These data show that the return of heparinized shed blood in animal T/HS models exacerbates ALI indirectly through the return of activated platelets and not the direct effects of heparin itself. As a molecule, heparin is evolutionarily conserved, and present in invertebrate species lacking a coagulation system, suggesting other primary roles, such as host defense, rather than anticoagulation.13,14
Though produced by endothelial cells, heparins are highly negatively charged glycans that have many other ionic interactions responsible for its mechanistic diversity. In spite of this fact, heparin has been widely accepted clinically as an effective anticoagulant, but few have questioned the multiple physiological effects of this molecule. A review of the literature favors heparin as protective in T/HS models,15,16
but heparin has also been implicated as an injurious molecule,4
further adding to the controversy17
of heparin use in T/HS models. Those involved in basic science research have therefore debated the use of heparin in animal models for these reasons. Yet, animal models utilizing the return of heparinized shed blood continue to be exploited.
Although a model not used in this study, we have employed heparin for the return of shed blood in our T/HS lung injury model with mesenteric lymph diversion18
, because the return of shed blood is critical for maintaining both oncotic pressure, due to the excessive protein loss in lymph diversion, and appropriate hemoglobin levels to maintain sufficient oxygen carrying capcity.19
Therefore, a study to determine the effects of heparin in these clinically relevant animal models of T/HS mediate lung injury was warranted. Initially, we found that heparin infusion in the setting of sham shock did not provoke ALI, but in the presence of shock and the return of shed blood, heparin exacerbated lung injury implicating the heparinized shed blood. Thus, shed blood was collected in 10 times the dose of heparin (800 Units/kg) to ensure adequate thrombin inhibition, but animals still developed ALI. Other anticoagulation methods of shed blood were employed, such as calcium chelation and factor dilution, and both were protective against ALI implicating a pathological process with heparin use in shed blood storage. Those involved with blood banking have abandoned the use of heparin for whole-blood storage due to increased platelet loss and hemolysis.20
The platelet loss was most likely secondary to the formation of platelet aggregates as observed in heparinized animal models in which “white” thrombi developed extracorporeally.21
Therefore, the shed blood was examined by obtaining peripheral smears, and demonstrated that although heparin was effective in inhibiting the fluid phase of coagulation, it was a poor antithrombotic agent, unable to prevent platelet activation/aggregation. This was confirmed by platelet function assays.
At baseline, PlateletMapping™
confirmed that 80 Units/kg of heparin was sufficient to prevent thrombin-mediated activation of platelets. However, shed blood collected during T/HS resulted in GPIIb/IIIa activation in spite of thrombin inhibition. Therefore, other endogenous mediators of T/HS must be involved in the activation of platelets. In-vivo platelet activation can occur via multiple platelet agonist/receptor interactions during T/HS; collagen (GP IV), thrombin (PAR1 and PAR4), ADP (P2Y1
), thromboxane A2
(TP), and epinephrine (α2A
Furthermore, when blood is removed and stored ex-vivo during T/HS, the endothelial-derived inhibitors of platelet activation (NO, PGI2
, and CD39-ADPase) are removed, further exacerbating platelet activation. Consequently, animals pretreated with a platelet P2Y12
receptor antagonist (clopidogrel), had a protective effect in spite of T/HS and the return of heparinized shed blood. Thus, implicating activated platelets in the pathogenesis of ALI and that heparin use for the storage of shed blood promotes platelet activation ex-vivo.
However, T/HS without the return of shed blood is sufficient to provoke ALI. Therefore, platelet activation must occur in-vivo during T/HS through potentially several different receptors. Growing evidence is showing that the platelet-endothelium interaction mediated through platelet P-selectin is vital in the pathogenesis of acute lung injury.23
Consequently, when heparinized shed blood is returned, containing an abundant source of activated platelets, the pathogenesis of ALI may be accelerated.
Calcium chelation and factor dilution appeared protective compared to the T/HS group who did not receive shed blood. Since coagulation and thrombosis are both dependent on extracellular calcium, as well as cytosolic calcium for platelet intracellular signaling, calcium chelation and dilution prevented platelet activation as evidenced by the platelet function assay. However, the protective effects of the T/HS citrate and T/HS blood dilution groups compared to the T/HS no SB group, are likely due to the return of shed blood (not containing activated platelets), ultimately improving their resuscitation through increased oxygen carrying capacity and oncotic pressures. As the T/HS + 800 Units/kg heparin group did not show any statistically significant decrease from the T/HS + 80 Units/kg heparin group, the citrate and blood dilution groups had no statistically change compared to the T/SS group. It is important to note that this study is likely underpowered to detect significant changes between these groups; however, these groups provided the necessary information to implicate platelets in ALI.
To determine the effects of activated platelets in the lung, histological sections were evaluated for microthrombi and were fluorescently labeled with antibodies against platelets, fibrinogen, and neutrophils. Animals undergoing T/HS with the return of heparinized shed blood had significantly higher levels of fibrinogen and neutrophils. Upon colocalization of all three microthrombi components (fibrinogen, platelets, and neutrophils), microthrombi were only significantly found in the T/HS group who received heparinized shed blood.
Minimal microthrombi were noted in the T/HS no shed blood group, which suggest that without the return of shed blood, only minimal thrombi were generated following NS resuscitation secondary to platelet and factor dilution. This raises the probability of other mechanisms for ALI in models not utilizing the return of shed blood, such as pulmonary ischemia secondary to anemia and decreased oxygen carrying capacity rather than microthrombi. However, microthrombi have been observed clinically in the setting of post-injury ALI/ARDS.10,24
This implies microthrombi formation may be essential for the development of lung injury, and suggests a critical role for platelets in post-traumatic organ failure. Therefore, a model utilizing the return of shed blood, which produces an outcome observed clinically (pulmonary microthrombi), is consistent and translates to known clinical scenarios.
To further address this question of mechanism, heparinized shed blood was transfused into a T/SS animal (exchange transfusion group), and no lung injury was detected. Therefore, activated platelets in shed blood were not sufficient for ALI, suggesting a two-event mechanism. During the initial insult, the pulmonary endothelium is activated allowing for neutrophil adhesion and emigration. A second insult is then required to activate the neutrophils, which triggers a cytokine release, resulting in endothelial leak and lung injury. Our MPO data show that an initial insult of T/HS is sufficient for pulmonary neutrophil sequestration, but not necessarily lung injury. It is the transfusion of heparinized shed blood which acts as the second-hit through activated platelets, triggering a cytokine release, and generating pulmonary capillary leak.
In antibody-mediated transfusion-related acute lung injury (TRALI), the Matthay group has described a two-event model in which neutrophil depletion, platelet depletion, or treatment with aspirin protected mice from TRALI.25
Although the mechanism remains unclear, this group suggested pulmonary platelet sequestration was dependent on neutrophils, but platelet depletion did not affect pulmonary neutrophil accumulation. Our study has shown similar results with platelet inhibition utilizing a P2Y12
receptor antagonist. However, in spite of a mild decrease in pulmonary neutrophil accumulation in the T/HS clopidogrel + hep group, these results were not significantly different.
Although pulmonary ischemia has some mechanistic merit, it does not fully describe the effects seen in returned shed blood models. However, if shock is prolonged in non-shed blood models, microthrombi may still develop.11
Thus, platelet activation may occur in-vivo, but at a slower rate than in ex-vivo storage. It is the transfusion of heparinized shed blood, supplying activated platelets, which accelerates the pathogenesis of ALI. Therefore, T/HS is necessary to prime the pulmonary endothelium, and activated platelets act as the “second-hit” enhancing pulmonary PMN sequestration and the formation of pulmonary microthrombi.
Since mesenteric lymph diversion attenuates ALI following T/HS, our laboratory continues to evaluate gut-derived factors in mesenteric lymph for the crucial mediators in the pathogenesis of ALI. Both protein and lipid substrates have been implicated, but have not been fully determined.18,26
Recently, heparin use, which is known to activate lipoprotein lipase,27
was found to increase lipase levels in mesenteric lymph following T/HS, which resulted in HUVEC toxicity.4
Although in-vitro data are suggestive, the clinical relevance remains to be determined. Similarly, as a limitation to all animal research, results of this study may not be extrapolated to the clinical sphere. However, clinical evidence currently supports no untoward effects of heparin use. Cardiac patients undergoing cardiopulmonary bypass procedures (a form of controlled trauma and shock), in spite of high dose heparin use, have a very low incidence of lung injury(0.5–1.0%).28,29
In contrast, the incidence of lung injury following trauma has been reported as high as 25%.30
In our recent study evaluating post-traumatic ICU patients with an injury severity score > 15, the incidence of lung dysfunction was greater than 70%.31
This phenomenon is unexpected since patients undergoing cardiopulmonary bypass (CPB) are older and have higher comorbidities, which are significant risk factors for multiple organ failure.32,33
Interestingly, CPB results in a 40–60% reduction in circulating platelets due to contact with the ex-vivo circulatatory system and decreases the sensitivity of the remaining platelets to aggregating agents.34
These data further implicate the significance of platelets in ALI. In contrast, a recent retrospective study from our institution has associated thrombocytopenia with MOF and worse outcomes.35
However, the relationship between lung injury and pulmonary platelet sequestration/aggregation in trauma patients, accounting for the thrombocytopenia, has been observed in animal models,36
as well as clinically.37
With the use of any drug in in-vivo models, unintentional effects must be assumed, and controlling for these effects is difficult. Although heparin may possess confounding properties, its use in T/HS models can generate a reproducible and accelerated model of ALI with similar pathological outcomes observed clinically. Investigators must have some understanding of these potential effects in order to properly interpret the results of studies utilizing heparin, but heparin use should not be automatically discounted. The return of heparinized shed blood in T/HS models most accurately simulates clinical resuscitative scenarios, and offers other benefits including maintenance of oncotic pressure and prevention of critical anemia. Furthermore, the return of heparinized shed blood likely enhances the clinical pathogenesis of ALI. It is important to note that heparin use does not directly activate platelets, but it is the failure of heparin to prevent platelet activation ex-vivo, which augments post-injury ALI. In spite of this, there are possible unintended consequences of heparin use, and investigators must determine if the use of heparin will confound outcomes in their own models.