Vagus nerve stimulation primes platelets and reduces bleeding in hemophilia A male mice

Abstract

Deficiency of coagulation factor VIII in hemophilia A disrupts clotting and prolongs bleeding. While the current mainstay of therapy is infusion of factor VIII concentrates, inhibitor antibodies often render these ineffective. Because preclinical evidence shows electrical vagus nerve stimulation accelerates clotting to reduce hemorrhage without precipitating systemic thrombosis, we reasoned it might reduce bleeding in hemophilia A. Using two different male murine hemorrhage and thrombosis models, we show vagus nerve stimulation bypasses the factor VIII deficiency of hemophilia A to decrease bleeding and accelerate clotting. Vagus nerve stimulation targets acetylcholine-producing T lymphocytes in spleen and α7 nicotinic acetylcholine receptors (α7nAChR) on platelets to increase calcium uptake and enhance alpha granule release. Splenectomy or genetic deletion of T cells or α7nAChR abolishes vagal control of platelet activation, thrombus formation, and bleeding in male mice. Vagus nerve stimulation warrants clinical study as a therapy for coagulation disorders and surgical or traumatic bleeding.

Fig. 1. Vagus nerve stimulation reduces traumatic blood loss and accelerates clot formation in hemophilia A mice.

a Circulating factor VIII activity in wild-type and factor VIII deficient (F8KO) hemophilia A mice. Data were presented as mean ± s.e.m. Sham (n = 7) and VNS (n = 7) mice per group. ****p < 0.0001 vs. control. Statistical significance was determined by unpaired two-tailed Student’s t-test. The figure represents pooled results from two experiments performed independently. b Hemophilia A (F8KO) mice received rFVIII (Advate®, 200 U/kg, r.o.) or vehicle before tail transection. Data were presented as mean ± s.e.m. Sham (n = 7) and VNS (n = 8) mice per group. *p < 0.018 vs. vehicle. Statistical significance was determined by unpaired two-tailed Student’s t-test. The figure represents pooled results from three experiments performed independently. c Hemophilia A (F8KO) mice received vagus nerve stimulation or sham stimulation before tail transection. Data were presented as mean ± s.e.m. WT (n = 6), Sham (n = 8), and VNS (n = 9) mice per group. *p < 0.05 vs. wild type. **p < 0.01 vs. sham. Statistical significance was determined by one-way ANOVA (Tukey). The figure represents pooled results from three experiments performed independently. d Hemophilia A (F8KO) mice received vagus nerve stimulation or sham stimulation before carotid artery injury. Data were presented as mean ± s.e.m. WT (n = 6), Sham (n = 6), and VNS (n = 6) mice per group. ****p < 0.0001 vs. wild type. ****p < 0.0001 vs. sham. Statistical significance was determined by one-way ANOVA (Tukey). The figure represents pooled results from three experiments performed independently. e Hemophilia A (F8KO) mice received vagus nerve stimulation or sham stimulation before blood collection to determine factor VIII activity. Data were presented as mean ± s.e.m. Sham (n = 8) and VNS (n = 11) mice per group. p = 0.97 vs. sham. Statistical significance was determined by unpaired two-tailed Student’s t-test. The figure represents pooled results from two experiments performed independently. f Representative images of lungs from hemophilia A (F8KO) mice after sham stimulation and tail transection. Experiments were performed independently at least three times with similar results. g Representative images of lungs from hemophilia A (F8KO) mice after vagus nerve stimulation and tail transection. Experiments were performed independently at least three times with similar results. Source data are provided as a Source Data File.

Fig. 2. Vagus nerve stimulation harnesses choline acetyltransferase-expressing T lymphocytes in the spleen to stimulate circulating platelets via α7nAChR.

a C57BL6/J mice underwent splenectomy or sham-splenectomy followed by vagus nerve stimulation or sham stimulation before tail transection. Data were presented as mean ± s.e.m. Sham SPX + Sham VNS (n = 7), Sham SPX + VNS (n = 6), SPX + Sham VNS (n = 9), SPX + VNS (n = 8) mice per group. **p = 0.009 vs. sham in sham-splenectomy group. p = 0.15 VNS vs. sham after SPX. Statistical significance was determined by unpaired two-tailed Student’s t-test. The figure represents pooled results from two experiments performed independently. b Wild-type BALB/c or T lymphocyte deficient (Foxn1^nu) mice received vagus nerve stimulation or sham stimulation before tail transection. Data were presented as mean ± s.e.m. WT + Sham VNS (n = 5), WT + VNS (n = 4), Foxn1^nu + Sham VNS (n = 9), Foxn1^nu + VNS (n = 10) mice per group. *p = 0.03 vs. sham in WT mice. p = 0.60 VNS vs. sham in Foxn1^nu mice. Statistical significance was determined by unpaired two-tailed Student’s t-test. The figure represents pooled results from two experiments performed independently. c T lymphocyte deficient (Foxn1^nu) mice were reconstituted with ChAT-eGFP⁺ or ChAT-eGFP^- cells followed by vagus nerve stimulation or sham stimulation before tail transection. Data were presented as mean ± s.e.m. ChAT-eGFP⁻ + Sham VNS (n = 8), ChAT-eGFP⁻ + VNS (n = 7), ChAT-eGFP⁺ + Sham VNS (n = 5), ChAT-eGFP⁺ + VNS (n = 6) mice per group. **p = 0.005 vs. sham in ChAT-eGFP⁺ cells. p = 0.85 VNS vs. sham in ChAT-eGFP^- cells. Statistical significance was determined by unpaired two-tailed Student’s t-test. The figure represents pooled results from two experiments performed independently. d Representative confocal microscopy image (10X) of spleen from ChAT-TdTomato mouse with immunostaining of CD41⁺ platelets (green), CD4⁺ T lymphocytes (purple), and ChAT-eGFP⁺ cells (red) throughout spleen but mostly in the peripheral white pulp (arrows). Scale bar = 200 µm. Experiments were performed independently at least three times with similar results. e Representative merged confocal microscopy image (left, 63X) of spleen from ChAT-TdTomato mouse showing CD4⁺ ChAT-eGFP⁺ T lymphocyte (red and pink, center) in direct contact with CD41⁺ platelets (green). Scale bar = 10 µm. Representative confocal microscopy of individual color channels (right, 63X) including CD41⁺ platelets (green), CD4⁺ T lymphocytes (purple), ChAT-eGFP⁺ cell (red), and DAPI (blue). Scale bars = 10 µm. Experiments were performed independently at least three times with similar results. f Wild-type or α7nAChR-deficient (α7KO) mice received vagus nerve stimulation or sham stimulation before tail transection. Data were presented as mean ± s.e.m. WT + Sham VNS (n = 7), WT + VNS (n = 7), α7KO + Sham VNS (n = 9), α7KO + VNS (n = 9) mice per group. *p = 0.025 vs. sham in WT mice. p = 0.87 VNS vs. sham in α7KO mice. Statistical significance was determined by unpaired two-tailed Student’s t-test. The figure represents pooled results from two experiments performed independently. g Wild-type or α7nAChR-deficient (α7KO) mice received vagus nerve stimulation or sham stimulation before carotid artery injury. Data were presented as mean ± s.e.m. WT + Sham VNS (n = 5), WT + VNS (n = 5), α7KO + Sham VNS (n = 5), α7KO + VNS (n = 5) mice per group. *p = 0.046 vs. sham in WT mice. p = 0.82 VNS vs. sham in α7KO mice. Statistical significance was determined by unpaired two-tailed Student’s t-test. The figure represents pooled results from two experiments performed independently. h Wild-type or α7nAChR-deficient (α7KO) mice received nicotine or vehicle before tail transection. Data were presented as mean ± s.e.m. WT + Vehicle (n = 4), WT + Nicotine (n = 5), α7KO + Vehicle (n = 8), α7KO + Nicotine (n = 7). **p = 0.0037 vs. vehicle in WT mice. p = 0.88 Nic vs. vehicle in α7KO mice. Statistical significance was determined by unpaired two-tailed Student’s t-test. The figure represents pooled results from two experiments performed independently. i α7nAChR-deficient (α7KO) mice were reconstituted with platelets from wild-type or α7KO mice, followed by treatment with nicotine or vehicle before tail transection. Data are presented as mean ± s.e.m. WT platelets + Vehicle (n = 6), WT Platelets + Nicotine (n = 7), α7KO Platelets + Vehicle (n = 5), α7KO Platelets + Nicotine (n = 5). *p = 0.029 vs. vehicle with WT platelets. p = 0.52 Nic vs. vehicle with α7KO platelets. Statistical significance was determined by unpaired two-tailed Student’s t-test. The figure represents pooled results from two experiments performed independently. Source data are provided as a Source Data File.

Fig. 3. Vagus nerve stimulation requires α7nAChR to increase platelet cytosolic calcium, enhance cellular activation, and accelerate local clot formation to reduce bleeding.

a C57BL6/J mice received vagus nerve stimulation or sham stimulation before harvesting circulating platelets to measure basal cytosolic calcium uptake. Data were presented as mean ± s.e.m. Sham (n = 5) and VNS (n = 6) mice per group. **p = 0.0073 vs. sham. Statistical significance was determined by unpaired two-tailed Student’s t-test. The figure represents pooled results from two experiments performed independently. b α7nAChR-deficient (α7KO) mice received vagus nerve stimulation or sham stimulation before harvesting circulating platelets to measure basal cytosolic calcium uptake. Data were presented as mean ± s.e.m. Sham (n = 4) and VNS (n = 4) mice per group. p = 0.32 vs. sham. Statistical significance was determined by unpaired two-tailed Student’s t-test. The figure represents pooled results from two experiments performed independently. c C57BL6/J mice received vagus nerve stimulation or sham stimulation before harvesting platelets for analysis of P-selectin (CD62P) expression. Data were presented as mean ± s.e.m. Sham (n = 13) and VNS (n = 12) mice per group. p = 0.98 vs. sham. Statistical significance was determined by unpaired two-tailed Student’s t-test. The figure represents pooled results from three experiments performed independently. d Representative FACS histogram of platelet P-selectin (CD62P) expression after vagus nerve stimulation or sham stimulation. Experiments were performed independently three times with similar results. e C57BL6/J mice received vagus nerve stimulation or sham stimulation before harvesting platelets, ex vivo thrombin stimulation, and analysis of P-selectin (CD62P) expression. Data were presented as mean ± s.e.m. Sham (n = 10) and VNS (n = 12) mice per group. *p = 0.029 vs. sham. Statistical significance was determined by unpaired two-tailed Student’s t-test. The figure represents pooled results from three experiments performed independently. f Representative FACS histogram of platelet P-selectin (CD62P) expression after vagus nerve stimulation or sham stimulation followed by ex vivo thrombin stimulation. Experiments were performed independently three times with similar results. g α7nAChR-deficient (α7KO) mice received vagus nerve stimulation or sham stimulation before harvesting platelets, ex vivo thrombin stimulation, and analysis of P-selectin (CD62P) expression. Data were presented as mean ± s.e.m. Sham (n = 11) and VNS (n = 11) mice per group. p = 0.57 vs. sham. Statistical significance was determined by unpaired two-tailed Student’s t-test. The figure represents pooled results from three experiments performed independently. h Platelets from uninjured C57BL6/J mice receiving vagus nerve stimulation or sham stimulation were transferred into naïve animals before tail transection. Data were presented as mean ± s.e.m. Sham (n = 9) and VNS (n = 8) mice per group. *p = 0.016 vs. sham. Statistical significance was determined by unpaired two-tailed Student’s t-test. The figure represents pooled results from two experiments performed independently. i C57BL6/J mice received vagus nerve stimulation or sham stimulation before tail transection and collection of systemic or local shed blood for measurement of (TAT) complexes. Data were presented as mean ± s.e.m. Sham VNS + Systemic (n-4), VNS + Systemic (n = 4), Sham VNS + Local (n = 3), VNS + Local (n = 3) mice per group. **p = 0.003 vs. sham in local blood. p = 0.42 VNS vs. sham in systemic blood. Statistical significance was determined by unpaired two-tailed Student’s t-test. The figure represents pooled results from two experiments performed independently. j Representative low power (left, scale bar = 100 µm) and high power (right, scale bar = 50 µm) lung images from C57BL6/J mice after sham stimulation and tail transection. Experiments were performed independently three times with similar results. k Representative low power (left, scale bar = 100 µm) and high power (right, scale bar = 50 µm) lung images from C57BL6/J mice after vagus nerve stimulation and tail transection. Experiments were performed independently three times with similar results. Source data are provided as a Source Data File.

Fig. 4. The Neural Tourniquet.

Electrical vagus nerve stimulation harnesses acetylcholine-secreting ChAT⁺ T lymphocytes in the spleen to stimulate platelet calcium uptake and alpha granule secretion via an α7nAChR-dependent mechanism. Vagus nerve stimulation primes circulating platelets to increase local thrombin production that accelerates clotting and reduces bleeding at sites of tissue injury.