Transmural triglycerides in acute myocardial ischaemia.

The effect of coronary artery occlusion on endogenous triglycerides of left ventricular subepicardium and subendocardium was studied in the open-chest anaesthetised dog. Under control conditions, the subepicardium was found to have a greater concentration of triglycerides than the subendocardium. Thirty minutes after acute coronary artery occlusion there was a decrease followed by a steady increase at 60, 120, and 240 min in subepicardial triglycerides of the ischaemic region. No change in triglycerides in the subendocardium of normal or ischaemic regions was observed. The initial decrease of subepicardial triglycerides in the ischaemic region was blocked by administration of propranolol or bevantolol (CI-775; a specific beta 1 antagonist) given 30 min before occlusion. It is concluded that the effect of coronary artery ligation on transmural endogenous triglycerides is biphasic with an initial period of increased mobilisation followed by a period of increased deposition.

Safety of plasmin in the setting of concomitant aspirin and heparin administration in an animal model of bleeding.

Plasmin is a direct thrombolytic which has been shown to have a strikingly favorable benefit to risk profile in comparison with plasminogen activators, notably tissue plasminogen activator (t-PA). As heparin is known to increase the risk of hemorrhage when co-administered with a plasminogen activator, we asked whether adjunct antithrombotic agents such as aspirin and heparin would affect the safety of plasmin. Three groups of rabbits were administered plasmin at a dose (4 mg kg-1) designed to induce significant decreases in antiplasmin, fibrinogen and factor (F)VIII, to about 25, 40 and 40%, respectively, of baseline values, but not cause prolongation of the ear puncture bleeding time. In a blinded and randomized trial, the results show that an intravenous aspirin bolus plus heparin administered as a bolus followed by a maintenance continuous infusion did not significantly prolong the bleeding time during plasmin infusion. These data indicate that in the rabbit, concomitant use of aspirin plus heparin does not affect the safety of a therapeutic dose of plasmin.

Plasmin induces local thrombolysis without causing hemorrhage: a comparison with tissue plasminogen activator in the rabbit.

The direct fibrinolytic enzyme, plasmin, was compared with tissue plasminogen activator (TPA) in rabbit models of local thrombolysis and fibrinolytic hemorrhage. Plasmin was produced by solid-phase urokinase activation of plasminogen and purified on benzamidine Sepharose. Applied as an intra-arterial infusion into the thrombosed abdominal aorta under conditions of unimpeded blood flow, plasmin (4 mg/kg) and TPA (2 mg/kg) achieved equivalent clot dissolution and flow restoration. Using the model of restricted blood flow into the thrombosed aorta, which limits local plasminogen supply, plasmin was superior to TPA in clot lysis and vascular reperfusion. Using similar dosages of plasmin (2 or 4 mg/kg) and TPA (1 or 2 mg/kg) in the earpuncture rebleed model. TPA induced rebleeding in a dose-dependent manner from prior puncture sites in 9 of 10 animals, while none of the 10 animals exposed to plasmin rebled from these sites. These results suggest that plasmin is an effective, unique thrombolytic agent, distinguished from the plasminogen activators in current usage by its striking safety profile.

Effect of propranolol and nitroglycerin plus methoxamine on transmural creatine kinase activity after acute coronary occlusion.

Transmural creatine kinase activity was determined 5 hours after acute occlusion of the left anterior descending coronary artery in 27 open chest anesthetized dogs. In seven dogs, propranolol, 2 mg/kg, was given intravenously over a 10 minute period 10 minutes after occlusion. In 10 dogs, nitroglycerin, 300 microgram/min, was infused intravenously for 1 hour 10 minutes after occlusion. Methoxamine, 300 to 500 microgram, was administered to return blood pressure and heart rate to prenitroglycerin levels. In untreated dogs, there was a distinct transmural gradient of creatine kinase activity in the ischemic region from subepicardium to subendocardium: nonischemic subepicardium 1,187 +/- 50 international units (IU)/g versus ischemic subepicardium 1,054 +/- 46 IU/g and nonischemic subendocardium 1,170 +/- 53 IU/g versus ischemic subendocard;um 766 +/- 42 IU/g, respectively. Administration of propranolol did not affect the transmural creatine kinase gradient after 5 hours of occlusion. In contrast, nitroglycerin plus methoxamine significantly (P less than 0.05) decreased subendocardial creatine kinase depletion after 5 hours of occlusion (776 +/- 42 versus 978 +/- 47 IU/g). These findings demonstrate the unique capability of nitroglycerin plus methoxamine to protect the subendocardium during ischemic insult.

Thromboxane-blocked swine as an experimental model of severe intravascular inflammation and septic shock.

The cardiopulmonary response elicited by intravenous bacteria or endotoxin is well characterized in swine and has two major components. The first represents the acute pulmonary and broncho-constrictive phase (0-2 h) and the second phase (3-8 h) represents increased microvascular permeability, hypotension, and enhanced leukocyte-endothelial adhesion. The pulmonary vasoconstriction and bronchoconstriction of phase 1 results in acute pulmonary hypertension and airway dysfunction, which may result in rapid mortality. Because this acute pulmonary response may not mimic the development of human septic shock, we sought to block this early phase and examine the role of tumor necrosis factor in the latter septic phase (3-8 h). Employing a thromboxane A2 (TXA2) receptor antagonist (BAY U 3405) in the presence of LD100 Escherichia coli challenge, we blocked the acute pulmonary hypertensive phase and prevented early mortality, however, TXA2 blockade did not affect the latter development of septic shock and death. This latter lethal phase, characterized by prolonged leukopenia, was blocked in a dose-dependent manner by tumor necrosis factor monoclonal antibody. We conclude that the TXA2-blocked E. coli-challenged swine may provide a novel animal model in which to investigate the pathophysiology of acute septic shock.

Steroid pretreatment reduces interleukin-2 toxicity in sheep.

Recombinant interleukin-2 (rIL-2) has shown promise in the treatment of patients with advanced cancer. However, toxicity of this therapy remains a major problem with its use in some patients. In this study we examined whether steroids could reduce the adverse cardiopulmonary effects of rIL-2. Seven sheep were surgically prepared with vascular catheters and lung lymph fistulas. Each sheep received either a single dose of rIL-2 (100 micrograms/kg) or rIL-2 plus methylprednisolone (30 mg/kg) followed by the reverse treatment 1 week later. Lung lymph flow increased markedly after rIL-2 with a peak QL of 140% +/- 30% (above baseline). Steroid pretreatment significantly reduced this lymph flow increase with peak lung lymph flow being only 40% +/- 16% (p less than 0.004). The lymph/plasma protein ratio tended to increase after rIL-2, but these changes were not statistically significant. After rIL-2, cardiac output, heart rate, core temperature, and mean pulmonary artery pressure increased (p less than 0.05), whereas systemic vascular resistance and arterial PO2 decreased (p less than 0.05). These changes did not occur with steroid pretreatment. The results of this study demonstrate that steroids reduced the adverse cardiopulmonary effects of rIL-2. We believe that rIL-2 induces activation of arachidonic acid metabolism, which leads to the production of multiple inflammatory mediators that cause increased microvascular permeability and the adverse cardiopulmonary effects of rIL-2.

Tumor necrosis factor's effects on lung mechanics, gas exchange, and airway reactivity in sheep.

The macrophage- and monocyte-produced cytokine tumor necrosis factor alpha (TNF alpha) has been proposed as a major mediator of endotoxin-induced injury. To determine if TNF alpha could reproduce the effects of endotoxin on the lung, we intravenously administered 10 micrograms/kg of human recombinant TNF alpha into five chronically instrumented unanesthetized sheep on two occasions to characterize the TNF alpha response and its reproducibility. We assessed changes in lung mechanics, pulmonary and systemic hemodynamics, gas exchange, and the number and type of peripheral blood leukocytes. We also determined airway reactivity by use of aerosolized histamine before and after TNF alpha infusion. Pulmonary arterial pressure (Ppa) peaked within 30 min of initiating the TNF alpha infusion [47.7 +/- 2.2 vs. 15.9 +/- 0.4 (SE) cmH2O at base line] and then returned toward base line over 4 h. There was a brief decline in left atrial pressure after TNF alpha. Pulmonary hypertension was accompanied by leukopenia, neutropenia, and increases in the alveolar-arterial O2 difference (AaDO2). Dynamic lung compliance (Cdyn) declined after TNF alpha, reaching a nadir within 15 min of the initiation of the TNF alpha infusion [0.045 +/- 0.007 vs. 0.093 +/- 0.007 (+/- SE) l/cmH2O at base line]. Resistance to airflow across the lung (RL) increased from 1.2 +/- 0.2 cmH2O.l-1.s at base line, peaking at 5.4 +/- 1.3 cmH2O.l-1.s 30 min after the start of the TNF alpha infusion. Alterations in Cdyn and RL persisted for 4 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Human recombinant tumor necrosis factor alpha infusion mimics endotoxemia in awake sheep.

The macrophage-derived cytokine tumor necrosis factor alpha (TNF alpha) has been proposed as the major mediator of endotoxin-induced injury. To examine whether a single infusion of human recombinant TNF alpha (rTNF alpha) reproduces the pulmonary effects of endotoxemia, we infused rTNF alpha (0.01 mg/kg) over 30 min into six chronically instrumented awake sheep and assessed the ensuing changes in hemodynamics, lung lymph flow and protein concentration, and number of peripheral blood and lung lymph leukocytes. In addition, levels of thromboxane B2, 6-ketoprostaglandin F1 alpha, prostaglandin E2, and leukotriene B4 were measured in lung lymph. Pulmonary arterial pressure (Ppa) peaked within 15 min of the start of rTNF alpha infusion [base-line Ppa = 22.0 +/- 1.5 (SE) cmH2O; after 15 min of rTNF alpha infusion, Ppa = 54.2 +/- 5.4] and then fell toward base line. The pulmonary hypertension was accompanied by hypoxemia and peripheral blood and lung lymph leukopenia, both of which persisted throughout the 4 h of study. These changes were followed by an increase in protein-rich lung lymph flow (base-line lymph protein clearance = 1.8 +/- 0.4 cmH2O; 3 h after rTNF alpha infusion, clearance = 5.6 +/- 1.2), consistent with an increase in pulmonary microvascular permeability. Cardiac output and left atrial pressure did not change significantly throughout the experiment. Light-microscopic examination of lung tissue at autopsy revealed congestion, neutrophil sequestration, and patchy interstitial edema. We conclude that rTNF alpha induces a response in awake sheep remarkable similar to that of endotoxemia. Because endotoxin is a known stimulant of TNF alpha production, TNF alpha may mediate endotoxin-induced lung injury.

A murine skeletal muscle ischemia-reperfusion injury model: differential pathology in BALB/c and DBA/2N mice.

Ischemia-reperfusion injuries can occur with diseases such as myocardial infarction and stroke and during surgical procedures such as organ transplantation and correction of aortic aneurysms. We developed a murine model to mimic abdominal aortic aneurysm repair with cross-clamping of the aorta distal to the renal artery. After model development, we compared the normal complement BALB/c mouse with the C5-deficient DBA/2N mouse. To assess quantitative differences, we measured neuromuscular function up to 72 h after ischemia with a subjective clinical scoring system, as well as plasma chemistries, hematology, and histopathology. There were significant increases in clinical scores and creatine phosphokinase, lactate dehydrogenase, and muscle histopathology scores in BALB/c mice compared with those in DBA/2N mice and sham-surgery mice. Muscle histopathology scores of the cranial tibialis and quadriceps correlated well with clinical signs, creatine phosphokinase, and lactate dehydrogenase, and indicated the greatest pathology in these muscle groups. We developed a murine model of skeletal muscle ischemia-reperfusion injury that can utilize the benefits of murine genetic and transgenic models to assess therapeutic principles of this model. Additionally, we have shown a significant reduction in clinical signs, plasma muscle enzyme concentrations, and muscle pathology in the C5-deficient DBA/2N mouse in this model.

Cardiorespiratory and cellular changes with interleukin 2 infusion in sheep.

Recombinant interleukin 2 (rIL-2) administration, a new form of therapy for patients with far-advanced cancer, is associated with a "third space" syndrome, i.e., pulmonary edema, respiratory distress, and hypoxemia, which limits the dose and duration of treatment. To extend our knowledge regarding this toxicity, we established a sheep chronic lung lymph fistula model and measured hemodynamics, arterial blood gases, caudal mediastinal (lung) lymph flow (QL), and blood and lung lymph cellular changes before, during, and after (recovery) a 3-day continuous rIL-2 infusion (9 x 10(5) U/kg). Moderate systemic hypotension, mild pulmonary hypertension, and an increase in alveolar-arterial PO2 gradient was present on day 3 of rIL-2 infusion. QL increased from a base line of 1.9 +/- 0.2 to a maximum of 4.3 +/- 1.1 ml/15 min on day 3 of rIL-2 infusion. At no time was there a change in lymph-to-plasma protein ratio. The leukocyte count increased significantly to 16.1 +/- 4.5 x 10(3) cells/mm3 at recovery day 1. The percentage of blood lymphocytes decreased significantly by day 1 of rIL-2 infusion, returned to base-line levels on day 3, and significantly increased on day 2 of recovery. Lung lymph lymphocytes decreased significantly on days 1 and 2 of rIL-2 infusion. There was a shift in their size; i.e., their area increased from 32 +/- 7 to 57 +/- 19 micron 2 (P less than 0.05) by day 2 of rIL-2 infusion. By day 1 of recovery, lung lymph lymphocyte counts increased significantly.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic interleukin-2 treatment in awake sheep causes minimal or no injury to the lung microvascular barrier.

Interleukin-2 (IL-2) is reputed to cause a "vascular leak syndrome." We studied pulmonary hemodynamics and lymph dynamics in six sheep treated for 7 days with IL-2 (1.8 million IU/kg twice daily or 1.8 million IU/kg each day as a continuous infusion). Lung lymph flow increased from 4.8 +/- 2 ml/15 min pre-IL-2 to 14.4 +/- 6.8 ml/15 min on the seventh day of IL-2. The lymph-to-plasma protein concentration ratio was unchanged (0.70 +/- 0.06 vs. 0.63 +/- 0.13). The plasma-to-lymph equilibration half-time of radiolabeled albumin was 2.0 +/- 0.6 h pre-IL-2 and 1.0 +/- 0.7 h on day 7 of IL-2. Pulmonary arterial pressure was 24 +/- 7 cmH2O pre-IL-2, increased to 32 +/- 4 cmH2O on the fourth day of IL-2, and returned to 29 +/- 5 cmH2O on the seventh day of IL-2. Extravascular lung water was normal (4.07 +/- 0.25 g/g dry lung). To clearly determine whether the increase in lung lymph flow was due to hemodynamic changes or to increased leakiness of the microvascular barrier, we volume loaded six sheep with lactated Ringer solution before and after 3 days of IL-2 treatment (1.8 million IU/kg twice daily). Lung lymph flows increased fivefold during 4 h of crystalloid infusion compared with baseline and were higher after 3 days of IL-2. However, lymph-to-plasma protein concentration ratios decreased to the same low levels pre-and post IL-2 (0.39 +/- 0.06 vs. 0.41 +/- 0.10), indicating and intact microvascular barrier. Extravascular lung water was elevated (5.56 +/- 0.39 g/g dry lung) but was not different from lung water in three volume-loaded control sheep (4.87 +/- 0.53 g/G dry lung). We conclude that IL-2 causes minimal or no injury to the pulmonary microvascular barrier and that volume expansion during IL-2 treatment can cause hydrostatic pulmonary edema.

Effects of interleukin-2 on the pulmonary microvasculature in anesthetized sheep.

Interleukin-2 (IL-2) is reputed to cause pulmonary microvascular injury. We studied the pulmonary and splanchnic microcirculation of anesthetized sheep after one dose (1.8 x 10(6) IU/kg) of IL-2 (n = 9) and after six doses (1.8 x 10(6) IU.kg-1.dose-1) of IL-2 over 3 days (n = 9). Seven control sheep received only 5% dextrose diluent. We measured hemodynamics and lymph dynamics in anesthetized sheep after the final dose of IL-2 or diluent. After one dose of IL-2, caudal mediastinal node (mainly pulmonary) lymph flow was stable, whereas thoracic duct lymph flow increased from a baseline of 54 +/- 6 to 124 +/- 22 ml/h. After 3 days of IL-2, the caudal mediastinal node lymph flow increased from 7.7 +/- 5.5 to 19.0 +/- 14.8 ml/h 5-6 h after the final dose of IL-2, and thoracic duct lymph flow increased from 84 +/- 43 to 143 +/- 42 ml/h. The lymph-to-plasma protein concentration ratio increased after IL-2 for thoracic duct but not for caudal mediastinal node lymph. The equilibration rate of 125I-albumin from plasma to caudal mediastinal node lymph did not change, whereas plasma-to-thoracic duct lymph equilibration was faster after both one dose and 3 days of IL-2. Positron emission tomography showed no increase in the pulmonary transcapillary escape rate for 68Ga-labeled transferrin or in extravascular lung water (n = 4). We conclude that IL-2 at doses two to three times those used clinically does not significantly injure the pulmonary microcirculation of sheep.

Monoclonal antibody to tumor necrosis factor alpha attenuates cardiopulmonary dysfunction in porcine gram-negative sepsis.

Tumor necrosis factor (TNF) is implicated in the pathophysiology of gram-negative sepsis. This study examined physiologic and biochemical effects of pretreatment with an anti-TNF alpha monoclonal antibody immediately before the onset of sepsis. Three groups of anesthetized ventilated pigs were studied for 300 minutes. Groups 1 (n = 12) and 2 (n = 6) received a 1-hour infusion of live Pseudomonas aeruginosa. Group 2 was pretreated with anti-TNF alpha monoclonal antibody (15 mg/kg). Group 3 (n = 8) received intravenous sterile saline. Group 1 exhibited a significant rise in plasma TNF activity, which was abolished in group 2. Cardiac index was reduced in both groups 1 and 2 in the first hour but recovered in group 2 (3.3 +/- 0.4 l/min per square meter at 300 minutes in group 2 vs 1.3 +/- 0.2 L/min per square meter in group 1). Metabolic acidosis was attenuated (arterial pH, 7.39 +/- 0.01 in group 2 vs 7.16 +/- 0.03 at 300 minutes in group 1). Increased extravascular lung water was also attenuated (5.9 +/- 0.7 in group 2 vs 13.2 +/- 1.5 mL/kg at 300 minutes in group 1). However, pulmonary hypertension and hypoxemia, which are known cyclooxygenase effects, were not affected. In the early phase of the study, plasma thromboxane B2 levels were elevated in both groups 1 and 2. We conclude that anti-TNF alpha monoclonal antibody offered significant protection against the effects of sepsis, but that other mediators may be responsible for the early changes seen in this model.

Delayed tumor necrosis factor alpha blockade attenuates pulmonary dysfunction and metabolic acidosis associated with experimental gram-negative sepsis.

OBJECTIVE: To ascertain the effect of delayed tumor necrosis factor alpha (TNF-alpha) on the evolution of systemic and pulmonary injury after the onset of sepsis. DESIGN: Prospective controlled trial. INTERVENTION: Anesthetized swine were made septic with a 1-hour infusion of live Pseudomonas aeruginosa, following which a treatment group received an infusion of anti-TNF-alpha monoclonal antibody (5 mg/kg). Control animals received 0.9% saline. RESULTS: Delayed anti-TNF-alpha treatment had no effect on septic pulmonary hypertension or decline in cardiac output. Late recovery in systemic arterial hypotension was associated with a reversal of arterial acidosis (P < .05 by t test and analysis of variance with Tukey's Studentized Range Test) compared with unprotected septic animals. Septic animals had a significant increase in mean (+/- SEM) plasma lactate levels at 5 hours compared with baseline values (3.8 +/- 0.7 vs 2 +/- 0.4, P < .05), but remained unchanged from baseline following anti-TNF-alpha treatment (1.5 +/- 0.1 vs 1.6 +/- 0.2, not significant). Characteristic septic neutropenia was dramatically reversed by anti-TNF-alpha treatment and was associated with downregulation (P < .05 by t test and analysis of variance) of polymorphonuclear neutrophil (PMN) leukocyte CD18 adhesion receptors and reduction (P < .05 by t test and analysis of variance) in lung PMN sequestration measured by myeloperoxidase activity. The mean (+/- SEM) decrease in bronchoalveolar lavage protein indicated an attenuated permeability injury in anti-TNF-alpha animals (septic animals at 5 hours compared with baseline value, 1044 +/- 270 vs 149 +/- 28 micrograms/mL; control animals at 5 hours compared with baseline value, 217 +/- 83 vs 129 +/- 19 micrograms/mL; P < .05 by t test and analysis of variance). CONCLUSIONS: These data show that delayed anti-TNF-alpha treatment reversed metabolic acidosis associated with sepsis. Furthermore, anti-TNF-alpha treatment reversed septic neutropenia, reduced PMN sequestration, and was associated with attenuated lung injury in a model of fulminant sepsis. This supports evidence of PMN-mediated tissue injury in sepsis and suggests mechanisms for potential therapeutic benefit of anti-TNF-alpha treatment in clinical practice.

Fat emulsion catabolism in vitro and in vivo--sex related differences.

The removal rates of an intravenously administered 10% fat emulsion (Intralipid) from plasma in male and female conscious rats are described. The plasma concentration of fat emulsion particles at various time intervals following a bolus administration (0.2 g/kg) was measured by nephelometry. At the dose employed, the removal of fat emulsion from the plasma followed first order kinetics, ie, a constant fraction was removed from the plasma per unit of time, K2 (%/min). Females exhibited a significantly greater fractional removal rate (K2) than comparably aged males (21.0 +/- 1.0 vs 15.0 +/- 1.4, p less than 0.05). Postheparin lipoprotein lipase, measured using fat emulsion as substrate, also was significantly greater in female rats compared with males. Our results demonstrate that, in rats, fat emulsion (Intralipid) is catabolized more rapidly in females than in males and a greater lipoprotein lipase activity in female rats may be the causative factor.

In vivo biology of recombinant interleukin-2 infusion in sheep: cardiopulmonary manifestations of an intravascular immune-inflammatory response.

We have described the physiologic changes that occur in the conscious sheep during five days of dosing with rIL-2. Major effects noted were a decrease in systemic vascular resistance (vasodilation) accompanied by an increase in cardiac output (blood flow) and heart rate and a mild increase in pulmonary vascular resistance (vasoconstriction). Body temperature increased and may have contributed to the peripheral vasodilation. Lung microvascular fluid and protein flux increased following rIL-2 administration, as evidenced by an increase in lung lymph flow and protein clearance, and circulating levels of leukocytes decreased. These phenomena were temporally related. Since the composite biologic response that occurred following rIL-2 administration was characterized by changes in vascular caliber and blood flow, increased vascular permeability, and margination of leukocytes, we propose that the response be viewed as an intravascular inflammatory reaction.

Tumor necrosis factor monoclonal antibody prevents alterations in leukocyte populations during cardiopulmonary bypass.

Tumor necrosis factor-alpha (TNF-alpha) has been implicated as causing the systemic inflammatory response to cardiopulmonary bypass (CPB) that contributes to the postoperative sequelae of coagulopathy, increased capillary permeability, leukocytosis, fever, and multiple organ dysfunction. To define the role of TNF-alpha on leukocyte populations during CPB, pigs (n = 6) were pretreated with 20 mg TNF-alpha monoclonal murine antibody before normothermic CPB (2 hr) in a blinded prospective randomized study with saline used as a control (n = 6). The leukocyte response to CPB was measured at 10, 30, 60, and 120 min during CPB and at 60 and 120 min after CPB. Repeated measures analysis of variance was performed and the null hypothesis was discarded at the 5% level. The control group displayed the typical leukocyte profile associated with CPB: and initial leukopenia (36% reduction) followed by leukocytosis (11% increase, P = 0.0001). The initial leukopenia was due to a fall in both polymorphonuclear neutrophils (33% reduced, P < 0.05) and monocytes (37% reduced, P < 0.05). In the TNF-alpha monoclonal murine antibody group the total leukocyte profile did not change significantly from baseline, (8.7% reduction to a 16% increase, P = 0.24) nor were there significant changes in populations including neutrophils and lymphocytes. In the treatment group the initial reduction in monocytes was prevented and total circulating monocytes increased during bypass. The experimental data suggest that TNF-alpha may play an important role in the early alterations in leukocyte populations associated with CPB, and TNF-alpha monoclonal murine antibody pretreatment ameliorates the leukocyte response.

Thromboelastography measurements of whole blood from factor VIII-deficient mice supplemented with rFVIII.

The rotational thromboelastography (ROTEG) assay system allows the real-time analysis of clot formation (fibrin formation) in a whole-blood assay format. The ROTEG system provides significant advantages over the current plasma-based assay systems as it includes the important interactions between cellular and plasmatic coagulation factors. We have employed the ROTEG system to characterize clot formation dynamics in factor VIII- deficient mouse whole blood and examined the ability of recombinant FVIII (rFVIII) supplementation to restore the normal phenotype. The ability to generate a clear dose-response relationship by adding rFVIII to FVIII-deficient murine whole blood (FVIII-/-) demonstrates the feasibility of this approach. A dose-response from 1 U to 0.00001 U mL(-1) demonstrates the enhanced sensitivity of the ROTEG system. Further characterization of this experimental approach may provide a potential tool for comparing the activity of FVIII concentrates and/or evaluating FVIII mutants.