Rapid in vivo induction of leukocyte tissue factor mRNA and protein synthesis following low dose endotoxin administration to rabbits.

INTRODUCTION: Disseminated intravascular coagulation in humans is frequently associated with Gram-negative bacterial sepsis. Therefore, to examine the role and time frame of the in vivo induction of tissue factor (TF) by bacterial endotoxin, a reverse transcription polymerase chain reaction and a solid-phase ELISA assay were developed to monitor the in vivo production in rabbits, of TF mRNA and TF antigen by peripheral blood leukocytes (PBL). METHODS: : Healthy rabbits were injected intravenously with either 1, 10 or 50 microg/kg of Salmonella endotoxin. Blood samples were obtained both before endotoxin administration and at various time points thereafter, up to 24 h. Some experiments were also done to determine whether all-trans retinoic acid would ameliorate the signs of the endotoxin-induced disseminated intravascular coagulation. RESULTS: PBL counts dropped significantly within 2 h of rabbits receiving the endotoxin, recovering to baseline levels by 24 h. Platelet counts decreased gradually over this same time frame. Fibrin deposition was noted in renal glomerular capillaries at 24 h. An increase (P<0.001) in PBL-associated TF mRNA levels was observed 2 h post-endotoxin (10 microg/kg, n = 8), followed by a gradual decline over the subsequent 24 h. The average increase in TF mRNA at 2 h was approximately 4.6-fold (P<0.001) over that seen at time 0. The amount of mononuclear cell associated TF antigen demonstrated a peak at 2 h post-endotoxin (10 microg/kg, n = 13), with levels approximately 9.6-fold greater than (P<0.001) baseline. Pre-treatment of rabbits with all-trans retinoic acid significantly (P<0.001) ameliorated the PBL-associated increase in TF mRNA and TF antigen levels. CONCLUSION: These results suggest that low dose endotoxin (10 microg/kg) faithfully reproduces the non-overt activation of coagulation observed in primates and human volunteers, supporting the hypothesis that TF expression is involved in the in vivo initiation and propagation of disseminated intravascular coagulation. Moreover, all-trans retinoic acid may be effective in modulating in vivo the TF transcription induced by endotoxin.

o-raffinose cross-linked hemoglobin improves the hemostatic defect associated with anemia and thrombocytopenia in rabbits.

Several different preparations of cross-linked hemoglobin (CLHb) are being evaluated for their efficacy and safety as red cell substitutes in a variety of preclinical and clinical settings. Because CLHb is known to sequester nitric oxide (NO) and inhibit NO-mediated processes, we hypothesized that CLHb would have a hemostatic effect by enhancing platelet reactivity, inducing vasoconstriction, or both. Infusion of o-raffinose CLHb shortened the prolonged microvascular bleeding time and decreased blood loss from ear incisions in rabbits rendered anemic and thrombocytopenic. Moreover, this hemostatic effect persisted for at least 24 hours after infusion. Phenylephrine induced a degree of vasoconstriction similar to that induced by CLHb but did not shorten the bleeding time or decrease blood loss, suggesting that vasoconstriction alone cannot account for the hemostatic effect of CLHb. There was no evidence of CLHb-induced activation of coagulation in vivo, since infusion of CLHb did not increase circulating levels of thrombin-antithrombin complex. In vitro, CLHb abolished the inhibitory effect of the NO donor 3-morpholinosydnonimine on platelet aggregation and enhanced the aggregation of stimulated but not resting platelets. This potentiating effect was not attenuated by the addition of superoxide dismutase or catalase. To evaluate the potential arterial thrombogenicity of CLHb, a model of carotid artery thrombosis was developed in rabbits without thrombocytopenia or anemia. Compared with albumin infusion, CLHb infusion shortened the time to complete carotid occlusion. These data suggest that CLHb may shift the thromboregulatory balance toward clot formation, resulting in decreased bleeding in anemic and thrombocytopenic rabbits and possibly increasing arterial thrombogenicity in normal rabbits.

The procoagulant state of the VX-2 tumor in rabbit lung in vivo--relative accumulation of fibrinogen, prothrombin, plasminogen, antithrombin and heparin cofactor II within the tumor.

During their growth, many malignant solid tumors elicit a hemostatic response in the host. In this report, the fluxes of various rabbit plasma hemostatic proteins into pulmonary VX-2 tumors have been measured in vivo. VX-2 cells were contained within a small rabbit plasma clot which was injected intravenously into rabbits. At 28 d, each rabbit was injected intravenously with two radiolabeled (131I, 125I) proteins selected from fibrinogen, prothrombin, antithrombin-alpha, heparin cofactor II, or plasminogen-I or -II. After allowing the labeled proteins to circulate for 10-200 min, each rabbit was perfused with Krebs-Henseleit solution and the lungs excised. Solid tumors were isolated, weighed and measured for radioactivity content. A mean of 14 tumors/pair of lungs with a mean tumor weight of 0.3 g was obtained. Radioactivity per g of tumor was related to radioactivity/ml of blood at exsanguination for each protein at each time interval. Maximum flux rates were calculated (as pmol/g of tumor/min): Fibrinogen, 65.0; prothrombin, 53.0; antithrombin-alpha, 24.1; HCII, 17.2; plasminogen-II, 5.0; and plasminogen-I, 3.2. Using immunocytochemical staining, fibrin(ogen) was distributed heterogeneously within the VX-2 tumor, appearing largely in the perinodular region and in the necrotic core. From the net fluxes of these proteins, the profile of chronic hemostasis associated with the VX-2 tumor was shown to differ markedly from the hemostatic response that occurs after an acute vascular injury.

Psoralen-mediated photodecontamination of platelet concentrates: inactivation of cell-free and cell-associated forms of human immunodeficiency virus and assessment of platelet function in vivo.

BACKGROUND: Treatment of platelet concentrates (PCs) with psoralens and broad-band ultraviolet A (UVA) radiation is being examined for the elimination of pathogens that might be present in donated blood. Previous studies have demonstrated the inactivation of cell-free viruses and the maintenance of platelet integrity with common in vitro assays. STUDY DESIGN AND METHODS: Human immunodeficiency virus (HIV) in three forms-cell-free, activity replicating, and latently infected cell lines-was added to PCs and treated with 50-microgram per mL of 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT), 0.35 mM rutin, and broad- and narrow-band UVA light (320-400 nm and 360-370 nm [UVA1], respectively). The inactivation of added HIV was assessed in tissue culture; platelet hemostatic activity was assessed in thrombocytopenic rabbits. RESULTS: Each form of HIV was inactivated completely (> or = 10(5) infectious units) on treatment with 30 J per cm2 of UVA1 light. Similar results were obtained on treatment of 2.5 mL of PCs in test tubes or intact PC units (50 mL) in blood bags. Latently infected cell lines were substantially more sensitive than cell-free HIV or HIV that was actively replicating. Human platelets treated with 40 J per cm2 of UVA1 light had a fully corrected bleeding time shortly after treatment or after 5 days' storage, as assessed in thrombocytopenic rabbits. Platelet hemostatic function began to decrease with 81 J per cm2 of UVA1 light and was abolished with 113 J per cm2. At similar fluences, broad-band UVA light was more injurious to platelets than was UVA1 light. CONCLUSION: HIV transmission might be eliminated by PCs after treatment with AMT and UVA1 light and without a reduction in platelet hemostatic function.

Treatment of T. cruzi infected human platelet concentrates with aminomethyltrimethyl psoralen (AMT) and ultraviolet A (UV-A) light: preliminary results.

The present measures adopted to prevent transfusion-associated Chagas' disease include screening of blood donors, and/or the inactivation of T. cruzi in collected blood using gentian violet (GV), as a trypanocidal agent. In this study, we investigated the efficacy of the combined use of AMT and UV-A in inactivating T. cruzi in infected human platelet concentrates. Human platelet concentrates were infected with T. cruzi (2 x 10(8)/ml) of the Y strain, transfered to PL 269 (Fenwal Laboratories) containers, and treated with GV (250 micrograms/ml), and ascorbic acid (1 mg/ml); GV, ascorbic acid and UV-A; GV and UV-A; AMT (40 microG/ml) and ascorbic acid; AMT, ascorbic acid and UV-A; AMT and UV-A; UV-A alone; and untreated (control). All UV-A treated platelet concentrates were exposed to UV-A doses of 24, 92, 184, 276, 368 and 644 kJ/m2, and the microscopical research of active T. cruzi was performed, using the microhematocrit technique, 1, 6 and 24 hours after each treatment. A high number of active forms of T. cruzi was observed in all condictions, except when GV was used as the trypanocidal agent, providing evidence of the failure of AMT and UV-A in inactivating T. cruzi in infected human platelet concentrates.

Prevention of transfusion-associated Chagas' disease: efficacy of white cell-reduction filters in removing Trypanosoma cruzi from infected blood.

BACKGROUND: Transfusion-associated Chagas' disease (TA-CD) is a worldwide problem. Measures adopted to prevent TA-CD include the clinical and serologic screening of blood donors and/or the inactivation of Trypanosoma cruzi present in collected blood, using gentian violet as the trypanocidal agent. This study investigated the efficacy of white cell-reduction filters in removing T. cruzi from infected blood. STUDY DESIGN AND METHODS: Human blood was contaminated with 2 or 150 T. cruzi parasites per mL and then left unfiltered or filtered with white cell-reduction filters that provided either 2, 3, or 6 log10 white cell removal. The efficacy of the parasite removal of these filters was evaluated by microscopic enumeration of active forms of T. cruzi both in vivo and in vitro. The in vivo experiments were done in Swiss mice that had been intraperitoneally inoculated with T. cruzi-infected human blood. The in vitro experiments were performed with fresh human blood that had been deliberately contaminated with T. cruzi. RESULTS: The number of parasites seen in mice inoculated with unfiltered blood containing 2 or 150 parasites per mL was significantly higher than the number of parasites seen in mice inoculated with blood from the same sample, but filtered with white cell-reduction filters providing 3 or 6 log10 white cell removal. Fifty to 70 percent of the mice given T. cruzi-infected (2 parasites/mL) filtered blood did not develop T. cruzi infection. In vitro, the use of white cell-reduction filters, providing 2, 3, or 6 log10 white cell removal, significantly reduced the number of parasites seen in culture. CONCLUSION: The present experimental data provide evidence that white cell-reduction filters are effective in reducing the number of parasites in T. cruzi-infected blood and that this efficacy depends, in part, on the concentration of parasites in the artificially infected blood. Properly designed clinical studies of known carriers of T. cruzi must be conducted to determine whether the use of white cell-reduction filters may be an alternative method of reducing the incidence of TA-CD.

Growth enhancement of established tumors by allogeneic blood transfusion in experimental animals and its amelioration by leukodepletion: the importance of the timing of the leukodepletion.

We had reported previously (Blood 81:1880, 1993) that allogeneic blood transfusions (ABT) administered before the infusion of tumor cells in both inbred and outbred experimental animals promote tumor growth and that this effect can be ameliorated by leukodepletion. To better reproduce the human situation, we evaluated, in this present study, the effect of ABT in animals with established tumors using enumeration of pulmonary metastatic nodules as the end point. The role of allogeneic blood component transfusions in promoting tumor growth and the relative efficacy of prestorage versus poststorage leukodepletion of the ABT in preventing tumor growth enhancement were also evaluated. In an inbred murine animal model, C57Bl/6J mice were administered nonleukodepleted allogeneic (ABT), leukodepleted allogeneic (LD-ABT), or syngeneic (SBT) blood transfusions after the intravenous infusion of syngeneic methylcholanthrene-induced fibrosarcoma cells using two different protocols. A significant increase in the number of pulmonary nodules was observed in those mice that received ABT, in both protocols, compared to animals transfused with SBT or LD-ABT. Significantly higher numbers of pulmonary nodules were also seen in mice transfused with allogeneic buffy-coat leukocytes compared with mice that received either nonleukodepleted allogeneic plasma or LD-ABT. In an outbred animal (rabbit) model, recipient rabbits were administered either nonleukodepleted ABT, prestorage LD-ABT, poststorage LD-ABT, or SBT on days +4 and +9 after the infusion of syngeneic epithelial tumor cells. A significant increase in the number of pulmonary nodules was seen in rabbits that received nonleukodepleted ABT compared to animals transfused with SBT. Significantly lower numbers of pulmonary nodules were observed in rabbits that received prestorage LD-ABT compared to animals transfused with poststorage LD-ABT, but no significant difference was seen in rabbits that received poststorage LD-ABT compared with animals transfused with nonleukodepleted ABT. These studies show that ABT promote tumor growth of established animal tumors, that the ABT-induced tumor growth effect is related to the presence of donor allogeneic leukocytes, and that this effect can be ameliorated by prestorage leukodepletion. The present results also provide evidence for the lack of efficacy of poststorage leukodepletion in preventing ABT tumor growth promotion.(ABSTRACT TRUNCATED AT 400 WORDS)

The contribution of the haematocrit to thrombocytopenic bleeding in experimental animals.

Clinical studies in anaemic uraemic patients have shown that increasing the haematocrit with either red blood cell (RBC) transfusions or erythropoietin corrects the prolonged bleeding time (BT) often seen in such individuals. In this present study we evaluated experimentally the effect of the haematocrit on the BT using a microvascular BT technique in New Zealand White rabbits. The correlation between haematocrit and BT was studied in both normal and thrombocytopenic rabbits. In non-thrombocytopenic animals the microvascular BT varied inversely with the haematocrit (r = -0.799); animals with haematocrit levels above 35% having significantly shorter BTs than animals with haematocrit values lower than 35% (P < 0.001). To assess the role of the haematocrit on the BT in thrombocytopenic animals, thrombocytopenia was induced by a combination of gamma-irradiation and heterologous platelet antiserum. Such experiments showed that anaemic rabbits had significantly longer BTs than non-anaemic animals with a similar degree of thrombocytopenia (P = 0.0001). These data thus provide evidence that anaemia contributes significantly to the prolonged BT in both thrombocytopenic and non-thrombocytopenic rabbits, and that RBC transfusions are capable of shortening the BT in thrombocytopenic anaemic animals. While results obtained from animal models cannot necessarily be extrapolated to the clinical situation, the fact that haematocrit influences the BT must be taken into account in the assessment of anaemic patients, particularly those who may have an associated haemostatic disorder.

Platelet concentrates stored for 5 days in a reduced volume of plasma maintain hemostatic function and viability.

BACKGROUND: Platelet concentrates prepared from whole blood are generally suspended in a standard volume of 50 to 60 mL of plasma and can be stored thus at 20 to 24 degrees C for up to 5 days. In vitro studies suggested that this plasma volume could be reduced to 30 to 35 mL without impairing platelet function. STUDY DESIGN AND METHODS: This study evaluated whether platelets stored for 5 days in a reduced volume (30-35 mL) of plasma maintained their in vivo viability, hemostatic function, and recovery in recipients. Paired autologous platelet survival studies were done in 20 adult volunteers to assess platelet viability. A rabbit ear bleeding-time model was used to compare the hemostatic effectiveness of human platelet concentrates stored for 5 days in the standard or reduced volume of plasma. Platelet recovery was compared in thrombocytopenic hospital patients. RESULTS: Paired platelet survival studies indicated no significant difference between the values in platelet concentrates stored for 5 days in the reduced volume of plasma and the values in those stored in the standard volume. In the animal model, there was no significant difference in the bleeding times achieved by either set of platelet concentrates. The platelet count increments in thrombocytopenic patients were measured. The platelet count increments in patients who received reduced-volume platelet concentrates were as good as the increments achieved in patients given standard-volume concentrates. CONCLUSION: The in vivo viability, recovery, and hemostatic function of platelets collected in polyvinylchloride plastic containers and stored in 30 to 35 mL of plasma for 5 days are maintained as well as those of platelets stored in 50 to 60 mL of plasma.

Assessment of the hemostatic effectiveness of human platelets treated with aminomethyltrimethyl psoralen and UV A light using a rabbit ear bleeding time technique.

The photochemical aminomethyltrimethyl psoralen (AMT), in conjunction with UV A light (UVA), has been shown to inactivate human immunodeficiency virus-1 and model viruses in platelet suspensions under conditions that have only a minimal effect on in vitro platelet properties. A rabbit ear bleeding time technique was used to assess the hemostatic effectiveness of human platelet suspensions treated with AMT/UVA. New Zealand White rabbits were made thrombocytopenic by a combination of irradiation and heterologous antirabbit platelet antiserum. Reticuloendothelial function in these rabbits was suppressed by the intravenous administration of ethyl palmitate. The hemostatic function of 1- and 5-day-old human platelet suspensions (14.5% plasma) that had been treated on day 1 with 40 micrograms/mL AMT and 24 kJ/m2 UVA (1 x UVA) was evaluated by measuring microvascular bleeding times after a standard incision. Comparable bleeding times were observed after infusion with both control and AMT/UVA-treated platelets stored for either 1 or 5 days. With the transfusion of AMT/1 x UVA-treated platelets stored for 5 days, the mean (+/- SD) bleeding time was 156.3 +/- 39.2 seconds (n = 10). With untreated platelets (no AMT/no UVA), stored for 5 days, the mean bleeding time was 189.2 +/- 36.4 seconds (n = 10). Neither AMT nor 1 x UVA treatment alone influenced the observed bleeding times. In contrast, the hemostatic effectiveness of human platelet suspensions was diminished if they were exposed to three times the standard UVA dose (72 kJ/m2) on day 1 and stored for 4 more days, regardless of whether AMT was present, with the mean bleeding time increasing to 442.2 +/- 122.6 seconds (n = 15, AMT present) or 396.0 +/- 45.9 seconds (n = 10, AMT absent). These results are consistent with data obtained from in vitro studies and indicate that virucidal AMT/1 x UVA treatment does not influence platelet hemostatic function. However, the final conditions to achieve these results must be carefully controlled.

Experimental animal model of refractoriness to donor platelets: the effect of plasma removal and the extent of white cell reduction on allogeneic alloimmunization.

Platelet transfusion effectiveness may be limited in multiply transfused patients by the development of the refractory state. White cell (WBC)-reduction filters with variable efficiency (1-3 log10 reduction) are available and have been shown to be effective in reducing the incidence of platelet alloimmunization. However, the threshold number of WBCs below which alloimmunization would no longer occur is yet to be determined. A previously established animal model was used to examine the relative efficiency of second- and third-generation filters in reducing the frequency of refractoriness to allogeneic platelets. In this model, California Black rabbits are used as blood donors and New Zealand White rabbits as transfusion recipients. Eight weekly transfusions of either second-generation or third-generation WBC-reduced blood resulted in no difference between the two groups in mean platelet survival and rate of refractoriness to allogeneic platelets. To evaluate the possible incremental benefit of removing supernatant plasma to prevent platelet refractoriness, experiments were performed in which groups of animals were given transfusion(s) with red cell suspensions that had been WBC-reduced or both plasma-depleted and WBC-reduced. A significantly lower rate of allogeneic platelet refractoriness was seen in the rabbits that received WBC-reduced and plasma-depleted red cells than in those that received red cells that had been WBC-reduced only. These data provide evidence that the combined use of plasma depletion and WBC reduction can decrease still further the frequency of refractoriness produced by allogeneic blood transfusions.

Allogeneic blood transfusion-induced enhancement of tumor growth: two animal models showing amelioration by leukodepletion and passive transfer using spleen cells.

Allogeneic blood transfusions have been reported to induce immunomodulation in recipients of blood products. While the mechanism(s) of this immunomodulatory effect is unknown, it has been suggested that this effect of allogeneic blood transfusions could adversely affect patients with a malignant disorder. These concerns have been supported by a number of nonrandomized, mainly retrospective, clinical studies which indicate that allogeneic blood transfusions can adversely affect prognosis following the surgical treatment of oncology patients. Recently, we have shown that allogeneic blood transfusions enhance primary tumor growth and increase metastatic pulmonary nodule formation in inbred mice. The tumor growth-promoting activity of allogeneic blood transfusions was studied also using outbred rabbits. In this present study, we demonstrate that the tumor growth-promoting effect of allogeneic blood transfusions is mediated by donor leukocytes and that this effect can be abolished by their removal before transfusion. We show also that the allogeneic blood transfusion tumor growth-promoting effect can be passively transferred to naive animals (both mice and rabbits) using spleen cells from allogeneically transfused animals. In these experiments, numbers of metastatic pulmonary nodules were significantly increased in both mice and rabbits that had received spleen cells from allogeneically transfused animals compared with those that had received spleen cells from syngeneically transfused animals, or from animals that had been transfused with leukodepleted allogeneic blood.

An animal model of allogeneic donor platelet refractoriness: the effect of the time of leukodepletion.

Approximately 50% of multi-transfused individuals become refractory to random donor platelets. Recent clinical data suggest that those patients receiving leukocyte-depleted blood products are less likely to become refractory to random donor platelets than recipients of non-leukocyte-depleted products. Leukocyte depletion can be performed immediately after collection of a unit of whole blood before its storage (prestorage leukodepletion) or just before the transfusion of the blood product to a recipient, after its storage (poststorage leukodepletion). However, the most appropriate time for the leukodepletion of blood products has not been established. The present study was undertaken to establish an animal model of allogeneic platelet refractoriness, and to compare the effect of prestorage and poststorage leukodepletion on the frequency of refractoriness to allogeneic donor platelets. In this model, two strains of rabbits were used: California Black rabbits were used as blood donors, while New Zealand White rabbits were used as recipients. Eight weekly infusions of nonleukodepleted allogeneic fresh blood resulted in an allogeneic platelet refractory rate of 91.2% (31/34). The prestorage leukodepletion of the donor blood was associated with a significantly higher allogeneic platelet survival and lower refractory rate (33.3%) to allogeneic platelets than poststorage leukodepletion (66.7%). Furthermore, the data suggest that cell-free plasma products are capable of inducing refractoriness to allogeneic donor platelets; the stored plasma having a greater likelihood of inducing such refractoriness than fresh plasma. Thus, these data provide evidence that the prestorage leukodepletion of allogeneic donor blood is associated with a lower frequency of refractoriness and better allogeneic platelet survival than poststorage leukodepletion.