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. 2017 Jul;187(5-6):815-825.
doi: 10.1007/s00360-017-1081-x. Epub 2017 Mar 23.

Temperature Effects on the Activity, Shape, and Storage of Platelets From 13-lined Ground Squirrels

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Free PMC article

Temperature Effects on the Activity, Shape, and Storage of Platelets From 13-lined Ground Squirrels

Scott Cooper et al. J Comp Physiol B. .
Free PMC article

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 the shear rates of 50 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 was 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 and 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.

Keywords: Collagen; Fibrinogen; Hypothermia; Tubulin; Von Willebrand factor.

Conflict of interest statement

Disclosures

No conflicts of interest, financial or otherwise, are declared by the author(s).

Figures

Figure 1
Figure 1
Adhesion and aggregation of platelets. Platelets adhere to a damaged vessel wall through binding of their glycoprotein Ib (GpIbα) receptor to von Willebrand Factor (vWF) which in turn binds to exposed collagen. Platelet aggregation occurs when two platelets bind to a single molecule of fibrinogen through their glycoprotein IIb/IIIa receptors (GpIIb/IIIa). Glycosylated proteins on the surface of the platelets also contain sialic acid caps, and the removal of these lead to clearance through Ashwell Morell receptors on hepatocytes.
Figure 2
Figure 2
Platelet fibrinogen binding. Platelets were isolated from non-hibernating squirrels and incubated with Alexa 488 fluorescently labeled fibrinogen. Some samples were activated with different agonists including ADP, TRAP, and PAR4 activating peptide for 5 minutes. Samples were then fixed with paraformaldehyde, analyzed on a flow cytometer, and normalized to matched unstimulated platelet fibrinogen binding. N=5 samples run in triplicate and analyzed by ANOVA, bars with different letters were significantly different, p<0.05.
Figure 3
Figure 3
Effects of temperature and plasma on platelet binding. Platelets were isolated from humans, non-hibernating ground squirrels (NH), and those in an interbout arousal (IBA). Different aliquots of the washed platelets were then incubated for two days in either plasma from the non-hibernating or IBA squirrels for two days at 4°C or room temperature. Platelets were washed, and immunoblotted for vWF, size bar is 50μm on the large images and 10μm on the insets (panel A). Band densities were quantified and normalized to the To platelets (panel B). N=5 samples analyzed by student t-test, bars with different letters were significantly different, p<0.05.
Figure 4
Figure 4
Effects of temperature and shear rate on platelet binding to collagen. Whole blood samples from humans, non-hibernating ground squirrels, and those in an IBA were incubated at 15°C or 35°C for 30 minutes. Platelets were labeled with DiOC6, and perfused over a 250μm strip of type I collagen for 5 minutes at 15°C or 35°C and shear stresses of 50 s−1 or 300 s−1 followed by fixative (panel A). Platelets were then fixed and quantified on a confocal microscope. N=5 samples analyzed by student t-test, bars with different letters were significantly different, p<0.05.
Figure 5
Figure 5
Platelet microtubule shape changes at 4°C. Panel A: Ground squirrel platelet rich plasma was incubated at 37°C for 2 hours and then place on ice. At each time point, aliquots were removed, centrifuged onto polylysine coated microscope slides, fixed and the percentage of platelets in a rod shape counted. The cold storage curve fit best to a hyperbolic curve, F= 0.312*x/(14.67+x). N=6 samples run in duplicate and time points at 30, 60, 90 and 120 minutes compared by student t-test. Panel B: Taxol treated platelets showed increased rod formation even at 37°C, while no changes were observed in control or nocodazole treated platelets. Panel C: Taxol treated platelets form rods and will not revert to rings upon rewarming to 37°C unlike control or nocodazole treated platelets. N=6 samples run in duplicate and analyzed by student t-test, bars with different letters were significantly different, p<0.05.
Figure 6
Figure 6
Effects of splenectomy on platelet levels during hibernation. Ground squirrels received either a splenectomy or sham surgery during the summer. Blood samples were collected pre-and post-surgery samples, upon entrance into hibernation and during an IBA, and platelets counted on a Hemavet. During torpor, blood smears were collected without waking the animal and counted manually. N= 10 animals, analyzed by student t-test, bars with different letters were significantly different, p<0.05.
Figure 7
Figure 7
Platelets are observed in extrasinusoidal regions of liver during torpor. Frozen sections of liver were prepared from non-hibernating, torpid, and IBA animals. The sections were incubated with rabbit anti-GpIbα and goat anti-rabbit HRP antibodies followed by the chromogenic substrate AEC. Sections were then counterstained with hematoxylin, size bars are 100μm (n=3).

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  • Cardiovascular resistance to thrombosis in 13-lined ground squirrels.
    Bonis A, Anderson L, Talhouarne G, Schueller E, Unke J, Krus C, Stokka J, Koepke A, Lehrer B, Schuh A, Andersen JJ, Cooper S. Bonis A, et al. J Comp Physiol B. 2019 Feb;189(1):167-177. doi: 10.1007/s00360-018-1186-x. Epub 2018 Oct 13. J Comp Physiol B. 2019. PMID: 30317383 Free PMC article.

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