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Angiostatin (AST), a polypeptide with potent antiangiogenic properties, is released proteolytically from plasminogen in vivo. Plasminogen exists naturally in plasma as two glycoforms (PLGs), I and II. Recently it was shown with the use of a chick-embryo chorioallantoic membrane (CAM) assay that rabbit PLG-I and -II yield distinct ASTs-AST-I and -II, respectively-with different antiangiogenic activities. AST glycoforms were of similar molecular weight, approximately 30 to 32,000 kD, and probably consisted of kringles 1 to 3 only. AST has now been identified in the interpleural effusate released from VX-2 lung tumors in rabbits. Effusate was collected from six rabbits with high tumor burdens and fractionated by means of lysine-Sepharose chromatography. The epsilon-aminohexanoic acid-eluted protein of all effusates contained AST (kringles 1-3) at a mean concentration of 1.2 microg/mL of effusate; with regard to AST content, 97% was AST-II. A CAM assay revealed that the lysine-Sepharose-bound fraction from all interpleural effusates contained potent antiangiogenic activity. Blood and urine from rabbits with high burdens of VX-2 contained essentially only AST-II, at mean concentrations of 145 and 4 ng/mL, respectively. AST was absent from the blood of control rabbits. In an attempt to compare their uptake by VX-2, iodine 125-labeled AST-I and iodine 131-labeled AST-II were injected intravenously into tumor-bearing rabbits. AST-I entered the tumor 1.6 times faster than AST-II. As a means of accounting for the preponderance of AST-II in the interpleural effusate, we postulate that VX-2 cells release proteolytic activity to activate plasminogen but that of the two PLGs, PLG-II may be the preferred substrate for AST formation in vivo.

A septic reaction occurring during or following the transfusion of cellular blood components was one of the earliest recognized complications of allogeneic blood transfusions. The presence of bacteria in blood products has been a problem for many decades and currently it is probably the most common microbiological cause of transfusion-associated morbidity and mortality. Transfusion-associated sepsis due to contaminated platelet concentrates appears to be much more common than those due to red cells. The overall prevalence of contaminated cellular blood products (red cells and platelets) is approximately one in 3000; however, the transfusion to a recipient of a contaminated blood product may not necessarily be associated with clinically evident morbidity. This is because the majority of contaminated blood product units contain only few bacteria. In other instances, contaminated units may contain large numbers of virulent bacteria as well as endotoxins, and their transfusion may be associated with significant morbidity and even be lethal to the recipient. The prevalence of severe episodes of transfusion-associated sepsis has not been clearly established, but is probably of the order of one in 50,000 per platelet unit and one in 500,000 per red cell unit transfused. As a result of the increased recognition that such transfusion-associated episodes can occur, a variety of measures have been proposed to try to prevent and/or control the risk of transfusion-associated septic reactions.

Severe transfusion reactions associated with bacteria and/or their products, during or following a blood transfusion, were one of the earliest recognized complications of allogeneic blood transfusions. Bacterial contamination of blood products has thus been a problem for many decades and at present is likely the most common microbiological cause of transfusion-associated morbidity and mortality. Transfusion-associated sepsis due to contaminated platelet concentrates appears to be much more common than that due to contaminated red blood cells. The overall incidence of contaminated cellular blood products is approximately 1 in 3,000. However, transfusion to a recipient of a contaminated platelet unit may not necessarily be associated with clinically apparent morbidity, because the majority of contaminated platelet units contain relatively few organisms. In a minority of instances, contaminated units contain large numbers of potentially virulent bacteria, as well as endotoxins, and their transfusion is often associated with significant recipient morbidity and mortality. The incidence of severe septic episodes has not been clearly established, but is probably of the order of 1 per 50,000 platelet units transfused. With heightened awareness in recent years of the possibility that platelet transfusion-associated septic episodes can occur, a variety of measures have been proposed, and in some cases implemented, to try to prevent and control this transfusion risk., (Copyright 2001 by W.B. Saunders Company.)

In the human circulation, factor VII is present in relatively low plasma concentration (0.01 microM) and has been reported to have a short half-life (t(1/2); 6 h). In contrast, prothrombin is present in a relatively high plasma concentration (2 microM) and has a relatively long catabolic half-life (t(1/2) = approximately 2-3 days). This report examines the metabolic characteristics of purified rabbit plasma factor VII and prothrombin, radiolabeled with (125)I and (131)I, respectively, in healthy young rabbits. From the plasma clearance curves of protein-bound radioactivities, fractional catabolic rates and compartmental distributions were calculated using a three-compartment model. Turnover of factor VII within the intravascular space (2.95 days) exceeded that of prothrombin (1.9 days). However, the whole body fractional catabolic rate of factor VII (0.34 days(-1); catabolic t(1/2) = 2.04 days) was significantly slower than that of prothrombin (0.53 days(-1); t(1/2) = 1.31 days). Furthermore, the fractional distributions of factor VII in the intravascular (0.14) and extravascular compartments (0.76) differed from those of prothrombin (0.29 and 0.53). Absolute quantities of factor VII and prothrombin catabolized by a 3-kg rabbit amounted to 0.18 and 24.0 mg/day, respectively (molar ratio of prothrombin to factor VII = 100). The molar ratio of catabolism was compared with the release rates of factor VII and prothrombin from rabbit livers perfused ex vivo. After correction for uptake of factor VII and prothrombin by the liver, the molar ratio of released prothrombin to factor VII in the perfusate was approximately 293:1 over a 0.25- to 3-h interval. These results indicate that, compared with prothrombin, factor VII in the healthy rabbit circulates as a relatively long-lived protein. This behavior does not reflect that reported for factor VII in the human circulation.

Background: Correcting the decrease in oxygen delivery from anemia using allogeneic RBC transfusions has been hypothesized to help with increased oxygen demands during weaning from mechanical ventilation. However, it is also possible that transfusions hinder the process because RBCs may not be able to adequately increase oxygen delivery. In this study, we determined whether a liberal RBC transfusion strategy improved outcomes related to mechanical ventilation., Methods: Seven hundred thirteen patients receiving mechanical ventilation, representing a subgroup of patients from a larger trial, were randomized to either a restrictive transfusion strategy, receiving allogeneic RBC transfusions at a hemoglobin concentration of 7.0 g/dL (and maintained between 7.0 g/dL and to 9.0 g/dL), or to a liberal transfusion strategy, receiving RBCs at 10.0 g/dL (and maintained between 10.0 g/dL and 12.0 g/dL). The larger trial was designed to evaluate transfusion practice rather than weaning per se., Results: Baseline characteristics in the restrictive-strategy group (n = 357) and the liberal-strategy group (n = 356) were comparable. The average durations of mechanical ventilation were 8.3 +/- 8.1 days and 8.3 +/- 8.1 days (95% confidence interval [CI] around difference, - 0.79 to 1.68; p = 0.48), while ventilator-free days were 17.5 +/- 10.9 days and 16.1 +/- 11.4 days (95% CI around difference, - 3.07 to 0.21; p = 0.09) in the restrictive-strategy group vs the liberal-strategy group, respectively. Eighty-two percent of the patients in the restrictive-strategy group were considered successfully weaned and extubated for at least 24 h, compared to 78% for the liberal-strategy group (p = 0.19). The relative risk (RR) of extubation success in the restrictive-strategy group compared to the liberal-strategy group, adjusted for the confounding effects of age, APACHE (acute physiology and chronic health evaluation) II score, and comorbid illness, was 1.07 (95% CI, 0.96 to 1.26; p = 0.43). The adjusted RR of extubation success associated with restrictive transfusion in the 219 patients who received mechanical ventilation for > 7 days was 1.1 (95% CI, 0.84 to 1.45; p = 0.47)., Conclusion: In this study, there was no evidence that a liberal RBC transfusion strategy decreased the duration of mechanical ventilation in a heterogeneous population of critically ill patients.

Despite the many advances in the safety, processing, and storage of conventional 22 degrees C liquid-stored allogeneic platelet concentrates, there still are significant drawbacks to the use of such products. Efforts to overcome these shortcomings have resulted in an array of novel platelet products, substitutes, and alternatives; which are currently at various stages of development. This review summarizes the recent developments in the frozen and cold storage of platelets; their pathogen inactivation; as well as the status of lyophilized platelets, infusible platelet membranes (IPMs), red cells bearing arginine-glycine-aspartic acid (RGD) ligands, fibrinogen-coated albumin microcapsules, and liposome-based agents; as potential alternatives to the use of conventional platelet transfusions. Pre-clinical studies have been encouraging for several of these novel products; however, to date, very few have entered human trials. Nonetheless, with the ongoing development of diverse products, those properties that may be necessary for their hemostatic effectiveness will become apparent. However, safety and efficacy must be demonstrated in pre-clinical and phase I to III clinical trials before these novel agents, substitutes and alternatives can be used clinically for patients with thrombocytopenia.

Although the hemoglobin level of 100 g/L has been used for many years as the allogeneic red blood cell (RBC) transfusion trigger, current evidence indicates that for most patients a more restrictive transfusion strategy is at least as effective as and possibly superior to a liberal transfusion strategy. Moreover, the available data indicate that the use of smaller volumes of allogeneic RBCs may be associated with decreased risk of morbidity and mortality. Thus several recent studies indicate that the use of more restrictive triggers than 100 g/L does not appear to adversely affect patient outcomes. Indeed, the majority of recently published RBC transfusion guidelines recommend a more conservative and cautious approach to allogeneic RBC transfusion practice, primarily to reduce the risk of transfusion-related adverse effects. However, the available transfusion trigger studies do not provide sufficient data to allow the claim that the improved outcomes observed are the sole result of the transfusion strategy used. It is possible that the results are the consequence of effects yet to be defined clearly. Additional studies will be necessary to determine the effects of RBC storage time and the presence of allogeneic leukocytes in allogeneic RBC transfusion practice. Nonetheless, the available data, together with detailed information about alternatives to blood product transfusions, will enable physicians to improve outcomes in transfused patients.

Objective: To compare a restrictive red blood cell transfusion strategy with a more liberal strategy in volume-resuscitated critically ill patients with cardiovascular disease., Setting: Twenty-two academic and three community critical care units across Canada., Study Design: Randomized controlled clinical trial., Study Population: Three hundred fifty-seven critically ill patients with cardiovascular diseases from the Transfusion Requirements in Critical Care trial who had a hemoglobin concentration of <90 g/L within 72 hrs of admission to the intensive care unit., Interventions: Patients were randomized to a restrictive strategy to receive allogeneic red blood cell transfusions at a hemoglobin concentration of 70 g/L (and maintained between 70 and 90 g/L) or a liberal strategy to receive red blood cells at 100 g/L (and maintained between 100 and 120 g/L)., Results: Baseline characteristics in the restrictive (n = 160) and the liberal group (n = 197) were comparable, except for the use of cardiac and anesthetic drugs (p <.02). Average hemoglobin concentrations (85 +/- 6.2 vs. 103 +/- 6.7 g/L; p <.01) and red blood cell units transfused (2.4 +/- 4.1 vs. 5.2 +/- 5.0 red blood cell units; p <.01) were significantly lower in the restrictive compared with the liberal group. Overall, all mortality rates were similar in both study groups, including 30-day (23% vs. 23%; p = 1.00), 60-day, hospital, and intensive care unit rates. Changes in multiple organ dysfunction from baseline scores were significantly less in the restrictive transfusion group overall (0.2 +/- 4.2 vs. 1.3 +/- 4.4; p =.02). In the 257 patients with severe ischemic heart disease, there were no statistically significant differences in all survival measures, but this is the only subgroup where the restrictive group had lower but nonsignificant absolute survival rates compared with the patients in the liberal group., Conclusion: A restrictive red blood cell transfusion strategy generally appears to be safe in most critically ill patients with cardiovascular disease, with the possible exception of patients with acute myocardial infarcts and unstable angina.

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.

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 first epidermal growth factor-like domain (EGF-1) of factor VII (FVII) provides the region of greatest contact during the interaction of FVIIa with tissue factor. To understand this interaction better, the conformation-sensitive FVII EGF-1-specific monoclonal antibody (mAb) 231-7 was used to investigate the conformational effects occurring in this region upon both FVII activation and active site occupation. The binding affinity of mAb 231-7 was approximately 3-fold greater for the zymogen state than for the active state; a result affected by the presence of both calcium and the adjacent Gla domain. Once activated, active site inhibition of FVIIa with a variety of chloromethyl ketone inhibitors resulted in a 10-fold range of affinities of FVIIai molecules to mAb 231-7. Gla domain removal eliminated this variation in affinity, suggesting the involvement of a Gla/EGF-1 interaction in this conformational effect. In addition, the binding of mAb 231-7 to FVIIa EGF-1 stimulated the amidolytic activity of free FVIIa. Taken together, these results imply an allosteric interaction between the FVIIa active site and the EGF-1 domain that is sensitive to variation in active site occupant structure. Thus, these present studies indicate that the conformational change associated with FVII activation and active site occupation involves the EGF-1 domain and suggest potential functional consequences of these changes.

Antithrombin (AT) is a plasma protein with four sites of N-linked glycosylation. Asn 135 is incompletely glycosylated, and the resulting 3-glycan AT is cleared more rapidly in vivo than the 4-glycan form. The Asn codons in each of the four sites of glycosylation were altered in turn, to create four mutant rabbit AT cDNAs. Permanently transfected CHO cell lines were generated following transfection of the resulting constructs, encoding either the wild-type rabbit AT (AT-WT) or one of the four underglycosylated variants (AT-N96Q, AT-N135Q, AT-N155Q, and AT-N155Q). Comparison of the five resulting recombinant AT proteins revealed that the major AT species of each variant co-migrated on SDS gels, and migrated more rapidly than the major form of AT-WT. The shift in mobility, from 60 to 57 kDa, was consistent with the loss of one fully sialylated complex N-linked glycan. Neither the amount of AT secreted (range: 1.25 to 4.2 microg/10(6) cells/day) nor the kinetics of secretion differed significantly between cell lines expressing AT-WT or any of the AT variants. All forms of recombinant rabbit AT were capable of forming denaturation-resistant complexes with thrombin. Purification and radioiodination of each of the five recombinant AT proteins permitted pharmacokinetic analysis of their individual clearance in rabbits. While neither the equilibration half-life (t(0.5)alpha) nor the terminal catabolic half-life (t(0. 5)beta) differed significantly between plasma-derived rabbit AT and AT-WT, the t(0.5)beta of all the underglycosylated variants was decreased relative to that of AT-WT (maximum reduction in mean: from 70.1+/-3.2 h to 52.4+/-2.5 h). These results suggest that the overall extent of glycosylation, rather than the location within AT of the glycan chains, is a primary determinant of AT clearance.

To facilitate studies of the in vivo role of the extrinsic pathway of coagulation in experimental hemostasis and thrombosis, a full-length cDNA-encoding rabbit factor VII was isolated using polymerase chain reaction-mediated DNA amplification from plaque-purified lambda gt11 phage. Repeated DNA sequencing of both full-length rabbit factor VII cDNA and shorter cDNA fragments verified four changes in the previously reported amino acid sequence of mature rabbit factor VII, now predicted to be 405 amino acids in length. Rabbit factor VII cDNA was transfected into human embryonic kidney 293 cells and a cell line that permanently expressed rabbit recombinant factor VII was established. Rabbit recombinant factor VII was purified from tissue culture media using a combination of barium citrate precipitation, DEAE-sepharose FF chromatography, benzamidine agarose, and affinity chromatography using a sheep antirabbit factor VII polyclonal antibody. The purity and authenticity of rabbit recombinant factor VII was confirmed by polyacrylamide gel electrophoresis and Western blot analysis. Homogeneous rabbit recombinant factor VII was fully active biologically as determined by prothrombin time assay in factor FVII-depleted plasmas, of both human and rabbit origin, using either human or rabbit thromboplastin. Rabbit recombinant factor VII should prove useful for future in vivo investigations of experimental coagulopathies.

Despite many advances in the safety, processing and storage of conventional 22 degrees C liquid-stored allogeneic platelet concentrates, there are still significant drawbacks to standard platelet concentrates used in transfusions for patients with thrombocytopenia. Efforts to overcome these shortcomings have been undertaken in both academic and commercial settings, resulting in an array of novel platelet products and substitutes that are currently at various stages of development. This review summarises the recent developments in lyophilised platelets, infusible platelet membranes (IPM), red cells bearing arginine-glycine-aspartic acid (RGD) ligands, fibrinogen-coated albumin microcapsules and liposome-based agents as putative alternatives to conventional transfusions involving allogeneic platelet concentrates. These various products are designed to replace the use of allogeneic donor platelets with modified or artificial platelets, to augment the function of existing platelets and/or provide a procoagulant material capable of achieving primary haemostasis in patients with thrombocytopenia. Preclinical studies have been encouraging for several of these platelet substitutes and novel platelet products, however, to date, only a few of these products have entered human trials. With the ongoing development of these diverse products, properties necessary for haemostatic effectiveness will become apparent. Safety and efficacy, however, must be demonstrated in preclinical and Phase I - III clinical trials, before these novel agents can be used clinically for patients with thrombocytopenia.

To determine whether the factor V Leiden mutation is associated with decreased bleeding in individuals with severe hemophilia A, factor concentrate utilization, maximum annual number of bleeding episodes, and the prevalence of hemophilic arthropathy between carriers and non-carriers of the factor V Leiden mutation were compared. Heterozygosity for the factor V Leiden mutation was found in 6 of 137 subjects (4.4%). Carriers of the factor V Leiden mutation utilized less factor concentrate (geometric mean: 310 vs. 1185 units/kg/year) and had fewer bleeding episodes than non-carriers (proportion with 10 or fewer bleeding episodes in their worst year: 50 vs. 11%). However, the factor V Leiden mutation was not associated with the absence of arthropathy. The intron 22 inversion mutation of the factor VIII gene was tested for in a subgroup of 80 subjects, but it was not found to be a significant variable for any of the bleeding endpoints. The results of this small study are consistent with the hypothesis that the factor V Leiden mutation imparts a protective effect; however, a larger confirmatory study in which the factor VIII molecular defects can be controlled for is needed. Furthermore, most severe hemophiliacs who used fewer than 200 units/kg/year of factor concentrate or who had experienced 10 or fewer bleeding episodes per year did not carry the factor V Leiden mutation, suggesting that the proportion of severe hemophiliacs whose mild clinical course can be attributed to the factor V Leiden mutation is small.

Many experimental approaches have been explored to produce hemostatically active novel human platelet products and substitutes capable of long-term storage. These include: platelet storage in the frozen state; storage in the cold (4 degrees C) in the liquid state; photochemical methods for the inactivation of viruses, bacteria and protozoa; infusible platelet membranes; and rehydrated lyophilized platelets. In addition to products using human platelets as their primary manufacturing source, other approaches have been used to make non-platelet-derived substitutes that might be capable of in vivo hemostasis. These include production of red blood cells, or liposomes, bearing hemostatically active agents on their surfaces; and fibrinogen-coated albumin microcapsules or microspheres. The development of platelet substitutes is an increasing scientific and technological endeavor and there are many reasons to believe that platelet substitutes will become more efficacious and safer to use over time. Ultimately, such work will result in safe and effective platelet substitutes being available for clinical use in thrombocytopenic or other patients with increased risk for bleeding.

Transfusion-associated septic reactions occurring during or following the transfusion of cellular blood components was one of the earliest recognised complications of allogeneic blood transfusions. The presence of bacteria in cellular blood products thus has been a problem for many decades and currently it is the most common microbiological cause of transfusion-associated morbidity and mortality. Transfusion-associated septic reactions due to contaminated platelet concentrates appear to be much more common than those due to red cell concentrates. The prevalence of contaminated cellular blood products (red cells and platelets) is approximately 1 in 2,000. However, the transfusion to a recipient of a contaminated blood product may not be associated with morbidity, because many contaminated blood product units contain only few bacteria and such transfusions may be innocuous to the recipient. In other instances, contaminated blood product units may contain large numbers of virulent bacteria and endotoxins, and their transfusion may be associated with significant morbidity and may even be lethal to the recipient. The prevalence of severe episodes of transfusion-associated sepsis has not been clearly established, but is probably of the order of 1 in 50,000 for platelet units and 1 in 500,000 for red blood cell units transfused. As a result of the increased recognition that such transfusion-associated septic episodes can occur, a variety of measures have been proposed to try to prevent and/or control the rate of contamination of blood products.

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.

Background: To determine whether a restrictive strategy of red-cell transfusion and a liberal strategy produced equivalent results in critically ill patients, we compared the rates of death from all causes at 30 days and the severity of organ dysfunction., Methods: We enrolled 838 critically ill patients with euvolemia after initial treatment who had hemoglobin concentrations of less than 9.0 g per deciliter within 72 hours after admission to the intensive care unit and randomly assigned 418 patients to a restrictive strategy of transfusion, in which red cells were transfused if the hemoglobin concentration dropped below 7.0 g per deciliter and hemoglobin concentrations were maintained at 7.0 to 9.0 g per deciliter, and 420 patients to a liberal strategy, in which transfusions were given when the hemoglobin concentration fell below 10.0 g per deciliter and hemoglobin concentrations were maintained at 10.0 to 12.0 g per deciliter., Results: Overall, 30-day mortality was similar in the two groups (18.7 percent vs. 23.3 percent, P= 0.11). However, the rates were significantly lower with the restrictive transfusion strategy among patients who were less acutely ill -- those with an Acute Physiology and Chronic Health Evaluation II score of < or =20 (8.7 percent in the restrictive-strategy group and 16.1 percent in the liberal-strategy group; P=0.03) -- and among patients who were less than 55 years of age (5.7 percent and 13.0 percent, respectively; P=0.02), but not among patients with clinically significant cardiac disease (20.5 percent and 22.9 percent, respectively; P=0.69). The mortality rate during hospitalization was significantly lower in the restrictive-strategy group (22.3 percent vs. 28.1 percent, P=0.05)., Conclusions: A restrictive strategy of red-cell transfusion is at least as effective as and possibly superior to a liberal transfusion strategy in critically ill patients, with the possible exception of patients with acute myocardial infarction and unstable angina.

We describe a novel, de novo point mutation in one antithrombin (AT) allele resulting in type I AT deficiency and thrombophilia. Low plasma AT activity as well as low plasma AT antigen were documented in the propositus, but not in the parents, or in a male sibling. AT gene analysis by sequencing polymerase chain reaction-amplified genomic DNA from exon 5 of the propositus revealed a novel point mutation, GAG-->TAG at codon 271, resulting in a stop codon (Glu271STOP). This mutation was not demonstrable in the other members of his immediate family. DNA marker polymorphism analysis indicated the expected parentage. Based on allele frequency data for Caucasians in the United States the cumulative paternity index, or CPI, for the propositus and his father is 219,077. This corresponds to a probability of paternity of 99.9995% based on a prior probability of 50%. Included in this analysis is a linkage analysis of a trinucleotide repeat in intron 5 of the AT gene of the various family members, which also confirmed maternity and paternity. These studies provide documentation of the first spontaneous mutation of an AT gene in a thrombophilic individual, resulting in a type I AT deficiency.

The initiation of a denuding injury to the vascular endothelium rapidly leads to a deposition of platelets and fibrin at the site of injury. We have measured previously the responses of rabbit fibrinogen, prothrombin, and antithrombin to a deendothelializing balloon-catheter injury to the rabbit aorta in vivo. In this study, rabbit iodine 125-labeled HCII and iodine 125-labeled AT were coinjected intravenously into anesthetized rabbits 5 minutes before deendothelialization of the thoracic aorta. The rabbit was exsanguinated at 5 to 60 minutes after injury, the aorta was excised, and the accumulation of each radiolabeled protein in each layer of aorta wall was determined relative to the concentration of the respective native protein in circulating blood at exsanguination. The maximum flux rates into the aorta wall (i.e., platelet layer and intima-media) in the first minute after injury were calculated from the uptake data; approximately 2.8 molecules of AT accumulated for each HCII molecule. By comparison with previous measurements, the maximum flux rate of AT was similar to that of prothrombin. Further, the molar ratio of accumulated prothrombin/AT + HCII) in the aorta wall was 0.75. Detergent extracts of the injured aorta intima-media contained unreacted HCII and HCII complexes; the uninjured aorta contained only unreacted HCII. By contrast, high molecular weight AT complexes and unreacted AT were extracted from the uninjured, and in greater quantity from the injured, aorta wall. We conclude that, of the plasma antithrombins, AT accumulated more rapidly than HCII in vivo and appeared to be the more active inhibitor at the site of vascular injury. HCII may play a relatively minor role as an antithrombin and possibly only after injury.

Thrombin is inhibited by its cognate plasma inhibitor antithrombin, through the formation of covalent thrombin-antithrombin (TAT) complexes that are found as ternary complexes with vitronectin (VN-TAT). To determine whether the metabolism of VN-TAT ternary complexes is different from that previously reported for binary TAT complexes, plasma clearance studies were done in rabbits using human VN-TAT. 125I-VN-TAT was shown to be cleared rapidly from the circulation (t1/2alpha = 3.8 min) in a biphasic manner mainly by the liver. 125I-TAT had a similar initial clearance (t1/2alpha = 5.3 min) but had a significantly faster beta-phase clearance (t1/2beta = 42.8 min versus 85.4 min for VN-TAT; p = 0.005). Protamine sulfate and heparin abolished the rapid initial alpha-phase of 125I-VN-TAT clearance and reduced its liver-specific association and in vivo degradation. Heparin also reduced the alpha-phase clearance of 125I-TAT and was associated with the appearance of high molecular weight complexes, suggesting enhanced complex formation between VN and TAT. 125I-VN-TAT binding to HepG2 cells was reduced by competition with VN-TAT or heparin but to a much lesser extent in the presence of TAT. The binding of VN-TAT to HepG2 cells was not inhibited by competition with the low density lipoprotein receptor-related protein ligand, methylamine-alpha2-macroglobulin. 125I-VN-TAT binding was also inhibited by treating HepG2 cells with heparinase or by growing the cells in the presence of beta-D-xyloside. Finally, both heparin and chloroquine, but not methylamine-alpha2-macroglobulin, reduced the internalization and degradation of VN-TAT by HepG2 cells. Taken together, these data indicate the importance of VN in TAT metabolism and demonstrate that VN-TAT binds to liver-associated heparan sulfate proteoglycans, which mediate its internalization and subsequent intracellular degradation.

Recognition that allogeneic transfusion associated immunomodulation can increase morbidity in allogeneically transfused individuals has become a major concern for those involved in transfusion medicine. However, whether allogeneic blood transfusions predispose recipients to increased risk for cancer recurrence or to bacterial infections is still unproven. In contrast, data from studies in experimental animal models suggest that allogeneic blood transfusion associated immunomodulation is an immunologically mediated biological effect which is associated primarily with the infusion of allogeneic leukocytes. Moreover, the available experimental animal data suggest that pre-storage leukoreduction, as opposed to post-storage leukodepletion, is effective in ameliorating this tumor growth-enhancing effect of allogeneic blood. While considerable data have accumulated in an attempt to unravel the mechanism of the immunomodulatory effect of allogeneic blood transfusions, the precise mechanism of this effect has not yet been elucidated.

We have previously described a kindred with factor VII (FVII) deficiency whose members exhibited reduced procoagulant activity relative to FVII antigen concentration. In this report, the molecular genetic basis of the FVII defect has been determined to be a heterozygous substitution of Asp for Asn at position 57 in the first epidermal growth factor (EGF) domain. Recombinant FVII (N57D) cDNA was created by site-directed mutagenesis and transiently expressed in human 293 cells. The transfected cells synthesized an immunoprecipitable protein with an apparent molecular weight of 50 kD. Quantitation of expression by FVII enzyme-linked immunosorbent assay indicated that mutant protein yields were consistently low, typically 10% to 30% of wild-type FVII. FVII (N57D) protein did not accumulate intracellularly, and Northern blot analysis indicated equivalent FVII mRNA levels in 293 cells expressing either wild-type FVII or FVII (N57D). Secreted FVII (N57D) protein did not bind tissue factor, exhibited no procoagulant activity, and failed to bind a conformation-dependent monoclonal antibody specific for the first EGF domain of FVII. Molecular modeling of the first EGF domain of FVII predicted that the N57D amino acid substitution would disrupt tertiary bonding structure. We conclude that the N57D mutation affects folding of the first EGF domain of FVII resulting in decreased cellular secretion of a mutant FVII molecule, which is unable to bind tissue factor and is therefore biologically inactive.

Previous studies have shown that alloxan-induced diabetes in rabbits effects a slower release of plasma proteins from the liver, a slower synthesis of 35S-glycosaminoglycan in the extracellular matrix of the arterial wall, and a concurrent reduction in the fractional catabolic rates of several plasma proteins. In the present study, the catabolism of two hemostatic proteins, prothrombin and antithrombin, are compared in alloxan-induced diabetic rabbits (of 6 months' duration) and age-matched control rabbits. Differentially radiolabeled prothrombin and antithrombin were injected intravenously, and arterial blood was sampled over a 7-day period to measure the clearance from plasma. A three-compartment model was used to determine the fractional catabolic rate and compartmental distribution of the two proteins. As observed for other plasma proteins, the whole-body fractional catabolic rates (jt) for prothrombin and antithrombin were significantly less in diabetic rabbits (prothrombin, 0.33 d-1; antithrombin, 0.27 d-1) than in control rabbits (prothrombin, 0.37 d-1; antithrombin, 0.30 d-1; P < .001 and P < .005, respectively). In absolute terms, the catabolism of antithrombin and prothrombin in diabetic rabbits was 5.1 and 6.2 mg.kg-1.d-1, respectively, equivalent to a molar ratio for antithrombin to prothrombin of 0.94. For the control rabbits, catabolism accounted for 6.3 mg.kg-1.d-1 of antithrombin and 7.3 mg.kg-1.d-1 of prothrombin, equivalent to a molar ratio of 1.01. The fractional distribution of these proteins was not significantly different within the intravascular and extravascular spaces in diabetic and control rabbits. The decreased catabolic rates observed for prothrombin and antithrombin in diabetic rabbits conform with results obtained previously for other plasma proteins, and probably reflect a generally decreased rate of plasma protein production by diabetic rabbit liver compared with control liver.

During experiments to identify putative hepatic receptors for thrombin-antithrombin (TAT) complexes, a 45-kDa protein was identified by ligand blotting. Following gel purification, amino acid sequencing revealed the 45-kDa TAT-binding polypeptide to be cytokeratin 18 (CK18). The presence of CK18 on the surface of intact rat hepatoma cells was demonstrated by binding of 125I-anti-CK18 antibodies. Anti-CK18 antibodies reduced the binding and internalization of 125I-TAT by rat hepatoma cells. Immunocytochemical analysis, to determine the location of CK18 in vivo, revealed a periportal gradient of CK18 staining; with hepatocytes around the portal triads demonstrating striking pericellular staining. In addition, anti-CK18 IgG associated with perfused livers to a significantly greater extent than preimmune IgG. Taken together, these data provide evidence that CK18 is found on the extracellular surface of hepatocytes and could play a role in TAT removal. Finally, these data, in conjunction with recent reports of CK8 (Hembrough, T. A., Li, L., and Gonias, S. L. (1996) J. Biol. Chem. 271, 25684-25691) and CK1 cell membrane surface expression (Schmaier, A. H. (1997) Thromb. Hemostasis 78, 101-107), indicate a novel role for these proteins as putative cellular receptors or cofactors to cellular receptors.

Antithrombin (AT) is a serpin capable of trapping thrombin (IIa) in a stable and covalent complex. Complex formation is prevented by leukocyte elastase (LE) cleavage near the AT reactive centre. We mutated the known LE cleavage sites of AT to explore the possibility of producing an LE-resistant AT molecule. Initially, six rabbit AT variants differing only at residue 390 (P4) were generated in a cell-free system, and gel-based assays were used to assess IIa-mediated complex formation and LE-mediated cleavage of the variants. Substitution of charged residues (Glu or Arg) reduced complex formation by 50-60%, while the Ser variant was incapable of inhibiting thrombin; LE reactivity was less affected. The least (Trp) and most (Ser) affected variants were expressed in COS-1 cells. Again, the Ser variant was incapable of detectably reducing the rate of thrombin-mediated amidolysis while the Trp variant inhibited thrombin at a slightly reduced rate (-28%). LE inactivated the Trp variant and the wild-type AT to a similar extent. Recreation of the Trp mutation in COS-derived human AT showed similar results. Since retention of LE-sensitivity could have arisen due to cleavage at Val389 (P5), we produced and characterized a human AT substitution mutant with Trp at both P4 and P5. This variant showed a slight reduction in thrombin inhibitory activity (-22%), but remained susceptible to LE inactivation. These results suggest either that LE cleaves at secondary sites if its primary cleavage sites are blocked, or that the substrate specificity of LE differs in polypeptides as compared to peptide substrates.

The metabolic characteristics of two rabbit plasma thrombin inhibitors, heparin cofactor II (HCII) and antithrombin (AT), have been compared in healthy young rabbits. Purified HCII and AT-alpha were differentially radiolabeled (125I, 131I) and injected intravenously; blood samples were taken at prescribed intervals over 7 days. From the plasma clearance curves of protein-bound radioactivities, fractional catabolic rates and compartmental distributions were calculated using a three-compartment model. The whole body fractional catabolic rate for HCII (jt, 0.43/day, equivalent to t1/2 = 1.61 days) was significantly faster than for AT (jt, 0.37/day; t1/2 = 1.89 days; P < 0.005). The fractional distribution of HCII in the intravascular compartment (Ap, 0.20) and in the extravascular compartment (Ac, 0.63) differed significantly from AT (Ap, 0.30; Ac, 0.56). From the catabolic data and blood concentrations, absolute quantities of HCII and AT catabolized by a 3-kg rabbit amounted to 12.8 and 19.9 mg/day, respectively, equivalent to a molar ratio, AT/HCII, of 1.7. The catabolic molar ratio was compared with the relative release rates of HCII and AT from perfused rabbit livers. Both proteins were released from the liver, the molar ratio in the perfusate rising to approximately 1.4 at 2.5 h. This report increases our understanding of the in vivo dynamics of these two proteins.

We have identified cis-acting elements and trans-acting factors that regulate constitutive expression of the human antithrombin gene. The activity of the sequences flanking the first exon of the gene was investigated using a luciferase-based reporter assay in transiently transfected HepG2, COS1, BSC40, and HeLa cells. Deletion analysis allowed the mapping of two elements able to promote antithrombin gene transcription in HepG2 and COS1 cells. The first element is located upstream of the first exon (-150/+68 nucleotides). The second element is in the first intervening sequence (+300/+700 nucleotides) and functions in an orientation opposite to that of the first. Footprint analysis showed three protected areas in the 5' upstream element at -92/-68 (element A), -14/+37 (element B), and -126/-100 nucleotides (element C). These elements acted as enhancers in luciferase reporter assays. Gel retardation analysis demonstrated that two liver-enriched transcription factors, hepatocyte nuclear factor 4 (HNF4) and CCAAT enhancer-binding protein (C/EBPa), bound to the 5' upstream element. HNF4 bound to elements A and C, whereas C/EBPa bound to element B. Element A also interacted with the ubiquitous nuclear hormone receptors chicken ovalbumin upstream promoter transcription factor 1 (COUP-TF1), thyroid hormone receptor alpha (TRalpha), peroxisome proliferator-activated receptor alpha(PPARalpha), and retinoid X receptor alpha (RXRalpha). In HepG2 and BSC40 cells, HNF4, C/EBPalpha, and RXRalpha activated luciferase expression from a reporter construct containing the 5'-upstream minimal antithrombin gene promoter, while COUP-TF1, TRalpha, and HNF3 (alpha or beta) repressed such expression. Our results show that constitutive expression of the human antithrombin gene depends in part upon the interplay of these transcription factors and suggest that signaling pathways regulated by these factors can modulate antithrombin gene transcription.

A competitive enzyme-linked immunoadsorbent assay (ELISA) technique has been developed to facilitate quantitative analysis of the earliest step in the initiation of the extrinsic pathway of coagulation, i.e., complex formation of factor VII/VIIa with tissue factor. The ELISA measures the binding of biotinylated human plasma factor VII to relipidated recombinant human tissue factor. Quantitation of the relative affinity (expressed as IC50) of any factor VII molecular population or structural analogue for tissue factor can be determined by competitive binding. Subnanomolar concentrations of both wild-type recombinant human factor VII (rFVII) and rFVII(R152Q), a mutation at the FVII activation site, competed effectively with biotinylated plasma-derived factor VII in binding to tissue factor. In contrast, the affinity of rFVII(R79Q), a mutation in the first epidermal growth factor-like domain, was 12-fold lower. Following activation of rFVII(R79Q), its affinity for tissue factor and enzymatic activity increased 4-fold and 6-fold, respectively. For wild-type rFVII, enzymatic activity rose significantly following activation. However, its affinity for tissue factor was unchanged. We conclude that both the activation state of factor VII and the mutation of amino-acid residues within the first epidermal growth factor-like domain may alter the affinity of factor VII for tissue factor.