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. 2025 Feb 26;15(1):6855.
doi: 10.1038/s41598-025-91181-y.

Inhibition of autophagy in platelets as a therapeutic strategy preventing hypoxia induced thrombosis

Propanna Bandyopadhyay  1 , Yash T Katakia  1 , Sudeshna Mukherjee  1 , Syamantak Majumder  1 , Shibasish Chowdhury  1 , Rajdeep Chowdhury  2
Affiliations

Inhibition of autophagy in platelets as a therapeutic strategy preventing hypoxia induced thrombosis

Propanna Bandyopadhyay et al. Sci Rep. .

Abstract

Hypoxia triggers activation of platelets, leading to thrombosis. If not addressed clinically, it can cause severe complications and fatal consequences. The current treatment regime for thrombosis is often palliative and include long-term administration of anticoagulants, causing over-bleeding risk and other secondary effects as well. This demands a molecular understanding of the process and exploration of an alternative therapeutic avenue. Interestingly, recent studies demonstrate that platelets exhibit functional autophagy. This cellular homeostatic process though well-studied in non-platelet cells, is under-explored in platelets. Herein, we report autophagy activation under physiologically relevant hypoxic condition (10% O2; associated with high altitude) in ex-vivo platelets and in vivo as well. We show that autophagy inhibition using chloroquine (CQ), a repurposed FDA-approved drug, can significantly reduce platelet activation, both in ex-vivo and in-vivo settings. Further, surgical ligation of inferior vena cava (IVC) was performed to induce thrombus formation. Interestingly, CQ pre-treated rats showed reduced clotting ability in surgical animals as well. Importantly, thrombosis inhibitory dose of CQ was considerably lower than the currently used drug-acetazolamide; CQ was also found to be non-toxic to the tissues. Hence, we propose that repurposing of CQ can attenuate hypoxia-induced thrombosis through inhibition of autophagy and can be explored as an effective therapeutic alternative.

Keywords: Autophagy; Chloroquine; Hypobaric hypoxia; IVC ligation; Platelet aggregation.

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Conflict of interest statement

Declarations. Competing interests: The authors declare no competing interests. Ethical approval: All animal experiments carried out for this study were approved by Institutional Animal Ethics Committee (IAEC), BITS Pilani (Reg. No.417/PO/ReBi/2001/CPCSEA) and were performed in accordance to IAEC and ARRIVE guidelines and regulations. The animals were anesthetized using isoflurane for blood collection. Further, euthanasia by cervical dislocation post inferior vena cava (IVC) ligation and hypoxia exposure for thrombus and organ extraction to minimize their contamination by chemical agents was executed according to American Veterinary Medical Association (AVMA) guidelines for the euthanasia of animals (2020).

Figures

Fig. 1
Fig. 1
Hypoxia (10%) induces pro-thrombotic features in platelets. (a) Phase-contrast microscopic images and graphical representation of platelet aggregates under normoxic (Cntrl; 21% oxygen) or hypoxic condition (Hyp; 10% oxygen; 30 min exposure) (Scale bar: 1 μm). (b) Graphical representation of light transmission aggregometry showing fold change in light absorbance owing to exposure to normoxia or hypoxia. (c) Scanning electron microscopy (SEM) images of platelets showing cytoskeletal extensions from platelet surface (denoted by black arrows) under hypoxia compared to normoxia (50,000 × magnification). Additional acquisition details of SEM images are provided in the ‘equipment and settings’ Section. (d) Phalloidin staining of cells depicting filamentous bridges (denoted by white arrows) formed between platelets under low oxygen conditions (10% oxygen for 30 min) (Scale bar: 10 μm). Details of image acquisition settings are mentioned in the ‘equipment and settings’ section. (e) Mean fluorescence intensity of P-Selectin as analyzed by flow cytometry 30 min post-exposure to 21% oxygen (Cntrl) or 10% oxygen (Hyp). **p < 0.01, ***p < 0.001.
Fig. 2
Fig. 2
Hypoxia induces autophagy in platelets. (a) Immunoblot of autophagy-associated proteins LC3II and p62 in platelets exposed to normoxia or hypoxia for 30 min. (b) Immunofluorescence of p62 protein (Red) in platelets upon subjecting them to hypoxia (30 min) compared to normoxia. Cells were co-stained with phalloidin (Green) (Scale bar: 20 μm). (c) Graphical representation of lysosomes in platelets under hypoxia measured with lysotracker by flow cytometry. (d) Immunofluorescence of LAMP1 (Red) in platelets exposed to varying oxygen conditions- Normoxia (Cntrl; 21%) or Hypoxia (Hyp; 10%) for 30 min. Cells were co-stained with phalloidin (Green) (Scale bar: 20 μm). Inset depicts zoomed image. Details of image acquisition settings are mentioned in equipment and settings Section. (e) Immunoblot of LAMP2A expressed by platelets when exposed to hypoxia for 30 min. All the original uncropped blots are provided in the supplementary Fig. 5. *p < 0.05, ***p < 0.001.
Fig. 3
Fig. 3
Chloroquine inhibits platelet activation. (a) Immunoblot of LAMP2A in platelets exposed to either hypoxia (10% oxygen; Hyp) or hypoxia plus chloroquine (CQ; 2 μM) for 30 min. (b) Immunoblot of autophagy-associated markers-LC3II and p62 in platelets treated with or without CQ under hypoxia for 30 min. All the original uncropped blots are provided in the Supplementary Fig. 7. (c) Immunofluorescence of LAMP1 (Red) in platelets subjected to CQ and hypoxia post 30 min exposure. Cells were co-stained with phalloidin (Green) (Scale bar: 20 μm). Details of image acquisition settings are mentioned in the ‘equipment and settings’ Section. (d) Fold change in lysotracker staining in platelets when exposed to hypoxia in presence or absence of CQ. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig. 4
Fig. 4
Chloroquine attenuates hypoxia-induced platelet activation ex-vivo. (a) Images and bar graph showing platelet aggregates under hypoxia (10% oxygen; 30 min) in presence or absence of CQ (2 μM) (Scale bar: 1 μm). (b) Graphical representation of light absorbed by platelets under hypoxia (Hyp) in presence or absence of CQ (Hyp + CQ). (c) Phalloidin staining of cytoskeletal filaments in platelets exposed to CQ under hypoxia (Scale bar: 10 μm). Details of image acquisition settings are mentioned in the ‘Equipment and settings’ section. (d) Scanning electron microscopy of platelets after CQ treatment under hypoxia (30,000 × magnification). Black arrows represent observed filamentous protrusions. Additional acquisition details are provided in the ‘Equipment and settings’ Section. (e) Graphical representation of fluorescence intensity of platelet activation mark-P-Selectin under hypoxia in presence or absence of CQ, analyzed through flow cytometry. ***p < 0.001.
Fig. 5
Fig. 5
Chloroquine inhibits platelet activation in-vivo. (a) Graphical representation of fold change in bleeding time in CQ pre-treated (5 mg/kg body weight; intraperitoneal; 30 min) hypoxia-exposed animals (10% oxygen; 24 h). (b) Graphical representation showing fold change in blood volume collected from CQ-treated animals under hypoxic condition. (c) Graphical representation of light transmission aggregometry based fold change in light absorbance in platelets obtained from in-vivo conditions. (d) Graph representing fold change in fluorescence intensity of P-Selectin in rats exposed to hypoxia (24 h) pre-injected with intra-peritoneal CQ. (e) Immunoblot of p62 and LC3II expressed by in-vivo derived platelets when rats were subjected to hypoxia for 24 h post intra-peritoneal CQ injection. All the original uncropped blots are provided in the Supplementary Fig. 10. **p < 0.01, ***p < 0.001.
Fig. 6
Fig. 6
Autophagy inhibition causes decrease in thrombus formation in flow restriction model. (a) Thrombus images collected 24-h post ligation of inferior vena cava (IVC) with or without CQ pre-treatment (5 mg/kg body weight; intraperitoneal; 30 min). (b) IVC region collected from SHAM animal (non-ligated) model post 24-h of surgery showing absence of thrombus in it. (c) Graphical representation of thrombus length (cm) obtained post-surgery from CQ-treated or untreated animals. (d) Graphical representation of wet weight of isolated thrombus from CQ-treated or untreated animals. (e) Histology of IVC showing blood vessel blockage in CQ untreated ligated animals, compared to a clean vessel obtained post-24-h surgery with CQ treatment (Scale: 100 μm). **p < 0.01.
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