Introduction
Reperfusion following the return of spontaneous circulation (ROSC) after complete whole-body ischemia is an unnatural pathophysiological state created by successful cardiopulmonary resuscitation (CPR). Systemic ischemia/reperfusion response induces generalised tissue damage with a release of reactive oxygen species and endothelial-leukocyte interaction, resulting in a systemic inflammatory response [
1,
2], endothelial activation and injury [
3‐
5], and coagulation abnormalities [
6‐
8]. This so-called post-cardiac arrest syndrome shares many features with severe sepsis and may complicate the clinical course of resuscitated patients at the ICU [
1,
9].
Microparticles (MPs) are small vesicles, which typically range in size from 0.1 to 1.5 μm [
9], shed from the plasma membrane into the extracellular space by most eukaryotic cells undergoing activation or apoptosis. They result from translocation of phosphatidylserine from the inner to the outer leaflet of the cell membrane where they express antigens characteristic of their cell of origin [
10]. MPs are considered to act as diffusible messengers [
11], to transport bioactive agents, and to initiate and mediate coagulation [
12], inflammation and cell-cell interactions [
13].
Monocyte-derived microparticles (MMPs) contain organized membrane receptors including ß2-integrins, like Mac-1 (CD11b/CD18). Therefore, they are capable of binding endothelial cells [
11] and acting as competent inflammatory agonists by stimulation of cytokine release and up-regulation of endothelial adhesion molecules [
14]. Additionally, MMPs play a crucial role in the initiation of endothelial dysfunction, endothelial thrombogenicity and apoptosis [
14‐
16] and are capable of exposing a highly coagulant tissue factor [
17]. Accordingly, elevated numbers of MMPs have so far been reported in patients with multiorgan failure, who developed severe disseminated intravascular coagulation [
17,
18], and in acute coronary syndromes, including acute myocardial infarction [
19,
20], underlining their inflammatory and procoagulant potential.
The second group, the platelet-derived microparticles (PMPs) are released by activated platelets [
21] and are able to activate platelets and endothelial cells [
22], monocyte adhesion [
23] and the release of inflammatory cytokines [
24] and induce endothelial apoptosis [
25]. Consequently, elevated numbers of PMPs have been found in patients suffering from diseases associated with an increased risk of thromboembolic processes and vascular damage, including acute coronary syndromes [
26], ischemic cerebrovascular disease [
27] and peripheral arterial disease [
28].
Endothelial-derived microparticles (EMPs) have been shown to be elevated after cardiopulmonary resuscitation [
5] and in various states of disturbed endothelial function. EMPs have been shown to interact with monocytes or platelets to form circulating conjugates [
29,
30]. EMP-monocyte conjugates have been shown to be sensitive markers of disease activity in many disorders, including inflammatory and procoagulant conditions such as severe sepsis [
31], multiple sclerosis [
32], systemic lupus erythematosus, antiphospholipid syndrome [
33] and venous thromboembolism [
34]. An enumeration of EMP-platelet conjugates has been considered as a marker of activation of coagulation and endothelial cells as well as endothelial damage [
30]. High numbers of these conjugates have been detected in the peripheral blood of patients with stable coronary disease [
30].
Intention of the study
In the present study, we aimed to investigate the effect of whole-body ischemia/reperfusion after successful CPR on the occurrence of different MPs and their conjugates in peripheral blood of resuscitated patients and their influence on patients' survival. Additionally the effect of MPs of resuscitated patients on endothelial cells was evaluated. As these MPs indicate and maintain inflammatory, coagulation and endothelial injuring processes, they are possible players in the field of post-cardiac arrest syndrome and may be indicators to predict outcome or possible targets of future therapeutic interventions.
Discussion
Our study demonstrates that different subtypes of (annexin V+) MPs and their conjugates are elevated and may influence the clinical course of patients after successful CPR. The main findings were a marked elevation of MMPs and EMP-monocyte conjugates immediately and in the following 24 hours after ROSC compared with control patients hospitalized for cardiac causes and healthy subjects. Additionally, we found increasing levels of procoagulant PMPs in the 24 hour follow up after CPR and a significant increase of EMP-platelet conjugates early after ROSC compared with both control groups. These results suggest an early onset of inflammation, an ongoing process of endothelial activation and a procoagulatory state and thereby, an involvement of (annexin V+) MPs in the development of the post-cardiac arrest syndrome. Additionally, we found MMP levels of 1.0 MMPs/μL or more on the second day after ROSC to be a prognostic value to predict outcome at 20 days after cardiac arrest.
As MPs are known to be elevated in CAD patients [
20,
30], we compared resuscitated subjects with patients with stable cardiac disease, mostly presenting CAD and undergoing coronary catherization in a similar proportion, to exclude possible effects caused by CAD or coronary intervention in the resuscitation group. Additionally, we included a smaller population of healthy volunteers.
The most impressive finding of the study is the strong elevation of MMPs and EMP-monocyte conjugates after CPR. The considerable increase immediately after ROSC was followed by persistently high levels in the first 24 hours after ROSC. Formation of leukocyte-derived MPs in general is enhanced by inflammatory stimuli and MMPs are known to be important
in vivo markers of neutrophil activation [
43]. They are capable of expression of selectins and integrins, which makes them candidates for playing a crucial role in inflammation and cell signalling [
44], suggesting a distinct inflammatory process occurring in patients after CPR. Additionally, MMPs are competent inflammatory mediators to induce endothelial activation and cytokine production [
14], thus potentially contributing to endothelial activation and dysregulation [
5,
15,
45]. On the other hand, EMPs, shed by the activated endothelium, have been shown to be elevated after CPR [
5] and are known to bind to monocytes in a time- and concentration-dependant manner [
29] to form EMP-monocyte conjugates. Once bound to monocytes, they activate them to enhanced expression of integrins and migration through the endothelial cell layer [
29,
32]. Our results are in line with various reports of an inflammatory in patients after CPR [
1‐
4]. Accordingly, IL-6 levels were also significantly elevated in resuscitated patients at both time points, whereas the preserved elevation of MMPs and EMP-monocyte conjugates in our study provides evidence for an ongoing inflammatory state in the first 24 hours of reperfusion. This is also reflected by a persistent elevation of inflammatory markers such as leukocyte count and C-reactive protein immediately and 24 hours after CPR compared with control patients. We could not observe any correlation between monocyte count and number of MMPs, or EMP-monocyte conjugates so that the results give the impression that (annexin V+) MP and conjugate counts are independent markers of inflammation and not only elevated arising from a general elevation of leukocytes or monocytes in resuscitated patients.
Other players in this field of activated coagulation and leukocyte/endothelial interaction are PMPs. As the phospholipid-dependent procoagulant activity of PMPs is limited to the annexin V positive subpopulation [
46], we measured procoagulant PMPs in this study, which were also elevated after CPR. PMPs in general are formed by the attachment of platelets to the vascular wall. Besides their procoagulant properties they are able to directly activate platelets [
22]. Furthermore, they are able to increase the adherence of monocytes to endothelial cells by the up-regulation of adhesion molecules [
23]. Therefore, Jy et al. suggested a possible role of PMPs as an activator of neutrophils in ischemic injury, thrombosis and inflammation [
47]. Our data match studies which revealed an increase of procoagulant PMPs in patients with ischemia/reperfusion, vascular injury and a risk of thromboembolism such as acute coronary syndromes [
20,
26] or ischemic stroke [
27]. As levels of procoagulant PMP were elevated compared with controls presenting CAD in a similar proportion to the resuscitated, we can exclude the possibility that CAD alone contributed to this effect.
Procoagulant PMPs were significantly elevated immediately after CPR compared with healthy subjects, but failed to show a significant difference with respect to the cardiac patients. This could be due to application of GPIIb/IIIa receptor antagonists during coronary intervention in a substantial amount of patients (42 vs. 10% in controls) [
20]. In point of fact, the subgroup of patients receiving GPIIb/IIIa receptor antagonists had a trend towards lower counts of procoagulant PMP, but there was no statistical difference between the two subgroups (data not shown). Another possible explanation is given by Nomura et al., who could not detect elevated levels of free PMPs in patients with multiple sclerosis and supposed that this was due to an absorbance of most of the free PMPs to leukocytes, contributing to the increased platelet-leukocyte conjugates in their study [
24]. Finally, high shear stress can initiate both platelet aggregation and shedding of procoagulant-containing MPs [
48] and the observed elevation could therefore also possibly be due to shear stress occurring during mechanical resuscitation.
Finally, our study revealed higher EMP-platelet conjugates immediately after CPR. These conjugates originate from binding of EMPs to activated platelets and have so far only been described by Héloire et al. in patients with CAD. The authors hypothesise that these conjugates originate from strongly activated and damaged endothelial cells and activated platelets and are able to contribute to thrombus formation by undergoing sequestration in peripheral vessels [
30]. In synopsis with our previous findings of a severe endothelial damage early after resuscitation [
5], the elevation of EMP-platelet conjugates in resuscitated patients could reflect endothelial injury and microcirculatory disorders occurring after CPR.
Interestingly, patients presenting with less than 1.0 MMP/μL on the second day after CPR showed a better 20-day survival after CPR compared with those with 1.0 or more MMPs/μL. Accordingly, we found this in trend too for patients with less than 10.0 procoagulant PMPs/μL immediately and 24 hours after CPR, although there was no statistical significant difference. Therefore, we suggest a potential prognostic value of MMP counts on the second day after CPR. It seems likely that the small number of subjects in each subgroup is due to the fact that these notable differences did not reach statistically significant differences.
Additionally, we provide evidence for enhanced apoptosis of endothelial cells under the influence of MPs of resuscitated patients
ex vivo. This is in line with other publications reporting of endothelial apoptosis under the influence of different MP subpopulations, such as
in vitro generated MMPs [
16] or EMPs [
49]. Additionally Gambim et al. showed that endothelial cells exposed to PMPs isolated from patients with septic shock undergo apoptosis [
25]. In comparing the effect of MPs with the effect of plasma of the same patients, we can exclude an apoptotic effect of certain plasma compounds occurring after CPR. As we tested the whole amount of circulating MPs in CPR patients we cannot identify a certain subpopulation contributing to or even initiating apoptosis in our study.
A limitation of the study could be the sample size, which is relatively small (n = 36) with a considerable heterogeneity of the patients, particularly in the CPR group. Although statistical analysis reveals clear differences between the groups, these results should be validated in a larger study.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
KF was responsible for the conception and the design of the study, for the acquisition, analysis, and interpretation of data as well as for the writing of the manuscript. LF recruited patients, drew blood samples and acquired data for MP measurements. MS was involved in conception and the design of the study, as well as analysis and interpretation of data and helped in trouble-shooting. NB acquired data and helped in patients' recruitment. TH revised the manuscript critically and gave important advices for completion and in the major revision process. CB contributed important intellectual content and gave final approval for the version to be published. TS contributed to the writing of the manuscript and was responsible for revising it critically. HJB participated substantially in the conception of the study, analysis, and interpretation of data as well as in the writing of the manuscript. All authors read and approved the final manuscript for publication.