Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Mar 26;8(6):1550-1566.
doi: 10.1182/bloodadvances.2023011734.

Plasma growth factors maintain constitutive translation in platelets to regulate reactivity and thrombotic potential

Jeremy G T Wurtzel  1 , Sophia Lazar  1 , Shayan Askari  1 , Xuefei Zhao  1 , Jenna Severa  1 , Francis Ayombil  2 , James V Michael  1 , Rodney M Camire  2   3 , Steven E McKenzie  1 , Timothy J Stalker  1 , Peisong Ma  1 , Lawrence E Goldfinger  1
Affiliations

Plasma growth factors maintain constitutive translation in platelets to regulate reactivity and thrombotic potential

Jeremy G T Wurtzel et al. Blood Adv. .

Abstract

Mechanisms of proteostasis in anucleate circulating platelets are unknown and may regulate platelet function. We investigated the hypothesis that plasma-borne growth factors/hormones (GFHs) maintain constitutive translation in circulating platelets to facilitate reactivity. Bio-orthogonal noncanonical amino acid tagging (BONCAT) coupled with liquid chromatography-tandem mass spectrometry analysis revealed constitutive translation of a broad-spectrum translatome in human platelets dependent upon plasma or GFH exposure, and in murine circulation. Freshly isolated platelets from plasma showed homeostatic activation of translation-initiation signaling pathways: phosphorylation of p38/ERK upstream kinases, essential intermediate MNK1/2, and effectors eIF4E/4E-BP1. Plasma starvation led to loss of pathway phosphorylation, but it was fully restored with 5-minute stimulation by plasma or GFHs. Cycloheximide or puromycin infusion suppressed ex vivo platelet GpIIb/IIIa activation and P-selectin exposure with low thrombin concentrations and low-to-saturating concentrations of adenosine 5'-diphosphate (ADP) or thromboxane analog but not convulxin. ADP-induced thromboxane generation was blunted by translation inhibition, and secondary-wave aggregation was inhibited in a thromboxane-dependent manner. Intravenously administered puromycin reduced injury-induced clot size in cremaster muscle arterioles, and delayed primary hemostasis after tail tip amputation but did not delay neither final hemostasis after subsequent rebleeds, nor final hemostasis after jugular vein puncture. In contrast, these mice were protected from injury-induced arterial thrombosis and thrombin-induced pulmonary thromboembolism (PE), and adoptive transfer of translation-inhibited platelets into untreated mice inhibited arterial thrombosis and PE. Thus, constitutive plasma GFH-driven translation regulates platelet G protein-coupled receptor reactivity to balance hemostasis and thrombotic potential.

PubMed Disclaimer

Conflict of interest statement

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Figures

None
Graphical abstract
Figure 1.
Figure 1.
Plasma-driven broad-spectrum protein synthesis in human platelets. (A) Platelet-rich plasma was treated with vehicle (−) or azidohomoalanine (AHA) in the absence (−) or presence (+) of puromycin (Puro, 10 μg/mL), and platelets were isolated at the indicated times, washed, and lysed. NSPs were labeled with biotin-alkyne and detected with streptavidin conjugate, and membranes were counter-stained with β-actin antibodies, as shown. (B) Washed human white blood cell/red blood cell–depleted platelets were either resuspended in autologous plasma–EDTA (platelet-poor plasma [PPP]; EDTA added to inhibit platelet activation) or SB followed by feedback with AHA and plasma or buffer as indicated, then harvested and processed for NSP detection as described earlier. (C) AHA (0.5 mg/g) with/without Puro (20 mg/kg) was injected into tail veins of wild-type (WT) mice, and blood was extracted after 1 hour for platelet isolation and platelet NSP detection. (D) Puromycin was administered as in panel C in the absence of AHA, platelets were collected at the indicated times, and platelet lysates were subject to immunoblotting with puromycin-specific antibodies. Each panel representative of 3 independent experiments.
Figure 2.
Figure 2.
Translation-initiation signaling by plasma GFHs in human platelets. (A) Human washed platelets were resuspended in SB for the indicated times, and whole-platelet lysates were immunoblotted with antibodies as indicated. p, phosphorylated; t, total. (B) Platelets maintained in SB for 120 minutes, as in panel A, were pelleted by gentle centrifugation and resuspended in autologous platelet-poor plasma for 5 minutes, then pelleted, lysed, and subject to immunoblotting with the indicated antibodies. (C) Platelets were harvested directly from platelet-rich plasma (Fresh), or otherwise resuspended in SB for 120 minutes, then treated as in panel B for 5 minutes with platelet-poor plasma (PPP) or each indicated GFH, then pelleted for immunoblotting with the indicated antibodies. Vascular endothelial growth factor (VEGF), 100 pg/mL; insulin-like growth factor-1 (IGF-1), 200 ng/mL; insulin, 500 pM; EGF, 2 ng/mL; high mobility group box protein 1 (HMGB1), 2 ng/mL; epinephrine (Epi), 500 pM; serotonin (5-hydroxytryptamine [5-HT]), 1 nM. Fold change in phosphorylated (phospho) to total proteins from immunoblots as measured by densitometric analysis are shown to the right ± standard error of the mean, normalized to Fresh (1) and Starved (0). All were significantly increased over starved levels (P < .05) except for the following: IGF-1: p-4E-BP1; Epi: p-ERK, pAKT, p-eIF4E, p-4E-BP1; 5-HT: p-ERK, p-AKT, and p-4E-BP1; n = 5.
Figure 3.
Figure 3.
Modulation of platelet reactivity by suppression of translation in circulating platelets. WT mice were dosed by tail vein injection with vehicle (saline, volume per volume), puromycin (20 mg/kg), or cycloheximide (120 mg/kg), 18 hours and again 4 hours before platelet collection. (A-D) Platelets from treated mice were subject to agonist stimulation as indicated in the presence of fluorophore-conjugated antibodies against activated αIIbβ3 integrin (Jon/A) and Cd62p, and measured by flow cytometry; n = 9. P < .03 compared with vehicle control except if indicated otherwise; other P values are shown compared with vehicle control; n.s., not significant compared with vehicle control; MFI, mean fluorescence intensity, shown ± standard error of the mean. Vehicle, green circles and green lines; puromycin, red triangles and red lines; cycloheximide, blue squares and blue lines.
Figure 4.
Figure 4.
Translation inhibition in vivo suppresses murine platelet ex vivo aggregation by inhibition of thromboxane generation. (A-C) Platelet aggregation in response to agonists as indicated. In panel C, platelets were pretreated with vehicle, apyrase (1 U/mL), and/or aspirin (1 mM) for 30 minutes before stimulation with 0.0025 U/mL thrombin; n = 3; ∗P < .01, ∗∗P < .03. AUC, area under the curve. (D) Washed platelets (2 × 108) from vehicle- or puromycin-treated mice were suspended in 0.4 mL of buffer and stimulated with 10 μM ADP for 3 minutes, and thromboxane B2 in platelet-depleted releasates was measured as described in “Methods”; n = 5. Vehicle, green lines or bars; puromycin, red lines or bars.
Figure 5.
Figure 5.
Inhibition of translation in circulating platelets delays but does not compromise hemostasis in mice. (A) Time to initial stoppage of bleeding after 1-mm diameter tail amputation in C57Bl/6J mice subject to 2 doses of either vehicle or puromycin as in Figure 3. Data are replotted as box and whisker plots to the right. (B) Time to hemostasis after rebleeds in the same mice as in panel A. The experiment was stopped at 1200 seconds; n = 9. P < .28 (not significant) for final hemostasis (log-rank test). (C-H) Bleeding times and clot features were monitored under microscopic observation in C57Bl/6J mice treated as earlier, after 150-μm puncture to the jugular vein; n = 10. (C) Time to hemostasis. The experiment was stopped at 300 seconds. (D) Replots of data from panel C. (E) Representative micrographs of intraluminal sides of injury sites, resected after the experimental end point, showing platelet volumes (Cd41, purple), Cd62p (P-selectin, green), and fibrin (blue); collagen is shown in gray. (F) Cd41 volumes. (G) Cd62p mean intensities. (H) Fibrin mean intensities. (C-H) no significant differences were observed. Vehicle, green closed circles and solid green lines or light green or green bars; puromycin, red open circles and dotted red lines or red bars.
Figure 6.
Figure 6.
Inhibition of thrombosis by blockade of plasma-driven translation in murine platelets. (A) Time to cessation of respiration in mice treated as indicated, after bolus administration of 900 U/kg thrombin to induce pulmonary thromboembolism; n = 6; P < .01. (B) Naïve mice were transfused with 1 × 108 washed platelets from vehicle- or puromycin-treated mice, and subject to thrombin administration and monitoring as in panel A. Time to cessation of respiration is shown. Veh, vehicle; puro, puromycin. n = 3; P < .03. (C) Representative images of hematoxylin/eosin-stained lung sections from mice from panel B, perfused with saline immediately after the experimental end point, before lung resection. Photographs of the resected lungs before sectioning are shown below. Lungs from mice receiving vehicle-treated platelets appeared blood-filled, indicating thrombosis preventing saline perfusion, whereas lungs from mice receiving puromycin-treated platelets appeared to contain less blood, indicating reduced thrombosis. (D) Carotid arteries in WT mice treated with vehicle or puromycin as indicated were injured with 7.5% FeCl3. Flow rates through the carotid artery downstream of the injury site were monitored with a Doppler probe. Time to cessation of blood flow is shown. Two representative tracings showing blood flow before, during, and after injury per treatment are shown to the right; n = 9 vehicle, n = 6 puromycin; P < .005. (E) WT untreated (naïve) mice transfused with 1 × 108 washed platelets freshly isolated from vehicle- or puromycin-treated mice were subject to FeCl3 carotid arterial injury as in panel D; n = 5 naïve + vehicle platelets, n = 6 naïve + puromycin platelets; P < .006. Vehicle or vehicle platelet-infused, green closed circles and solid green lines; puromycin or puro platelet-infused, red open circles and dotted red lines.
Figure 7.
Figure 7.
Clot dynamics after laser injury to cremaster muscle arterioles in puromycin-treated mice. (A) Representative images from hemostatic clots formed in vehicle- and puromycin-treated mice, 100 seconds after injury. Cd41, red; Cd62p, blue; fibrin, green. (B) Platelet accumulation, (C) Cd62p exposure, and (D) fibrin deposition were monitored by confocal intravital fluorescence microscopy; 32 injuries in 3 vehicle-treated mice, and 46 injuries in 3 puromycin-treated mice. Graphs in panels B-D shows the AUC for each. The lines and error bars indicate median and interquartile ranges. Vehicle, green lines and green closed circles; puromycin, red lines and red open circles.
See this image and copyright information in PMC

Comment in

Similar articles

See all similar articles

Cited by

References

    1. Warshaw AL, Laster L, Shulman NR. Protein synthesis by human platelets. J Biol Chem. 1967;242(9):2094–2097. - PubMed
    1. Kieffer N, Guichard J, Farcet JP, Vainchenker W, Breton-Gorius J. Biosynthesis of major platelet proteins in human blood platelets. Eur J Biochem. 1987;164(1):189–195. - PubMed
    1. Mills EW, Green R, Ingolia NT. Slowed decay of mRNAs enhances platelet specific translation. Blood. 2017;129(17):e38–e48. - PMC - PubMed
    1. Clancy L, Freedman JE. The role of circulating platelet transcripts. J Thromb Haemost. 2015;13(suppl 1):S33–39. - PubMed
    1. Schubert S, Weyrich AS, Rowley JW. A tour through the transcriptional landscape of platelets. Blood. 2014;124(4):493–502. - PMC - PubMed
-