Evidence of Platelet Activation at Medically Used Hypothermia and Mechanistic Data Indicating ADP as a Key Mediator and Therapeutic Target
Objective—Hypothermia is used in various clinical settings to inhibit ischemia-related organ damage. However, prothrombotic effects have been described as potential side effects. This study aimed to elucidate the mechanism of hypothermia-induced platelet activation and subsequent prothrombotic events and to develop preventative pharmacological strategies applicable during clinically used hypothermia.
Methods and Results—Platelet function was investigated ex vivo and in vivo at clinically used hypothermia (28°C/18°C). Hypothermic mice demonstrated increased expression of platelet activation marker P-selectin, platelet-leukocyte aggregate formation, and thrombocytopenia. Intravital microscopy of FeCl3-injured murine mesenteric arteries revealed increased platelet thrombus formation with hypothermia. Ex vivo flow chamber experiments indicated increased platelet-fibrinogen adhesion under hypothermia. We show that hypothermia results in reduced ADP hydrolysis via reduction of CD39 (E-NTPDase1) activity, resulting in increased levels of ADP and subsequent augmented primary and secondary platelet activation. In vivo administration of ADP receptor P2Y12 antagonists and recombinant soluble CD39 prevented hypothermia-induced thrombus formation and thrombocytopenia, respectively.
Conclusion—The platelet agonist ADP plays a key role in hypothermia-induced platelet activation. Inhibition of receptor binding or hydrolysis of ADP has the potential to protect platelets against hypothermia-induced activation. Our findings provide a rational basis for further evaluation of novel antithrombotic strategies in clinically applied hypothermia.
Cooling as a means of reducing oxygen requirements of organs was initially reported in 1950 by Bigelow et al1 This concept was first implemented in cardiac surgery in 1952, when John Lewis and his team used total-body hypothermia for the closure of an atrial septal defect.2 Since then, hypothermia has been routinely used in cardiac surgery as an adjunct to extracorporeal circulation (ECC) with the aim of protecting organs against ischemia-related damage. Temperatures applied are usually between 28°C and 32°C. Furthermore, during deep hypothermic circulatory arrest (which is used either in congenital cardiac surgery, for operations on the thoracic aorta in adults, or in neurosurgical operations for the treatment of cerebral aneurysms), temperatures lower than 20°C are used. Mild hypothermia is also used successfully in emergency and intensive care medicine to improve survival and neurological outcomes of patients after cardiac arrest.3–6
Potential prothrombotic events of medically used hypothermia are of major concern. In fact, increased activation, aggregation, and sequestration of platelets and (micro)vascular thrombus formation have all been seen in both in vitro and in vivo settings at hypothermia.4,7–17 An increased platelet response to activation at temperatures less than 37°C could be considered part of human physiology. It has been proposed that platelets act as thermosensors, being less responsive to thrombogenic stimuli at the core body temperature of the central circulation, where coronary or cerebral thrombus formation could be lethal. However, at the lower temperatures of external body surfaces, the sites most susceptible to bleeding throughout evolutionary history, platelets would be primed for activation.18,19 Under clinical conditions, hypothermia-induced platelet aggregation is associated with an added risk of cognitive dysfunction in patients undergoing cardiac surgery, probably caused by microvascular occlusion in the brain.20 Hypothermia used during ECC has also been identified as an independent predictor of late thrombocytopenia following cardiac operations.21 In addition, fatal intravascular thrombus formation in hypothermic patients has been reported.22 Therefore, hypothermia-associated thromboembolic complications potentially leading to substantial disability and mortality are a significant problem. The mechanism of hypothermia-induced thrombosis is not understood, and therefore no therapeutic approach to preventing or treating this potentially deleterious effect has been established.
Our study systematically evaluates mechanistic changes in platelet function under hypothermia using ex vivo and in vivo platelet function assays. (1) Platelet activation as measured by P-selectin expression on the platelet surface, binding of fibrinogen to the platelet glycoprotein (GP) IIb/IIIa receptor, and expression of the platelet GPIb receptor.23,24 (2) Binding of platelets to von Willebrand factor (vWF), which mediates tethering of platelets to extracellular matrix on vascular injury,25 and adhesion of platelets to fibrinogen, which typically mediates binding of platelets to each other on activation and aggregate formation.23,24 (3) Platelet aggregation and thrombus formation as evaluated by intravital microscopy26, platelet-leukocyte aggregate formation as determined with flow cytometry,27 and hypothermia-associated changes in platelet and leukocyte counts. Furthermore, temperature-dependent changes of enzymatic activity of the ADP-metabolizing enzyme ecto-nucleoside triphosphate diphosphohydrolase 1 (E-NTPDase1/CD39)28,29 and temperature-dependent degradation of the platelet agonist ADP were investigated. Using these assays, we provide evidence for platelet activation at hypothermia, describe a novel pathomechanism, and, most importantly, report therapeutic strategies with the potential of preventing prothrombotic complications during medically applied hypothermia.
Materials and Methods
A full description of all methods can be found in the Supplemental Data, available online at http://atvb.ahajournals.org.
All in vivo experiments were performed with anesthetized wild-type C57BL/6 mice. The rectal temperature was monitored using a digital thermometer and maintained at either 37°C or 28°C using a heat pad.
Blood Sampling in Human Subjects
Blood was collected by venipuncture from nonmedicated healthy subjects into tubes with preloaded heparin (final concentration, 3 IU/mL), citric acid (final concentration, 10.6 mmol/L), or EDTA.
Analysis of Platelet Activation, Platelet-Leukocyte Aggregate Formation, and Blood Count at Hypothermia
Blood was sampled from mice with body temperatures of 37°C or 28°C, and platelet activation, platelet-leukocyte aggregates, and blood counts were evaluated.
Binding of Oregon Green–labeled fibrinogen to human platelets at 37°C or 18°C was analyzed by flow cytometry. Citrated human whole blood was incubated at 37°C, 28°C, or 18°C for 30 minutes. Expression of platelet surface receptors GPIbα was analyzed by flow cytometry as previously described.10,30
Ex Vivo Flow Studies
Flow studies were performed as previously described31 using microcapillary tubes coated with vWF or fibrinogen. Heparinized human blood was treated as either control or with platelet antagonists and perfused through the tubes at 37°C, 28°C, or 18°C on either vWF for 2 minutes (shear rate: 1800 seconds−1) or on fibrinogen for 5 minutes (shear rate: 150 seconds−1). Platelet-surface interactions were visualized in real time using differential interference contrast microscopy.
In Vivo Thrombosis Model
Intravital microscopy of mesenteric arteries from mice with body temperatures of either 37°C or 28°C was performed as previously described.26 All mice received heparin (0.25 IU/g of body weight) to simulate conditions of cardiac surgery, in which all patients undergoing routine ECC procedures are anticoagulated with heparin. Furthermore, antiplatelet agents (2-MeSAMP [3.2 μg/g of body weight] bolus injection only or in combination with bolus injection of MRS2179 [4.2 μg/g of body weight] or cangrelor solution [40 ng/μL] administered as initial bolus of 50 μL followed by continuous infusion of 4 ng/g of body weight per minute) or saline (control) were administered. After vessel injury with ferric chloride, thrombus formation of rhodamine-labeled platelets was monitored for 40 minutes or until full occlusion. The following parameters were examined: thrombus surface in relation to vessel surface 5 minutes after injury and embolization rate of thrombi over a period of 1 minute.
Production, Characterization, and Administration of Soluble CD39
Soluble (sol) CD39 was cloned into a mammalian expression vector and transfected into Hek293F cells. Transfected cells were cultured for 7 to 9 days before being purified via a polyhistidine tag by fast protein liquid chromatography. Enzyme activity was measured at 3 different temperatures (18°C, 28°C, and 37°C) using a bioluminometric assay as previously described.32 For in vivo studies with solCD39, mice with body temperatures of either 37°C or 28°C received either PBS as a control substance (vehicle) or solCD39 via the left jugular vein, and blood cell count was measured after 30 minutes.
ADP Degradation at Hypothermia and Effect on Platelet Activation
To investigate temperature-dependent differences of ADP degradation, ADP (2 μmol/L) was added to cell-free plasma samples at 37°C or 18°C. After 10 minutes, ADP concentrations were conserved by EDTA addition. Platelet-rich plasma was resuspended in these samples, and platelet P-selectin expression was analyzed by flow cytometry. ADP levels in cell-free plasma samples were determined using a previously described bioluminometric assay.32
Nucleotide Release From Blood Cells and Platelet Activation in Hypothermia
EDTA-anticoagulated whole blood was incubated at 3 different temperatures. After 60 minutes, P-selectin expression on single platelets was measured, and nucleotide plasma levels above nucleotide background concentration (directly after blood taking) were measured as previously described.32 In short, ADP was converted to ATP by the pyruvate kinase reaction, and total ATP concentration was determined in a bioluminescence assay on a microplate luminometer (Microlumat.Plus, Berthold).
Data are depicted as means and SEM unless otherwise indicated. Differences between 2 data sets were evaluated using unpaired 2-tailed t tests for unmatched data and using paired t tests for matched data. If not otherwise indicated, differences among 3 or more data sets were evaluated by repeated-measures ANOVA with the indicated post hoc tests for matched data and using 1-way ANOVA with the indicated post hoc tests for unmatched data. A probability value of <0.05 was defined to indicate a statistically significant difference.
Prothrombotic Effects of Hypothermia on Platelet Function In Vivo
Hypothermia Induces Platelet Activation, Platelet-Leukocyte Aggregate Formation, Thrombocytopenia, and Leukocytopenia In Vivo
To evaluate the in vivo effect of hypothermia on platelet function, expression of the activation marker P-selectin on nonstimulated platelets was determined in mice with body temperatures of 28°C and 37°C using flow cytometry (see Figure 1A and 1B for representative dot plot and histograms). At 28°C, platelet P-selectin expression was significantly increased compared with 37°C (P<0.05, Figure 1C). Furthermore, in vivo platelet-leukocyte aggregate formation was significantly increased at hypothermia (P<0.05, Figure 1D). Analysis of whole blood counts revealed that at hypothermia, platelet count (P<0.05, Figure 1E) and white blood cell count (P<0.05, Figure 1F) were significantly lower compared with 37°C. No effect of hypothermia on red blood cell counts was observed (Figure 1G).
Hypothermia Increases In Vivo Platelet Thrombus Formation and Thrombus Stability
Intravital microscopy allows direct visualization and quantification of the dynamic process of platelet accumulation and thrombus formation at injured or diseased vessels.33 Five minutes after application of ferric chloride to murine mesenteric arteries, the surface area of platelet thrombi in mice that were cooled to 28°C was increased (2.2-fold, P<0.001) compared with mice with a body temperature of 37°C (Figure 2A). Disaggregation analysis of thrombi revealed that the thrombus dissolving rate was 5-fold lower at 28°C compared with 37°C (P<0.0001) indicating stronger binding of platelets and therefore decreased thrombus dissolvement at hypothermia. (Figure 2B). Supplemental Video I shows representative examples of thrombus formation and disaggregation at 37°C and 28°C.
Effect of Hypothermia on GPIbα Expression and Platelet-vWF Adhesion Under Flow
The interaction of the platelet receptor GPIbα with vWF plays a key role in promoting initial platelet tethering to the vessel wall.23 To evaluate a potential role of hypothermia on platelet-vWF interaction, we analyzed the platelet surface expression of GPIbα and platelet binding on a vWF matrix under ex vivo flow conditions. GPIbα expression was unchanged at 28°C and showed a small reduction at 18°C in comparison to 37°C (Supplemental Figure I), which is consistent with decreased expression of GPIbα via receptor shedding34 on platelet activation at hypothermia. The evaluation of platelet tethering on a vWF matrix under flow conditions did not reveal a significant difference between 18°C and 37°C (Supplemental Video II).
Hypothermia Increases Fibrinogen Binding to Platelets and Platelet Adhesion on Fibrinogen Under Flow Conditions
Binding of fibrinogen to platelets is an important mechanism mediating platelet aggregate formation and thrombus growth. Fibrinogen binding to platelets was 1.6-fold higher (P<0.05) at hypothermia compared with normothermia (Figure 3A). Furthermore, on a fibrinogen matrix under ex vivo flow conditions, the mean number of adherent platelets was increased 2.2-fold (P<0.001) at both 28°C and 18°C (Figure 3B and 3C) relative to experiments performed at 37°C.
Potential Mechanisms for Prothrombotic Effects of Hypothermia
Hypothermia Inhibits Activity of Soluble CD39, Thereby Increasing ADP Levels and Subsequently Causing Platelet Activation
It has previously been postulated that the platelet agonist ADP may be involved in hypothermia-induced platelet activation and aggregation.4 However, this has not been shown in vivo and the underlying mechanism of this phenomenon has not yet been elucidated. The E-NTPDase CD39, which hydrolyzes extracellular nucleotides ATP and ADP to AMP, is expressed by the endothelium and thereby presents a physiologically important platelet-inhibitory surface.35 For our experiments, we generated CD39 in soluble form (solCD39), for which enzymatic properties have been described previously in detail.36 We tested the specific activity of solCD39 at different temperatures in vitro using a bioluminometric assay.32 We found a significant decrease of solCD39 activity at 28°C and 18°C compared with 37°C (Figure 4A).
ADP Degradation in Plasma Is Decreased At Hypothermia
It has previously been reported that ADP is metabolized in whole blood and plasma.28,37 This is certainly of central importance for the regulation of platelet function. To investigate whether the described effect of hypothermia on ADP metabolism is also present in plasma, we examined ADP degradation in plasma at 37°C compared with 18°C. In these experiments, ADP (2 μmol/L) was added to plasma samples, which were then exposed to a temperature of 37°C or 18°C for 10 minutes. Measurement of ADP concentrations at the end of this period demonstrated that samples that had been exposed to a temperature of 18°C contained a significantly higher ADP concentration (mean, 1.75±0.35 μmol/L) compared with samples that had been exposed to a temperature of 37°C (mean, 0.94±0.22 μmol/L; P<0.01; Figure 4B). The concentration of 2 μmol/L ADP was chosen according to a study by Valles et al, which, after collagen stimulation, found ADP concentrations in releasates in the range of 1 μmol/L for platelet-rich plasma and in the range of 7 μmol/L for platelet-rich plasma plus red blood cells.38 Furthermore, we used P-selectin expression on the platelet membrane as a sensitive marker of platelet activation. The increased amount of ADP in samples that had been exposed to a temperature of 18°C was associated with an increase (P<0.001) in P-selectin expression on platelets that had been resuspended in the respective plasma samples (Figure 4C).
CD39 is found on endothelial cells, leukocytes, and microparticles.39 Previous reports have demonstrated that exogenous ADP is hydrolyzed in human cell-free plasma because of ADPase activity.37 Altogether, the in vitro conversion of ADP in plasma samples in our experiments can therefore be related to the action of CD39 on microparticles, as well as to soluble plasma phosphatases. Nevertheless, in the in vivo setting of blood circulation, CD39 is proposed to be the major ADP hydrolyzing enzyme on the basis of its broad expression on endothelial cells and leukocytes.35,39
Hypothermia Does Not Induce Nucleotide Leakage
To investigate whether nucleotides (ATP and ADP) spontaneously leak from whole blood cells at hypothermia, whole blood was incubated for 60 minutes at 3 different temperatures (37°C, 28°C, 18°C). Potential differences of nucleotide hydrolysis rates at different temperatures were negated by EDTA addition. Samples were treated as controls or with a combination of P2Y12 and P2Y1 receptor blockers to avoid active ADP release resulting from platelet activation. To analyze a potential direct effect of hypothermia on platelet degranulation, expression of the platelet activation marker P-selectin and ADP and ATP plasma levels were investigated.
A decrease in temperature resulted in an increase of P-selectin expression and nucleotide plasma concentrations in control samples (Figure 4D and 4E). In the presence of ADP receptor blockers, P-selectin expression and nucleotide levels were not significantly different among the 3 temperatures (Figure 4F and 4G). To closely explore potential hypothermia-induced nucleotide leak by platelets alone, the same assay (including P2Y receptor blockade) was repeated with washed platelets alone. Under these conditions, platelets also did not secrete ADP to a greater extent at hypothermia (data not shown).
Therapeutic Implications for the Prevention of Hypothermia-Induced Prothrombotic Effects
Blockade of P2Y12 and P2Y1 Decreases Hypothermia-Induced Platelet-Fibrinogen Binding Under Ex Vivo Flow Conditions
The pathomechanistic role of ADP that has been described provides the unique opportunity to develop novel therapeutic approaches with the aim of preventing hypothermia-induced platelet activation. Therefore, we systematically investigated whether inhibition of platelet ADP receptors could potentially be used therapeutically. First, we used the established flow chamber model. Heparinized whole blood samples, which had been treated with the P2Y12 antagonist 2-MeSAMP in combination with the P2Y1 antagonist MRS2179 (P2Y block), were perfused over fibrinogen at 37°C, 28°C, or 18°C at a shear rate of 150 seconds−1. In this model, at 37°C, no inhibitory effect of ADP receptor blockade was observed. However, at both 28°C and 18°C, the combination of P2Y12 and P2Y1 inhibition significantly decreased platelet adhesion (28°C, Figure 5A, P<0.001; 18°C, Figure 5B, P<0.001), returning the levels of platelet adhesion to values close to those observed at 37°C. This finding indicates that in our ex vivo flow model, ADP-induced platelet activation is of much greater importance at hypothermia than at normothermia. This further emphasizes the essential role of ADP as key platelet agonist in hypothermia.
In Vivo Blockade of ADP Receptors Decreases Hypothermia-Induced Platelet Thrombus Formation in Mice
ADP receptor blockade has proven to be clinically highly beneficial in patients with coronary artery disease, and major drug developments are providing a plethora of various ADP receptor blockers, including the short-acting intravenously applicable cangrelor.40 To provide proof of concept for a potential therapeutic approach in preventing hypothermia-induced platelet activation, we evaluated ADP receptor blockade in intravital microscopy. Again, the P2Y12 antagonist 2-MeSAMP alone and in combination with the P2Y1 antagonist MRS2179 were used as experimentally available ADP receptor blockers. In addition, the P2Y12 antagonist cangrelor, which has been developed as a promising therapeutic agent,40,41 was administered intravenously to mice before mesenteric artery injury. At a body temperature of 28°C, 2-MeSAMP and cangrelor significantly decreased (P<0.01) thrombus surface in mesenteric arteries after ferric chloride injury (Figure 6A). The disaggregation rate of platelet thrombi at 28°C was also significantly (P<0.01) increased after treatment with ADP receptor blockers including cangrelor (Figure 6B). The finding that cangrelor displayed a favorable potency in comparison with the experimental P2Y1/ P2Y12 blockade supports the therapeutic potential of this short-acting ADP receptor blocker.
SolCD39 Inhibits Hypothermia-Induced Platelet Aggregation and Thrombocytopenia
We aimed to evaluate the potential effect of solCD39 administration on hypothermia-induced platelet dysfunction. First, we tested whether solCD39 would inhibit platelet aggregation at hypothermic temperatures in vitro. Our results indicate that solCD39 indeed inhibits ADP-induced platelet aggregation at hypothermia, but clearly less efficiently than at 37°C (Supplemental Figure II). This finding confirms our statement that hypothermia has an inhibitory effect on CD39 function. However, a significant antiaggregatory effect of solCD39 is still present at 28°C and 18°C. To investigate the effect of solCD39 in vivo, we administered it to mice at body temperatures of 37°C and 28°C. Our results indicate that treatment with solCD39 abolishes hypothermia-induced platelet loss (see Figure 6C). These findings support the suitability of solCD39 as a potential agent for the prevention of platelet activation and thrombosis at hypothermia.
This systematic ex vivo and in vivo study on platelet function reports on platelet activation and prothrombotic effects and provides mechanistic data indicating ADP as a key mediator and important therapeutic target in medically used hypothermia.
Primary platelet activation by potent agonists, such as thrombin, ADP, or collagen, causes the release of the platelet agonist ADP from platelet dense granules.42 As a secondary activating effect, ADP recruits additional circulating platelets and amplifies activation of platelets within the platelet clot (ADP augmentation pathway).43 We postulate a potential pathomechanism indicating primary and secondary effects of ADP in hypothermia-induced platelet activation. At hypothermia, ADP hydrolysis and especially the activity of the main ADP-metabolizing enzyme, CD39 (NTPDase1),28,35 are significantly decreased, resulting in a lower rate of hydrolysis and thus higher levels of the platelet agonist ADP. This can promote primary platelet activation as reflected by platelet adhesion on fibrinogen under flow and by increased platelet P-selectin expression. Once platelets have become activated, more ADP is actively released into plasma via the ADP augmentation pathway. The increase in plasma ADP concentration, which will again be amplified by decreased ADP hydrolysis at hypothermia, will in turn cause further activation of platelets, resulting in the systemic activation of platelets that is observed at hypothermia. This hypothesis is supported by our finding that platelet ADP receptor P2Y12 blockade decreases hypothermia-induced platelet adhesion and aggregation under ex vivo and in vivo conditions. Additional support for the important role of ADP in platelet function at hypothermia is provided by our finding that administration of solCD39 inhibits hypothermia induced decreases of platelet and leukocyte counts. Our findings are also supported by an in vitro study by Xavier et al4 demonstrating that hypothermia-induced platelet aggregation is inhibited by ADP-receptor blockade.
With the identification of ADP as the major mediator of hypothermia-associated platelet activation, other parameters of platelet physiology may be influenced by hypothermia as well. Although it is possible that changes in membrane fluidity, receptor clustering, or microhemodynamics may occur under hypothermia, our findings that ADP hydrolysis or P2Y12 blockade can prevent hypothermia-induced platelet activation does not suggest a sufficient role for these mechanisms.44 Also notably, the inhibition of the thromboxane pathway by aspirin, which inhibits thromboxane A2 synthesis, is reported not to inhibit hypothermia-induced platelet aggregation.9
Shear stress during ECC as used in cardiac surgery results in substantial ADP release from platelets45 and erythrocytes46 in amounts that are sufficient to induce platelet aggregation.47 ADP-mediated platelet activation may therefore be of particular importance during ECC, which is often used together with hypothermia. In addition, the artificial ECC surface is a strong inducer of platelet activation.27 Elevated ADP levels at hypothermia may significantly enhance the prothrombotic side effects of ECC. Therefore, the approach of specifically inhibiting ADP-induced platelet activation via inhibition of P2Y12 at hypothermia may be of particular benefit during ECC.
The platelet ADP receptor P2Y12 is an important target for antiplatelet drugs, particularly in the setting of stent implantation in coronary arteries.35 Using a whole set of ex vivo and in vivo experiments, we have demonstrated that hypothermia-induced prothrombotic effects are reversed by pharmacological P2Y12 inhibition. The direct-acting and reversible P2Y12 antagonist cangrelor, which can be administered intravenously and which has a half-life of less than 5 minutes, has been developed for clinical application. Cangrelor's short half-life offers a major advantage in comparison to other antiplatelet agents.40,41 With the idea of exploiting this advantage, cangrelor is currently under clinical trial (BRIDGE trial; http://clinicaltrials.gov ID: NCT00767507) to enable safe bridging from oral P2Y12 blockade to coronary bypass graft surgery. Based on our finding that cangrelor inhibits hypothermia-induced platelet activation, this intravenously applicable P2Y12 blocker with a short half-life appears to be an ideal candidate for platelet protection during medically induced hypothermia. Particularly in cardiac surgical procedures using hypothermia, the concept of administering a short-acting P2Y12 antagonist only during the phase of hypothermia is very attractive, providing protective antithrombotic effects during hypothermia (as well as during ECC and during surgical procedures with increased risk of thrombosis) but ensuring fully functional platelets after rewarming at the end of the operation when a fully functional hemostasis is needed to prevent postoperative bleeding complications.
In animal models, administration of solCD39 has been shown to minimize platelet deposition and leukocyte recruitment at sites of vascular endothelial injury and to reduce cerebral infarct size.36,48,49 Our experiments demonstrate that solCD39 can prevent hypothermia-induced reduction of platelet and leukocyte counts. Therefore, administration of solCD39 may represent an alternate pharmacological approach to inhibit hypothermia-associated platelet dysfunction. Hypothermia-induced thrombocytopenia and leukocytopenia can be explained by the fact that hypothermia induces a prothrombotic state characterized by in vivo platelet/platelet and platelet/leukocyte aggregation and cell sequestration, as well as consecutive microinfarctions in liver and pancreas.50
Besides several literature reports, which indicate deleterious prothrombotic effects of hypothermia.30,51 it has also been reported that under clinical conditions, hypothermia can induce coagulopathy and bleeding tendency.52–55 In addition to functional impairment of plasmatic coagulation enzymes at hypothermia, the hypothermia-associated decrease in platelet counts, as observed in our experiments and in the clinical setting, may contribute to an increased bleeding tendency.55–57 Nevertheless, our results indicate that the main effect of hypothermia on platelets is the induction of prothrombotic events, namely typical signs of platelet activation (ie, GPIIb/IIIa activation and P-selectin expression) and augmented formation of platelet thrombi with increased stability at sites of endothelial injury.
Although our in vitro data have been obtained using human platelets, our in vivo data were obtained in mice. Therefore, there is a certain general caveat that the latter data may not be fully transferable to the human situation. Nevertheless, our findings warrant further testing of our hypothesis and potential therapeutic approaches in patients during perioperatively applied hypothermia.
In conclusion, our ex vivo and in vivo findings indicate that medically applied hypothermia induces platelet activation and thrombus formation/stabilization. The platelet agonist ADP plays a central role in hypothermia-induced platelet activation. Decreased NTPDase1 (CD39) activity and thus decreased ADP hydrolysis contribute to platelet activation at hypothermia. Inhibition of receptor binding or hydrolysis of ADP has the potential to protect platelets against hypothermia-induced activation. Our findings provide a rational basis for further evaluation of this novel antithrombotic approach in clinically applied hypothermia.
Sources of Funding
Dr Straub was supported by a grant from the German Research Foundation (STR 687/1-1); Dr Peter was supported by the Australian Research Council; and Drs Bassler, Jackson, Hickey, and Peter were supported by the National Health and Medical Research Council of Australia.
- Received February 9, 2010.
- Accepted April 5, 2011.
- © 2011 American Heart Association, Inc.
- Foltan M,
- Philipp A,
- Birnbaum D
- Kim F,
- Olsufka M,
- Longstreth WT Jr.,
- Maynard C,
- Carlbom D,
- Deem S,
- Kudenchuk P,
- Copass MK,
- Cobb LA
- Lindenblatt N,
- Menger MD,
- Klar E,
- Vollmar B
- Straub A,
- Schiebold D,
- Wendel HP,
- Hamilton C,
- Wagner T,
- Schmid E,
- Dietz K,
- Ziemer G
- Harker LA,
- Malpass TW,
- Branson HE,
- Hessel EA II.,
- Slichter SJ
- Savides EP,
- Hoffbrand BI
- Hall MW,
- Hopkins RO,
- Long JW,
- Mohammad SF,
- Solen KA
- Jackson SP
- Sachs UJ,
- Nieswandt B
- Goschnick MW,
- Lau LM,
- Wee JL,
- Liu YS,
- Hogarth PM,
- Robb LM,
- Hickey MJ,
- Wright MD,
- Jackson DE
- Massberg S,
- Brand K,
- Gruner S,
- Page S,
- Muller E,
- Muller I,
- Bergmeier W,
- Richter T,
- Lorenz M,
- Konrad I,
- Nieswandt B,
- Gawaz M
- Coade SB,
- Pearson JD
- Valles J,
- Santos MT,
- Aznar J,
- Marcus AJ,
- Martinez-Sales V,
- Portoles M,
- Broekman MJ,
- Safier LB
- Meadows TA,
- Bhatt DL
- Li Z,
- Delaney MK,
- O'Brien KA,
- Du X
- Reimers RC,
- Sutera SP,
- Joist JH
- Belayev L,
- Khoutorova L,
- Deisher TA,
- Belayev A,
- Busto R,
- Zhang Y,
- Zhao W,
- Ginsberg MD
- Alty JE,
- Ford HL
- Schneider DJ,
- Taatjes DJ,
- Sobel BE
- Mossad EB,
- Machado S,
- Apostolakis J