Erik B. Kistler

Infection by SARS-CoV-2 and subsequent COVID-19 can cause viral sepsis and septic shock. Several complications have been observed in patients admitted to the intensive care unit (ICU) with COVID-19, one of those being bacterial superinfection. Based on prior evidence that dysregulated systemwide proteolysis is associated with death in bacterial septic shock, we investigated whether protease activity and proteolysis could be elevated in COVID-19-induced sepsis with bacterial superinfection. In particular, we sought to assess the possible implications on the regulation of protein systems, such as for instance the proteins and enzymes involved in the clotting cascade. Blood samples collected at multiple time points during the ICU stay of four COVID-19 patients were analyzed to quantify: a) the circulating proteome and peptidome by mass spectrometry; b) plasma enzymatic activity of trypsin-like substrates and five clotting factors (plasmin, thrombin, factor VII, factor IX, factor X) by a fluorogenic assay. Of the four patients, one was diagnosed with bacterial superinfection on day 7 after beginning of the study and later died. The other three patients all survived (ICU length-of-stay 11.25±6.55 days, hospital stay of 15.25±7.18 days). Spikes in protease activity (factor VII, trypsin-like activity) were detected on day 7 for the patient who died. Corresponding increases in the total intensity of peptides derived by hydrolysis of plasma proteins, especially of fibrinogen degradation products, and a general reduction of coagulation proteins, were measured as well. A downregulation of endogenous enzymatic inhibitors, in particular trypsin inhibitors, characterized the non-surviving patient throughout her ICU stay. Enzymatic activity was stable in the patients who survived. Our study highlights the potential of multiomics approaches, combined with quantitative analysis of enzymatic activity, to i) shed light on proteolysis as a possible pathological mechanism in sepsis and septic shock, including COVID-19-induced sepsis; ii) provide additional insight into malfunctioning protease-mediated systems, such as the coagulation cascade; and iii) describe the progression of COVID-19 with bacterial superinfection.

OBJECTIVES: Refractory vascular failure due to the inability of vascular smooth muscle to respond to vasoconstrictors such as phenylephrine is a final common pathway for severe circulatory shock of any cause, including trauma/hemorrhagic shock. Increased inflammation, Toll-like receptor 4 activation, and decreased response of the alpha-1 adrenergic receptors which control vascular tone have been reported in trauma/hemorrhagic shock. HYPOTHESIS: In trauma/hemorrhagic shock, Toll-like receptor 4 activation contributes to vascular failure via decreased bioavailability of adrenergic receptors. DESIGN AND MEASUREMENTS: Trauma/hemorrhagic shock was induced in Wistar rats (laparotomy combined with mean arterial pressure at 40 mm Hg for 90 min followed by 2 hr resuscitation with Lactated Ringers solution). To inhibit Toll-like receptor 4, resatorvid (TAK-242) and resveratrol were used, and plasma was collected. Smooth muscle cells were incubated with lipopolysaccharide (10 ng/mL) or plasma. Inflammatory cytokines were screened using dot-blot. Toll-like receptor 4 and nuclear factor κB activation and cellular localization of the alpha-1 adrenergic receptor were measured by immunofluorescence imaging and Western blot analysis. Clustered regularly interspaced short palindromic repeats/Cas9 was used to knock out Toll-like receptor 4, and calcium influx following stimulation with phenylephrine was recorded. MAIN RESULTS: Trauma/hemorrhagic shock caused a decreased response to phenylephrine, whereas Toll-like receptor 4 inhibition improved blood pressure. Trauma/hemorrhagic shock plasma activated the Toll-like receptor 4/nuclear factor κB pathway in smooth muscle cells. Double labeling of Toll-like receptor 4 and the alpha-1 adrenergic receptor showed that these receptors are colocalized on the cell membrane. Activation of Toll-like receptor 4 caused cointernalization of both receptors. Calcium influx was impaired in cells incubated with trauma/hemorrhagic shock plasma but restored when Toll-like receptor 4 was knocked out or inhibited. CONCLUSIONS: Activation of the Toll-like receptor 4 desensitizes vascular smooth muscle cells to vasopressors in experimental trauma/hemorrhagic shock by reducing the levels of membrane alpha-1 adrenergic receptor.

Proteomic technologies have identified 234 peptidases in plasma but little quantitative information about the proteolytic activity has been uncovered. In this study, the substrate profile of plasma proteases was evaluated using two nano-LC-ESI-MS/MS methods. Multiplex substrate profiling by mass spectrometry (MSP-MS) quantifies plasma protease activity in vitro using a global and unbiased library of synthetic peptide reporter substrates, and shotgun peptidomics quantifies protein degradation products that have been generated in vivo by proteases. The two approaches gave complementary results since they both highlight key peptidase activities in plasma including amino- and carboxypeptidases with different substrate specificity profiles. These assays provide a significant advantage over traditional approaches, such as fluorogenic peptide reporter substrates, because they can detect active plasma proteases in a global and unbiased manner, in comparison to detecting select proteases using specific reporter substrates. We discovered that plasma proteins are cleaved by endoproteases and these peptide products are subsequently degraded by amino- and carboxypeptidases. The exopeptidases are more active and stable in plasma and therefore were found to be the most active proteases in the in vitro assay. The protocols presented here set the groundwork for studies to evaluate changes in plasma proteolytic activity in shock.

Hemorrhagic Shock (HS) after Traumatic Brain Injury (TBI) is common and lethal among military and civilians. Blood transfusion has been the resuscitation gold standard procedure for many years. However, only 3% of age‐eligible individuals donate blood, which limits blood availability and cannot meet current blood demands. This study was compared the efficacy to resuscitate from HS after TBI via infusion of Lactated Ringer’s solution (LR, electrolyte solution used to restore the loss of blood volume), fresh blood (Blood, autologous blood drawn during the hemorrhage), or Polymerized Hemoglobin [PolyHb, a Hemoglobin Based Oxygen Carrier (HBOC) that shows promise in increasing oxygenation]. Fifteen Wistar rats (350–400g) were instrumented with catheters in the femoral artery and vein and subjected to TBI through a pneumatic controlled cortical impact (CCI) (Leica Biosystems, Vista, CA). To induce TBI, a 5mm craniotomy over the right cerebral cortex was performed, and the scalp was impacted at a velocity of 5m/s and dwell time of 200ms. The scalp was subsequently closed and animals were given 10 min to stabilize before performing HS. HS was induced by blood withdrawal from the femoral artery catheter to achieve a mean arterial pressure (MAP) between 35–40mmHg, and the hypotensive state was maintained for 90 min before resuscitation. Animals were separated into 3 groups based on the resuscitation solution: Blood, LR, or PolyHb. Animals were monitored for an additional 120 min before euthanasia. Measurements were taken at baseline (BL), 90 min into HS (HS), and 30 min (R1) and 2 hours (R2) after resuscitation. The MAP, systolic blood pressure (SBP), and diastolic blood pressure (DBP) were decreased equally in all groups during HS. Blood and PolyHb groups increased MAP compared to the LR group at R1. The MAP was not different between Blood and PolyHb at R2. The SBP was increased only for the Blood group as compared to the LR group at R1. Moreover, DBP was increased for both Blood and PolyHb compared to the LR group at R1 and R2. As expected, hematocrit decreased in all groups after HS, with only the Blood group recovering hematocrit after resuscitation. Total hemoglobin (tHb) decreased after HS, and increased after resuscitation for the Blood and PolyHb groups but not in the LR group. Oxygen saturation was decreased for the Blood and PolyHb groups compared to the LR group at R1 and R2. The oxygen saturation was increased on the Blood group compared to PolyHb group at R2. After HS all groups increased lactate, after R1 just Blood and LR group recovered lactate to BL levels, and PolyHb did not decreased lactate concentration at R2 compared to the blood group. In conclusion, our results show that PolyHb was effective to resuscitation from HS after TBI, restoring blood pressure similarly to Blood group and could be a good alternative to transfusion for field resuscitation from HS after TBI. Support or Funding Information: This work was supported by the NIH Heart Lung and Blood Institute under Grant, R01‐HL126945, and the DOD DMRDP under Grants W81XWH‐18‐1‐0059, W81XWH‐17‐2‐0047

Objective: Fluid resuscitation with blood products is standard of care in the current management of trauma/hemorrhagic shock (T/HS). However, the availability of whole blood is rare in civilian prehospital care, and thus blood component therapy and other fluid replacement options are required. This study sought to differentiate and compare the effects of lactated Ringer’s solution (LR), plasma, washed red blood cells (RBC), and whole blood (Control) as resuscitation fluids for hemodynamic restoration after T/HS. Methods: Wister male rats (400g) were randomly divided into four groups: LR, Plasma, RBC, and Control, anesthetized with isoflurane and subjected to experimental T/HS. A mid‐line laparotomy incision was made, and blood was slowly withdrawn to maintain a mean arterial blood pressure (MAP) of 35–40 mmHg for 90 minutes. After the 90‐min shock period, intravenous fluids corresponding to the experimental group were started with the goal of achieving a MAP ≥ 65 mmHg, the clinical target for adequate pressure. Animals were monitored for 120 minutes following start of reperfusion. Vital signs were continuously measured, as well as arterial blood gases, lactate, and hemoglobin. Results: MAP in animals that received RBCs was similar to that of the whole‐blood Control group (70.4 ± 4.0 mmHg vs 83.0 ± 5.8 mmHg, RBC vs Control, NS), and significantly greater than MAPs of Plasma (59.9 ± 4.4 mmHg, p=0.03) and LR (45.6 ± 4.8 mmHg, p2 saturation (88.3 ± 0.5% vs. LR: 93.9 ± 4.7%, Plasma: 94.8 ± 0.6%, WB: 93.5 ± 4.5%), yet PaO ₂ was comparable between groups. Conclusion: In our model of experimental T/HS, reperfusion fluids carrying red blood cells substantially improved hemodynamic stability by returning the mean arterial blood pressure to survivable values. The efficacy of RBC and whole blood resuscitation may be largely the result of improvements in diastolic blood pressure, suggesting increased contribution from systemic vascular resistance in these groups. Support or Funding Information: DOD W81XWH‐17‐2‐0047

Background: Septic shock (SS) and cardiogenic shock (CS) are two types of circulatory shock with a different etiology. Several studies have described the molecular alterations in SS patients, whereas the molecular factors involved in CS have been poorly investigated. We aimed to assess in the whole blood of CS and SS patients, using septic patients without shock (SC) as controls, transcriptomic modifications that occur over 1 week after ICU admission and are common to the two types of shock.Methods: We performed whole blood RNA sequencing in 21 SS, 11 CS, and 5 SC. In shock patients, blood samples were collected within 16 h from ICU admission (T1), 48 h after ICU admission (T2), and at day 7 or before discharge (T3). In controls, blood samples were available at T1 and T2. Gene expression changes over time have been studied in CS, SS, and SC separately with a paired analysis. Genes withpvalue < 0.01 (Benjamini-Hochberg multiple test correction) were defined differentially expressed (DEGs). We used gene set enrichment analysis (GSEA) to identify the biological processes and transcriptional regulators significantly enriched in both types of shock.Results: In both CS and SS patients, GO terms of inflammatory response and pattern recognition receptors (PRRs) were downregulated following ICU admission, whereas gene sets of DNA replication were upregulated. At the gene level, we observed that alarmins, interleukin receptors, PRRs, inflammasome, and DNA replication genes significantly changed their expression in CS and SS, but not in SC. Analysis of transcription factor targets showed in both CS and SS patients, an enrichment of CCAAT-enhancer-binding protein beta (CEBPB) targets in genes downregulated over time and an enrichment of E2F targets in genes with an increasing expression trend.Conclusions: This pilot study supports, within the limits of a small sample size, the role of alarmins, PRRs, DNA replication, and immunoglobulins in the pathophysiology of circulatory shock, either in the presence of infection or not. We hypothesize that these genes could be potential targets of therapeutic interventions in CS and SS.Trial registration: ClinicalTrials.gov,NCT02141607. Registered 19 May 2014.

The mechanisms for cardiac injury after hemorrhagic shock (HS) are unresolved. We hypothesize that remote organ damage can be caused by uncontrolled pancreatic proteolytic activity, as enteral protease inhibition improves outcomes in experimental HS. Uncontrolled proteolysis in the heart may disrupt cardiac metabolism and adrenergic control with subsequent deleterious outcomes. To test this hypothesis, the heart rate–pressure product (RPP) as an index of myocardial oxygen consumption and the levels of fatty acid transporter proteins CD36 and FATP6 as surrogates for metabolic activity in the heart were measured in rats subjected to experimental HS (n = 6/group) with and without the enteral protease inhibitor tranexamic acid (TXA). Plasma troponin I and heart fatty acid-binding protein (HFABP) concentrations were measured as indices of myocardial damage. Expression of the adrenergic receptors β₁, α_1D, and β₂ was also measured in the heart to determine the possible effects of shock with and without enteral TXA on the adrenergic control of heart function. Hemorrhagic shock was induced by reduction in mean arterial blood pressure to 35 mm Hg for 2 hours before reperfusion of shed blood. The RPP was maintained in shocked animals treated enterally with TXA but not in those subjected to HS alone; this group also demonstrated decreased HFABP and plasma troponin I levels. Serine protease (trypsin, chymotrypsin, and elastase) and matrix metalloproteinase (MMP)-2 and MMP-9 activity was elevated in cardiac tissue and plasma after HS and abrogated by enteral TXA. Levels of CD36, FATP6, β₁, α_1D, and β₂ were also increased after HS in cardiac tissue, and the increases were mitigated by TXA treatment. These results suggest that increased proteolytic activity may contribute to cardiac injury after HS. Enteral TXA prevents these changes, indicating a potential therapeutic option in the management of shock with resultant cardiac injury.