Reversible thrombocytopenia during hibernation originates from storage and release of platelets in liver sinusoids

Abstract

Immobility is a risk factor for thrombosis due to low blood flow, which may result in activation of the coagulation system, recruitment of platelets and clot formation. Nevertheless, hibernating animals—who endure lengthy periods of immobility—do not show signs of thrombosis throughout or after hibernation. One of the adaptations of hemostasis in hibernators consists of a rapidly reversible reduction of the number of circulating platelets during torpor, i.e., the hibernation phase with reduction of metabolic rate, low blood flow and immobility. It is unknown whether these platelet dynamics in hibernating hamsters originate from storage and release, as suggested for ground squirrel, or from breakdown and de novo synthesis. A reduction in detaching forces due to low blood flow can induce reversible adhesion of platelets to the vessel wall, which is called margination. Here, we hypothesized that storage-and-release by margination to the vessel wall induces reversible thrombocytopenia in torpor. Therefore, we transfused labeled platelets in hibernating Syrian hamster (Mesocricetus auratus) and platelets were analyzed using flow cytometry and electron microscopy. The half-life of labeled platelets was extended from 20 to 30 h in hibernating animals compared to non-hibernating control hamsters. More than 90% of labeled platelets were cleared from the circulation during torpor, followed by emergence during arousal which supports storage-and-release to govern thrombocytopenia in torpor. Furthermore, the low number of immature platelets, plasma level of interleukin-1α and normal numbers of megakaryocytes in bone marrow make platelet synthesis or megakaryocyte rupture via interleukin-1α unlikely to account for the recovery of platelet counts upon arousal. Finally, using large-scale electron microscopy we revealed platelets to accumulate in liver sinusoids, but not in spleen or lung, during torpor. These results thus demonstrate that platelet dynamics in hibernation are caused by storage and release of platelets, most likely by margination to the vessel wall in liver sinusoids. Translating the molecular mechanisms that govern platelet retention in the liver, may be of major relevance for hemostatic management in (accidental) hypothermia and for the development of novel anti-thrombotic strategies.

Hibernation and hemostasis

“…The rapid return of platelets in circulation after arousal, in the absence of large numbers of circulating immature platelets, suggests that hibernator platelets are stored during the cold and released into circulation during rewarming. Removal of the spleens of Syrian hamsters and 13lined ground squirrels before or during torpor did not affect the platelet storage and release indicating that spleen is not required for storage as originally proposed (Reddick et al, 1973;Cooper et al, 2012;de Vrij et al, 2021). Rather, platelet storage during torpor occurs in the sinusoids of the liver, consistent with this organ being responsible for storing platelets (Cooper et al, 2017;de Vrij et al, 2021).…”

“…Removal of the spleens of Syrian hamsters and 13lined ground squirrels before or during torpor did not affect the platelet storage and release indicating that spleen is not required for storage as originally proposed (Reddick et al, 1973;Cooper et al, 2012;de Vrij et al, 2021). Rather, platelet storage during torpor occurs in the sinusoids of the liver, consistent with this organ being responsible for storing platelets (Cooper et al, 2017;de Vrij et al, 2021). The mechanism of hepatic storage of platelets during torpor is currently unknown, potentially platelet margination may underlie the reversible storage and release in liver (de Vrij et al, 2021).…”

Hibernation and hemostasis

“…The rapid return of platelets in circulation after arousal, in the absence of large numbers of circulating immature platelets, suggests that hibernator platelets are stored during the cold and released into circulation during rewarming. Removal of the spleens of Syrian hamsters and 13lined ground squirrels before or during torpor did not affect the platelet storage and release indicating that spleen is not required for storage as originally proposed (Reddick et al, 1973;Cooper et al, 2012;de Vrij et al, 2021). Rather, platelet storage during torpor occurs in the sinusoids of the liver, consistent with this organ being responsible for storing platelets (Cooper et al, 2017;de Vrij et al, 2021).…”

“…Removal of the spleens of Syrian hamsters and 13lined ground squirrels before or during torpor did not affect the platelet storage and release indicating that spleen is not required for storage as originally proposed (Reddick et al, 1973;Cooper et al, 2012;de Vrij et al, 2021). Rather, platelet storage during torpor occurs in the sinusoids of the liver, consistent with this organ being responsible for storing platelets (Cooper et al, 2017;de Vrij et al, 2021). The mechanism of hepatic storage of platelets during torpor is currently unknown, potentially platelet margination may underlie the reversible storage and release in liver (de Vrij et al, 2021).…”

Enhanced Blood Clotting After Rewarming From Experimental Hypothermia in an Intact Porcine Model

“…In line with previous findings (Filseth et al, 2010;Valkov et al, 2019), the current data demonstrate a markedly depressed circulatory function during hypothermia. Such low-flow conditions can promote reversible adhesion of platelets and WBCs to the vessel wall (de Vrij et al, 2021;Russell et al, 2003;Sands et al, 1979), and possibly also lead to platetet aggregation and secretion (Li et al, 2000). The rheological changes during hypothermia include a significant hemoconcentration, which is evident from the increased RBC count, Hct and Hb in response to cooling in our study.…”

Liver transcriptomic and methylomic analyses identify transcriptional MAPK regulation in facultative hibernation of Syrian hamster

“…Both genes belong to cluster 4, which only contains 13 genes. These two genes are mainly expressed in platelets, and their upregulation is in line with the storage of platelets in liver during torpor 10 . Also 5-aminolevulinate synthase ( ALAS2 ) is a prominently upregulated gene in torpor compared to SE and AE (5-aminolevulinate synthase: 5-fold).…”

Hibernation and hemostasis

“…The rapid return of platelets in circulation after arousal, in the absence of large numbers of circulating immature platelets, suggests that hibernator platelets are stored during the cold and released into circulation during rewarming. Removal of the spleens of Syrian hamsters and 13lined ground squirrels before or during torpor did not affect the platelet storage and release indicating that spleen is not required for storage as originally proposed (Reddick et al, 1973;Cooper et al, 2012;de Vrij et al, 2021). Rather, platelet storage during torpor occurs in the sinusoids of the liver, consistent with this organ being responsible for storing platelets (Cooper et al, 2017;de Vrij et al, 2021).…”

“…Removal of the spleens of Syrian hamsters and 13lined ground squirrels before or during torpor did not affect the platelet storage and release indicating that spleen is not required for storage as originally proposed (Reddick et al, 1973;Cooper et al, 2012;de Vrij et al, 2021). Rather, platelet storage during torpor occurs in the sinusoids of the liver, consistent with this organ being responsible for storing platelets (Cooper et al, 2017;de Vrij et al, 2021). The mechanism of hepatic storage of platelets during torpor is currently unknown, potentially platelet margination may underlie the reversible storage and release in liver (de Vrij et al, 2021).…”

Enhanced Blood Clotting After Rewarming From Experimental Hypothermia in an Intact Porcine Model

“…In line with previous findings (Filseth et al, 2010;Valkov et al, 2019), the current data demonstrate a markedly depressed circulatory function during hypothermia. Such low-flow conditions can promote reversible adhesion of platelets and WBCs to the vessel wall (de Vrij et al, 2021;Russell et al, 2003;Sands et al, 1979), and possibly also lead to platetet aggregation and secretion (Li et al, 2000). The rheological changes during hypothermia include a significant hemoconcentration, which is evident from the increased RBC count, Hct and Hb in response to cooling in our study.…”

Liver transcriptomic and methylomic analyses identify transcriptional MAPK regulation in facultative hibernation of Syrian hamster

“…Both genes belong to cluster 4, which only contains 13 genes. These two genes are mainly expressed in platelets, and their upregulation is in line with the storage of platelets in liver during torpor 10 . Also 5-aminolevulinate synthase ( ALAS2 ) is a prominently upregulated gene in torpor compared to SE and AE (5-aminolevulinate synthase: 5-fold).…”

Hibernation and hemostasis

“…The rapid return of platelets in circulation after arousal, in the absence of large numbers of circulating immature platelets, suggests that hibernator platelets are stored during the cold and released into circulation during rewarming. Removal of the spleens of Syrian hamsters and 13lined ground squirrels before or during torpor did not affect the platelet storage and release indicating that spleen is not required for storage as originally proposed (Reddick et al, 1973;Cooper et al, 2012;de Vrij et al, 2021). Rather, platelet storage during torpor occurs in the sinusoids of the liver, consistent with this organ being responsible for storing platelets (Cooper et al, 2017;de Vrij et al, 2021).…”

“…Removal of the spleens of Syrian hamsters and 13lined ground squirrels before or during torpor did not affect the platelet storage and release indicating that spleen is not required for storage as originally proposed (Reddick et al, 1973;Cooper et al, 2012;de Vrij et al, 2021). Rather, platelet storage during torpor occurs in the sinusoids of the liver, consistent with this organ being responsible for storing platelets (Cooper et al, 2017;de Vrij et al, 2021). The mechanism of hepatic storage of platelets during torpor is currently unknown, potentially platelet margination may underlie the reversible storage and release in liver (de Vrij et al, 2021).…”

Enhanced Blood Clotting After Rewarming From Experimental Hypothermia in an Intact Porcine Model

“…In line with previous findings (Filseth et al, 2010;Valkov et al, 2019), the current data demonstrate a markedly depressed circulatory function during hypothermia. Such low-flow conditions can promote reversible adhesion of platelets and WBCs to the vessel wall (de Vrij et al, 2021;Russell et al, 2003;Sands et al, 1979), and possibly also lead to platetet aggregation and secretion (Li et al, 2000). The rheological changes during hypothermia include a significant hemoconcentration, which is evident from the increased RBC count, Hct and Hb in response to cooling in our study.…”

Liver transcriptomic and methylomic analyses identify transcriptional MAPK regulation in facultative hibernation of Syrian hamster

“…Both genes belong to cluster 4, which only contains 13 genes. These two genes are mainly expressed in platelets, and their upregulation is in line with the storage of platelets in liver during torpor 10 . Also 5-aminolevulinate synthase ( ALAS2 ) is a prominently upregulated gene in torpor compared to SE and AE (5-aminolevulinate synthase: 5-fold).…”