of exposure, VD was given to two vaping and two non-vaping groups for 2 \n101 months. The two vaping groups received VD at different dosages—one with a low dose (1000) \n102 and the other with a high dose (50,000 IU weekly). Additionally, two other rat groups will receive \n103 vitamin D without vape exposure, one with a high dose (50,000) and the other with a low dose \n104 (1000 IU daily).\n105 Animal management\n106 This study was an in vivo study which used 42 Wistar rats, aged 12 weeks, housed at Applied \n107 Science Private University in Amman, Jordan, for acclimatization over one week under standard \n108 laboratory conditions, including a temperature of 21–23 °C, relative humidity of 35–70%, and a \n109 12-hour light/dark cycle, with free access to food and water.\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint \n\n110 All experimental procedures involving animals were conducted in accordance with institutional \n111 and international guidelines for the care and use of laboratory animals. Ethical approval was \n112 granted by the Research and Ethics Committee of the Faculty of Pharmacy, ASU (Approval No.: \n113 2024-PHA-40).\n114 Rats were randomly assigned to six experimental groups (n = 7 per group) as follows: Control \n115 group (C): This group was not exposed to vaping nor VD treatment for the entire 12-week period \n116 (negative control). Vaping group (EC): This group was exposed to vaping only for the entire 12 \n117 weeks, 2 hours per day, 5 days a week, without receiving VD treatment (positive control). Vaping \n118 + low dose VD (ECD1000): This group was exposed to vaping for 4 weeks. Then, 1000 IU of VD \n119 was administered daily for 8 weeks while continuing vaping exposure. Vaping + high dose VD \n120 (ECD50,000): This group was exposed to vaping for 4 weeks. Then, 50,000 IU of VD was \n121 administered once weekly for 8 weeks while continuing vaping exposure. Low-dose VD (D1000): \n122 This group took 1000 IU of VD daily for 8 weeks without exposure to vaping. High dose VD \n123 (D50,000): This group took 50,000 IU of VD weekly for 8 weeks without exposure to vaping. \n124 The exposure settings\n125 The vaping groups were removed from their cages to a 50 cm (length) x 50 cm (width) smoking \n126 chamber shown in Figure.1, and back again to their cages after the end of the exposure. Rats were \n127 exposed to the vape twice daily in two separate sessions, one hour in the morning and one hour in \n128 the evening. After modification, an air pump was used to pull the vapor from the mouthpiece \n129 through plastic oxygen tubes directly connected to the smoking chamber. At the beginning, the \n130 chamber was filled with vapor (saturation phase). In the saturation phase, the pump was activated \n131 for 20 seconds. During this time, the fire button was pressed for 5 seconds, followed by 2-3 seconds \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint \n\n132 of rest to avoid coil and mouthpiece overheating. The second exposure phase began after the \n133 chamber was filled with vapor. During this phase, the pump was activated for 5 seconds, followed \n134 by 20 seconds of rest, mimicking real situations of typical user behavior. \n135 After the first hour of exposure, the pump was stopped, and the chamber remained closed until the \n136 rats inhaled the remaining vapor inside. Then, the chamber was opened and supplied with fresh air \n137 by opening a window directly beside the chamber, and the rats remained inside until the next hour \n138 of exposure. The exact process was repeated in the second hour of exposure.\n139 VD treatment \n140 VD was given orally by directly dropping the dose into the rat’s mouth after one month of vape \n141 exposure for 4 groups. It was administered in two doses: 1,000 IU daily and 50,000 IU weekly. \n142 The equivalent doses were calculated using this equation: animal equation dose = Human dose/60 \n143 × Km ratio, Km = 6.2 [12]. The equivalent dose of 50,000 IU was 5 drops from the commercially \n144 available Hi Dee® (2000 IU/5 drops) vial and 3 drops from the commercially available tera D® \n145 (400 IU/ml) vial for a 1000 IU dose. The vitamin was given in the morning.             \n146 Blood sampling \n147 Blood samples were collected in ethylenediamine tetraacetic acid (EDTA) tubes. The blood plasma \n148 was obtained and stored in Eppendorf tubes at -80 °C. \n149 Measurement of nicotine and cotinine levels \n150 Nicotine and cotinine concentrations were measured using LC-MS-8030 As LIQUID \n151 CHROMATOGRAPH MASS SPECTROMETER-Triple Quad MS (Shimadzu Corp.Japan). \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint \n\n152 Briefly, a C18  4.5 mmx15cm x 0.2um column was used as a stationary phase, with a mobile phase \n153 of 75% acetonitrile mixed with 0.05% formic acid and 25% distilled water, eluted isocratically at \n154 a flow rate of 0.3 ml/min for 4 minutes. The MS interface was electrospray ionization (ESI) \n155 running in positive ionization mode to generate [M+H]+ ions at m/z 162.23, 176.21 for nicotine \n156 and cotinine, respectively. \n157 Measurement of IL-6, D-dimer, TM, and FX levels \n158 Commercially available rat-specific ELISA kits (ELK Biotechnology, Wuhan, China) were used \n159 to detect IL-6, D-dimer, and TM, according to the manufacturer's instructions. Briefly, Plasma \n160 samples, standards, and controls were placed in wells pre-coated with particular capture antibodies, \n161 then incubated and washed. The detection antibodies were then coupled with horseradish \n162 peroxidase (HRP), and colorimetric detection was performed on a TMB substrate. The reaction \n163 ended with a sulfuric acid solution, and the absorbance was measured at 450 nm using a microplate \n164 reader. The concentrations were determined using standard curves developed from the established \n165 values included in each kit. Each sample was examined in duplicate. \n166 FX levels were evaluated using a mouse-specific sandwich ELISA kit (Mouse F10 ELISA Kit, \n167 Reed Biotech Ltd., Hubei, China), following the manufacturer's protocol, which was similar to the \n168 previously used method. Although the kit was designed for mouse samples, it was used due to \n169 documented cross-reactivity with rat plasma. Each sample was examined in duplicate. \n170 Measurement of ALT and Creatinine levels \n171 Serum alanine aminotransferase (ALT) and creatinine levels were measured using the BioSystems \n172 ALT and Creatinine kits (BioSystems S.A., Barcelona, Spain). ALT activity was assessed by \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint \n\n173 monitoring NADH oxidation at 340 nm via spectrophotometry, while creatinine levels were \n174 determined through the Jaffé reaction, which forms a colored complex measured at 500 nm.\n175 Histopathological analysis\n176 Lung, kidneys, and liver were harvested from three rats from each group after sacrifice. Organs \n177 were fixed in 10% formalin for 1 week. Then, the organs were transferred to Smart Lab to make \n178 the histopathological analysis. The organs were trimmed into cassettes and then a high \n179 concentration of alcohol was added to dehydrate the tissues. A clearing agent (xylene) was added \n180 to replace the alcohol. Then, melted paraffin wax was added to support the tissues. Using a \n181 microtome, the paraffin block was cut into 4-µm-thick sections and placed on a glass slide to be \n182 stained with hematoxylin and eosin for examination under the light microscope. \n183 Statistical analysis\n184 All results were expressed as mean ± standard deviation. Data were analyzed using the two-factor \n185 analysis of variance (ANOVA) in GraphPad PRISM, the tenth version of statistical software to \n186 calculate the statistical significance between the groups at different times. Tukey’s post hoc test \n187 was then used to compare the differences between the groups, considering a P value of <0.05 a \n188 statistically significant value. In addition, the Pearson test was used to detect the correlation \n189 between nicotine concentration and other parameters in the vaping groups.   \n190\n191 Results \n192 Nicotine and Cotinine\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint \n\n193 As shown in Fig. 2, after the first month of exposure, the nicotine and cotinine levels increased \n194 significantly from 0 ng/mL to 5.5 ng/mL ± 0.1 and 103 ng/mL ± 5, respectively. After 3 months, \n195 a significant increase occurred in both compounds’ mean levels to 12.45 ng/mL ± 0.55 in nicotine \n196 and 283.5 ng/mL ± 3.5 in cotinine, with a p-value of <0.0001 in the EC group.\n197 However, the ECD1000 group had nicotine concentration close to the one-month level, with a \n198 slight increase of 7.27%. Consequently, the cotinine levels increased significantly in three months \n199 to 120 ng/mL ± 9.5, with a p-value of 0.0010.\n200 Regarding the ECD50,000 group, the nicotine mean level was 8.25 ng/mL ± 0.25, and the cotinine \n201 mean level was 174 ng/mL ± 6, representing a statistically significant increase in nicotine and \n202 cotinine levels after three months of exposure to a p-value of <0.0001. Pearson test results \n203 indicated that there was no correlation between nicotine level and other parameters.\n204 Interleukin-6 (IL-6)\n205 At baseline, the mean IL-6 level was 71.843 pg/mL ±10.414. After 1 month, the IL-6 level was \n206 97.19 pg/mL ± 14.4294, representing a slight, non-significant increase of approximately 35%. \n207 After 3 months, no statistically significant difference was observed in the EC group. The mean IL-\n208 6 level was 115.1 pg/mL ± 10.6, representing 60% and 18% higher levels than the baseline and \n209 one-month levels, respectively.\n210 For the VD-treated groups, the mean value for the D1000 group was 108.175 pg/mL ± 28.325, \n211 which was 1.5-fold higher than the baseline level. The mean value for the D50,000 group was \n212 235.9 pg/mL ± 109.1, which was significantly higher than both the D1000 group and the C group, \n213 with a p-value of <0.0001. \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint \n\n214 Among the vape-exposed groups, the lowest IL-6 levels were in the ECD50,000 group, with a \n215 mean of 93.285 pg/mL ± 12.715, and the highest levels were in the ECD1000 group, with a mean \n216 of 122.23 pg/mL ± 46.67. Group ECD1000 had approximately 31% higher, non-significant IL-6 \n217 levels compared to group ECD50,000. \n218 As Figure 3 summarizes, group D50,000 had significantly higher IL-6 mean levels than the EC, \n219 ECD1000, ECD50,000, and C groups, with p-values of 0.0002, 0.0004, <0.0001, and <0.0001, \n220 respectively. Group ECD1000 had approximately 13% higher IL-6 than the D1000 group. \n221 D-dimer, FX, and TM\n222 At baseline, the mean D-dimer, FX, and TM levels were 1412.13 ng/mL ± 1072.1288, 0.8027 \n223 μg/mL ± 0.0945, and 7.606 ng/mL ± 1.6511, respectively. After 1 month, all the parameters \n224 increased significantly after the first month of exposure. The D-dimer levels increased to 4402.05 \n225 ng/mL ± 785.15, with a p-value of 0.0019. Moreover, the FX level increased to 1.8687 μg/mL ± \n226 0.3132 with a p-value of < 0.0001, and the TM level increased to 34.71 ng/mL ± 8.42 with a p-\n227 value of <0.0001. After 3 months, in the EC group, the mean D-dimer level decreased significantly \n228 with a p-value of 0.0016 from the one-month level to become higher by only 3% than the C group \n229 reading, with mean values of 1370.65 ng/mL ± 853.05 and 1332.25 ng/mL ± 376.85, respectively. \n230 The FX level was 0.904 µg/ml ±0.233, which was lower by 106.71% than the one-month level.  \n231 The mean level did not differ significantly from the C group, which had a mean value of 0.9975 \n232 µg/ml ± 0.3965. The mean level of TM was 13.475 ng/mL ± 2.125, which represented a \n233 statistically significant decrease with a p-value of <0.0001. This TM mean value is lower than in \n234 the C group by 5.79%, indicating a non-significant difference.\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint \n\n235 After VD treatment, D-dimer levels did not differ significantly between the VD-supplemented \n236 groups. Group D1000 had the highest D-dimer level compared to group D50,000 and the C group, \n237 with mean values of 2345.95 ng/ml ± 1585.65, 1785.05 ng/ml ± 1420.05, and 1332.25 ng/ml ± \n238 376.85, respectively. Group D1000 had approximately 31.42% higher levels than group D50,000 \n239 and 76.07% than the C group. In group D50,000, the d-dimer level was higher by 34% than in the \n240 C group.\n241 The mean D-dimer level in the ECD1000 group was 956.9 ng/mL ± 589.6, which decreased \n242 significantly from the one-month mean level with a p-value of 0.0003. The ECD50,000 group had \n243 a D-dimer mean level of 439.95 ng/mL ± 294.05, representing the lowest D-dimer level compared \n244 with the EC and ECD1000 groups. Although the difference between the group’s levels was not \n245 statistically significant, group ECD50,000 had 117.5% and 211.6% lower levels than the ECD1000 \n246 and EC groups, respectively. Both VD supplemented with vape exposure groups had lower D-\n247 dimer levels than the C group. The C group had approximately 39.23% higher levels than group \n248 ECD1000 and 202.8% than group ECD50,000.\n249 The analysis showed no statistically significant differences between the groups, as shown in \n250 Figure S4A. However, the D-dimer levels in the D1000 and D50,000 groups were higher than \n251 those in the ECD1000 and ECD50,000 groups by 145.17% and 305.78%, respectively. The lowest \n252 D-dimer levels were found in group ECD50,000 among all other groups.\n253 The highest increase in FX mean level among all the groups was found in group D50,000, which \n254 had a significant elevation from the baseline level to 1.5575 μg/mL ± 0.2335 and a p-value of \n255 <0.0001. A statistically significant difference between group D50,000  from group D1000 mean \n256 value of 0.9275 μg/mL ± 0.0235, and the C group mean value of 0.9975 μg/mL ± 0.3965, appeared \n257 after analysis with p-values of 0.0015 and 0.0062, respectively. In contrast, group D1000 had a \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint \n\n258 higher FX mean level of 15.56% from the baseline and a lower mean level of 7.55% from the C \n259 group. \n260 The FX mean level decreased significantly from the one-month mean value to 0.752 μg/mL ± \n261 0.245 with a p-value of <0.0001 in the ECD1000 group. a non-statistically significant decrease in \n262 FX mean level by 20.21% in the ECD1000 group compared to the EC group. The lowest FX mean \n263 level was found in group ECD50,000, which was equal to 0.647 μg/mL. Even not statistically \n264 significant, the ECD50,000 group had a 16.23% lower mean FX level than the ECD1000 and \n265 39.72% than the EC groups. Both the ECD1000 and the ECD50,000 groups had lower FX levels \n266 than the C group by 32.64% and 54.17% respectively.\n267 According to Figure S4C, group D50,000 had a significantly higher mean FX level than all the \n268 groups. When group D50,000 was compared with vaping groups, the results were different p-\n269 values of 0.0009 against the EC group, <0.0001 against group ECD1000, and <0.0001 against \n270 group ECD50,000. The C group had the second-highest mean FX level, followed by group D1000 \n271 and the EC group, which had close mean values. Group D1000 had a higher mean FX level of \n272 43.36% compared to group ECD50,000 and 23.34% compared to group ECD1000. However, \n273 group D1000 had a lower FX mean value than the EC group by 2.60%. \n274 Regarding TM, a slight, non-significant decrease of 23.95% compared to the baseline level was \n275 detected in group D1000, which had a mean value of 6.1365 ng/ml ± 2.4315. In contrast, group \n276 D50,000 showed an increase in TM level by 5.47% from baseline with a mean value of 8.022 \n277 ng/ml ± 1.251. Group D1000 had a lower TM mean value of 30.73% than group D50,000. The \n278 D1000 and D50,000 groups had no statistically significantly lower TM mean values than the C \n279 group, which had a mean level of 14.255 ng/ml ± 3.17002, by 132.32% and 77.68%, respectively.\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint \n\n280 In addition, the TM mean level decreased significantly in the ECD1000 and ECD50,000 IU VD \n281 groups, with mean values of 20.41 ng/mL ± 8.35 and 17.375 ng/mL ± 3.895, respectively, and \n282 corresponding p-values of 0.0002 and <0.0001. The highest TM mean level was in group \n283 ECD1000, which was higher than the EC group by 51% and by 17.47% compared to group \n284 ECD50,000. Both the ECD1000 and the ECD50,000 groups had higher, but non-significant, TM \n285 mean values compared to the C group, by 43.18% and 21.89%, respectively.\n286 When comparing all the groups, the group receiving D1000 had a significantly lower TM mean \n287 level than both the ECD1000 and ECD50,000 groups, with p-values of 0.0006 and 0.0106, \n288 respectively. Regarding group D1000, a non-statistically significant difference of 119.59% lower \n289 TM mean level than the EC group was found after analysis. The D50,000 group had a significantly \n290 lower mean value than group ECD1000 IU VD, with a p-value of 0.0037 and a non-significant \n291 lower mean level than group ECD50,000 IU VD by 116.64%. In addition, group D50,000 had a \n292 lower TM level of 67.99% than the EC group. Figure S4B visually summarizes those findings.\n293\n294 ALT and creatinine\n295 At baseline, the ALT and creatinine mean levels were 60.1 U/L ± 4.1605 and 0.645 µmol/L ± \n296 0.015, respectively. After 1 month, ALT level decreased significantly after one month of exposure \n297 to 42.7 U/L ± 3.8105, with a p-value of 0.0028. However, the Creatinine level was 0.69 µmol/L ± \n298 0.09, which was considered a non-significant increase by 6.98% from baseline levels. After 3 \n299 months, in the EC group, the ALT mean level increased significantly to 57.5 U/L ± 3.5 and a p-\n300 value of 0.0136, less by 4.33% than the baseline and 15.38% than the C group, which had a mean \n301 value of 67.95 U/L ± 1.65. A 3% decrease in creatinine levels occurred compared to the first \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint \n\n302 month’s levels to be 0.67 µmol/L. However, compared to the control’s mean level of 0.61 µmol/L \n303 ± 0.03, the EC group had 9.84% higher creatinine levels. \n304 After VD treatment in both groups, an increase in ALT levels appeared. Group D1000 had 15.35%, \n305 and group D50,000 had 13.71% higher values than the baseline, with mean values of 71 U/L ± 8.5 \n306 and 69.65 U/L ± 7.85, respectively. Compared with the C group, group D1000 and group D50,000 \n307 had higher mean ALT levels by 4.30% and 2.44%, respectively. Both groups, D1000 and D50,000 \n308 had very close mean values, with group D1000 having approximately 1.9% higher values.\n309 Regarding creatinine, group D1000 had the highest creatinine level of 0.665 µmol/L ± 0.005 by \n310 3.10% and 9.02%, respectively, compared with group D50,000 and the C group. When compared \n311 with the C group, group D50,000 had a mean level of 0.645 µmol/L ± 0.015, which was 5.74% \n312 higher. \n313 Both the ECD1000 and the ECD50,000 group's ALT mean levels increased compared to one \n314 month after VD treatment, with mean values of 75.25 U/L ± 16.15 and 51.9 U/L ± 5.2, respectively. \n315 This increase was significant in the ECD1000 group with a p-value of <0.0001, and non-\n316 statistically significant in group ECD50,000, with a 21.54% increase only. The ECD1000 group \n317 had the highest ALT mean level among the EC group and group ECD50,000, with p-values of \n318 0.0015 and 0.0001, respectively. Group ECD50,000 had a 9.74% lower ALT mean value than the \n319 EC group.\n320 Figure S5A showed that the highest ALT mean level was in group ECD1000 compared to all other \n321 groups. It had a significantly higher mean level of 75.25 U/L ± 16.15 than the EC group with a p-\n322 value of 0.0051. Both groups D1000 and D50,000 had lower ALT mean values of 5.65% and \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint \n\n323 7.44% than the ECD1000, respectively. In addition, group ECD1000 had a 9.70% higher mean \n324 ALT level than the C group.\n325 However, the group ECD50,000 mean ALT level was 9.74% lower than the EC group. Upon \n326 analysis, the ECD50,000 group had a significantly lower ALT mean value than the D1000, \n327 D50,000, and the C groups, with p-values of 0.0021, 0.0051, and 0.0145, respectively.\n328 The creatinine in the ECD1000 group was significantly higher than the EC group and the C groups, \n329 with a mean value of 0.78 μmol/L ± 0.01 and p-values of 0.0120 and <0.0001, respectively. In \n330 contrast, the ECD50,000 group had a mean creatinine level of 0.72 μmol/L ± 0.06, which was \n331 7.69% less than the group ECD1000 IU VD mean level and higher than the EC group by 7.46%. \n332 A significantly higher mean creatinine level with a p-value of 0.0120 was found between the \n333 ECD50,000 and the C group.\n334 A significantly higher mean creatinine level was observed in group ECD1000 than in the D1000 \n335 and D50,000 groups, with p-values of 0.0076 and 0.0011, respectively. Regarding group \n336 ECD50,000, it had an 8.27% higher mean creatinine level than group D1000 and 11.635% higher \n337 than group D50,000. There were very close mean values in the EC, D1000, and D50,000 groups. \n338 The EC group had 0.75% higher creatinine levels than group D1000 and 3.88% higher than group \n339 D50,000, as illustrated in Figure S5B.\n340 Histopathological examination\n341 Liver\n342 The EC and the C groups had a resemble hepato-microscopic examination with moderate \n343 lymphocytic infiltration in the portal tracts, and no significant fibrosis, steatosis, or cirrhosis was \n344 observed, as shown in Figure S6A and S6B.\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint \n\n345 The hepatic tissue in group D1000 resembled the C group by having mild chronic inflammation in \n346 the portal tracts, with a predominantly lymphocytic infiltrate without significant hepatocellular \n347 damage or fibrosis. As shown in Figure S6C. In contrast, group D50,000 had normal liver tissue \n348 without significant fibrosis or inflammation. As shown in Figure S6D.\n349 Histopathological examination reveals chronic inflammation with lymphocytic infiltration and \n350 occasional macrophages in the portal tracts in the ECD1000 group, without signs of fibrosis, \n351 necrosis, or steatosis, see Figure S6E. Moreover, in the ECD50,000 group, the liver showed \n352 evidence of chronic inflammation with mild lymphocytic infiltration around the portal tracts \n353 without significant hepatocellular damage or fibrosis, see Figure S6F.\n354\n355 Kidneys\n356 In the C group, the glomeruli demonstrate cellular expansion in both mesangial and endocapillary \n357 areas, with reduced capillary lumen size. Tubules show preserved morphology or mild nonspecific \n358 changes, as illustrated in Figure S7A. \n359 The glomeruli in the EC group exhibited diffuse mesangial hypercellularity and mild endocapillary \n360 proliferation, with some narrowing of capillary lumina. There was no significant \n361 glomerulosclerosis or crescent formation, and the tubules appeared largely preserved, as shown in \n362 Figure S7B. In group D1000, the glomeruli demonstrate mild mesangial expansion with focal \n363 endocapillary hypercellularity. Tubules are largely preserved, with minimal signs of atrophy and \n364 rare protein casts, as Figure S7C provides.\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint \n\n365 In group D50,000, Glomeruli showed mesangial and endocapillary hypercellularity with capillary \n366 lumen narrowing. There was no evidence of crescent formation or significant glomerular scarring. \n367 Tubules are unremarkable or show mild reactive changes, as Figure S7D provides.\n368 In the ECD1000 group, as shown in Figure S7E, glomeruli showed mesangial and endocapillary \n369 hypercellularity with capillary lumen narrowing. Tubules were unremarkable or showed mild \n370 reactive changes. Blood vessels were within normal limits, without evidence of vasculitis or \n371 thrombosis, which indicates glomerular inflammation or injury.\n372 The glomerular inflammation and injury were more severe in the ECD50,000 group, as shown in \n373 Figure S7F, which was characterized by the expansion of the mesangial matrix with focal crescent \n374 formation and endocapillary hypercellularity.\n375 Lungs\n376 In the C group, the lung tissues appeared with no significant inflammatory infiltrates, numerous \n377 blood cells, and preserved alveolar walls, as represented in Figure S8A. However, in the EC group, \n378 the alveolar spaces were filled with abundant neutrophils, and areas of necrosis were evident, \n379 consistent with necrotizing pneumonia. The alveolar walls exhibit marked disruption and extensive \n380 hemorrhage in the affected regions, as shown in Figure S8B.\n381 Diffuse interalveolar hemorrhage was evident in group D1000, with blood in the alveolar spaces \n382 and mild edema in the interstitium without any alveolar wall necrosis or significant inflammation \n383 observation, as evidenced by Figure S8C. In contrast, group D50,000 had normal lung tissue \n384 without hemorrhage, fibrosis, or inflammation, as evidenced by Figure S8D.\n385 Extensive red blood cells were present in the alveolar spaces in the ECD1000 group, indicating \n386 hemorrhage. The alveolar walls were intact, but blood-filled spaces were noted, with occasional \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint \n\n387 hemosiderin-laden macrophages. Mild interstitial edema was present without significant \n388 inflammation, as illustrated in Figure S8E.\n389 In the ECD50,000 group, the lung was more injured, as evidenced in Figure S8F, by having the \n390 alveolar spaces filled with neutrophils, indicating acute bacterial pneumonia. The alveolar walls \n391 showed mild inflammation, and a few alveolar septa were thickened due to edema. The \n392 mucopurulent exudate was present within the bronchioles, and the surrounding alveoli were \n393 congested.\n394 Discussion\n395 The extensive advertising of e-cigs as a safer and healthier alternative to traditional tobacco \n396 smoking has considerably boosted their popularity, particularly among teenagers and young adults. \n397 The appealing tastes and generally moderate odor of vaping devices have particularly attracted \n398 female consumers, emphasizing the importance of researching any potential risks linked with their \n399 usage. According to a global survey conducted in 2020, an estimated 68 million people are active \n400 e-cig users globally [13]. A survey conducted by a team of British scientists found that the majority \n401 of vape shop consumers are between the ages of 18 and 25, with females accounting for around \n402 41% of this population. Moreover, information received from vape shop workers suggested that \n403 fruit-flavored e-liquids were the most popular option among their customers [14]. According to \n404 emerging data from several research, tobacco smoking has a deleterious impact on VD status by \n405 interfering with key enzymes involved in its production and metabolism. Furthermore, smoking \n406 has been linked to increased liver damage indicators, which may inhibit VD synthesis and lead to \n407 a greater risk of deficiencies among tobacco users [15]. This study is particularly significant \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint \n\n408 because there is little information on the consequences of VD on e-cig users, particularly in terms \n409 of blood coagulability parameters and inflammatory markers.\n410 The current study's principal findings show that one month of exposure to e-cig vapor led in a \n411 substantial increase in blood levels of nicotine, TM, FX, D-dimer, and IL-6. These findings are \n412 consistent with previous studies that found similar short-term effects of vaping on coagulation \n413 indicators and inflammatory markers [16, 17]. The significant rise in certain clinical markers might \n414 be attributed to nicotine or the vape juice ingredients themselves. Many in vivo and in vitro \n415 investigations revealed that nicotine, flavoring ingredients, hygroscopic carriers, and metals \n416 emitted by the heated coil might cause cardiac toxicity and increase IL-6 and other inflammatory \n417 markers. likewise, previous research found that acute vape exposure raises the risk of \n418 cardiovascular disease via worsening endothelial dysfunction [18, 19]. However, during the third \n419 month of exposure, the levels of these biomarkers were decreased, except for nicotine in the vape-\n420 only group and IL-6, which remained high. Notably, nicotine concentrations were lower in both \n421 the vape + 1000 IU VD and vape + 50,000 IU VD groups, indicating a possible modulatory impact \n422 of VD supplementation. In support of this, Knihtilä et al. found that appropriate maternal VD levels \n423 during pregnancy were related with reduced cotinine concentrations in tobacco-exposed mothers, \n424 which contributed to better respiratory outcomes in their children [20]. The study also identified a \n425 significant interaction between cotinine and VD levels, though the underlying mechanism remains \n426 unclear. This interaction may be attributed to the anti-inflammatory and antioxidant properties of \n427 VD [15].\n428 The observed increases in IL-6 might be attributed to the increase in toxic substances generated \n429 by vaping and their buildup in the lungs. These irritating substances produce oxidative stress, \n430 which increases the production of white blood cells and cytokines. The observed decrease in blood \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint \n\n431 coagulation indicators after one month of exposure might be due to the rats' physiological ability \n432 to adapt and maintain homeostasis over time [21]. This conclusion is supported by Garrett's study, \n433 which examined the long-term effects of cigarette smoke exposure on coagulation in rats. In this \n434 study, rats exposed to tobacco smoke for 22 weeks showed no significant variations in plasma \n435 clotting times compared to the control group, indicating an adaptive response. Furthermore, the \n436 study revealed an age-dependent effect, with the prothrombotic effect of cigarette smoke being \n437 more prominent in older rats (24 months) than in younger rats (about 3 months), indicating that \n438 age may influence susceptibility to smoking-induced coagulopathy [22]. In research on the \n439 cardiovascular impacts of vaping, Dai et al. exposed 6-week-old rats to e-cig aerosol for 5 hours \n440 per day, 4 days per week, for 3 months. Their findings showed that this exposure regimen did not \n441 cause substantial changes in blood pressure or heart rate at the end of the research [23]. To further \n442 explore the temporal dynamics of cardiovascular adaptation to vaping, El-Mahdy et al. conducted \n443 a prolonged exposure study in which rats were subjected to e-cig vapor for 60 weeks, with pulse \n444 measurements recorded at multiple intervals. During the initial 8 weeks, pulse rates were elevated \n445 relative to baseline values, suggesting an acute physiological response. However, from weeks 8 to \n446 16, pulse rates declined, indicative of adaptive mechanisms. Following this period, from week 16 \n447 onward, pulse rates increased progressively and significantly, persisting through the remainder of \n448 the 60-week exposure period [24]. Additionally, Rafiq et al. confirmed an inverse connection \n449 between IL-6 and TM. In individuals with coronary artery disease, NF-kB was down-regulated \n450 whereas TM expression was up-regulated. In vitro and in mouse lung injury models, inhibiting \n451 NF-kB activity reduced cytokine-induced TM downregulation [25]. These prior findings are \n452 consistent with the findings of this investigation, which showed that TM and IL-6 levels were \n453 conflicting [26]. \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint \n\n454 Recently, in a vitro study conducted by Cirillo et al, tissue factor expression at gene and protein \n455 levels and the pro-coagulation activity were increased after incubating human umbilical vein \n456 endothelial cells with vape containing 18 mg/mL nicotine [27]. The inflammation that occurred in \n457 vaping groups increased the levels of IL-6, which consequently elevated platelet count and \n458 increased the tendency to blood coagulation and thrombosis. In addition, the presence of tissue \n459 factor leads to the acceleration in factor VIII conversion to its active form which will convert FX \n460 to its active form sequentially [28-30]. However, it has been approved by Cimmino et al, that VD \n461 can decrease tissue factor expression and atherosclerotic risk by modulating the nuclear factor \n462 kappa B in pre-incubated cells with VD. Although not all VD doses have the ability to decrease \n463 the activity of nuclear factor kappa B, the low dose VD of 1000 IU caused a reverse effect by \n464 increasing its activity in ulcerative colitis patients [31, 32]. That result clearly elucidates that 1000 \n465 IU of VD daily will cause an elevation in inflammatory biomarkers and increase the risk of \n466 thrombogenesis during stress conditions and inflammation. As the dose of VD increases, the \n467 activity of nuclear factor kappa B will be diminished in a dose-dependent manner during \n468 inflammation or stress conditions [33]. Those results explain the effect of 1000 IU and 50,000 IU \n469 on increasing and decreasing the inflammatory marker IL-6 and blood coagulation predictor levels \n470 in vape+50,000 IU VD and vape+1000 IU VD groups, respectively. In addition, the vape+1000 \n471 IU VD group had the highest creatine levels among all the groups with evidence of \n472 glomerulonephritis, which affected IL-6 clearance and led to its accumulation in the body.\n473 The immunomodulatory action of VD is dose-dependent and changes according to physiological \n474 circumstances. According to Bock et al., giving healthy people 140,000 IU VD once a month \n475 markedly increased regulatory T cell activity, suggesting that large dosages of VD stimulate the \n476 immune system [34]. In a similar vein, Bader et al. showed that high-dose VD (50,000 IU) caused \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint \n\n477 a cytokine storm and increased IL-6 levels in healthy individuals [35]. In contrast, under \n478 inflammatory or stress conditions, VD has been shown to reduce IL-6 levels in a dose-dependent \n479 manner [36]. \n480 D-dimer and IL-6 levels were shown to positively correlate in vaping-exposed groups, which is in \n481 line with research showing that fibrin breakdown products like D-dimer might increase vascular \n482 inflammation and IL-6 release [37]. Additionally, it has been shown that during inflammatory \n483 conditions, especially in lung infections as COVID-19, there is an inverse link between VD levels \n484 and D-dimer concentration [38]. The differences in D-dimer levels across groups might be \n485 explained by these interactions. The observed alterations in IL-6, D-dimer, TM, FX, ALT, and \n486 creatinine were probably caused by other components of the vape aerosol rather than nicotine \n487 alone, as evidenced by the notable lack of a significant link between nicotine and other measured \n488 parameters according to Pearson correlation analysis.\n489\n490 Regarding the histopathological findings, the necrotic results reported in the vape group lung tissue \n491 were consistent with an in vitro investigation conducted by Chastain Anderson et al, who \n492 discovered that vaping caused cell necrosis. Since TM is found in the alveolar epithelial cells, the \n493 drop in TM level that occurred after three months of exposure in the vape group was caused to \n494 necrotic pneumonia, which emerged after the histological examination as authorized by Boehme \n495 et al [39]. The Boehme et al. research discovered that TM levels increased dramatically before the \n496 start of cell injury, but the release was lost once cell damage occurred. These findings explain why \n497 the vape group had higher levels of IL-6 and lower levels of TM after one and three months, \n498 respectively [40]. The bacterial pneumonia that emerged in the vape+50,000 IU VD group may be \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint \n\n499 attributed to the absence of a VD receptor, which impacts the innate defense against bacterial \n500 infections in rodents and is confined to primates [41].\n501 As the hepatic regeneration capacity in Wistar rats reaches its maximum ability between 9 to 24 \n502 weeks of age [41]. This could strongly explain the histopathological examination of the liver, \n503 which showed no significant changes in the hepatic tissues after three months of exposure.  \n504 The glomerular histopathology examination showed that there was immune-related kidney injury. \n505 This damage was produced by inflammation and activation of the NF-kB pathway, which led to \n506 an increase in IL-6 levels. This pathway activation increases the synthesis of proinflammatory \n507 cytokines present in the nephritic glomeruli, resulting in glomerulonephritis in rats [42]. \n508 Conclusion\n509 In conclusion, our study shows that short-term nicotine vape exposure increases coagulability \n510 indicators and inflammatory cytokines in female rats, but longer exposure may cause physiological \n511 adaptation. High-dose VD supplementation (50,000 IU/week) protected vaping-exposed rats \n512 against these changes, whereas low-dose VD (1,000 IU/day) showed moderate efficacy. Notably, \n513 high-dose VD in non-vaping rats induced a proinflammatory response. These data indicate that \n514 VD may reduce vaping-induced coagulopathy and inflammation while also potentially aiding in \n515 nicotine detoxification. While based on an animal model, the findings emphasize the potential \n516 therapeutic benefit of VD in vape users.\n517 Declaration\n518 Acknowledgment\n519 The authors thank X-vape shop, Smart lab, and Al-Hikma lab.\n520 Funding\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint \n\n521 The Deanship of Scientific Research at the Applied Science University, Amman, Jordan, funded \n522 this research.  \n523 Data availability\n524 All relevant data can be found within the manuscript and in the supporting file. \n525 Author contributions\n526 Conceptualization: Muna Barakat and Mahmoud Abu-Samak. \n527 Methodology: Lujain F. Zghari, Aman M. Hammad.\n528 Writing - original draft: Aman M. 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Erem, C., et al., Blood coagulation, fibrinolysis and lipid profile in patients with primary \n594 hyperparathyroidism: increased plasma factor VII and X activities and D-Dimer levels. Exp Clin \n595 Endocrinol Diabetes, 2008. 116(10): p. 619-24.\n596 31. Cimmino, G., et al., Vitamin D inhibits Tissue Factor and CAMs expression in oxidized low-density \n597 lipoproteins-treated human endothelial cells by modulating NF-κB pathway.  Eur J Pharmacol, \n598 2020. 885: p. 173422.\n599 32. Karimi, S., et al., Inflammatory biomarkers response to two dosages of vitamin D supplementation \n600 in patients with ulcerative colitis: A randomized, double-blind, placebo-controlled pilot study. Clin \n601 Nutr ESPEN, 2020. 36 : p. 76-81.\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint \n\n602 33. Mathur, J., et al., A randomized clinical trial of vitamin D(3) (cholecalciferol) in ulcerative colitis \n603 patients with hypovitaminosis D(3). PeerJ, 2017. 5: p. e3654.\n604 34. Bock, G., et al., The effect of vitamin D supplementation on peripheral regulatory T cells and β cell \n605 function in healthy humans: a randomized controlled trial.  Diabetes Metab Res Rev, 2011. 27(8): \n606 p. 942-5.\n607 35. Bader, D.A., et al., The Effect of Weekly 50,000 IU Vitamin D(3) Supplements on the Serum Levels \n608 of Selected Cytokines Involved in Cytokine Storm: A Randomized Clinical Trial in Adults with \n609 Vitamin D Deficiency. Nutrients, 2023. 15(5).\n610 36. Sedaghat, K., et al., Regulatory effect of vitamin D on pro-inflammatory cytokines and anti-\n611 oxidative enzymes dysregulations due to chronic mild stress in the rat hippocampus and prefrontal \n612 cortical area. Mol Biol Rep, 2021. 48(12): p. 7865-7873.\n613 37. Lowe, G.D., et al., Interleukin-6, fibrin D-dimer, and coagulation factors VII and XIIa in prediction \n614 of coronary heart disease. Arterioscler Thromb Vasc Biol, 2004. 24(8): p. 1529-34.\n615 38. Popovska Jovičić, B., et al., Vitamin D, Albumin, and D-Dimer as Significant Prognostic Markers in \n616 Early Hospitalization in Patients with COVID-19. J Clin Med, 2023. 12(8).\n617 39. Boehme, M.W., P. Galle, and W. Stremmel, Kinetics of thrombomodulin release and endothelial \n618 cell injury by neutrophil-derived proteases and oxygen radicals. Immunology, 2002. 107(3): p. 340-\n619 9.\n620 40. Bergman, P., et al., Vitamin D3 supplementation in patients with frequent respiratory tract \n621 infections: a randomised and double-blind intervention study. BMJ Open, 2012. 2(6).\n622 41. Vázquez, C., J. Buján, and D. Vallejo, Blood coagulation variations induced by carbon tetrachloride \n623 inhalation in Wistar rats. Toxicol Appl Pharmacol, 1990. 103(2): p. 206-13.\n624 42. Sakurai, H., et al., Activation of transcription factor NF-kappa B in experimental glomerulonephritis \n625 in rats. Biochim Biophys Acta, 1996. 1316(2): p. 132-8.\n626\n627 Figure ligands\n628 Figure 1. (A) The timeline of the experiment, and (B) the vaping chamber. \n629 Figure 2. The (A) nicotine and (B) cotinine concentrations (ng/mL) among all vaping groups at \n630 baseline, one month, and two months.\n631 Figure 3. Summary of mean plasma IL-6 concentrations (pg/mL) among all the groups at baseline, \n632 one month, and three months. \n633 Figure S4. Summary of mean plasma (A) D-dimer concentrations (ng/mL), (B) TM concentrations \n634 (ng/mL), and (C) FX concentrations (µg/mL) among all the groups at baseline, one month, and \n635 three months. \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint \n\n636 Figure S5. (A) Summary of mean plasma ALT concentrations (U/L), and (B) Creatinine \n637 concentrations (µmol/L) among all the groups at baseline, one month, and three months. \n638 Figure S6. Histopathological appearance of liver tissue from all the groups under light \n639 microscopy. \n640 Figure S7. Histopathological appearance of kidney tissue from all the groups under light \n641 microscopy. \n642 Figure S8. Histopathological appearance of lung tissue from all the groups under light microscopy. \n643\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.25.708056doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}