Hyperthermia inhibits platelet hemostatic functions and selectively regulates the release of alpha-granule proteins

Abstract

Background: Hyperthermia is one of the main disturbances of homeostasis occurring during sepsis or hypermetabolic states such as cancer. Platelets are important mediators of the inflammation that accompanies these processes, but very little is known about the changes in platelet function that occur at different temperatures.

Objectives: To explore the effect of higher temperatures on platelet physiology.

Methods: Platelet responses including adhesion, spreading (fluorescence microscopy), α(IIb)β(3) activation (flow cytometry), aggregation (turbidimetry), ATP release (luminescence), thromboxane A(2) generation, alpha-granule protein secretion (ELISA) and protein phosphorylation from different signaling pathways (immunoblotting) were studied.

Results: Preincubation of platelets at temperatures higher than 37 °C (38.5-42 °C) inhibited thrombin-induced hemostasis, including platelet adhesion, aggregation, ATP release and thromboxane A(2) generation. The expression of P-selectin and CD63, as well as vascular endothelial growth factor (VEGF) release, was completely inhibited by hyperthermia, whereas von Willebrand factor (VWF) and endostatin levels remained substantially increased at high temperatures. This suggested that release of proteins from platelet granules is modulated not only by classical platelet agonists but also by microenvironmental factors. The observed gradation of response involved not only antiangiogenesis regulators, but also other cargo proteins. Some signaling pathways were more stable than others. While ERK1/2 and AKT phosphorylation were resistant to changes in temperature, Src, Syk, p38 phosphorylation and IkappaB degradation were decreased in a temperature-dependent fashion.

Conclusions: Higher temperatures, such as those observed with fever or tissue invasion, inhibit the hemostatic functions of platelets and selectively regulate the release of alpha-granule proteins.

© 2011 International Society on Thrombosis and Haemostasis.

Figures

Fig. 1

Heat exposure negatively regulated thrombin-induced αIIbβ3 activation. WPs were incubated at 37°, 38.5°, 40°, 41° or 42°C for 10 minutes and then stimulated with thrombin (0.05 U/ml) for 5 minutes. Analysis of PAC-1 and fibrinogen binding were performed by flow cytometry (n=6). *P < 0.05 vs. unstimulated control; #P < 0.05 vs. thrombin at 37°C.

Fig. 2

High temperature inhibits platelet spreading. WPs were incubated at 37°, 40° or 42°C for 10 minutes and then plated on BSA- or fibrinogen-coated slides and stained with TRITC-Phalloidin. Platelet spreading was visualized under fluorescent microscopy (original magnification 600×). Platelet perimeter and number per field were measured using Image-Pro software (n=4). #P < 0.01 vs. 37°C.

Fig. 3

Inhibition of platelet aggregation, ATP release and TXB2 formation at high temperatures. (A) WPs or (B) PRP were incubated at 37°, 40° or 42°C for 10 minutes and then aggregation was induced by the indicated agonists. Graphics are representative of 4 independent experiments. (C) PRP was incubated at 37°, 40° or 42°C for 10 minutes and then stimulated with ADP. Aggregation and ATP release were determined simultaneously in a Lumi-aggregometer (n=4). After 5 minutes stimulation, supernatants were collected to measure TXB2 levels by ELISA. *P < 0.05 vs. unstimulated control. #P < 0.05 vs. thrombin at 37°C (n = 3).

Fig. 4

Effect of hyperthermia on platelet alpha-granule secretion induced by thrombin. WPs were preincubated at 37° (dark line), 40° (grey line) or 42°C (dashed line) for 10 minutes and then stimulated with thrombin (0.05 U/ml) for 5 minutes (A) or at the indicated time (B). P-selectin was detected by flow cytometry (n=10). vWF, VEGF, and endostatin levels in the supernatants were quantified by ELISA (n=5). Since the basal levels of each protein release in unstimulated controls were not affected by the temperature results are expressed as fold increase vs. unstimulated controls at the same temperature. *P < 0.05 vs. unstimulated control; #P < 0.05 vs. thrombin at 37°C.

Fig. 5

Effect of temperature on platelet signaling. WPs were preincubated at 37°, 40° or 42°C for 10 minutes. (A) WPs were stimulated with the indicated concentration of thrombin for 5 minutes and lysates were immunoblotted with anti-pp38, pERK1/2, pHSP27 or pAKT antibody. (B) WPs were stimulated with thrombin for 2 minutes and lysates were immunoblotted with anti-pSyk and pSrc antibody. Each membrane was reprobed with anti-actin antibody to calculate the relative IOD using Gel-Pro software (n=3–5). *P

Fig. 6

Inhibition of p38 and NF-kappaB pathways mimics the effect of heat exposure on platelet alpha-granule secretion. WPs were preincubated with SB203580 (25 μM), RO1069920 (6 μM) or the combination of both for 10 minutes and then stimulated with thrombin (0.05 U/ml) for 5 minutes. P-selectin was detected by flow cytometry (n=5). vWF, VEGF, and endostatin levels in the supernatants were quantified by ELISA (n=4). Results are expressed as fold increase vs. unstimulated controls. *P #P < 0.05 vs. thrombin alone.