Temperature effects on the activity, shape, and storage of platelets from 13-lined ground squirrels

Abstract

The objective of this study is to determine how a hibernating mammal avoids the formation of blood clots under periods of low blood flow. A microfluidic vascular injury model was performed to differentiate the effects of temperature and shear rate on platelet adhesion to collagen. Human and ground squirrel whole blood was incubated at 15 or 37°C and then passed through a microfluidic chamber over a 250 μm strip of type I fibrillar collagen at that temperature and shear rates of 50 s−1 or 300 s−1 to simulate torpid and aroused conditions respectively. At 15°C, both human and ground squirrel platelets showed a 90–95% decrease in accumulation on collagen independent of shear rate. At 37°C, human platelet accumulation reduced by 50% at 50 s−1 compared to 300 s−1, while ground squirrel platelet accumulation dropped by 80%. When compared to platelets from non-hibernating animals, platelets from animals collected after arousal from torpor showed a 60% decrease in binding at 37°C and 300 s−1, but a 2.5-fold increase in binding at 15°C and 50 s−1. vWF binding in platelets from hibernating ground squirrels were decreased by 50% relative to non-hibernating platelets. The source of the plasma that platelets were stored in did not affect the results indicating that the decreased vWF binding was a property of the platelets. Upon chilling, ground squirrel platelets increase microtubule assembly leading to the formation of long rods. This shape change is concurrent with sequestration of platelets in the liver and not the spleen. In conclusion, it appears that ground squirrel platelets are sequestered in the liver during torpor, have reduced binding capacity for plasma vWF, and lower accumulation on collagen at low shear rates and after storage at cold temperatures, while still being activated by external agonists. These adaptations would protect the animals from spontaneous thrombus formation during torpor but allow them to restore normal platelet function upon arousal.

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

“…We demonstrate that liver sinusoids appear as the main compartment of platelet storage during torpor, from where platelets are released upon arousal. These findings match a recent study demonstrating increased amount of platelet glycoprotein Ib staining in liver of hibernating torpid ground squirrels, which reverses in arousal (Cooper et al 2017 ). By large-scale electron microscopy (nanotomy) analysis (Kuipers et al 2016 ; Sokol et al 2015 ) we determined that the platelet storage location in torpor was not in lung or spleen, since the number of platelets did not change in these organs.…”

“…By large-scale electron microscopy (nanotomy) analysis (Kuipers et al 2016 ; Sokol et al 2015 ) we determined that the platelet storage location in torpor was not in lung or spleen, since the number of platelets did not change in these organs. In accord, we previously excluded a role of spleen in platelet storage by demonstrating that splenectomy before or during torpor is without an effect on platelet dynamics in hibernating hamster (de Vrij et al 2014 ), which was recently corroborated in splenectomized squirrels (Cooper et al 2017 ). Finally, our results exclude thrombosis and trapping of platelets within immune complexes or rosette cell formation as a contributor to platelet storage, since (micro)thrombi were absent in liver and lung, levels of D-dimer remained low throughout torpor and arousal, and platelets in torpid liver sinusoids were not degranulated and did not form large activated aggregates, but rather non-activated accumulations.…”

“…Main arguments are that platelet count rapidly normalizes within a few hours of arousal, i.e., faster than accounted for by synthesis from megakaryocytes, and that the amount of newly synthesized platelets does not increase in arousal (Cooper et al 2012 ; de Vrij et al 2014 ). Recently, a histological analysis of hibernating ground squirrels points towards the storage and release of platelets in liver in torpid ground squirrels (Cooper et al 2017 ). Whether megakaryocyte rupture, recently discovered as a rapid platelet producing process (Nishimura et al 2015 ), might play a role in the swift recovery of platelet count during arousal is not yet known.…”

Cardiovascular resistance to thrombosis in 13-lined ground squirrels

“…In hibernating ground squirrels circulating platelets and vWF both drop 10-fold, while Factors VIII and IX drop 3-fold, leading to prolonged clotting times (Suomalainen and Lehto 1952, Svihla et al 1953, Smith et al 1954, Lechler and Penick 1963, Pivorun and Sinnamon 1981, de Vrij et al 2014. The duration of the periodic IBAs is important in primary hemostasis as the time frame is long enough for sequestered platelets to be released back into circulation, but too short to allow for new cell or protein synthesis based on measured post-arousal kinetics (Cooper et al 2012, Cooper et al 2017. Because ground squirrel platelets can withstand repeated cycling between 4°C and 37°C for several months, they are an intriguing model for platelet storage in the cold.…”

Does hypometabolism constrain innate immune defense?

“…Among other changes, hibernators have decreased plasma leukocyte concentrations 149,151–155 . It is not well understood to where these blood cells disappear, but it is likely they are sequestered into the liver, lung, and lymphoid tissues, but not the spleen 150,156,157 . Leukocyte sequestration could reduce the risk of triggering the inflammatory cascade, which may be damaging and could lead to cerebral ischemia 58 …”