ARTICLE https://doi.org/10.1038/s41467-020-17254-w OPEN The choline transporter Slc44a2 controls platelet activation and thrombosis by regulating mitochondrial function J. Allen Bennett1, Michael A. Mastrangelo1, Sara K. Ture1, Charles O. Smith 2, Shannon G. Loelius3, Rachel A. Berg4, Xu Shi5, Ryan M. Burke1, Sherry L. Spinelli4, Scott J. Cameron 1, Thomas E. Carey6, Paul S. Brookes 2, Robert E. Gerszten5, Maria Sabater-Lleal 7,8, Paul S. de Vries9, Jennifer E. Huffman 10, Nicholas L. Smith11,12, Craig N. Morrell1 & Charles J. Lowenstein 1 ✉ 1234567890():,; Genetic factors contribute to the risk of thrombotic diseases. Recent genome wide asso- ciation studies have identified genetic loci including SLC44A2 which may regulate thrombosis. Here we show that Slc44a2 controls platelet activation and thrombosis by regulating mito- chondrial energetics. We find that Slc44a2 null mice (Slc44a2(KO)) have increased bleeding times and delayed thrombosis compared to wild-type (Slc44a2(WT)) controls. Platelets from Slc44a2(KO) mice have impaired activation in response to thrombin. We discover that Slc44a2 mediates choline transport into mitochondria, where choline metabolism leads to an increase in mitochondrial oxygen consumption and ATP production. Platelets lacking Slc44a2 contain less ATP at rest, release less ATP when activated, and have an activation defect that can be rescued by exogenous ADP. Taken together, our data suggest that mitochondria require choline for maximum function, demonstrate the importance of mitochondrial meta- bolism to platelet activation, and reveal a mechanism by which Slc44a2 influences thrombosis. 1 Aab Cardiovascular Research Institute, Department of Medicine, University of Rochester Medical Center, Rochester, NY 14642, USA. 2 Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, NY 14642, USA. 3 Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY 14642, USA. 4 Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, NY 14642, USA. 5 Division of Cardiovascular Medicine, Beth Israel Deaconess Medical Center, Boston, MA, USA. 6 Department of Otolaryngology-Head and Neck Surgery, University of Michigan, Ann Arbor, MI, USA. 7 Cardiovascular Medicine Unit, Department of Medicine, Karolinska Institutet, Center for Molecular Medicine, Karolinska University Hospital, Stockholm, Sweden. 8 Genomics of Complex Diseases, Research Institute of Hospital de la Santa Creu i Sant Pau (IIB Sant Pau), Barcelona, Spain. 9 Human Genetics Center, Department of Epidemiology, Human Genetics, and Environmental Sciences, School of Public Health, The University of Texas Health Science Center at Houston, Houston, TX, USA. 10 Center for Population Genomics, MAVERIC, VA Boston Healthcare System, Jamaica Plain, MA, USA. 11 Department of Epidemiology, Cardiovascular Health Research Unit, University of Washington, Seattle, WA, USA. 12 Seattle Epidemiologic Research and Information Center, Department of Veterans Affairs Office of Research and Development, Seattle, WA, USA. ✉email: clowens1@jhmi.edu NATURE COMMUNICATIONS | (2020)11:3479 | https://doi.org/10.1038/s41467-020-17254-w | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-17254-w V enous thromboembolism (VTE) is a disease characterized lacking Slc44a2 (Fig. 1j, k). Taken together, our data suggest that by thrombosis in veins (deep vein thrombosis (DVT)) or Slc44a2 in hematopoietic cells promotes the development of in the lung (pulmonary embolism)1. VTE has a strong thrombosis and contributes to normal hemostasis. genetic basis, with the risk of disease increased 2.5-fold if a sibling The bone marrow transplantation experiments suggest but do is affected2. However, most of the genetic risk factors for not prove that Slc44a2 exerts its effects on thrombosis through a thrombosis are incompletely understood. The INVENT Con- cell derived from the bone marrow. In order to determine sortium used GWAS to identify genetic variants associated with whether or not this Slc44a2 effect is specific to platelets, we VTE in a cohort of over 60,000 human subjects, including a transfused washed platelets from WT and KO donors into KO genetic variant located within the gene SLC44A2 which was recipients. Slc44a2(KO) mice have shorter bleeding times when associated with a ~20% increased risk of thrombosis in replication receiving platelets from Slc44a2(WT) mice instead of from and discovery cohorts3,4. Slc44a2(KO) mice (Fig. 1l). (WT platelets improve the bleeding The biological and physiological roles of the protein SLC44A2 time in Slc44a2(KO) mice but do not fully restore the bleeding are not well understood5,6. The function of SLC44A2 is unknown, time to normal wild-type bleeding times, probably because there but it shares homology with choline transporters such as are still residual KO platelets within the transfused Slc44a2(KO) SLC5A77,8. GWAS studies have associated the SLC44A2 locus mice.) This suggests that at least part of the prothrombotic effect with human phenotypes including: hearing loss, Meniere’s dis- of Slc44a2 is due to its role in platelets. ease, and venous thrombosis3,9. Recent studies have explored the role of SLC44A2 in thrombosis10–13. Two studies found that Slc44a2 promotes thrombosis in a mouse model of laser injury or Slc44a2 increases platelet activation. We hypothesized that venous stenosis but did not identify the mechanisms underlying Slc44a2 directly regulates platelet activation, since the bleeding this phenomenon11,13. A search for mechanisms of Slc44a2 phenotype is dependent on the presence of Slc44a2 in bone affecting thrombosis found that Slc44a2 does not affect VWF marrow derived cells. We found that Slc44a2 is expressed in levels in mice13. Another study explored the influence of Slc44a2 human and murine platelets (Fig. 1b, c). We harvested platelets upon plasma proteins, and finding no difference in plasma pro- from Slc44a2(WT) and Slc44a2(KO) mice, exposed them to teins between wild-type and Slc44a2 null mice, concluded that vehicle or thrombin, and measured formation of platelet aggre- Slc44a2 must influence thrombosis through cellular based gates using light transmission aggregometry. Slc44a2(KO) plate- mechanisms12. lets aggregate less than Slc44a2(WT) platelets after thrombin We now show that Slc44a2 is a mitochondrial choline trans- treatment (Fig. 2a–d). In particular, the time to 50% maximal porter that regulates mitochondrial synthesis of ATP, platelet aggregation is prolonged, the rate of aggregation is slower, and activation and thrombosis. the percent of aggregation at 3 min is lower in Slc44a2(KO) pla- telets (Fig. 2b–d). We compared the response of Slc44a2(WT) and Slc44a2(KO) Results platelets to various agonists, such as thrombin which signals Slc44a2 promotes hemostasis and thrombosis. We first deter- through G protein coupled receptors, and convulxin which mined the expression of Slc44a2 using qPCR and immunoblot- interacts with glycoprotein VI and signals through a (hem) ting in murine and human tissues. Slc44a2 RNA is expressed in immunoreceptor tyrosine-based activation motif-dependent all tissues examined (Fig. 1a). Slc44a2 protein was detected in pathway14,15. Slc44a2(KO) platelets expose less P-selectin after human and murine platelets (Fig. 1b, c). Relative expression of thrombin stimulation (Fig. 2e). Slc44a2(KO) platelets expose less Slc44a2 is higher in the heart than in most other tissues for P-selectin than Slc44a2(WT) platelets when treated with various reasons that are unknown. Mice lacking Slc44a2, designated as agonists (Fig. 2f). Slc44a2(KO) platelets also have less activation of Slc44a2(KO) mice, are global Slc44a2 null mice that lack Slc44a2 GPIIbIIIA than Slc44a2(WT) platelets when treated with expression in all organs including platelets and bone marrow thrombin (Fig. 2g). However, Slc44a2(KO) and Slc44a2(WT) (Fig. 1c)8. platelets have similar levels of dense granule release, measured by SLC44A2 has been genetically linked to thrombosis in humans, CD63 translocation (Fig. 2h). These data suggest that Slc44a2 so we next characterized the effect of Slc44a2 on hemostasis and regulates platelet alpha-granule release and platelet GPIIbIIIa thrombosis in mice. We measured the time to hemostasis after activation. tail transection in Slc44a2(WT) and Slc44a2(KO) mice. Slc44a2 We next explored the mechanisms through which Slc44a2 (KO) mice have a greatly prolonged bleeding time, up to 50% regulates platelet activation. We hypothesized that Slc44a2 longer than wild-type mice, suggesting a defect in hemostasis regulates mitochondrial activity and ATP synthesis. Our hypoth- (Fig. 1d). We then used intravital microscopy to measure the time esis is based on the observations of others, showing that Slc44a2 is to formation of an occlusive thrombus in mesenteric arteries after a choline transporter, and mitochondria can use choline to FeCl3 treatment. Slc44a2(KO) mice have an increased time to generate betaine, in the process generating reactive oxygen species mesenteric artery thrombosis (Fig. 1e). Next we explored the role and ATP16–18. To explore the effect of Slc44a2 upon platelet of Slc44a2 in a murine model of DVT and found that Slc44a2 generation of ROS, we compared ROS production from Slc44a2 (KO) mice have decreased DVT formation following ligature (WT) and Slc44a2(KO) platelets after thrombin stimulation. constriction of the inferior vena cava (IVC) (Fig. 1f–h). Slc44a2(KO) platelets produce less ROS after thrombin activation than Slc44a2(WT) platelets (Fig. 2i). We found that mitochondria Slc44a2 in platelets increases hemostasis. We then explored the are the source of the oxidative burst after thrombin stimulation, effect of Slc44a2 in the bone marrow compartment and in pla- since inhibitors of mitochondrial enzymes abolish thrombin telets. The bleeding effect of Slc44a2 is dependent on Slc44a2 in stimulated ROS (Fig. 2j). We next confirmed that platelet derived bone-marrow-derived cells, since transplantation of bone marrow mitochondrial ROS mediate platelet activation. Inhibitors of from Slc44a2(KO) mice transfers the bleeding defect to Slc44a2 mitochondrial complexes that produce ROS decreased platelet (WT) mice (Fig. 1i). We used ultrasound to measure the time to activation (Fig. 2k). These studies suggest that mitochondria are formation of an occlusive thrombus in the carotid artery after the source of ROS which accompany platelet activation. Taken FeCl3 treatment. Carotid artery thrombosis times are delayed in together, our data suggest that Slc44a2 controls mitochondrial Slc44a2(WT) mice which have a bone marrow compartment production of ROS during platelet activation. 2 NATURE COMMUNICATIONS | (2020)11:3479 | https://doi.org/10.1038/s41467-020-17254-w | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-17254-w ARTICLE a4 SLC44A2 relative expression b c d WT bm KO bm WT plt KO plt 1200 3 2 μl 5 μl 1000 Bleeding time (s) 75 kD 75 kD 2 Slc44a2 800 Slc44a2 50 kD 600 50 kD 1 37 kD 400 GAPDH 37 kD GAPDH 200 0 0 n rt ey r ng Th n Pl s et ve u ai e ea el dn ym Lu le Br Li Slc44a2 (WT) Slc44a2 (KO) at H Sp Ki e Pre 1 min 5 min 10 min 15 min 20 min f g h Slc44a2 WT Slc44a2 KO 1200 Measuring stopped 0.010 Time for vessel occlusion 0.03 Thrombus mass (grams) 1000 0.008 IVC mass (grams) 800 0.02 0.006 600 0.004 400 0.01 200 0.002 0 0.00 0.000 Slc44a2 (WT) Slc44a2 (KO) Slc44a2 (WT) Slc44a2 (KO) Slc44a2 (WT) Slc44a2 (KO) i j 120% WT k l NS KO → KO 100% KO → WT WT → KO Measuring stopped 1200 1200 Time to cessation of flow (s) WT → WT 1200 80% 1000 % max flow 1000 1000 Bleeding time (s) Bleeding time (s) 800 60% 800 800 600 600 600 40% 400 400 400 20% 200 200 200 0 0% 0 0 WT to WT KO to WT KO to KO WT to KO 0 200 400 600 800 1000 1200 WT WT to WT WT to KO KO to WT KO to KO WT KO WT to KO KO to KO Time (s) Fig. 1 Slc44a2 is expressed in platelets and regulates hemostasis and thrombosis in mice. a RNA levels of Slc44a2 relative to ß-actin in murine organs were measured by qPCR (n = 3 ± biologically independent samples ±S.D.). b Protein levels of SLC44A2 in normal human platelets were measured by immunoblotting. c Protein levels of Slc44a2 in mouse platelets and mouse bone marrow were measured by immunoblotting. d The bleeding time of Slc44a2(WT) and Slc44a2(KO) mice was measured after tail transection (n = 6 WT and 8 KO mice ±S.D. and *P < 0.01 in a two-tailed Student’s t test). e The time for mesenteric arterial thrombosis after FeCl3 treatment was measured by intravital microscopy. *For WT vs. KO, the Fisher’s exact test statistic is 0.0001 and the result is significant at P < 0.05. f Representative image of inferior vena cava 6 h after IVC constriction, with WT above and KO below. g Quantification of IVC mass containing IVC segment and thrombus 6 h after IVC constriction (n = 13 WT and 15 KO mice ±S.D. and *P < 0.01 in a two- tailed Student’s t test). h Quantification of thrombus mass isolated from IVC 6 h after IVC constriction (n = 5 WT and 6 KO mice ±S.D. and *P < 0.01 in a two-tailed Student’s t test). i Bleeding times were repeated after bone marrow transplantation between Slc44a2(WT) and Slc44a2(KO) mice (n = 10, 7, 9, and 4 mice as shown in the chart ±S.D. and *P < 0.01 for WT–WT vs. KO–WT and *P < 0.01 for KO–KO vs. WT–KO in Tukey’s range test). Bone marrow from Slc44a2(KO) donor mice prolongs the bleeding time of recipient mice. j Percent maximal blood flow in carotid artery after treatment with FeCl3 was measured by ultrasound. k Quantitation of j. For WT–WT vs. KO–WT, the Fisher’s exact test statistic is 0.02 and the result is significant at P < 0.05. For WT–KO vs. KO–KO, the Fisher’s exact test statistic is 0.3 and the result is not significant at P < 0.05 Sample size includes: 10, 12, 7, 6, and 8 as shown in chart. l The bleeding time of Slc44a2(WT) and Slc44a2(KO) mice was measured after tail transection (WT and KO). (n = 6, 9, 9, and 8 mice as shown in the chart ±S.D. and *P < 0.01 for WT vs. KO). The bleeding time of Slc44a2(KO) mice after transfusion with platelets from Slc44a2(WT) or Slc44a2(KO) mice was measured after tail transection (WT to KO and also KO to KO). (n = 10 mice ±S.D. and *P < 0.01 for WT to KO vs. KO to KO compared in Tukey’s range test). Source data are provided as a Source Data file. Slc44a2 regulates mitochondrial function. Since our data indi- radiolabeled choline, or with radiolabeled choline in the presence cate that Slc44a2(KO) platelets have abnormal mitochondrial ROS of nonlabeled choline, washed the cells or mitochondria, and production, we next examined mitochondrial number and func- measured residual radioactivity. Slc44a2 mediates choline trans- tion in Slc44a2(KO) mice. We observed that platelets from port into mitochondria, but not into whole platelets (Fig. 3d, e). Slc44a2(KO) mice contain more mitochondria by measuring We performed mass spectrometry analysis of platelets from mtDNA (Fig. 3a, b). However, platelets from Slc44a2(WT) and Slc44a2(KO) and Slc44a2(WT) mice to measure choline meta- Slc44a2(KO) have similar numbers of alpha-granules and dense bolites (Fig. 3f). Mice lacking Slc44a2 have decreased choline and granules (Supplementary Fig. 1). We used a fractional immuno- one of its metabolites dimethylglycine (Fig. 3f). This suggests that blotting approach to show that Slc44a2 is expressed in mito- Slc44a2 transports choline into mitochondria, and that choline is chondria (Fig. 3c). Since Slc44a2 has functional homology with actively metabolized within mitochondria. choline transporter proteins, we characterized choline transport We next measured the effect of Slc44a2 upon mitochondrial into whole platelets and into mitochondria isolated from platelets. function. A primary function of mitochondria is oxidative We incubated platelets or purified mitochondria with phosphorylation and ATP production, processes which consume NATURE COMMUNICATIONS | (2020)11:3479 | https://doi.org/10.1038/s41467-020-17254-w | www.nature.com/naturecommunications 3 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-17254-w a Slc44a2 (WT) Slc44a2 (KO) b c d 0 0 0 0 200 10 4 200 80 10 4 Time to 50% aggregation (s) Aggregation (% at t = 180 s) 20 8 20 8 Aggregation (slope) 30 12 30 12 150 150 60 40 16 40 16 Percent OHMS Percent OHMS 50 20 50 20 60 24 60 24 100 100 40 70 28 70 28 80 32 80 32 90 36 90 36 50 50 20 100 40 100 40 1:00 2:00 3:00 4:00 5:00 6:00 1:00 2:00 3:00 4:00 5:00 0 0 Time (min:sec) 0 Time (min:sec) Slc44a2 (WT) Slc44a2 (KO) Slc44a2 (WT) Slc44a2 (KO) Slc44a2 (WT) Slc44a2 (KO) e Slc44a2 (WT) f Slc44a2 (WT) g Slc44a2 (WT) h 2000 1000 1000 80 Slc44a2 (WT) Slc44a2 (KO) Slc44a2 (KO) Slc44a2 (KO) P-selectin (geometric MFI) P-selectin (geometric MFI) Slc44a2 (KO) 800 800 CD63 (geometric MFI) MFI fibrinogen-FITC 1500 60 600 600 1000 40 400 400 500 200 200 20 0 0 0 0 0 0.1 0.25 0.5 1 Vehicle Thrombin ADP U44619 CVX 0.0 0.1 0.25 0.5 1.0 0.0 0.1 0.25 0.5 1.0 [Thrombin] (μ/mL) [Thrombin] (μ/mL) [Thrombin] (μ/mL) i j k Slc44a2 (WT) Slc44a2 (KO) Unstimuated Slc44a2 (WT) Slc44a2 (KO) 300 160 200 Thrombin P-selectin (geometric MFI) ROS (DCFDA oxidation) ROS (DCFDA oxidation) 140 150 200 120 100 100 100 50 0 80 0 Vehicle MITOTempo Cyanide Rotenone Vehicle MITOTempo Cyanide Rotenone Vehicle MITOTempo Cyanide Rotenone Vehicle MITOTempo Cyanide Rotenone Vehicle MITOTempo Cyanide Rotenone Vehicle MITOTempo Cyanide Rotenone Vehicle MITOTempo Cyanide Rotenone Vehicle MITOTempo Cyanide Rotenone WT KO WT KO Fig. 2 Slc44a2 regulates murine platelet activation ex vivo. a–d Platelet aggregation after treatment with 0.5 µ/mL thrombin was measured by light transmission aggregometry. n = 4 biologically independent samples ±S.D. *P < 0.05 in a two-tailed Student’s t test. e Platelet externalization of P-selectin after thrombin stimulation was measured by flow cytometry. n = 3 biologically independent samples ±S.D. *P < 0.05 in a two-tailed Student’s t test. f Platelet externalization of P-selectin after treatment with various agonists (0.5 µ/mL thrombin, 5 µM ADP or U46619, or 0.5 ng/mL convulxin). n = 3 biologically independent samples ±S.D. *P < 0.05 in a two-tailed Student’s t test. g Platelet activation of GPIIbIIIA after 0.5 µ/mL thrombin treatment was measured by flow cytometry of FITC-fibrinogen binding. n = 3 biologically independent samples ±S.D. *P < 0.05 in a two-tailed Student’s t test. h Platelet externalization of CD63 after 0.5 µ/mL thrombin treatment was measured by flow cytometry. n = 4 biologically independent samples ±S.D. *P < 0.05 in a two-tailed Student’s t test. i Platelet ROS production during stimulation with 0.5 µ/mL thrombin was measured by flow cytometry with DCF-DA. j Platelet ROS production was measured after treatment with mitochondrial inhibitors followed by 0.5 µ/mL thrombin treatment. n = 3 biologically independent samples ±S.D. *P < 0.05 in a two-tailed Student’s t test. k Platelet externalization of P-selectin was measured after mitochondrial inhibitor treatment followed by 0.5 µ/mL thrombin treatment. n = 3 biologically independent samples ±S.D. *P < 0.05 in a two-tailed Student’s t test. For j and k, all compounds tested at 5 µM with thrombin at 0.5 µ/mL. Source data are provided as a Source Data file. oxygen. Platelets from Slc44a2(KO) mice have a lower baseline of Slc44a2 increases mitochondrial production of ATP. Platelets oxygen consumption rate than Slc44a2(WT) mice platelets, as secrete ADP after stimulation by multiple extracellular agonists measured by the Seahorse analyzer (Fig. 3g before oligomycin). such as thrombin19. After its release, ADP then amplifies platelet Oxygen consumption rates following treatment with the ATP activation in an autocrine and paracrine manner through the synthase inhibitor oligomycin are similar between Slc44a2(KO) platelet P2Y12 receptor20. Since we observed decreased mito- and Slc44a2(WT) platelets, suggesting that they have similar rates chondrial function in Slc44a2(KO) platelets, we next compared of nonmitochondrial oxygen usage (Fig. 3g, after oligomycin ATP and ADP levels in isolated platelets. Platelets from Slc44a2 treatment). The maximum rate of ATP production is greater in (KO) mice contain less ADP and less ATP than platelets from Slc44a2(WT) platelets than in Slc44a2(KO) platelets, following Slc44a2(WT) mice (Fig. 4a, b). The ratio of ADP/ATP in Slc44a2 treatment with the membrane uncoupler FCCP (carbonyl- (KO) platelets is less than in Slc44a2(WT) platelets (Fig. 4c). We cyanide-4-(trifluoromethoxy) phenylhydrazone) (Fig. 3g after found that platelets from Slc44a2(KO) mice release less ATP after FCCP). When rotenone and antimycin A are added to inhibit thrombin stimulation than Slc44a2(WT) platelets (Fig. 4d). We electron transport, the oxygen consumption rate falls to equally tested the ability of choline to influence mitochondrial ROS and low levels for both Slc44a2(WT) and Slc44a2(KO) platelets ATP production: mitochondria from Slc44a2(WT) platelets pro- (Fig. 3g after Rot/AA). Thus, resting platelets lacking Slc44a2 duce additional ROS and ATP in response to choline, but mito- consume less oxygen, and display a decreased maximum oxygen chondria from Slc44a2(KO) platelets fail to make additional ROS consumption after FCCP treatment (Fig. 3g). Finally, we observed or additional ATP in response to choline (Fig. 4e, f). that the addition of choline produces a moderate increase in Decreased ATP production and release could explain why oxygen consumption in Slc44a2(WT) platelets, but not in Slc44a2 Slc44a2(KO) platelet activation is diminished, since ATP and (KO) platelets (Fig. 3h). These data suggest that choline plays a ADP released from platelets boosts platelet activation. If this is major role in mitochondrial respiration. true, then adding exogenous ADP should rescue the defective 4 NATURE COMMUNICATIONS | (2020)11:3479 | https://doi.org/10.1038/s41467-020-17254-w | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-17254-w ARTICLE a WT b c MtDNArelative copy number 4 Slc44a2 (WT) Total Cyto Slc44a2 WT Slc44a2 KO Mito 3 Empty lane 2 Slc44a2 (65 kD) 1 VDAC (25 kD) Slc44a2 (65 kD) 0.5 μm SDHC (20 kD) GAPDH (35 kD) 0 KO Slc44a2 (WT) Slc44a2 (KO) d e f Mitochondrial choline transport 0x choline 1000x choline 0x choline 1000x choline 15,000 (CPM/mg/ml protein/ 30min chloride chloride chloride chloride 60,000 0x choline 1000x choline 0x choline 1000x choline Cellular choline transport chloride chloride chloride chloride 4 CPM/50,00 cells Kynurenic acid 40,000 10,000 3 Reduced Glutathione Choline 5-MTHF -logP Dimethylglycine 20,000 5000 2 0.5 μm 1 0 Betaine 0 ADP Liver Spleen Platelet Liver Spleen Platelet Liver Spleen Platelet Liver Spleen Platelet Liver Spleen Platelet Liver Spleen Platelet Liver Spleen Platelet Liver Spleen Platelet ATP 0 0.1 1 10 Slc44a2 (+/+) Slc44a2 (–/–) Slc44a2 (+/+) Slc44a2 (–/–) Fold change g FCCP Rot/AA h Choline Oligomycin FCCP Rot/AA 80 OCR (pmol/min/2 × 107 platelets) 80 Oligomycin 60 60 Slc44a2 (WT) Slc44a2 (WT) Slc44a2 (KO) Slc44a2 (KO) OCR 40 40 20 20 0 0 20 40 60 80 0 0 20 40 60 80 100 Time (min) Time (min) Fig. 3 Slc44a2 regulates mitochondrial number and function. a Representative transmission electron microscopy images of platelets from Slc44a2(WT) and Slc44a2(KO) mice. Arrow indicates an individual mitochondrion. b Platelet mitochondria DNA copy number measured by qPCR. n = 4. *P < 0.05 in a two-tailed Student’s t test (c) Slc44a2 is present in lysate of mitochondria purified from platelets (left immunoblot) and Slc44a2 is enriched in the mitochondrial fraction of platelets but not in the cytosolic fraction of platelets (right immunoblot). d Slc44a2 does not regulate choline transport into whole cells, as measured by cellular uptake of radiolabeled choline competed with nonlabeled choline. (n = 3 biologically independent samples ±S.D. and *P < 0.05 for WT vs. KO). e Slc44a2 regulates transport of choline into isolated mitochondria, as measured by mitochondrial uptake of radiolabeled choline competed with nonlabeled choline. (n = 3 biologically independent samples ±S.D. and *P < 0.05 for WT vs. KO). f Metabolite profiles in platelets from Slc44a2(KO) mice relative to Slc44a2(WT) mice as measured by mass spectroscopy. (n = 2–3). g Mitochondrial oxygen consumption rate (OCR) of platelets from Slc44a2(KO) and Slc44a2(WT) mice: basal and uncoupled OCR are decreased in Slc44a2(KO) platelets (n = 4 biologically independent samples ±S.D.; *P < 0.05 WT vs. KO in a two-tailed Student’s t test). h Mitochondrial OCR during treatment with choline (n = 4 biologically independent samples ±S.D.; *P < 0.05 WT vs. KO in a two-tailed Student’s t test). For stress testing, Antimycin A = 1.0 µM, FCCP = 1.0 µM and Rotenone = 0.5 µM. Choline was added at 20 µM for all experiments. (For all panels, *P < 0.05 WT vs. KO in a two-tailed Student’s t test). Source data are provided as a Source Data file. activation of platelets lacking Slc44a2. To test this hypothesis, we platelet activation. One potential explanation for this defect in added exogenous nonhydrolysable ADP to platelets. As before, platelet activation is that Slc44a2 mediates choline transport thrombin failed to activate platelets from Slc44a2(KO) mice, but into mitochondria, where it is metabolized into substrates 2-me-ADP restores activation of Slc44a2(KO) platelets in for oxidative metabolism which generate ATP. Another possi- response to thrombin (Fig. 4g). We also tested a nonhydrolysable bility is that Slc44a2 mediates choline transport into mito- ATP analog, and this agonist slightly increases platelet activation chondria where it is metabolized into structural compounds as expected, and there is no difference between WT and KO such as phosphatidylcholine phospholipids that stabilize platelets (Supplementary Fig. 2). Taken together, our data suggest mitochondrial membranes which are necessary for oxidative that Slc44a2 controls intracellular levels of ATP and ADP, and phosphorylation16,17,21. Slc44a2 may also regulate platelet abnormally low platelet levels of ADP and ATP contribute to the activation by controlling mitochondrial production of ROS, impaired activation of platelets from Slc44a2(KO) mice. which are important for mediating platelet activation18,22–30. GWAS of human subjects have shown that genetic variants in the SLC44A2 locus are linked to abnormalities in risk of VTE3,4. Discussion Our data support these findings by demonstrating that Slc44a2 The major findings of our study are that choline is a substrate for regulates platelet activation and thrombosis in mice (Figs. 1 and 2). mitochondria, Slc44a2 regulates choline transport into mito- The human GWAS data also show that the genetic variants in the chondria, and choline plays a key role in production of ATP SLC44A2 locus are not associated with known hemostatic necessary for maximal platelet activation (Fig. 5). biomarkers3,4. We discovered that mitochondria in Slc44a2(KO) platelets Our study of Slc44a2 is supported by prior work. Others have are dysfunctional: their oxygen consumption rate at rest is low, also shown that Slc44a2 increases thrombosis in various murine and their maximal respiration rate when uncoupled is also low, models11,13. Searching for mechanistic pathways through which compared with wild-type controls. Mitochondrial dysfunction Slc44a2 affects thrombosis, one study excluded an effect of leads to decrease ATP content in platelets, decreased ATP Slc44a2 upon plasma proteins including coagulation factors, and release from platelets, and decreased extracellular ADP. proposed that Slc44a2 affects thrombosis through cellular path- Decreased ADP in turn leads to less paracrine and autocrine ways12. We confirm this prediction and extend this prior work, NATURE COMMUNICATIONS | (2020)11:3479 | https://doi.org/10.1038/s41467-020-17254-w | www.nature.com/naturecommunications 5 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-17254-w a b 35,000 c 0.4 d 10,000 Vehicle ATP release (Relative Light Units) 15,000 ATP (Relative Light Units) Thrombin ADP (relative light units) 8000 30,000 0.3 ADP/ATP ratio 6000 10,000 25,000 0.2 4000 5000 20,000 0.1 2000 0 15,000 0.0 0 Slc44a2 (WT) Slc44a2 (KO) Slc44a2 (WT) Slc44a2 (KO) Slc44a2 (WT) Slc44a2 (KO) Slc44a2 (WT) Slc44a2 (KO) e Slc44a2 (WT) f Vehicle g 2000 Vehicle 3000 Slc44a2 (KO) 1500 Thrombin Thrombin ROS production (RFUs) P-selectin (geometric MFI) 1800 ATS (AFUs) 2000 1600 1000 1400 1000 500 1200 0 Vehicle Choline Betanine DMG 1000 0 Slc44a2 (WT) Slc44a2 (KO) le e P P cl ic AD D hi eh )A ve T) i )v h O (W T) (K Slc44a2 (WT) O Slc44a2 (WT) ROS (DCFDA oxidation) (K 2000 (W 2 2 800 ROS (DCFDA oxidation) Slc44a2 (KO) 4a 4a 2 2 Slc44a2 (KO) 4a c4 4a c4 c4 Sl c4 Sl 1500 Sl 600 Sl 1000 400 500 200 0 0 e e le P id id AD ic n n e le e P h ya ya id id Ve AD ic n n h C C ya ya Ve P+ C C AD P+ AD Fig. 4 Slc44a2 and choline increase mitochondrial production of ATP and platelet ATP/ADP levels. a Platelets from Slc44a2(KO) mice contain less ADP than platelets from Slc44a2(WT) mice. n = 7 biologically independent samples ±S.D. *P < 0.05 in a two-tailed Student’s t test. b Platelets from Slc44a2(KO) mice contain less ATP than platelets from Slc44a2(WT) mice. n = 7 biologically independent samples ±S.D. *P < 0.05 in a two-tailed Student’s t test. c Platelets from Slc44a2(KO) mice have an altered ADP/ATP ratio compared to platelets from Slc44a2(WT) mice. n = 7 biologically independent samples ± S.D. *P < 0.05 in a two-tailed Student’s t test. d Platelets from Slc44a2(KO) mice release less ATP in response to 0.5 µ/mL thrombin. n = 4 biologically independent samples ±S.D. *P < 0.05 in a two-tailed Student’s t test. e Choline increases ROS production in a manner that depends upon Slc44a2 in purified mitochondria. n = 3 biologically independent samples ±S.D. *P < 0.05 in a two-tailed Student’s t test. f Choline increases ATP production in a manner that depends upon Slc44a2 in isolated mitochondria. Choline was added at 20 µM. n = 6 biologically independent samples ±S.D. *P < 0.05 in a two-tailed Student’s t test. g Exogenous ADP rescues platelet activation defect in Slc44a2(KO) platelets. Platelets were treated with 0.5 µ/mL thrombin and 5 µM 2-MeSADP. n = 3 biologically independent samples ±S.D. *P < 0.05 in a two-tailed Student’s t test. h ADP increases platelet ROS production. i ADP increases mitochondrial ROS production. n = 3 biologically independent samples ±S.D. *P < 0.05 in a two-tailed Student’s t test. Source data are provided as a Source Data file. Choline Slc44a2(KO) mice have activation defects (Fig. 2) and since pla- telet transfusion partially restores bleeding time in Slc44a2(KO) Slc44a2 Platelet mice (Fig. 1). However, other cells lacking Slc44a2 may also ATP Activation contribute to the hemostatic and thrombotic abnormalities in Choline Slc44a2(KO) mice. For example, knockout mice have lower ROS Mitochondria Thrombosis lymphocyte counts compared to wild-type mice (Supplementary Table 1), and lymphocytes can regulate thrombosis in vivo31–33. Platelet Changes in the number of lymphocytes can alter the kinetics and Fig. 5 Proposed model for choline transport and Slc44a2 regulation of composition of DVT formation as a “red thrombus” with a platelet activation. We propose that Slc44a2 transports choline into substantial portion of erythrocytes in the thrombus. As another mitochondria, where it is metabolized and regulates the production of ATP example, even though Slc44a2(KO) mice have neutrophil counts and release of ROS. ATP is released from platelets and hydrolyzed to ADP similar to WT mice (Supplementary Table 1), others have shown which acts upon platelet purinergic receptors and drives further platelet that neutrophils can influence thrombosis34–38. Changes in the activation. number of neutrophils can alter arterial thrombus, which appears as a platelet-rich “white thrombus.” It is possible that Slc44a2 expressed in leukocytes can partially influence thrombosis in the now demonstrating that Slc44a2 promotes thrombosis by reg- venous and arterial beds. Our data show that Slc44a2(KO) mice ulating platelet metabolism. have decreased hemostasis and thrombosis, and we show that Our study has several limitations. We show that Slc44a2 Slc44a2(KO) platelets have decreased activation, and transfusions directly affects platelet function, since platelets isolated from of platelets from Slc44a2(KO) mice fail to improve the bleeding 6 NATURE COMMUNICATIONS | (2020)11:3479 | https://doi.org/10.1038/s41467-020-17254-w | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-17254-w ARTICLE time. These data suggest that Slc44a2 in platelets is a major cause Mouse tail bleeding assay. Mouse tail bleeding time was measured as described41. of the hemostasis defect in Slc44a2(KO) mice. After IP anesthetization with ketamine and xylazine (80/12 mg/kg), the distal 3 mm of the tails of the mice were amputated and immersed immediately in 37 °C saline, In conclusion, our data suggest that Slc44a2 is a thrombosis and the time to visual cessation of bleeding for 30 s or continuous bleeding to regulator which does not lie within a well-studied thrombotic 20 min maximal duration, whichever occurs first, was recorded. pathway, and Slc44a2 may be an important therapeutic target. Mouse mesenteric thrombosis model and carotid thrombosis model. Throm- Methods bosis was measured as previously described42,43. For the mesenteric thrombosis Mice. Slc44a2(KO) mice were obtained from Dr. Thomas Carey (University of model, mice were anesthetized and platelets labeled with DyLight488-antibody to Michigan) and a colony was established at the University of Rochester8. These mice GPIb beta. A target area containing mesenteric arterioles (120–150 µm in diameter) are global Slc44a2 null mice that lack exons 3–10 of Slc44a28. All mice were treated was externalized for imaging. The arteriole flow was recorded for 3 min at resting in accordance with established protocols at the University of Rochester, which are condition. Then 1 mm2 of Whatman paper saturated with 7.5% FeCl3 solution was approved by the University Committee on Animal Research (UCAR). These applied to the arteriole for 3 min and the arteriole flow was continuously recorded approved protocols and all studies with mice complied with all ethical regulations for a total of 20 min The time to form a small thrombus (50-pixel diameter) and to for animal testing and research at the University of Rochester. This study received full vessel occlusion were recorded. Recording was terminated at the end of 20 min ethical approval by the UCAR of the University of Rochester. All experiments used if no occlusion were observed. For the carotid thrombosis model, mice were male mice aged 8–12 weeks, except for mesenteric thrombosis where mice 6 weeks sedated with 2.5% isoflurane and maintained anesthetized with 2% isoflurane. The of age were chosen to facilitate visualization of the mesenteric arteries or venules. common carotid arteries were exposed for a baseline flow recording using an Every effort was taken to minimize pain and discomfort in the mice used for MA1PRB Perivascular Flowprobe and a TS420 Flowmeter (Transonic Systems). breeding or experiments. Then 1 × 2 mm Whatman paper soaked with 1.5 µL 7.5% FeCl3 solution was applied to the ventral surface of the carotid upstream of the flowprobe for 3 min Flow measurement was resumed for a total of 20 min after FeCl3 wash off. We Antibodies and reagents. Primary antibodies against Slc44a2 were purchased define occlusion as the absence of blood flow (0 mL/min) for 3 min from Aviva Systems Biology (ARP44009_P050) and used at a dilution of 1:500. Primary antibody against GAPDH was purchased from Abcam (ab 8245) and used Mouse deep vein thrombosis model. For the DVT model, mice were anesthe- at a dilution of 1:2000. HRP-conjugated secondary antibody against rabbit primary tized, a midline incision was made. A ligature was secured around the IVC between antibody were purchased from GE (NA931) and used at a dilution of 1:5000. Anti- the renal veins and the bifurcation of the iliac veins, and a 28-gauge blunt needle CD62-PE and Anti-CD63-PE antibodies for flow cytometry were purchased from was placed between the ligature and the IVC. The suture was tied off, temporarily BioLegend (Catalog # 304905 and #143903). FITC-Fibrinogen was purchased from causing complete occlusion of the IVC, and the needle was removed from under Thermo Fisher (Catalog # RB-1924-R2). DCF-DA was purchased from Invitrogen. the suture, restoring partial luminal flow with a severe stenosis of the IVC. The All ROS enzyme inhibitors were purchased directly from Sigma-Aldrich. wound closed with sutures. Six hours following ligation, the mice were anesthetized and the IVC was isolated including any thrombus which had formed. Total mass Western blotting. Western blots were performed as previously described39. In was recorded, and the isolated IVCs deposited into 4% PFA for 24–48 h, and then brief, endothelial cells or platelets were lysed with Laemmli sample buffer (Bio- the thrombus was excised and the thrombus mass was recorded. Rad), boiled for 5 min at 95 °C, resolved on 4–20% Mini-PROTEAN TGX Precast gels (Bio-Rad), and transferred using a wet electrophoretic transfer unit (Bio-Rad) Bone marrow transplantation. Slc44a2(WT) and Slc44a2(KO) donor mice were onto nitrocellulose membranes. After 1-h blocking with 5% nonfat milk in PBS euthanized, and femurs were isolated under sterile conditions. Bone marrow was containing 0.05% Tween 20 at room temperature, the membranes were hybridized harvested, and then aspirated repeatedly to create single cell suspensions. Cell with primary antibodies followed by HRP-conjugated secondary antibodies and counts were manually performed and 107 cells were injected into each recipient enhanced chemiluminescence detection using a Bio-Rad Chemidoc Imager and mouse intravenously via the retro-orbital plexus on the same day as lethal irra- ImageLab software (Bio-Rad) (see Supplementary Fig. 3). diation of recipients. Eight-week-old mice were used as recipients and were lethally irradiated with an X-ray RS 2000 (Rad-sources) irradiator delivering a split dose of Quantitative real-time PCR. Murine tissue was harvested immediately after 1100 rad, delivered as 550 rad in the morning and then 550 rad in the afternoon the sacrifice and snap frozen on dry ice. Total RNA was isolated using an RNEasy kit day of transplantation. After marrow transplantation, mice were provided with following the manufacture’s protocol (Qiagen). The A260/A280 ratio of all samples water supplemented with sulfatrim for 2 weeks, and allowed to reconstitute for was between 1.9 and 2.1 as measured by spectrophotometry (NanoDrop; Thermo 6 weeks prior to tail bleeding assay or blood collection. Scientific). cDNA was synthesized using an iScript™ cDNA Synthesis kit (Bio-Rad). Quantitative real-time PCR was performed by PrimePCR Sybr Green gene Platelet transfusion. Platelets were isolated as above. Washed platelets were expression assay (Bio-Rad) for 40 cycles on an CFX Connect thermal cycler (Bio- transfused into recipient mice through a retro-orbital injection44. Rad). The qPCR primers were form a kit (Bio-Rad qMmuCID0026393 and qMmuCED0027497). Quantification was performed in triplicate for each sample, and tissues were harvested from at least n = 3 mice. Expression results were cal- Mitochondrial isolation and ATP production. Isolation of mitochondria was culated by ΔΔCT method and were normalized to the reference gene Gapdh. performed using a commercially available kit (Thermo Fisher #89801). Briefly, platelets are isolated as described above, lysed and then a series of sucrose gradient centrifugation steps performed to yield a pure layer of mitochondria, which was Platelet isolation and activation. Murine blood was obtained by retro-orbital then suspended for further experiments. For ATP production, mitochondria were bleeding of anesthetized animals into heparinized murine Tyrode's buffer (134 mM suspended in mitochondrial respiration buffer KCl (120 mM), sucrose (25 mM), NaCl, 2.9 mM KCl, 12 mM NaHCO3, 0.34 mM Na2HPO4, 20 mM HEPES, pH 7.0, HEPES (10 mM), EGTA (1 mM), KH2PO4 (1 mM), MgCl2 (5 mM), glutamate 5 mM glucose, and 0.35% bovine serum albumin) in Eppendorf tubes. The blood (15 mM), and malate (7.5 mM) pH 7.3, supplemented with vehicle or choline, and was then centrifuged to yield platelet-rich plasma which was then washed in new ATP quantified as above. tubes containing Tyrode's buffer with 1% PGE2 to prevent platelet activation. Platelets were then pelleted by 5 min 600 × g centrifugation at room temperature Choline transport. The rate of choline uptake was determined by measuring 3H- and the supernatant was discarded. The pelleted platelets were gently resuspended choline chloride (Perkin Elmer) uptake over time. Platelets or mitochondria were in Tyrode's buffer and kept at room temperature for further experiments within added into 1.5 mL tubes. One hour prior to uptake, media were removed and cells 2 h. For platelet activation, diluted platelet suspension was divided into 100 μL were washed with PBS before being incubated in Tyrode’s buffer until use. aliquots, treated with indicated drugs where appropriate (10 µM MitoTEMPO, Immediately prior to uptake, cells were washed again followed by the addition of 1 mM NAC, 100 µM sodium cyanide, or 10 µM rotenone), and then stimulated Tyrode’s buffer containing 1 μCi/mL of 3H-choline and were incubated at 37 °C for with PBS or indicated concentrations of thrombin for 15 min followed by staining desired time point (1–30 min). Following incubation, cells were washed twice with with 1 μL CD62P-PE/CD63-PE (BioLegend) or 1 µL Fibrinogen-Oregon Green Tyrode’s buffer, lysed in 150 μL of 0.1 M NaOH and an aliquot was used to (Thermo Fisher) for 30 min, and immediately fixed with 100 µL 2% formalin. For determine radioactivity by liquid scintillation counting. Total cellular protein was DCFDA experiments, platelets were loaded with 10 µM DCFDA at 37 °C for 1 h, determined using a BCA protein assay (Thermo Fisher Scientific) according to the and then treated/stimulated as above. The fluorescence intensity was measured on manufacturer’s instructions. Choline uptake was expressed as counts per minute an Accuri C6 Flow Cytometer (BD Biosciences). The data were analyzed with per mg protein (for mitochondria) or per 50,000 cells (for platelets). FlowJo software (Tree Star Inc.). ATP release was measured in poststimulation supernatant using an ATP Bioluminescent Assay Kit (Sigma-Aldrich). ADP/ATP contents of whole platelets was determined using an ADP/ATP Ratio kit, according Platelet mitochondrial function measurements. A 96-well format Seahorse to manufacturer instructions (Abcam). Aggregometry was performed using flow extracellular flux analyzer (Seahorse Bioscience, MA, USA) was used to measure cytometry as described40. Mitochondrial DNA copy number was measured in bioenergetics45,46. Platelets were diluted to a concentration of 2 × 107 in XF DMEM 50,000 platelets by qPCR using a commercial kit (Abcam), and normalized to WT assay buffer (DMEM with 1 mM pyruvate, 5.5 mM D-glucose, 4 mM L-glutamine, copy number. pH 7.4) and were seeded onto Cell-Tak coated XF96 microplates and NATURE COMMUNICATIONS | (2020)11:3479 | https://doi.org/10.1038/s41467-020-17254-w | www.nature.com/naturecommunications 7 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-17254-w mitochondrial stress test was performed as described45,46. For some Seahorse 3. Germain, M. et al. Meta-analysis of 65,734 individuals identifies TSPAN15 experiments, choline was injected at 10 µM prior to the stress test. and SLC44A2 as two susceptibility loci for venous thromboembolism. Am. J. Hum. Genet. 96, 532–542 (2015). Mass spectrometry. We used LC-MS/MS based methods to profile 147 analytes 4. Hinds, D. A. et al. Genome-wide association analysis of self-reported events in including amino acids, organic acids, bile acids, indoles, nucleotides, and sugars. 6135 individuals and 252 827 controls identifies 8 loci associated with We have previously used these platforms to characterize biochemical pathways thrombosis. Hum. Mol. Genet. 25, 1867–1874 (2016). implicated in metabolism47,48. Briefly, samples were deproteinized using extraction 5. Nair, T. S. et al. Identification and characterization of choline transporter-like solvent containing stable isotope labeled internal standards. Samples were vortexed protein 2, an inner ear glycoprotein of 68 and 72 kDa that is the target of and centrifuged, and aliquots were transferred to 2 mL autosampler vials with glass antibody-induced hearing loss. J. Neurosci. 24, 1772–1779 (2004). inserts for LC-MS analysis. In positive mode, normal phase hydrophilic interaction 6. Kommareddi, P. K. et al. Isoforms, expression, glycosylation, and tissue chromatography (HILIC) using a 2.1 × 150 mm 3 μm Atlantis column (Waters) distribution of CTL2/SLC44A2. Protein J. 29, 417–426 (2010). was coupled to a 4000 QTrap triple quadrupole mass spectrometer (Applied 7. Iwao, B. et al. Functional expression of choline transporter like-protein 1 Biosystems/Sciex) equipped with an electrospray ionization source for targeted (CTL1) and CTL2 in human brain microvascular endothelial cells. detection of 78 metabolites using a dynamic multiple reaction monitoring (dMRM) Neurochemistry Int. 93, 40–50 (2016). mechanism. In negative mode, HILIC chromatography using a 2.1 × 100 mm 8. Kommareddi, P. et al. Hair cell loss, spiral ganglion degeneration, and 3.5 μm Xbridge Amide column (Waters) was coupled to an Agilent 6490 triple progressive sensorineural hearing loss in mice with targeted deletion of quadrupole mass spectrometer equipped with an electrospray ionization source for Slc44a2/Ctl2. J. Assoc. Res. Otolaryngol. 16, 695–712 (2015). targeted detection of 69 metabolites using dMRM. Metabolite peak areas were 9. Nair, T. S. et al. SLC44A2 single nucleotide polymorphisms, isoforms, and integrated using Sciex MultiQuant software (positive mode) or Agilent Masshunter expression: association with severity of Meniere's disease? Genomics 108, Quantitative software (negative mode). All metabolite peaks were manually 201–208 (2016). reviewed for peak quality in a blinded manner. (See Supplementary Tables 2 and 3 10. Constantinescu-Bercu, A. et al. Activated alphaIIbbeta3 on platelets mediates for metabolites and metabolomic data.) flow-dependent NETosis via SLC44A2. Elife 9, https://doi.org/10.7554/ eLife.53353 (2020). Human platelet collection. Human blood collection was performed as previously 11. Tilburg, J. et al. SLC44A2 deficient mice have a reduced response in stenosis described using protocols approved by the Institutional Review Board at the but not in hypercoagulability driven venous thrombosis. J. Thrombosis University of Rochester Medical Center (IRB Protocol RSRB00028659)49. Normal Haemost. https://doi.org/10.1111/jth.14835 (2020). healthy blood donors were recruited. Subjects were excluded if they had used 12. Tilburg, J. et al. Plasma protein signatures of a murine venous thrombosis aspirin or any nonsteroidal anti-inflammatory agent within 10 days before the model and Slc44a2 knockout mice using quantitative-targeted proteomics. blood draw. Blood was collected by venipuncture into sodium citrate anticoagulant Thromb. Haemost. 120, 423–436 (2020). tubes. Whole blood was centrifuged at 180 × g for 15 min to isolate the top layer of 13. Tilburg, J. et al. Characterization of hemostasis in mice lacking the novel platelet-rich plasma, which was diluted 1:20 in room temperature Tyrode’s buffer thrombosis susceptibility gene Slc44a2. Thrombosis Res. 171, 155–159 (2018). (134 mM NaCl, 2.9 mM KCl, 12 mM NaHCO3, 0.34 mM Na2HPO4, 20 mM 14. Cherpokova, D. et al. SLAP/SLAP2 prevent excessive platelet (hem)ITAM HEPES, pH 7.0, 5 mM glucose, and 0.35% bovine serum albumin) and dispensed in signaling in thrombosis and ischemic stroke in mice. Blood 125, 185–194 100-µL volumes for treatment with various drugs. Western blotting was performed (2015). as previously described49. 15. Boulaftali, Y., Hess, P. R., Kahn, M. L. & Bergmeier, W. Platelet immunoreceptor tyrosine-based activation motif (ITAM) signaling and Statistics. Data with a normal distribution were analyzed by two-tailed Student’s t vascular integrity. Circ. Res. 114, 1174–1184 (2014). test for comparison of two groups, and by ANOVA to compare means of three or 16. Baykal, A. T., Jain, M. R. & Li, H. Aberrant regulation of choline metabolism more groups. Statistical significance was defined as P < 0.05. To compare the tail by mitochondrial electron transport system inhibition in neuroblastoma cells. bleeding time of two groups (bleeding time for Slc44a2(WT) and Slc44a2(KO) mice Metabolomics 4, 347–356 (2008). in Fig. 1e), a dichotomous measure of cessation of bleeding within 20 min (yes or 17. Mailloux, R. J. et al. Choline and dimethylglycine produce superoxide/ no) was used and compared in a 2 × 2 contingency table. Because of small sample hydrogen peroxide from the electron transport chain in liver mitochondria. sizes within some of the cells, the Fisher’s Exact Probability test was used to FEBS Lett. 590, 4318–4328 (2016). compare the two groups. To compare the thrombosis time of two groups (time for 18. Violi, F. & Pignatelli, P. Platelet NOX, a novel target for anti-thrombotic cessation of flow in the carotid artery for WT to KO and KO to KO mice in treatment. Thromb. Haemost. 111, 817–823 (2014). Fig. 1k), a dichotomous measure of thrombosis within 20 min (yes or no) was used 19. Conley, P. B. & Delaney, S. M. Scientific and therapeutic insights into the role and compared in a 2 × 2 contingency table. Because of small sample sizes within of the platelet P2Y12 receptor in thrombosis. Curr. Opin. Hematol. 10, some of the cells, the Fisher’s Exact Probability test was used to compare the two 333–338 (2003). groups. Tukey’s test was used for multiple comparisons (such as differences in 20. Hollopeter, G. et al. Identification of the platelet ADP receptor targeted by bleeding time between transfused hosts in Fig. 1l). antithrombotic drugs. Nature 409, 202–207 (2001). 21. Zeisel, S. H. Metabolic crosstalk between choline/1-carbon metabolism and Study approval. All in vivo procedures and usage of mice were approved by the energy homeostasis. Clin. Chem. Lab. Med. 51, 467–475 (2013). Division of Laboratory Animal Medicine at the University of Rochester Medical 22. Krotz, F., Sohn, H. Y. & Pohl, U. Reactive oxygen species: players in the Center. platelet game. Arterioscler Thromb. Vasc. Biol. 24, 1988–1996 (2004). 23. Leopold, J. A. & Loscalzo, J. Oxidative risk for atherothrombotic cardiovascular disease. Free Radic. Biol. Med. 47, 1673–1706 (2009). Reporting summary. Further information on research design is available in 24. Carrim, N. et al. Thrombin-induced reactive oxygen species generation in the Nature Research Reporting Summary linked to this article. platelets: a novel role for protease-activated receptor 4 and GPIbalpha. Redox Biol. 6, 640–647 (2015). Data availability 25. Akbar, H., Duan, X., Saleem, S., Davis, A. K. & Zheng, Y. RhoA and Rac1 The datasets generated during and analyzed during the current study are available from GTPases differentially regulate agonist-receptor mediated reactive oxygen the corresponding author on reasonable request. The dataset of the metabolomics species generation in platelets. PLoS ONE 11, e0163227 (2016). experiment is available in the Supplementary Material. The source data underlying 26. Mukerjee, S. A decade's tracking of cholera with bacteriophage. Indian J. Med. Figs. 1a–e, g–l, 2b–k, 3c–e, g, h, and 4a–i are provided as a Source Data file. Source Data Res. 55, 308–313 (1967). are provided with this paper. 27. Qiao, J. et al. Regulation of platelet activation and thrombus formation by reactive oxygen species. Redox Biol. 14, 126–130 (2018). 28. Delaney, M. K. et al. Differential roles of the NADPH-oxidase 1 and 2 in Received: 27 August 2018; Accepted: 15 June 2020; platelet activation and thrombosis. Arterioscler Thromb. Vasc. Biol. 36, 846–854 (2016). 29. Arthur, J. F. et al. TNF receptor-associated factor 4 (TRAF4) is a novel binding partner of glycoprotein Ib and glycoprotein VI in human platelets. J. Thrombosis Haemost. 9, 163–172 (2011). 30. Jang, J. Y. et al. Peroxiredoxin II is an antioxidant enzyme that negatively References regulates collagen-stimulated platelet function. J. Biol. Chem. 290, 1. Heit, J. A. Epidemiology of venous thromboembolism. Nat. Rev. Cardiol. 12, 11432–11442 (2015). 464–474 (2015). 31. Franks, Z. G., Campbell, R. A., Weyrich, A. S. & Rondina, M. T. Platelet- 2. Heit, J. A. et al. Familial segregation of venous thromboembolism. J. leukocyte interactions link inflammatory and thromboembolic events in Thrombosis Haemost. 2, 731–736 (2004). ischemic stroke. Ann. N. Y Acad. Sci. 1207, 11–17 (2010). 8 NATURE COMMUNICATIONS | (2020)11:3479 | https://doi.org/10.1038/s41467-020-17254-w | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-17254-w ARTICLE 32. Swystun, L. L. & Liaw, P. C. The role of leukocytes in thrombosis. Blood 128, (T.E.C.), R01 HL071158 (P.S.B.), a Miguel Servet contract from the ISCIII Spanish 753–762 (2016). Health Institute CP17/00142 and from the European Social Fund (M.S-L.), a gift from 33. Luther, N. et al. Innate effector-memory T-cell activation regulates post- Richard T. Aab and the Aab Cardiovascular Research Foundation, and the Paul N. Yu thrombotic vein wall inflammation and thrombus resolution. Circ. Res. 119, Professorship (C.J.L.). Electron Microscopy support was provided by the Electron 1286–1295 (2016). Microscopy Core Facility at University of Rochester. 34. Brill, A. et al. Neutrophil extracellular traps promote deep vein thrombosis in mice. J. Thrombosis Haemost. 10, 136–144 (2012). 35. Fuchs, T. A., Brill, A. & Wagner, D. D. Neutrophil extracellular trap (NET) Author contributions J.A.B. designed experiments, conducted experiments, acquired data, analyzed data, and impact on deep vein thrombosis. Arterioscler Thromb. Vasc. Biol. 32, wrote the manuscript. S.K.T. conducted experiments and trained lab workers in animal 1777–1783 (2012). procedures. M.A.M. conducted experiments, acquired data, and analyzed data. R.A.B. 36. Martinod, K. et al. Neutrophil elastase-deficient mice form neutrophil conducted experiments and trained lab workers in platelet analyses. S.J.C. designed extracellular traps in an experimental model of deep vein thrombosis. J. experiments, conducted experiments, acquired data, and analyzed data. T.E.C. provided Thrombosis Haemost. 14, 551–558 (2016). reagents and reviewed the manuscript. S.G.L. and R.A.B. and S.L.S assisted with the 37. Savchenko, A. S. et al. Neutrophil extracellular traps form predominantly platelet aggregation studies. P.S.B. and C.O.S. and J.A.B. designed and assisted in Sea- during the organizing stage of human venous thromboembolism horse analyzer studies. R.E.G. and X.S. designed and performed metabolomics studies. development. J. Thrombosis Haemost. 12, 860–870 (2014). R.M.B. conducted experiments. N.L.S. and M.S.-L. and P.S.d.V. and J.E.H. reviewed the 38. von Bruhl, M. L. et al. Monocytes, neutrophils, and platelets cooperate to manuscript. C.N.M. designed experiments, analyzed data, and wrote the manuscript. initiate and propagate venous thrombosis in mice in vivo. J. Exp. Med. 209, C.J.L. designed experiments, analyzed data, and wrote the manuscript. 819–835 (2012). 39. Matsushita, K. et al. Nitric oxide regulates exocytosis by S-nitrosylation of N- ethylmaleimide-sensitive factor. Cell 115, 139–150 (2003). Competing interests 40. De Cuyper, I. M. et al. A novel flow cytometry-based platelet aggregation The authors declare no competing interests. assay. Blood 121, e70–e80 (2013). 41. Morrell, C. N. et al. Glutamate mediates platelet activation through the AMPA receptor. J. Exp. Med. 205, 575–584 (2008). Additional information 42. Owens, A. P. 3rd et al. Towards a standardization of the murine ferric Supplementary information is available for this paper at https://doi.org/10.1038/s41467- chloride-induced carotid arterial thrombosis model. J. Thrombosis Haemost. 9, 020-17254-w. 1862–1863 (2011). 43. Eckly, A. et al. Mechanisms underlying FeCl3-induced arterial thrombosis. J. Correspondence and requests for materials should be addressed to C.J.L. Thrombosis Haemost. 9, 779–789 (2011). 44. Bergmeier, W. & Boulaftali, Y. Adoptive transfer method to study platelet Peer review information Nature Communications thanks Gary Siuzdak and the other function in mouse models of disease. Thrombosis Res. 133(Suppl 1), S3–S5 anonymous reviewer(s) for their contribution to the peer review of this work. Peer (2014). reviewer reports are available. 45. Chacko, B. K. et al. Methods for defining distinct bioenergetic profiles in platelets, lymphocytes, monocytes, and neutrophils, and the oxidative burst Reprints and permission information is available at http://www.nature.com/reprints from human blood. Lab. Investig. 93, 690–700 (2013). 46. Schoenwaelder, S. M. et al. 14-3-3zeta regulates the mitochondrial respiratory Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in reserve linked to platelet phosphatidylserine exposure and procoagulant published maps and institutional affiliations. function. Nat. Commun. 7, 12862 (2016). 47. Kimberly, W. T. et al. Metabolite profiling identifies anandamide as a biomarker of nonalcoholic steatohepatitis. JCI Insight 2, https://doi.org/ Open Access This article is licensed under a Creative Commons 10.1172/jci.insight.92989 (2017). Attribution 4.0 International License, which permits use, sharing, 48. Wang, T. J. et al. Metabolite profiles and the risk of developing diabetes. Nat. adaptation, distribution and reproduction in any medium or format, as long as you give Med. 17, 448–453 (2011). appropriate credit to the original author(s) and the source, provide a link to the Creative 49. Cameron, S. J. et al. Platelet extracellular regulated protein kinase 5 is a redox Commons license, and indicate if changes were made. The images or other third party switch and triggers maladaptive platelet responses and myocardial infarct material in this article are included in the article’s Creative Commons license, unless expansion. Circulation 132, 47–58 (2015). indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ Acknowledgements licenses/by/4.0/. This work was supported by NIH NHLBI R01 HL134894 (C.J.L. and N.L.S.), R61 HL141791 (C.J.L. and N.L.S.), R01 HL124018 (C.N.M.), K08 HL128856 and NIH HL12020 (S.J.C.), R01 HL139553 (N.L.S.), R01 CA194536 (T.E.C.), U01 DE025184 © The Author(s) 2020 NATURE COMMUNICATIONS | (2020)11:3479 | https://doi.org/10.1038/s41467-020-17254-w | www.nature.com/naturecommunications 9