Fig 2: 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.

Fig 3: Proposed model for choline transport and Slc44a2 regulation of platelet activation.We propose that Slc44a2 transports choline into mitochondria, where it is metabolized and regulates the production of ATP and release of ROS. ATP is released from platelets and hydrolyzed to ADP which acts upon platelet purinergic receptors and drives further platelet activation.

Fig 4: 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.

Fig 5: 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.