Recombinant tissue factor pathway inhibitor enhances the binding of factor Xa to human monocytes.

Tissue factor pathway inhibitor (TFPI) is a kunitz-type inhibitor of activated factor X (Xa). TFPI was reported to mediate Xa binding to a few of carcinoma cell lines. In this study it was observed that the Xa activity associated with human peripheral blood mononuclear cells (PBMC) incubated with Xa in the presence of recombinant TFPI (rTFPI) was much higher than with Xa alone. Xa activity on PBMC was also observed after whole blood was incubated with pre-formed Xa/TFPI complex. Further studies with flow cytometric analysis demonstrate that rTFPI enhances the binding of Xa to human monocytes. Western blot analysis showed that rTFPI was cleaved into a few of fragments after its incubation with monocytes either in the presence or absence of Xa. Based on these results and the observations reported by others, we speculate that Xa/TFPI complex may bind to human monocytes by a yet unidentified mechanism. The recovery of Xa activity from Xa/TFPI complex on PBMC may be related to the cleavage of rTFPI by Xa and/or monocyte proteases. This observation suggests a new mechanism by which monocytes become procoagulant in some pathological conditions in addition of the well known tissue factor expression on proinflammatic monocytes.

Endothelial cell protein C receptor plays an important role in protein C activation in vivo.

Endothelial cell protein C receptor (EPCR) augments protein C activation by the thrombin-thrombomodulin complex about 5-fold in vitro. Augmentation is EPCR concentration dependent even when the EPCR concentration is in excess of the thrombomodulin. EPCR is expressed preferentially on large blood vessel endothelium, raising questions about the importance of protein C-EPCR interaction for augmenting systemic protein C activation. In these studies, this question was addressed directly by infusing thrombin into baboons in the presence or absence of a monoclonal antibody to EPCR that blocks protein C binding. Activated protein C levels were then measured directly by capturing the enzyme on a monoclonal antibody and assaying with chromogenic substrate. Blocking protein C-EPCR interaction resulted in about an 88% decrease in circulating activated protein C levels generated in response to thrombin infusion. Leukocyte changes, fibrinogen consumption, fibrin degradation products, and vital signs were similar between the animals infused with thrombin alone and those infused with thrombin and the anti-EPCR antibody. The results indicate that EPCR plays a major role in protein C activation and suggest that defects in the EPCR gene might contribute to increased risk of thrombosis.

Peritonitis in the baboon: a primate model which stimulates human sepsis.

The physiological, hemostatic, and immunological responses of 12 chronically instrumented conscious baboons with sepsis due to Escherichia coli peritonitis were compared with that of similarly instrumented controls. Chronic indwelling cannulae were placed in the aorta and pulmonary artery to monitor pressure, cardiac output, and obtain blood samples. At t = 0 a sterile or E. coli-laden fibrin clot containing 1.9-6.7 x 10(11) CFU/kg was introduced into the peritoneal cavity. The control animals were group 1 (n = 3). The animals with peritonitis were divided into three groups depending on their clinical response. Group 2 animals (n = 3) were clinically well at the time of sacrifice (day 14), group 3 (n = 4) survived but were obviously sick on day 14, and group 4 (n = 5) died of sepsis. Implantation of a sterile fibrin clot was well tolerated with little hemodynamic change and a transient minimal inflammatory response in group 1. Implantation of an E. coli-containing clot elicited a hyperdynamic cardiovascular response and evoked a marked inflammatory reaction and a disseminated intravascular coagulopathy. Five of 12 (42%) E. coli animals died from sepsis. In general, the physiological, hemostatic, and immunological disturbances tended to be greatest in these animals. Autopsy revealed residual peritoneal inflammation and varying degrees of inflammation in the lungs, adrenal, spleen, liver, and kidneys in all the animals that received E. coli with the inflammatory infiltrate increasing in severity from group 2 through group 4. Tissue necrosis was observed only in the latter group. We conclude that the cardiovascular, hemostatic, and immunological responses of baboons with sepsis due to E. coli peritonitis exhibit a variable course that resembles the clinical manifestations of gram-negative sepsis in humans.

Comparison of the capacity of rhTNF-alpha and Escherichia coli to induce procoagulant activity by baboon mononuclear cells in vivo and in vitro.

The procoagulant activity of mononuclear cells (MNCs) may play an important role in the disseminated intravascular coagulation seen in septic shock. This study compares the capacity of Escherichia coli (E. coli) and recombinant human TNF-alpha (rhTNF-alpha) to induce procoagulant activity by baboon MNCs. In vivo studies showed that MNC procoagulant activity was significantly increased at T + 120 min after LD100 E. coli infusion into baboons. Most of this procoagulant activity was attributable to tissue factor. In contrast, a bolus infusion of rhTNF-alpha (150 micrograms/kg) and a monoclonal antibody to activated protein C (2 mg/kg) did not induce any increase of MNC procoagulant activity at T + 120 min even though the plasma TNF-alpha level was 10 times higher than that seen following infusion of E. coli. In vitro studies showed that E. coli at concentrations comparable to that observed in the vivo study and LPS at a concentration of 2.5 ng/mL induced more intense tissue factor expression by both human and baboon monocytes than rhTNF-alpha in the concentrations ranging from 10 to 1,000 ng/mL. These results suggest that TNF-alpha alone is not sufficient to induced noticeable MNC procoagulant activity, at least, in the early stage of this septic shock model.

Functional assay of plasma antithrombin using polyethylene glycol (PEG) defibrinated plasma.

Polyethylene glycol(PEG) was used to precipitate fibrinogen to prepare defibrinated plasma in the two stage clotting assay of antithrombin activity. Five percent PEG-8000 precipitated fibrinogen from plasma without loss of antithrombin activity in the defibrinated plasma. Fibrin degradation products(FDP) as high as 640 ug/ml did not interfere the two stage clotting assay using PEG defibrinated plasma possibly because part of FDP was precipitated by PEG in the process of plasma defibrination. The two stage clotting assay was very sensitive to the changes of antithrombin activity in the range of 60%-100% of normal level. The assay was reproducible and correlated with chromogenic assay. The decrease of plasma antithrombin activity in a baboon septic shock model was demonstrated with this assay.

Recombinant E. coli-derived tissue factor pathway inhibitor reduces coagulopathic and lethal effects in the baboon gram-negative model of septic shock.

Excessive coagulation is a typical response to the vascular injury occurring in gram negative sepsis. This study evaluated the pharmacological effects of the use of a recombinant Escherichia coli derived form of tissue factor pathway inhibitor (ala-TFPI) in a baboon model of septic shock. Several doses of ala-TFPI were administered either 30 or 120 min after the initiation of a lethal intravenous infusion of E. coli into baboons. Treatment at 30 min with either 2.7 or 7.4 mg/kg of ala-TFPI resulted in the same survival rates and attenuation of both the coagulation response and cellular injury, as measured by clinical chemistry. When administration of ala-TFPI was delayed for 120 min, a dose of ala-TFPI protein continued to provide a benefit to survival. Ala-TFPI reduced the drop in mean systemic arterial pressure compared to control baboons in addition to partially attenuating the coagulopathic response. Baboons given ala-TFPI also maintained lower levels of plasma interleukin-6 (IL-6) and thrombin-antithrombin. These results suggest that the site of action of the protein may involve the later stage components of the coagulation and inflammatory pathways.

Lethal Staphylococcus aureus-induced shock in primates: prevention of death with anti-TNF antibody.

A successful experimental treatment for gram-positive sepsis to our knowledge has not been achieved. The objectives of this study were to develop a nonhuman primate model of lethal gram-positive sepsis employing the micro-organism Staphylococcus aureus and to determine the efficacy of treatment using monoclonal antibody (MAb) to tumor necrosis factor alpha (TNF). The antibody was administered intravenously, 15 mg/kg, 30 minutes after the beginning of a 2-hour infusion of S. aureus, 4 x 10(10) colony forming units/kilogram. The baboons infused with S. aureus demonstrated the release of the cytokines TNF and interleukin-6 (IL-6), but endotoxin was not observed in the plasma at any time. Treatment with antibody to TNF abolished the rise in serum TNF levels and reduced the increased levels of IL-6. Treatment with MAb to TNF prevented multiple organ failure and achieved permanent (> 7 day) survival of all baboons.

DEGR-factor Xa blocks disseminated intravascular coagulation initiated by Escherichia coli without preventing shock or organ damage.

One of the aims of research in the area of thrombosis has been to design an effective anticoagulant that would function in a predictable and direct manner. In evaluating the role of coagulation in sepsis we used factor Xa blocked in the active center with [5-(dimethylamino)1-naphthalenesulfonyl]-glutamylglycylarginyl+ ++ chloromethyl ketone (DEGR-Xa). We infused 1 mg/kg of DEGR-Xa together with LD100 concentrations of Escherichia coli (4 x 10(10) organisms/kg) into five baboons. As controls, we infused E coli alone into five baboons. The inflammatory, coagulant, and cell injury responses to E coli of both the treated and control groups were lethal and were similar in every respect except for the complete inhibition of the consumption of fibrinogen in the DEGR-Xa group. The half life of DEGR-Xa was approximately 10 hours and 2 hours, as determined by isotopic and enzyme-linked immunosorbent assays, respectively. These results for the first time demonstrate that, although coagulation occurs in E coli sepsis, fibrin formation per se did not influence the lethal outcome in this model. These results also show the effectiveness of DEGR-Xa as an anticoagulant and raise the possibility that it could serve as an alternative to anticoagulants currently in use.

Survival of primates in LD100 septic shock following therapy with antibody to tumor necrosis factor (TNF alpha).

The purpose of this study was to determine the efficacy of treatment with anti-TNF monoclonal antibody in preventing the deleterious effects of sepsis in a nonhuman primate. Experiments were carried out on anesthetized baboons intravenously infused with a lethal dose of Escherichia coli (E. coli). Twelve baboons (six control and six experimental) received 2 hr infusions of E. coli. The experimental group was administered a bolus of anti-TNF antibody, 15 mg/kg, 30 min after beginning the E. coli infusion. Control baboons lived an average of 19 hr (12-34 hr). All antibody-treated baboons survived more than 7 days with a significantly improved quality of life compared to the control group. Although some adverse changes occurred during the monitoring period in surviving baboons, they maintained nearly normal arterial pressures, and serum urea nitrogen and creatinine concentrations. The severe histopathologic changes in lungs, liver, adrenals, kidneys, and spleen documented at death in baboons receiving E. coli only were absent after 7 days in baboons given E. coli and early post-treatment with antibody to TNF.

Staphylococcus aureus-induced shock: a pathophysiologic study.

The purpose of the present study was to determine the effects of lethal intravenous infusions of Staphylococcus aureus (SA) in adult dogs. Animals were maintained under anesthesia for 6 hr and observed until death following the 1-hr infusions of SA organisms. Findings unique to SA administration compared to those with Escherichia coli were the absence of significant necrosis of the mucosal intestinal glands of the small and large intestines; widespread intravascular colonization of bacteria in lung, heart, kidney and adrenal tissues often associated with neutrophil sequestration, microabscess formation, and necrosis; relative constant blood pressure, fibrinogen, fibrin degradation products (FDP), serum glutamic pyruvic transaminase (SGPT), blood (serum) urea nitrogen (BUN), creatinine, pH, and PO2, all of which remained relatively unchanged for 6 hr. Rapid early increases were observed in temperature, respiration rate, lactate, and hematocrit, while PCO2, platelet and white blood cell concentrations decreased. Results suggest unique qualitative differences in responses to Staphylococcal-induced shock compared to those caused by gram-negative bacteria.