Mechanisms of platelet-neutrophil interactions and effects on cell filtration in septic shock.

ABSTRACT-We examined the mechanisms and the adhesive molecules mediating platelet-neutrophil adhesion in patients with septic shock. Neutrophils, platelets, and platelet poor plasma (NPPP) were isolated from 12 normal volunteers. Platelets and neutrophils were stimulated with platelet poor plasma (SPPP) removed from 12 patients in septic shock. Cell adhesion was assessed by filtration through 5-microm pore filters and by flow cytometry. Blocking monoclonal antibodies were used against the platelet and neutrophil surface receptors glycoprotein complex IIb/IIla, P-selectin, ICAM-2, CD11a, CD11b, and CD18. The filtration pressure (Pi) of cells suspended in SPPP was significantly greater than that of cells suspended in NPPP (24 +/- 1.0 mmHg vs. 14 +/- 1.0 mmHg; P< 0.05). The difference between the Pi of cells suspended in SPPP or NPPP (deltaPi SPPP-NPPP) in the presence of monoclonal antibodies anti-CD41, anti-CD62P, abciximab, anti-CD11a, anti-CD11b, and anti-CD18 was significantly less than the APi SPPP-NPPP of cell suspensions without the addition of these monoclonal antibodies (P < 0.01). The greatest reduction in Pi occurred when platelet receptor P-selectin was blocked simultaneously with the CD11b receptor on the neutrophil as compared to all other single blocking monoclonal antibodies or combinations of monoclonal antibodies. The mean fluorescence of activated platelet CD63-PE binding to neutrophils suspended in SPPP was significantly greater than that of cells suspended in NPPP (780 +/- 130 Ifu vs. 295 +/- 35 Ifu; P < 0.05). The greatest attenuation in mean fluorescence occurred by blocking the P-selectin receptor on the platelet simultaneously with CD11b receptor on the neutrophil. We conclude that platelet-neutrophil aggregation is increased in septic shock. This aggregation is mediated by the interaction of multiple platelet and neutrophil surface receptors. The platelet receptor P-selectin and the neutrophil receptor CD11b/CD18 appear to play the most important role in these interactions.

Lipopolysaccharide- and superantigen-modulated superoxide production and monocyte hyporesponsiveness to activating stimuli in sepsis.

The effects of acute and prior exposure to lipopolysaccharide (LPS) and staphylococcal enterotoxin B (SEB) on superoxide release by monocytes were examined in control subjects and in patients with sepsis and septic shock during the acute stage and recovery. High doses of LPS, PMA (phorbol 12-myristate 13-acetate), and SEB stimulated monocyte superoxide release in control subjects (P < 0.05). Pretreatment of normal monocytes with these doses of LPS, PMA, and SEB induced significant hyporesponsiveness to subsequent challenge (P < 0.01), and evidence of cross-tolerance was observed. Monocytes isolated from patients with sepsis and septic shock demonstrated high spontaneous superoxide release compared with those of control subjects (P < 0.05). Stimulation of patient monocytes with LPS or SEB resulted in less superoxide production than that spontaneously released by controls (P < 0.01). In patients recovering from their initial infection, spontaneous superoxide release was less than that released during acute stage. In addition, the superoxide release in response to the same stimuli was significantly increased when compared with release during the acute stage (P < 0.05). These data demonstrate that both LPS and SEB induce hyporesponsiveness to LPS- or SEB-stimulated superoxide release. A similar pattern of hyporesponsiveness was observed during sepsis that may represent a mechanism for modulating the inflammatory response during severe infections.

The effect of resuscitation fluids on neutrophil-endothelial cell interactions in septic shock.

Fluid resuscitation is essential in the treatment of septic shock. This study examined the effect of resuscitative fluids (RFs) on sepsis-induced neutrophil-endothelial cell interactions. The RFs studied were 0.9% saline (NS), Ringer's lactate (RL), 7.5% saline and dextran-70 (DHS), 5% albumin (AL), and 6% hydroxyethyl starch (HS). Platelets and neutrophils were obtained from normal volunteers, and plasma was obtained from patients with septic shock. Microslides coated with human umbilical endothelial vein cell (HUVEC) and platelet-neutrophil solutions were primed with septic plasma with/without the RF. Neutrophil rolling velocity, leukoaggregation, and neutrophil adherence were determined. Separately, platelet-neutrophil solutions and endothelial cells were exposed to septic plasma with/without RFs, and cellular activation, neutrophil superoxide production, and endothelial cell E-selectin expression were assessed. Ringer's lactate decreased neutrophil rolling velocity and increased aggregation and adherence. Normal saline had no effect on these parameters. Hydroxyethyl starch and AL increased neutrophil rolling velocity and decreased adherence and aggregation when HUVECs were preincubated with the RF. Dextran-70 and 7.5% saline decreased neutrophil-endothelial cell interactions in both HUVECs and platelet/neutrophil preincubated experiments. Ringer's lactate increased activation of neutrophils and platelets, whereas AL decreased their activation. Other than NS, all the RFs increased neutrophil superoxide production. Ringer's lactate increased endothelial cell E-selectin release, whereas AL and HS both decreased its release. These data suggest that fluids used in the resuscitation of septic shock vary in their effects on sepsis-induced neutrophil-endothelial cell interactions. Ringer's lactate amplifies the effects of sepsis, while NS appears to have minimal impact. Dextran-70 and 7.5% saline, AL, and HS in varying degrees decrease sepsis-related neutrophil-endothelial cell interactions and activation.

Augmentation of platelet and endothelial cell eNOS activity decreases sepsis-related neutrophil-endothelial cell interactions.

NO is an important mediator of microvascular patency and blood flow. The purpose of this study was to examine the role of enhanced eNOS activity in attenuating sepsis-induced neutrophil-endothelial cell interactions. Microslides coated with human umbilical vein endothelial cells were stimulated with plasma from patients with septic shock. Neutrophil and platelets from control subjects were also stimulated with plasma from patients in septic shock and perfused over stimulated endothelial cells. l-Arginine (LA) with and without NG-monomethyl l-arginine (LNMMA), a nonselective NOS inhibitor, and N-(3-(aminomethyl) benzyl acetamide) ethanimidamide dihydrochloride (1400W), a highly selective iNOS inhibitor, were added to the septic plasma. The number of neutrophils adherent to endothelial cells, neutrophil rolling velocity, and the number of neutrophil aggregates were determined. Cell activation and the formation of platelet-neutrophil aggregates were assessed by flow cytometry. Separate experiments were done with isolated platelets using platelet aggregometry. l-Arginine significantly decreased sepsis-related neutrophil adhesion and aggregation and increased rolling velocity. The addition of LNMMA to LA and cell suspensions reversed the effects of LA on these parameters, whereas the addition of 1400W had no effect on LA-related changes. Platelet-neutrophil aggregation, platelet aggregation, platelet activation, and neutrophil activation induced by septic plasma were also significantly decreased by LA. Again, the addition of LNMMA reversed the effects of LA on these parameters, whereas 1400W had no effect on LA-related changes. These data suggest that enhancement of platelet and endothelial cell eNOS activity decreases sepsis-induced neutrophil-endothelial cell interactions and may play a role in maintaining microvascular patency in septic shock.

The effect of anticoagulants and the role of thrombin on neutrophil-endothelial cell interactions in septic shock.

To examine the effects of anticoagulants and the role of thrombin on neutrophil-platelet-endothelial cell interactions in septic shock. Controlled experiments using phase-contrast microscopy to study neutrophil, platelet, and endothelial cell interactions in flowing cell suspensions under simulated physiologic conditions. University research laboratory. Adult patients with septic shock and normal volunteers. Microslides were coated with human umbilical vein endothelial cells. Neutrophils and platelets removed from control subjects were stimulated with plasma from patients in septic shock and perfused over endothelial cells. Heparin (H), argatroban (A), antithrombin III (ATIII), and recombinant human activated protein C (rhAPC) with and without thrombin were added to cells suspended in septic plasma and normal plasma. The number of neutrophils adherent to endothelial cells, neutrophil rolling velocity, and the number of neutrophils in aggregates were determined. Flow cytometric analysis of cells was used to identify cell activation and the formation of platelet-neutrophil aggregates. Heparin, A, ATIII, rhAPC all significantly decreased neutrophil adhesion and aggregation, and increased rolling velocity of neutrophils suspended in septic plasma. These results are similar to those observed with normal plasma but present greater absolute changes. Platelet-neutrophil aggregation, platelet activation, and neutrophil activation were significantly decreased by each of the anticoagulants. The addition of thrombin to cell suspensions containing anticoagulants reversed the effects of H, A, ATIII, rhAPC on neutrophil adhesion, adherence, and rolling velocity. In addition, thrombin attenuated the effects of each of these agents on platelet-neutrophil aggregation, platelet activation, and neutrophil activation. These data suggest that H, A, ATIII, and rhAPC decrease sepsis-induced neutrophil-endothelial cell interactions. The reversal of this effect by thrombin suggests that these agents alter neutrophil-endothelial interactions through their anticoagulant effects and the resulting decrease in thrombin activity.

Comparison of cardiovascular effects of tiletamine-zolazepam, pentobarbital, and ketamine-xylazine in male rats.

We allocated 35 male Sprague-Dawley rats into 7 groups and anesthetized each by using one of the following regimens: ketamine 50 mg+xylaxine 5 mg; ketamine 75 mg+xylazine 5 mg; pentobarbital 45 mg; and Telazol 30, 40, 50, and 60 mg/kg; supplemental doses were used as required. Respiratory rate, heart rate, mean arterial pressure, cardiac index, and stroke index were measured every 30 min for 4 h. The Telazol groups showed a dose-dependent increase in duration of anesthesia. Duration of anesthesia was significantly shorter for the ketamine and pentobarbital groups than for any of the Telazol doses. Heart rate showed a dose-dependent decrease among the Telazol groups, but overall heart rate in these groups was higher than in the ketamine and pentobarbital groups. Mean arterial pressure in the Telazol 40 and 50 groups was significantly higher than the pentobarbital and higher ketamine groups yet lower than that of the Telazol 60 group. Overall animals anesthetized with Telazol showed the highest cardiac index, ketamine intermediate, and pentobarbital the lowest; cardiac index was higher in the Telazol 50 group than in either the Telazol 30 or pentobarbital groups. The pentobarbital group exhibited the lowest stroke index, whereas ketamine-treated animals had an intermediate stroke index. These differing effects of anesthetics on cardiovascular parameters must be considered when choosing an anesthesia regimen or comparing data from different studies. In our model, the Telazol 40 and 50 groups appeared to exhibit the fewest adverse cardiovascular effects.

Effect of recombinant activated protein C and low-dose heparin on neutrophil-endothelial cell interactions in septic shock.

OBJECTIVE: To examine the effects of recombinant activated protein C (rhAPC) and low-dose heparin on neutrophil-platelet-endothelial cell interactions in septic shock. DESIGN: Controlled experiments using phase contrast microscopy to study neutrophil, platelet, and endothelial cell interactions in flowing cell suspensions under simulated physiologic conditions. SETTING: University research laboratory. PATIENTS: Adult patients with septic shock and normal volunteers. INTERVENTIONS: Neutrophils and platelets removed from control subjects were stimulated with plasma and serum from 21 patients in septic shock and perfused over endothelial cells. Activated protein C, low-dose heparin, and low-dose heparin with rhAPC were added to cells suspended in septic plasma. Neutrophil rolling velocity and the number of neutrophils adherent to endothelial cells and in aggregates were determined. Flow cytometric analysis of CD11b/CD63 cells was used to identify platelet-neutrophil aggregates. MEASUREMENTS AND MAIN RESULTS: Activated protein C significantly decreased neutrophil adhesion and aggregation and increased rolling velocity in cells stimulated with both septic serum and septic plasma. Significant decreases in platelet-neutrophil aggregates induced by septic plasma were also observed. Low-dose heparin alone had no effects on these variables. The addition of low-dose heparin to cells suspended in septic plasma and rhAPC attenuated the benefits observed with rhAPC alone in each of these variables. CONCLUSIONS: These data suggest that the in vitro addition of rhAPC decreases sepsis-induced interactions between isolated platelets, neutrophils, and endothelial cells. Low-dose heparin attenuates the benefits observed with rhAPC. The changes in neutrophil-endothelial cell interactions demonstrated with rhAPC may play a role in preserving microvascular patency in patients with septic shock.

The experience at St. Vincent's Hospital, Manhattan, on September 11, 2001: preparedness, response, and lessons learned.

St. Vincent's Hospital in New York City was the primary recipient of patients after the 1993 bombing of the World Trade Center. This experience prompted the drafting of a formal disaster plan, which was implemented during the terrorist attack on the World Trade Center on September 11, 2001. Here, we outline the Emergency Management External Disaster Plan of St. Vincent's Hospital and discuss the time course of presentation and medical characteristics of the critically injured patients on that day. We describe how the critical care service adapted to the specific challenges presented and the lessons that we learned. We hope to provide other critical care systems with a framework for response to such large-scale disasters.

Impaired mitochondrial function induced by serum from septic shock patients is attenuated by inhibition of nitric oxide synthase and poly(ADP-ribose) synthase.

OBJECTIVE: The purpose of this study was to determine the role of nitric oxide and poly(ADP-ribose) synthase on impaired mitochondrial function in septic shock. DESIGN: Human umbilical vein endothelial cells were incubated with serum from ten healthy controls, 20 patients with septic shock, and seven critically ill patients who were not septic. The experiment was repeated after pretreatment with 3-aminobenzamide, a poly(ADP-ribose) synthase inhibitor, or N(G)-methyl-L-arginine, a nonspecific nitric oxide synthase inhibitor. MEASUREMENTS: Mitochondrial respiration was measured using a modified MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay. SETTING: Research laboratory. MAIN RESULT: Endothelial cell mitochondrial respiration was significantly depressed by septic serum and averaged 61% +/- 6% of control values (p <.05). Incubation with septic serum as compared with control serum also significantly decreased cellular adenosine triphosphate levels (6.7 +/- 1.2 nM vs. 13.5 +/- 1.9 nM, p<.01). The level of mitochondrial respiration in endothelial cells exposed to septic serum did not correlate with arterial lactate concentration but was correlated with both cardiac output (r(s) =.52, p<.05) and mixed venous oxygen saturation (r(s) =.61, p<.05). Pretreatment with N(G)-methyl-L-arginine significantly increased mitochondrial respiration in endothelial cells treated with septic serum from 63% +/- 6% of normal to 88% +/- 6% (p <.05) of normal values. Similarly, pretreatment with 3-aminobenzamide increased mitochondrial respiration in endothelial cells treated with septic serum from 64% +/- 6% to 100% +/- 4% (p <.01) of normal values. Endothelial cells incubated with serum from nonseptic critically ill patients did not demonstrate a significant decrease in mitochondrial respiration. CONCLUSION: In vitro mitochondrial respiration was significantly depressed by septic serum. The addition of N(G)-methyl-L-arginine, a nitric oxide synthase inhibitor, and 3-aminobenzamide, a blocker of the poly(ADP-ribose) synthase pathway, significantly attenuated this suppression. These data suggest that nitric oxide and poly(ADP-ribose) synthase activation may play an important role in the inhibition of mitochondrial respiration in septic shock.

Importance of platelets and fibrinogen in neutrophil-endothelial cell interactions in septic shock.

OBJECTIVE: To examine the role of platelets, fibrin, and adhesion molecules in mediating neutrophil-endothelial cell interactions in septic shock. DESIGN: Controlled experiments using phase contrast microscopy to examine neutrophil, platelet, and endothelial cell interactions in flowing cell suspensions under simulated physiologic conditions. SETTING: University research laboratory. PATIENTS: Adult patients with septic shock and normal volunteers. INTERVENTIONS: Microslides were coated with human umbilical vein endothelial cells. Neutrophils were removed from control subjects and patients in septic shock and were perfused over endothelial cells at rates representing a range of physiologic shear stresses. In an attempt to examine the effects of fibrin deposition on neutrophil-endothelial cell interactions, neutrophils, with and without platelets, were suspended in plasma and serum was removed from patients in septic shock. In addition, blocking monoclonal antibodies against the platelet receptor P-selectin and neutrophil receptor CD11b/CD18, and a platelet glycoprotein IIb/IIIa inhibitor, were incubated with cells suspended in plasma. Phase contrast video microscopy was used to count the number of neutrophils/mm adherent to endothelial cells during cessation of flow. Neutrophil rolling velocity was calculated as the time required for neutrophils to move across a 1-mm field (mm/sec). Leukoaggregation was defined as the number of neutrophils in aggregates (three or more nuclei) across a 1-mm field. MEASUREMENTS AND MAIN RESULTS: Normal neutrophils exposed to plasma from patients with septic shock demonstrated significant increases in aggregation and endothelial cell adherence with associated decreases in neutrophil rolling velocity. These changes were significantly enhanced in the presence of platelets and significantly attenuated in the presence of serum, which is fibrinogen depleted. Preincubation with antibodies to the surface receptors P-selectin, CD11b/CD18, and glycoprotein IIb/IIIa abrogated the changes in neutrophil aggregation, adhesion, and rolling velocity. CONCLUSIONS: These data suggest that platelets and fibrinogen play an important role in mediating neutrophil-endothelial cell adherence in septic shock.

Practice parameters for hemodynamic support of sepsis in adult patients: 2004 update.

OBJECTIVE: To provide the American College of Critical Care Medicine with updated guidelines for hemodynamic support of adult patients with sepsis. DATA SOURCE: Publications relevant to hemodynamic support of septic patients were obtained from the medical literature, supplemented by the expertise and experience of members of an international task force convened from the membership of the Society of Critical Care Medicine. STUDY SELECTION: Both human studies and relevant animal studies were considered. DATA SYNTHESIS: The experts articles reviewed the literature and classified the strength of evidence of human studies according to study design and scientific value. Recommendations were drafted and graded levels based on an evidence-based rating system described in the text. The recommendations were debated, and the task force chairman modified the document until <10% of the experts disagreed with the recommendations. CONCLUSIONS: An organized approach to the hemodynamic support of sepsis was formulated. The fundamental principle is that clinicians using hemodynamic therapies should define specific goals and end points, titrate therapies to those end points, and evaluate the results of their interventions on an ongoing basis by monitoring a combination of variables of global and regional perfusion. Using this approach, specific recommendations for fluid resuscitation, vasopressor therapy, and inotropic therapy of septic in adult patients were promulgated.