Consumptive coagulopathy is associated with organ dysfunction during PICS

Abstract

Patients who survive the acute phase of sepsis can progress to persistent inflammation, immunosuppression, and catabolism syndrome (PICS). Although sepsis is characterized by early hypercoagulability and delayed hypocoagulability, coagulopathy during chronic critical illness is not fully understood. The objective of this study was to determine whether sepsis-induced PICS is associated with coagulation abnormalities. Using our previously described murine PICS model, outbred mice underwent cecal ligation and puncture, and coagulability was characterized after 8 days. We found that during PICS the spleen became markedly enlarged with increased splenocytes and splenic megakaryocytes without a concomitant increase in circulating platelets. Microscopy revealed a nearly sevenfold increase in pulmonary microvascular thrombi in PICS mice, along with significantly decreased pulmonary tidal volumes and inspiratory times and with significantly increased respiratory rates. Thromboelastometry showed that PICS mice had significantly delayed clot initiation time but increased clot firmness. Finally, PICS mice displayed delayed thrombin production and decreased overall thrombin concentrations. All together, these data demonstrate a general dysregulation of coagulation resulting in microthrombus formation and compromised lung function. On the basis of these findings, we propose that consumptive coagulopathy constitutes another cardinal feature of PICS and may contribute to the ongoing tissue damage and multiple organ failure that can occur in chronic critical illness.

INTRODUCTION

Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection (27). In the United States, sepsis affects upward of 1.5 million people per year with an in-hospital mortality rate of 30% (25). Some sepsis survivors develop persistent inflammation, immunosuppression, and catabolism syndrome (PICS), marked by recurrent nosocomial infections, inadequate nutrition, poor wound healing, neurocognitive deficits, and the need for long-term skilled nursing. Physiologically, this syndrome is characterized by ongoing inflammation, suppressed host immunity, and loss of lean muscle mass (10, 26). Many of these patients have prolonged recovery times, utilize immense hospital resources, and often progress to chronic critical illness (CCI) with less than 50% of patients surviving one year after hospital discharge (23, 24). Unfortunately, the only treatment currently utilized for PICS is supportive care. Therefore, efforts to better understand this process are crucial to improving outcomes for these patients.

Sepsis and coagulopathy are closely intertwined. Between 50% and 70% of septic patients manifest coagulation abnormalities, which range from fairly benign laboratory changes to disseminated intravascular coagulation (DIC) (16). The development of sepsis-associated coagulopathy is driven by the upregulation of inflammatory mediators and the downregulation of endogenous anticoagulant pathways. This imbalance produces a prothrombotic state that leads to vasodilation, endothelial damage, tissue ischemia, and organ dysfunction (12, 19). In severe cases, sepsis-associated coagulopathy is associated with longer intensive care unit and hospital lengths of stay, with an in-hospital mortality rate of greater than 50% (17).

By contrast, the relationship between PICS and coagulopathy is not fully understood. Retrospective clinical data demonstrate that, even after infection is controlled, immune and hemostatic markers persist until hospital discharge. This suggests that coagulation abnormalities may identify patients at high risk for end-organ dysfunction and death (12, 33). Murine data focused on coagulopathy and PICS are also limited. We recently reported that, after cecal ligation and puncture (CLP), mice were acutely hypercoagulable, but 16 h after injury, they became hypocoagulable (32). These data raise the question of whether coagulation abnormalities continue even after acute infection or sepsis subsides.

The objective of the present study was to characterize coagulation in a murine PICS model. We hypothesized that PICS involves a consumptive coagulopathy marked by the depletion of platelets, formation of microthrombi, and presence of profound coagulation abnormalities that contribute to the end-organ dysfunction seen in PICS. If this is the case, then consumptive coagulopathy could be used as a clinical marker to stratify those patients at greatest risk for ongoing tissue injury and progressive organ failure.

MATERIALS AND METHODS

Cecal ligation and puncture model.

Male CD-1 mice aged 6–8 wk (Charles River Laboratories, Wilmington, MA) were housed in standard environmental conditions and allowed to acclimate for 1–2 wk before experiments. A standard pellet diet and water ab libitum were provided. All animal protocols were approved by the Institutional Animal Care and Use Committee at the University of Cincinnati (Protocol 10-05-10-01). To induce acute polymicrobial sepsis, mice underwent 33% cecal ligation with a single, full-thickness, 25-gauge needle puncture under 2.5% isoflurane general anesthesia, as previously described (26, 32). All surgeries were performed between 8 AM and 12 PM. After CLP, mice received 1 ml of subcutaneous normal saline resuscitation and recovered on a 37°C heating pad for 1 h. Cohort sizes were chosen based on previously published data to account for variability and mortality (26). The mortality rate of this moderate CLP injury is 25% to 33%, paralleling the 10% to 40% mortality rate observed in human sepsis (26).

Persistent inflammation, immunosuppression, and catabolism syndrome model.

Eight days after CLP, all surviving mice displayed all the characteristics of PICS, including increased circulating myeloid cells, lymphocyte depletion, and loss of lean muscle, as previously described (26). Thus, PICS mice refer to those that survived 8 days after CLP injury and were used in experimental studies. Experiments were planned and performed in series, meaning different cohorts of mice that underwent CLP and developed PICS were used in experiments without any randomization or selection bias. On the basis of pilot data indicating that untouched and sham mice had near-identical levels of systemic inflammation and coagulation parameters 8 days after sham surgery, untreated mice were used as controls (data not shown). Figure 1 provides a pictorial depiction of the creation of our animal model and experimental workflow.

Fig. 1.

Consort diagram depicting experimental design and defining the persistent inflammation, immunosuppression, and catabolism syndrome (PICS) phenotype. H&E, hematoxylin and eosin; MSB, Martius Scarlet Blue; CAT, calibrated automated thrombogram.

Histology.

Spleen and lungs were harvested from PICS and healthy mice and stored in 10% formalin. Spleens were stained with hematoxylin and eosin, and lungs were stained with Martius Scarlet Blue (MSB), a polychrome stain that can simultaneously detect fibrin, erythrocytes, and collagen (15). Microscopy was performed on the Imager. M2 microscope (Zeiss Thornwood, NY), and images were photographed at equivalent exposure times per unit of magnification. Two blinded observers quantified the number of microthrombi per lung field. The mean proportion of microthrombi relative to total vessels per field (%) was then calculated for three ×40 images per mouse.

Splenic cell enumeration and characterization.

Spleens were harvested from PICS and healthy mice, weighed, and then homogenized in RPMI. Splenocytes were enumerated with a cell counter (Beckman Coulter, Brea, CA). Analysis of cell surface antigens was performed with flow cytometry on the Attune Flow Cytometer (Life Technologies, Foster City, CA) with the following antibodies: CD41 (clone MWReg30, BD Biosciences), c-kit (clone 2B8, BD Biosciences), Sca-1 (clone D7, BD Biosciences), and CD62P (clone PSel.K02.3, BD Biosciences). Splenocytes were gated based on high forward scatter and high CD41 levels. To differentiate megakaryocytes from platelets, which do not contain DNA, DAPI solution (BD Biosciences) was used as a fluorescent nucleic acid stain. Megakaryocytes were subsequently identified as DAPI(+)/CD41(+).

Blood count determination.

Whole blood was collected by cardiac puncture from PICS and healthy mice and then anticoagulated 1:10 with 3.2% buffered sodium citrate (Medicago, Quebec City, QC, Canada). Platelet counts were obtained with the Coulter AcT 10 Hematology Analyzer (Beckman Coulter, Brea, CA).

Rotational thromboelastometry.

To characterize abnormalities in coagulation, rotational thromboelastometry (ROTEM) (TEM Systems, Durham, NC) was performed according to the manufacturer’s instructions. Whole blood was collected by cardiac puncture from both healthy and PICS mice, immediately anticoagulated 1:10 with 3.2% buffered sodium citrate (Medicago), and analyzed within 10 min of collection. Native thromboelastometry (NATEM) was used to examine overall coagulation. EXTEM was used to identify aberrations in the extrinsic coagulation pathway. FIBTEM was used to determine fibrin’s role in coagulation independent of platelet contribution. For EXTEM and FIBTEM tests, 20 μl of thromboplastin was added to 300 μl of citrated blood to initiate clot formation. For FIBTEM, cytochalasin D was used to inhibit platelet activation. Clotting time (CT; seconds), clot formation time (CFT; seconds), and maximum clot firmness (MCF; mm) were determined for each test, as previously described (14, 21).

Fibrinogen quantification.

Whole blood was collected by cardiac puncture and anticoagulated 1:10 with 3.2% buffered sodium citrate. Samples were centrifuged at 4°C to separate plasma and immediately stored at −80°C. Plasma levels of fibrinogen were measured by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s protocol (MyBioSource, San Diego, CA).

Thrombin quantification.

Whole blood was collected by cardiac puncture and immediately anticoagulated 1:10 with 3.2% buffered sodium citrate. Samples were double-centrifuged at room temperature to generate platelet-poor plasma and then loaded in triplicate onto the Thrombinoscope BV (Stago Group, The Netherlands) for calibrated automated thrombogram (CAT) assay. Variables of interest included lag time (min), time to peak (min), endogenous thrombin potential (ETP; nM/min), and peak thrombin concentration (nM).

Quantification of breathing parameters.

Assessment of lung parameters was performed with the Buxco QT Digital Preamplifier with FinePointe software (Data Sciences International, St. Paul, MN). Mice were restrained and placed head-out into chambers. After a brief acclimation period, breathing was monitored, and the following parameters were recorded: frequency (breaths per minute), tidal volume (ml), inspiratory time (seconds), and minute ventilation (ml/min). Data were reported as the subject’s mean output during the cumulative monitoring period of 8 min.

Statistical analyses.

Analyses were performed with Prism 6 (GraphPad Software, La Jolla, CA). Descriptive data are reported as means ± SE. Student’s t-tests were used to compare groups, and P values ≤ 0.05 were considered statistically significant.

RESULTS

Previous data indicated that spleen mass increased during PICS (26). To confirm these findings, spleen size and mass differences were quantified in PICS compared with healthy mice (Fig. 2, A and B). Figure 2C demonstrates that differences in spleen mass also corresponded to a significant splenocyte increase during PICS. Next, to determine whether PICS involved the simultaneous expansion of other cell types, we used histology and flow cytometry to enumerate splenic megakaryocytes. Figure 2, D and E demonstrate that splenic megakaryocytes significantly increased during PICS. However, this was not associated with an increase in platelet counts in the peripheral blood (Fig. 2F).

Fig. 2.

Mice with persistent inflammation, immunosuppression, and catabolism syndrome (PICS) demonstrate splenomegaly with increased numbers of splenocytes and splenic megakaryocytes without correspondingly elevated peripheral platelet counts. PICS mice and untouched mice were euthanized and spleens and blood were harvested. A–C: PICS spleens were evaluated for size (A), weight (B), and number (C) of splenocytes compared with untouched mice (PICS, n = 7; untouched, n = 4). D: histologic architecture of spleens from untouched mice and PICS mice was evaluated using hematoxylin and eosin staining. E: flow cytometry was then used to evaluate the number of megakaryocytes in the spleen (PICS, n = 7; untouched, n = 4). F: a cell counter was used to identify the number of platelets in the blood of PICS (n = 34) and untouched (n = 31) mice. *P < 0.05 vs. untouched.

Since splenic megakaryocytes increase without concomitant thrombocytosis, and acute sepsis is associated with a prothrombotic state, we evaluated whether PICS also involves the development of microvascular thrombi. Blinded quantification of lung sections stained with MSB revealed a nearly sevenfold increase in pulmonary microvascular thrombi in PICS compared with control mice (Fig. 3). To determine whether these findings affected respiratory physiology, PICS mice and healthy mice were evaluated for various breathing parameters. PICS mice had significantly decreased tidal volumes and inspiratory times but significantly increased respiratory rates compared with healthy mice (Fig. 4). Minute ventilation, the product of respiratory rate and tidal volume, was similar between groups.

Fig. 3.

Lungs having persistent inflammation, immunosuppression, and catabolism syndrome (PICS) have increased microvascular thrombi. PICS (n = 10) and untouched (n = 8) mice were euthanized, and lungs were harvested. A: Martius Scarlet Blue (MSB) stain was used to identify fibrin (red, blue) and erythrocytes (yellow). B: number of microthrombi per lung field was counted by two blinded-observers with a median of three ×40 images per animal. The mean proportion of microthrombi relative to total vessels per field (%) is displayed. *P < 0.05 vs. untouched.

Fig. 4.

Mice with persistent inflammation, immunosuppression, and catabolism syndrome (PICS) demonstrate altered respiratory dynamics. Head-out restrained evaluation of breathing parameters was performed on PICS (n = 6) and untouched (n = 7) mice to evaluate tidal volume (A), respiratory rate (B), inspiratory time (C), and minute ventilation (D). *P < 0.05 vs. untouched.

Next, ROTEM and CAT assays were performed to determine whether the consumption of platelets and formation of microthrombi correlated with global changes in coagulation. First, NATEM was performed to investigate the collective coagulation process. This assay demonstrated that CT and MCF were significantly increased during PICS, whereas CFT was relatively unchanged (Fig. 5). Second, CAT assays were conducted to assess whether the delay to initiation of clotting was related to deficient thrombin generation. Compared with healthy mice, PICS mice had significantly increased lag time and time to peak thrombin generation (Fig. 6, A and B). PICS mice also had significantly reduced total thrombin production (ETP, Fig. 6C). However, PICS and healthy mice showed similar peak thrombin concentrations (Fig. 6D). Third, to evaluate the extrinsic coagulation pathway in isolation, EXTEM was performed. After the exogenous addition of thromboplastin to stimulate coagulation, PICS mice demonstrated significantly decreased CT and CFT but increased MCF compared with healthy mice (Fig. 7). Finally, fibrin contribution to clot strength was investigated through FIBTEM, an assay that measures whole blood coagulation after inhibition of platelets, in addition to ELISA quantification of plasma fibrinogen. Both of these assays revealed that, during PICS, fibrinogen levels and fibrin contribution to clot strength were significantly augmented compared with those of healthy mice (Fig. 8). In summary, ROTEM and CAT data show that PICS mice take longer to initiate hemostasis but the strength of thrombi is ultimately greater than that of healthy mice. When the extrinsic pathway of coagulation was isolated, PICS mice trended toward a hypercoagulable state following a prothrombotic stimulus.

Fig. 5.

Native thromboelastometry shows delayed clot initiation, but greater clot strength during persistent inflammation, immunosuppression, and catabolism syndrome (PICS). Blood from PICS (n = 27) and untouched (n = 28) mice was obtained and evaluated for various aspects of native clot formation using thromboelastometry: clotting time (A), clot formation time (B), and maximum clot firmness (C). *P < 0.05 vs. untouched.

Fig. 6.

Calibrated automated thrombogram assay indicates that delayed clot initiation parallels deficient thrombin production in persistent inflammation, immunosuppression, and catabolism syndrome (PICS). Blood from PICS (n = 26) and untouched (n = 16) mice was obtained. After generation of platelet-poor plasma, samples were loaded onto the Thrombinoscope. Thrombin generation was evaluated according to lag time or the number of minutes before thrombin generation begins (A), time to peak thrombin concentration (B), endogenous thrombin potential or the total concentration of thrombin produced in whole blood (C), and peak thrombin concentration (D). *P < 0.05 vs. untouched.

Fig. 7.

Extrinsic thromboelastometry indicates a shift toward hypercoagulability with tissue factor exposure in persistent inflammation, immunosuppression, and catabolism syndrome (PICS). Blood from PICS (n = 7) and untouched (n = 9) mice was anticoagulated. Thromboplastin was added to initiate clot formation to determine the role of the extrinsic coagulation pathway by measuring clotting time (A), clot formation time (B), and maximum clot firmness using thromboelastometry (C). *P < 0.05 vs. untouched.

Fig. 8.

Mice with persistent inflammation, immunosuppression, and catabolism syndrome (PICS) demonstrate increased plasma fibrinogen concentrations and greater fibrin contribution to clot strength. Blood was obtained from PICS and untouched mice. A: plasma fibrinogen concentrations were measured using ELISA (PICS, n = 11; untouched, n = 7). B: in a separate set of experiments, cytochalasin D was added to whole blood to inhibit platelet activation, and maximum clot firmness was measured using thromboelastometry (PICS, n = 11; untouched, n = 14). *P < 0.05 vs. untouched.

DISCUSSION

Using a murine model of PICS, we demonstrated profound splenomegaly and splenic megakaryocyte accumulation without a concomitant increase in circulating platelets. This was accompanied by increased pulmonary microthrombus formation and altered respiratory dynamics. We also identified numerous anomalies in whole blood coagulability, signifying the general dysregulation of coagulation during PICS. Overall, these data indicate that a prolonged state of consumptive coagulopathy may contribute to systemic tissue damage, which may be a major contributing factor to the state of persistent inflammation that occurs in PICS.

Over the past several decades, critical illness paradigms have evolved dramatically as the diagnosis and management of sepsis have improved. Although inpatient and short-term mortality rates have declined, up to 40% of septic patients fail to make a full recovery (10). Sepsis survivors are plagued by long-term morbidity, poor quality of life, and an estimated three-year mortality rate of 71% (23). The term CCI applies to those patients with protracted clinical courses in intensive care units, multiple organ failure (MOF), recurrent infections, malnutrition, cognitive impairments, poor rehabilitation, and indolent death. More recently, PICS was proposed as an underlying mechanism for a subset of CCI patients who fail to reestablish immunologic homeostasis after sepsis subsides (7). Given the lack of current therapeutic interventions beyond antibiotics and supportive care, as well as the dismal survival of patients with CCI, it is imperative to more clearly define the PICS phenotype and develop approaches for stratifying patients at greatest risk for morbidity, MOF, and death.

The spleen is the largest secondary lymphoid organ in the body and plays a crucial role in the immune system, including the differentiation and proliferation of lymphocytes in response to invading pathogens. Mice that survive CLP-induced polymicrobial sepsis have been found to develop persistent splenomegaly and expansion of splenocytes (3, 26, 30). Consistent with these previous studies, we found that PICS mice developed profound splenomegaly, but also that the spleen became a site of extramedullary megakaryocyte accumulation. The precise contributions of megakaryocytes and platelets to immunosuppression are not completely understood, but they may induce splenocyte apoptosis through increased granzyme B protein expression, resulting in direct lymphotoxicity (6). Interestingly, the rise in splenic megakaryocytes did not translate to increased peripheral platelet counts, as might be expected. The discrepancy between splenic megakaryocytes and circulating platelets could be the result of insufficient local growth factors for nuclear proliferation and megakaryocyte development. The process of megakaryocyte maturation and release into the systemic circulation takes 2–3 days; thus, it is also conceivable that our PICS model lends to nonoptimal sample collection that precludes the identification of thrombocytosis (18). Alternatively, we hypothesized that peripheral platelet counts normalize during PICS from the recruitment of platelets and the formation of microvascular thrombi. Indeed, we identified a greater than sixfold increase in microthrombi in PICS lungs, explaining the discrepancy between splenic megakaryocyte accumulation and circulating platelet counts.

Immunothrombosis describes the production of thrombin and formation of microthrombi during sepsis that results from endothelial injury. This process promotes the containment and clearance of pathogens by immune cells, and the simultaneous production of thrombin helps activate platelets (5, 28). Although immunothrombosis bolsters host defense during sepsis, unchecked production of thrombi in the microcirculation may lead to the consumption of coagulation factors, bleeding, and tissue damage. Excessive accumulation of pulmonary microvascular thrombi may eventually lead to DIC and/or acute respiratory distress syndrome (ARDS), manifestations of sepsis-associated end-organ dysfunction. In severe cases of acute lung injury/ARDS, pulmonary artery thrombi and microvascular filling defects create ventilation to perfusion mismatch, which may cause atelectasis, increased pulmonary vascular resistance, and death (11).

Given that patients who survive sepsis also suffer long-term pulmonary impairments, including tracheostomy and mechanical ventilation, we next explored whether pulmonary microthrombi were associated with altered respiratory dynamics during PICS (8). These studies revealed significant decreases in tidal volume and inspiratory time compensated for by a significant increase in respiratory rate. Minute ventilation, the product of respiratory frequency and tidal volume and a surrogate for air exchange, did not differ between PICS and healthy mice (29). Without arterial blood gas or oxygen saturation measurements or true plethysmography, it is difficult to draw definitive conclusions about the impact of excessive fibrin deposition on pulmonary compliance, function, and gas exchange. It is possible that the observed alterations in respiratory dynamics resulted from diaphragm muscle catabolism rather than from pulmonary microthrombi; however, if that was the case, one might expect reductions in both frequency and minute volume signifying respiratory muscle fatigue. We also speculate that, even if microvascular thrombi and associated changes in respiratory mechanics are not themselves deleterious, they may contribute to vulnerability to secondary bacterial pulmonary infection during PICS and cause a multifactorial reduction in pulmonary function (26). Because acute lung injury can be associated with progressive organ dysfunction, identification of even the earliest aberrations in respiratory rate or tidal volume may alert clinicians to impending MOF (22, 31).

Although sepsis-associated coagulopathy is very well described, PICS coagulopathy is less clear and further complicated by the variability of terminology used to describe both critical illness and coagulation disorders. Clinical data suggest that coagulopathy is a negative predictor of survival after infection and sepsis (13, 17, 20, 33), and CCI patients are known to have coagulopathy as a part of MOF (8, 9), but murine studies are scarce. Coagulation is initiated by subendothelial collagen exposure or interaction with tissue factor, with downstream conversion of fibrinogen to fibrin by thrombin. In turn, fibrin recruits activated platelets to assist with crosslinking and clot strength. We found that, during PICS, CT was prolonged and thrombin formation and thrombus polymerization were delayed. Ultimately, PICS mice had a greater MCF, likely a testament to their higher plasma fibrinogen levels and greater fibrin contribution to coagulation as detected by FIBTEM. Although the constellation of pulmonary microvascular thrombi and associated delay in coagulation appear similar to DIC, DIC usually involves the overproduction of both tissue factor and thrombin, findings not observed in this study. In fact, delayed CT was actually paralleled by decreased total thrombin production in PICS mice. Only when whole blood was exposed to tissue factor during EXTEM did PICS mice shift toward a hypercoagulable phenotype, suggesting that they are primed to form thrombi when provided the correct substrate. Further studies are needed to more fully understand coagulation abnormalities, platelet function, and fibrinolysis in PICS mice.

There are several limitations to the current study. First, PICS was modeled on outbred, albeit young adult mice. Ongoing studies will utilize older mice to reflect the aging patient population most susceptible to sepsis and CCI (1, 24). Second, although the current study proposes a novel phenotype in PICS, the underlying mechanisms remain elusive. It is possible that indolent infection, ongoing systemic inflammation, and myelopoiesis can perpetuate prothrombotic pathways during PICS. Thus, a logical next step would be to test this mechanism by treating PICS mice with immune modulating agents to determine whether this improves consumptive coagulopathy. A preliminary experiment evaluating the effect of splenectomy on native coagulability was unsuccessful in mitigating coagulopathy during PICS (data not shown). However, further data on the timing and development of coagulation abnormalities are required before definitive conclusions can be drawn. Finally, because our primary interest was to explore coagulation during PICS by using our specific model, we did not quantify pulmonary microthrombi or measure coagulation at any time point besides 8 days after CLP. Thus, on the basis of the presented data, we cannot determine when changes occurred or at what time point intervention could have been most efficacious.

In conclusion, there is splenic megakaryocyte accumulation, delayed initiation of coagulation, enhanced clot strength bolstered by elevated fibrinogen levels, and pulmonary microthrombus formation during PICS. On the basis of these findings, we propose that consumptive coagulopathy contributes to the pathogenesis of PICS, particularly as it relates to organ damage. Taken together, these data suggest that the early identification of coagulation abnormalities could help predict and define the development of PICS, as well as stratify the patients at greatest risk for subsequent MOF and death.

GRANTS

This work was supported by National Institute of General Medical Sciences Grant K08-GM-131284-01.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

C.C.C. and V.N. conceived and designed research; L.K.W., N.B., and R.A.V. performed experiments; L.K.W., N.B., R.A.V., C.C.C., and V.N. analyzed data; L.K.W., N.B., M.D.G., C.C.C., and V.N. interpreted results of experiments; L.K.W. prepared figures; L.K.W., C.C.C., and V.N. drafted manuscript; L.K.W., N.B., R.A.V., M.D.G., C.C.C., and V.N. edited and revised manuscript; L.K.W., N.B., R.A.V., M.D.G., C.C.C., and V.N. approved final version of manuscript.