Mechanisms of hemorrhage in dengue without circulatory collapse.

To characterize the molecular basis for the hemostatic defects of dengue infections, a study was conducted in Bangkok, Thailand. Febrile children (n = 68) hospitalized with suspected dengue were enrolled before their clinical syndromes were classified as either dengue fever (DF) or dengue hemorrhagic fever (DHF). Hospital course and outcome were recorded; blood was obtained during the febrile illness (S1), after defervescence (S2), and 1 month after onset of disease (S4). Patients were classified as DF (n = 21) and DHF grades 1, 2, and 3; (DHF1, n = 8; DHF2, n = 30; and DHF3, n = 9). All had marked thrombocytopenia. Bleeding scores were assigned on the basis of bleeding site. Although there was no correlation between bleeding scores and pleural effusion index (a measure of vascular leakage) or bleeding scores and platelet counts, there was a correlation between pleural effusion index and platelet counts. Bleeding scores did not correlate with hemostatic data. Activated partial thromboplastin time was prolonged, with trends toward decreased fibrinogen and increased levels of prothrombin fragment F1.2 in the acute-phase samples. However, no factor level was dramatically decreased. We conclude that most patients with DF or DHF, even without overt hemorrhage, have consumptive coagulopathy. Nevertheless, hemorrhage in dengue without circulatory collapse is most likely due to activation of platelets rather than coagulopathy, which is well compensated. Our data suggest that vascular alteration may be the principal factor involved in the association of thrombocytopenia and hemorrhage with disease severity.

Correlation of in vivo and in vitro functions of fresh and stored human platelets.

BACKGROUND: Long-term storage of human platelets has been hindered by the loss of function of the platelets stored under current protocols. Novel preservation methods have encouraged examination of platelet function of cells preserved by cooling and freezing. The function of the platelets was assessed by using both in vitro assays and an in vivo rabbit bleeding model. STUDY DESIGN AND METHODS: Human platelets were stored in the presence or absence of 2 microM: cytochalasin B and 80 microM: EGTA/AM at 4 degrees C for 14 days or by freezing in the presence or absence of 5 percent DMSO. After the storage period, the platelets were resuspended in normal saline and infused into rabbits. Platelet function was assessed in vivo in a kidney bleeding model and in vitro by platelet-induced clot retraction and by platelet aggregation. RESULTS: Platelets stored at either 4 degrees C or -145 degrees C exhibited shorter survival times in the rabbit circulation than did fresh platelets. Platelets cooled to 4 degrees C, in both the presence or absence of cytochalasin B and EGTA/AM treatment, or frozen in the absence of DMSO were not effective in halting bleeding. However, frozen DMSO-treated platelets were as effective as fresh platelets in stopping bleeding. In vitro assays showed that cooled platelets treated with cytochalasin B and egtazic acid/AM and frozen DMSO-treated platelets retained 30 to 40 percent platelet function, while the cooled and frozen control samples exhibited no platelet-induced clot retraction. With thrombin as the agonist, only frozen DMSO-treated platelets exhibited a tendency to aggregate, although at only 22 percent of the aggregation function of fresh platelets. CONCLUSION: It is possible to freeze platelets and retain in vivo efficacy if the cryopreservative DMSO is included in the preparation. In vitro responses were greatly reduced by all of the storage protocols, but it may not be necessary to retain 100 percent in vitro function to have a platelet substitute or storage product that functions satisfactorily in vivo.

Reduction of blood loss by infusion of human platelets in a rabbit kidney injury model.

BACKGROUND: As a first step toward testing the efficacy of stored platelets or platelet substitutes in vivo, a kidney injury model was developed to assess the hemostatic properties of human platelets in normal and thrombocytopenic rabbits. STUDY DESIGN AND METHODS: New Zealand white rabbits were made thrombocytopenic by two consecutive injections of busulfan. Two weeks later, human platelets were transfused to animals whose reticuloendothelial systems were inhibited by the administration of ethyl palmitate. The left kidney was exposed and a slice excised from the anterior pole. The blood was contained in a parafilm boat and absorbed by preweighed gauze to assess blood loss. The percentage of human platelets transfused to the rabbit was determined by flow cytometry on blood collected from the cut site using anti-CD42a (marker for human platelets). The degree of activation of the human platelets was determined using anti-CD62a (marker specific for human p-selectin). RESULTS: Blood loss was similar in normal animals treated with saline alone (35.4 +/- 5.8 g; n = 4); ethyl palmitate and saline (42.5 +/- 5.7 g; n = 6, p = 0.4); or ethyl palmitate and fresh human platelets (45.7 +/- 7.9 g; n = 6, p = 0.3). Bleeding in thrombocytopenic rabbits infused with saline was increased (75.6 +/- 3.9 g; n = 7) as compared with nonthrombocytopenic animals. A significant reduction in blood loss was noted in thrombocytopenic rabbits given fresh human platelets (51.6 +/- 4.5 g; n = 6, p = 0.0023). Transfusion of human platelets to rabbits did not cause activation of the platelets. Furthermore, transfusion of thrombin-activated platelets (60-98% activated) to thrombocytopenic rabbits reduced blood loss (54 +/- 7.3 g; n = 7) to the same extent as fresh platelets. CONCLUSIONS: This is the first report describing a kidney injury model developed to assess the efficacy of fresh and activated human platelets in reducing blood loss in thrombocytopenic rabbits. This model could monitor the efficacy of human platelets prepared by various preservation protocols in suppressing bleeding in rabbits.

Physiologic responses of cross-linked hemoglobin in endotoxin-treated rats.

Purified human cross-linked hemoglobin (alpha alpha Hb) as well as recombinant human hemoglobin is undergoing clinical trials in the setting of acute blood loss and perioperative hemodilution. We have previously demonstrated that in rabbits with circulating plasma Hb, such as alpha alpha Hb, infusion of endotoxin (LPS) impairs myocardial contractility which results in hypotension, tissue hypoperfusion and increased mortality. The untoward cardiovascular effects occurring after the combined infusion of LPS and alpha alpha Hb in this model are similar to those reported for other agents that inhibit nitric oxide (NO) availability. To determine if the deleterious effects of alpha alpha Hb and LPS were species specific, we performed similar studies in rats. Anesthetized Sprague-Dawley rats received LPS (4 mg/kg or 40 mg/kg) alone or in combination with alpha alpha Hb (0.7 g/kg). Mean arterial blood pressures (MAP) increased in the group that received alpha alpha Hb alone (105 +/- 8 to 120 +/- 7 mm Hg, p = 0.2) and a decrease was noted in the groups that received low dose LPS (4 mg/kg, p = 0.5) and high dose LPS (40 mg/kg, p = 0.016). MAP in rats treated with the LPS at either dose combined with alpha alpha Hb remained unchanged. Levels of urine nitrite, which was measured as a surrogate marker for plasma NO, were significantly decreased at 2 hr in groups that received the combination of alpha alpha Hb and LPS at 4 mg/kg (p = 0.022) and 40 mg/kg (p = 0.003). No significant decrease was observed in animals treated only with alpha alpha Hb (p = 0.21) or LPS (4 mg/kg; p = 0.78 and 40 mg/kg; p = 0.65). Survival was evaluated during 72 hr in animals that were infused with high dose LPS (40 mg/kg) alone or in combination with alpha alpha Hb and then allowed to recover. The survival of rats treated with LPS alone or the combination was 29% at the end of 24 hr and was 100% for rats receiving only alpha alpha Hb. The data suggest that the toxicity of alpha alpha Hb appears to be a species specific phenomenon.

Hemostatic efficacy of a fibrin sealant dressing in an animal model of kidney injury.

OBJECTIVE: The purpose of this study is to determine the hemostatic efficacy of a fibrin sealant dressing compared with a standard collagen control dressing in an animal model of kidney injury. METHODS: Twenty adult male Sprague-Dawley rats were administered general anesthesia and underwent partial nephrectomy with heparin anticoagulation (300 U/kg intravenous). Treatment of the cut surface of the kidney was randomized to three groups: group I, no hemostatic agent; group II, collagen dressing; and group III, fibrin sealant dressing. RESULTS: Blood loss was significantly less in group III (3.39+/-0.63 mL) than in group I (8.64+/-2.26 mL) and group II (8.63+/-1.72 mL; p < 0.001). The percentage decrease in the mean arterial pressure was significantly less in group III (34.09+/-15.58%) than in group I (59.66+/-16.19%) and group II (60.35+/-15.66%; p=0.015). CONCLUSION: Fibrin sealant dressings provide effective hemostasis and are superior to collagen dressings in an animal model of kidney injury. Additional development of fibrin sealant dressings for potential clinical use is warranted.

Survival of fresh human platelets in a rabbit model as traced by flow cytometry.

BACKGROUND: In the event of hemorrhage and blood loss, platelets play a vital role in the coagulation process. However, there are currently no acceptable protocols for long-term storage of platelets. As a first step toward testing the efficacy of stored platelets or platelet substitutes in vivo, a flow cytometric technique was developed to detect human platelets in rabbit blood. STUDY DESIGN AND METHODS: Human platelets were transfused to rabbits whose reticuloendothelial system was inhibited by the administration of ethyl palmitate. Because human and rabbit platelets display surface molecules with different epitopes, human platelets were selectively labeled with antibodies specific for glycoprotein IX (CD42a). As this antibody does not label rabbit platelets, it allows discrimination of human from rabbit platelets in samples of rabbit blood containing both types of platelets. RESULTS: Survival of human platelets in rabbits was monitored by flow cytometry and fluorescence microscopy in blood drawn at various times after the platelet transfusion. Fresh human platelets transfused to untreated control rabbits (n = 3) were removed from circulation within 10 minutes of the completion of the transfusion. Fresh platelets (1 day old) transfused to rabbits treated with ethyl palmitate (n = 5) survived for 24 hours with an average half-life of 8.6 hours. In contrast, 8-day-old platelets were cleared from the circulation sooner with an average half-life of 2.9 hours (n = 4). CONCLUSION: This report describes a rapid and efficient method of assessing the survival of human platelets in a rabbit model using flow cytometry. This technique will enable the monitoring in rabbits of human platelets prepared by various preservation protocols.

Cardiovascular toxicity of human cross-linked hemoglobin in a rabbit endotoxemia model.

OBJECTIVE: To determine the possible adverse effects of human cross-linked hemoglobin in endotoxemia. DESIGN: Prospective, controlled, laboratory trial. SETTING: Animal research laboratory. SUBJECTS: New Zealand white rabbits. INTERVENTIONS: Conscious rabbits received intravenous infusions of either lipopolysaccharide (LPS) alone (10 micrograms/kg, Escherichia coli 0111:B4), human hemoglobin cross-linked between the alpha chains (alpha alpha Hb, 0.7 g/kg), or both LPS and alpha alpha Hb. The cardiovascular effects of alpha alpha Hb and LPS as single agents or administered together were then studied in anesthetized rabbits. MEASUREMENTS AND MAIN RESULTS: Mortality in conscious animals that received alpha alpha Hb followed by LPS 4 hrs later (n = 5), or LPS and alpha alpha Hb at the same time (n = 6) was 60% and 67%, respectively. In anesthetized animals, infusion of both LPS and alpha alpha Hb (n = 6) resulted in hypoxia, lactic acidosis, ventricular arrhythmias, and decreased myocardial contractility and left ventricular pressure. In contrast, anesthetized rabbits that received alpha alpha Hb (n = 5) or LPS (n = 5) alone did not develop hypoxia, acidosis, alteration in myocardial contractility, or arrhythmias. Furthermore, death did not occur in any of the conscious animals that received either LPS (n = 7) or alpha alpha Hb (n = 4) as single agents. CONCLUSIONS: In an animal model of nonlethal endotoxemia, infusion of alpha alpha Hb significantly increases mortality. Our data suggest that mortality may be due to the acute increased cardiopulmonary toxicity of alpha alpha Hb in animals with underlying endotoxemia.

Diagnosis of heparin-induced thrombocytopenia in the vascular surgery patient.

BACKGROUND: Recent studies have shown that patients with heparin-induced thrombocytopenia (HIT) form immunoglobulin G (IgG) and/or IgM antibodies directed against a complex of platelet factor 4 (PF4) and heparin. This recognition has resulted in the development of enzyme-linked immunosorbent assays (ELISAs) that use the heparin/PF4 complex as the antigen. This study describes the use of a standardized ELISA to assess antibody formation in five patients suspected of having HIT. METHODS: Five patients received heparin for treatment of arterial or venous thrombotic disorders. All patients had the ELISA performed to detect IgG or IgM antibodies directed against heparin-PF4, as well as the 14C serotonin release assay, when HIT was clinically suspected. RESULTS: HIT was diagnosed in four patients and ruled out in a fifth by using the ELISA. All patients had a 40% decrease in platelet count that returned to normal after heparin cessation. Only one of the four patients who tested positive by ELISA for IgG antibodies also tested positive by the 14C serotonin release assay. Treatment was significantly altered by the ELISA results in all five patients. CONCLUSIONS: It is likely that the ELISA is more sensitive in the diagnosis of HIT than the more traditional aggregation tests, and it may emerge as a new gold standard. Prospective studies in which serial laboratory testing is combined with measurement of clinical outcomes are needed and will eventually provide a greater understanding of the full spectrum of HIT and the clinical settings that precipitate thrombosis in the vascular surgery patient.

A compartmental model depicting short-term kinetic changes in selenium metabolism in ewes fed hay containing normal or inadequate levels of selenium.

Changes in Se metabolism were studied in ewes fed hay containing normal or inadequate levels of Se. After intravenous injection of 75Se-sodium selenite, blood, feces and urine were collected at different times, and the concentrations of labeled and unlabeled Se were determined. Ewes were killed 1, 5, 9 or 14 d after tracer injection, and tissues were obtained for determination of radioactivity and Se concentration. The data were fitted to a compartmental model using the SAAM/CONSAM computer program, and kinetic parameters and steady-state transport rates were estimated. Daily Se intake (Vi) and fecal excretion (VF) were significantly (P < 0.001) higher in the ewes fed normal hay (6.06 +/- 1.09 and 3.36 +/- 0.88 mumol/d, respectively) than in those fed Se-deficient hay (0.64 +/- 0.18 and 0.26 +/- 0.15 mumol/d). The net absorption (Va) of Se was significantly higher in ewes fed normal hay [3.19 +/- 0.82 mumol/d by the balance method, Va = Vi-(VF -Vf) (Vf = endogenous fecal Se) and 1.05 +/- 0.38 mumol/d by using the model (plasma entry rate, U(1))] than in those fed hay deficient in Se [0.57 +/- 0.33 mumol/d (balance) and 0.28 +/- 0.08 mumol/d (model)]. The efficiency of absorption [alpha = U(1) divided by Vi] was significantly higher (0.46 +/- 0.19) in ewes fed Se-deficient hay than in those fed normal hay (0.18 +/- 0.09). Simultaneous fitting of the tracer data of both the groups showed that changes in hepatic extraction and urinary and fecal excretion were sufficient and necessary to account for the kinetic differences observed between treatments.

Recognition and management of heparin-induced thrombocytopenia (HIT) and thrombosis.

An immune response to heparin, which is clinically manifested by the development of thrombocytopenia with or without thrombosis, is stimulated by a complex of heparin with platelet factor 4 (PF4). The primary thrombotic events in patients with heparin-induced thrombocytopenia (HIT) are more frequently venous than arterial. The development of antibodies, however, does not always result in thrombocytopenia or in catastrophic events. The antibodies, which are of the IgG, IgM, and IgA isotypes, can be easily measured by an ELISA that contains a complex of heparin-platelet factor 4 (PF4). Initial antibody formation can be greatly reduced by limiting the exposure to unfractionated heparin or by the use of low-molecular-weight heparin. For those patients who require anticoagulation and who have antibodies to heparin-PF4, danaparoid (Orgaran), a low-molecular weight heparinoid that does not react with the antibodies, is now commercially available; argatroban, a thrombin-specific inhibitor, can also be obtained for compassionate use. The use of these agents during anticoagulation with warfarin is preferable to the simple discontinuation of heparin and intitiation of warfarin, because the latter treatment can result in ongoing thrombosis.

PAI-1-resistant t-PA: low doses prevent fibrin deposition in rabbits with increased PAI-1 activity.

The present study compared the activities of recombinant tissue-type plasminogen activator (t-PA) and a plasminogen activator inhibitor-1 (PAI-1)-resistant variant t-PA (Kt-PA, KHRR 296-299 AAAA) in preventing renal fibrin deposition in rabbits with elevated PAI-1 activity. In this model, all rabbits were infused with endotoxin (10 micrograms/kg), followed by initiation of thrombin (130 U/kg) 4 hours later, when plasma PAI-1 activity was greater than 200 arbitrary units (AU)/mL (baseline, < 3 AU/mL). Thirty minutes after completion of the 1-hour thrombin infusion, rabbits were killed and the kidneys fixed and stained for identification of fibrin deposition. Rabbits received one of the following treatments initiated 30 minutes before thrombin and continued during the 1-hour thrombin infusion: (1) saline (n = 7); (2) low-dose t-PA (17 micrograms/kg, n = 4); (3) higher-dose t-PA (170 micrograms/kg, n = 4); or (4) low-dose Kt-PA (17 micrograms/kg, n = 6). Fibrin deposition occurred in 86% and 100% of the rabbits receiving saline or low-dose t-PA, respectively. Fibrin deposition did not occur in any of the rabbits receiving low-dose Kt-PA or higher-dose t-PA. Low-dose Kt-PA and higher dose t-PA also caused a reduction in fibrin deposition when infused after thrombin administration had been completed. The data provide in vivo evidence that Kt-PA is more effective than t-PA in preventing fibrin deposition in an animal model that combines thrombogenic stimulation with increased PAI-1 activity.

Inhibitory effects of lysine analogues on t-PA induced whole blood clot lysis.

The lysine analogues epsilon-aminocaproic acid (EACA) and trans-4-amino-methyl cyclohexane carboxylic acid (AMCA) are used to prevent excessive bleeding in patients with coagulopathies, such as hemophilia and thrombocytopenia, or in those who have received tissue plasminogen activator (t-PA). However, their relative efficacy in inhibiting lysis of clots that have been formed in the presence of exogenous t-PA or that have been formed and then exposed to exogenous t-PA has not been well characterized. The present study utilized blood from normal volunteers and 125I-fibrinogen in a dilute whole blood clot assay to determine the relative concentrations of lysine analogues required for inhibition of clot lysis induced by exogenous t-PA. AMCA (0.06 mM) and EACA (0.6 mM) were effective in prolonging clot lysis if (1) whole blood clots were formed and then exposed to a lysine analogue and exogenous t-PA or if (2) whole blood clots were formed in the presence of exogenous t-PA and a lysine analogue. However, their inhibitory effect was markedly reduced if clots were formed in the presence of t-PA and then exposed to either of the lysine analogues. The analogues did not inhibit the initial binding of t-PA to fibrin. They did inhibit binding of plasminogen to fibrin as well as the activation of plasminogen by t-PA in the absence of fibrin. The data suggest that lysine analogues, even at low concentrations, reduce the rate of t-PA induced whole blood clot lysis by several mechanisms.

Role of plasminogen activator inhibitor-1 in promoting fibrin deposition in rabbits infused with ancrod or thrombin.

The role of defective fibrinolysis caused by elevated activity of plasminogen activator inhibitor-1 (PAI-1) in promoting fibrin deposition in vivo has not been well established. The present study compared the efficacy of thrombin or ancrod, a venom-derived enzyme that clots fibrinogen, to induce fibrin formation in rabbits with elevated PAI-1 levels. One set of male New Zealand rabbits received intravenous endotoxin to increase endogenous PAI-1 activity followed by a 1-hour infusion of ancrod or thrombin; another set of normal rabbits received intravenous human recombinant PAI-1 (rPAI-1) during an infusion of ancrod or thrombin. Thirty minutes after the end of the infusion, renal fibrin deposition was assessed by histopathology. Animals receiving endotoxin, rPAI-1, ancrod, or thrombin alone did not develop renal thrombi. All endotoxin-treated rabbits developed fibrin deposition when infused with ancrod (n = 4) or thrombin (n = 6). Fibrin deposition occurred in 7 of 7 rabbits receiving both rPAI-1 and ancrod and in only 1 of 6 receiving rPAI-1 and thrombin (P < .01). In vitro, thrombin but not ancrod was inactivated by normal rabbit plasma and by purified antithrombin III or thrombomodulin. The data indicate that elevated levels of PAI-1 promote fibrin deposition in rabbits infused with ancrod but not with thrombin. In endotoxin-treated rabbits, fibrin deposition that occurs with thrombin infusion may be caused by decreased inhibition of procoagulant activity and not increased PAI-1 activity.

Association of protein S deficiency with thrombosis in a kindred with increased levels of plasminogen activator inhibitor-1.

OBJECTIVE: A single kindred in North America with venous thrombosis was described as having defective fibrinolysis because of increased levels of plasminogen activator inhibitor-1 (PAI-1). Our study describes the discovery of protein S deficiency in this kindred and its association with venous thromboembolism. DESIGN: A family study. SETTING: Community. PARTICIPANTS: Twenty-eight adults (ages 21 to 71 years) from three generations of the kindred; seven had a history of venous thromboembolism. MEASUREMENTS: Plasma levels of total and free protein S antigen, as well as the activities of protein S, protein C, PAI-1, and antithrombin III. RESULTS: Six of 7 persons (86%) with a history of venous thromboembolism were deficient in total and free protein S; of 21 asymptomatic members, 9 were deficient in protein S (P = 0.08). When compared with these 9 asymptomatic family members, the 6 persons with protein S deficiency and a history of thrombosis tended to smoke (P = 0.01) and to have higher triglyceride levels (P = 0.001). Overall, the mean PAI-1 activity in the 7 persons who had thrombosis was 7.9 kAU/L (AU/mL) and was 9.3 kAU/L (AU/mL) in the 21 persons who did not have thrombosis (95% CI, -9.9 to 7.0). CONCLUSIONS: In this kindred, a deficiency of total and free or functional protein S is the cause of thrombosis. Measurement of PAI-1 activity was not useful in the evaluation of familial thrombosis. The utility of the routine measurement of PAI-1 activity in the evaluation of familial thrombosis has not been established.

Application of models to determination of nutrient requirements: experimental techniques employing tracers.

This paper concerns some issues involved in the design of tracer experiments for the development of whole-body compartmental models of nutrient metabolism. It focuses on tracer administration protocol, use of multiple tracers, sampling strategy, measurement types and experiment duration in a pragmatic approach to obtaining data suitable for analysis and interpretation with use of models.

Effect of plasminogen activator inhibitor-1 on tissue-type plasminogen activator-induced fibrinolysis.

The effect of fibrin on the interaction of human recombinant single-chain tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) was studied in normal rabbit plasma and in plasma with high levels of native PAI-1. t-PA was added to diluted plasma containing calcium (10 mM) and 125I-fibrinogen at 37 degrees C. Clotting was initiated with human thrombin, and lysis was monitored both turbidimetrically and by release of 125I-fibrin degradation products (fdp). The activity of t-PA (50 IU/ml) was rapidly reduced to 15% of the initial value in plasma containing PAI-1 (23 AU/ml). When thrombin and t-PA were added simultaneously to the plasma, more than 70% of the activity was retained through incorporation of t-PA into the fibrin clot. t-PA-induced fibrinolysis in PAI-1 enriched plasma was further delayed when the temperature was reduced from 37 to 25 degrees C. Turbidimetric and 125I-fdp release data provided complementary information. The former technique traced fiber dissolution, while the latter reflected network integrity. These results indicate that t-PA-induced fibrinolysis in PAI-1 enriched plasma is modulated by the presence of fibrin and by temperature.

Enhancement of tissue plasminogen activator-induced fibrinolysis by activated protein C in endotoxin-treated rabbits.

Endotoxin-treated rabbits produce high levels of plasminogen activator inhibitor-1 (PAI-1), which inhibits fibrinolysis by neutralizing endogenous tissue-type plasminogen activator (t-PA). These animals will develop renal fibrin deposition when infused with ancrod, an enzyme that acts directly on fibrinogen. In normal rabbits with an intact fibrinolytic system, ancrod induces hypofibrinogenemia without fibrin deposition. Rabbit PAI-1 activity can be neutralized by recombinant human t-PA or by bovine activated protein C. The present study determined the efficacy of these two agents used alone or in combination in neutralizing increased PAI-1 activity and in preventing renal fibrin deposition in a rabbit model. Male New Zealand rabbits first received intravenous endotoxin to increase PAI-1 activity. Ancrod was infused intravenously during hour 4 to 5, and the kidneys were examined at hour 5.5. Renal fibrin deposition occurred in 100% (6 out of 6) of the endotoxin-treated rabbits that received ancrod; this was reduced to 14% (1 out of 7) for rabbits receiving t-PA (170 micrograms/kg) before and during the ancrod infusion. Fibrin deposition occurred in only 12% (1 out of 8) of the rabbits that received a 10-fold lower dose of t-PA (17 micrograms/kg) combined with activated protein C (1 mg/kg) before and during the ancrod. Activated protein C at this dose completely neutralized plasma PAI-1 activity. However, low-dose t-PA and activated protein C did not prevent fibrin deposition when used as single agents, with fibrin deposition occurring in 75% and 100% of rabbits, respectively. The data indicate that activated protein C can neutralize plasma PAI-1 activity in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetic modeling of selenium metabolism in nonpregnant ewes.

The kinetics of selenium metabolism in three nonpregnant ewes were studied by the intravenous injection of 75Se-sodium selenite and measurement of radioactivity responses in blood, tissues and excreta. Stable selenium measurements were also made to determine selenium intake, excretion in feces and urine, and mass of selenium in tissues. Immediately following tracer injection, there was a rapid disappearance of radioactivity from plasma reflecting the uptake of the element by the liver and blood cells. The decrease in plasma radioactivity ceased abruptly by 30-45 min, and was followed by an increase to a peak by 3-4 h and a more gradual biphasic decline thereafter. A kinetic model of selenium metabolism in the whole animal was constructed employing the SAAM/CONSAM computer program. The multiphasic response of plasma radioactivity during a physiological steady state was explained on the basis of rapid hepatic uptake of selenium and its subsequent reappearance in the circulation in protein-bound form followed by further metabolism and excretion of the element. The model provides reference parameter values for 75Se-sodium selenite kinetics in selenium-replete, mature nonpregnant ewes for comparison with the kinetics in animals whose selenium status may be altered.