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BACKGROUND: Anemia may be an important factor contributing to an increased risk of bleeding, particularly in patients with thrombocytopenia. STUDY DESIGN AND METHODS: A multicenter, single-blinded pilot randomized controlled trial (RCT) was performed to evaluate the feasibility of conducting a larger RCT to determine the effect of the hemoglobin (Hb) concentration on bleeding risk. Patients with acute leukemia receiving induction chemotherapy or those undergoing stem cell transplantation were assigned to one of two treatment groups: standard transfusion strategy (transfusion of 2 units of red blood cells [RBCs] when their Hb level was less than 80 g/L) or an augmented transfusion strategy (transfusion of 2 units of RBCs when their Hb level was less than 120 g/L). RESULTS: Sixty patients were enrolled: 29 in the control group and 31 in the experimental group. The proportions of patients experiencing clinically significant bleeding and the time to first bleed were not significantly different between the control and experimental groups. The experimental group received more RBC transfusions (transfusions/patient-day) than the control group (0.233 vs. 0.151; relative risk, 1.56; 95% confidence interval, 1.16-2.10; p = 0.003). The proportion of patient-days with platelet (PLT) transfusions was not different between the experimental and control groups. The mean number of donor exposures (PLT and RBC transfusions) was not different between experimental and control groups. Bleeding symptoms were systematically documented. CONCLUSION: This pilot study thus indicated that it would be feasible to enroll the required number of patients to enable the performance of a large RCT to investigate the effect of Hb on bleeding risk in thrombocytopenic patients. [ABSTRACT FROM AUTHOR]

BACKGROUND: Prestorage pooling of whole blood-derived platelets (PLTs) would simplify bacterial detection. This study evaluated the in vivo effect of the prestorage pooling of PLTs stored for up to 5 days, by assessing the corrected count increment (CCI) 18 to 24 hours after transfusion of the product. STUDY DESIGN AND METHODS: A randomized block noninferiority design was used. Eligible patients had chemotherapy-induced thrombocytopenia and were considered likely to need at least six PLT transfusions. For every block of two transfusion events, one consisted of PLTs stored individually and then pooled before transfusion, and the other was a product pooled before storage. The primary outcome was categorized as a successful (>4.5) or unsuccessful (

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.

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.

Previous studies have reported that pre-operative plasmas of patients over the age of 40 years who developed post-operative deep vein thrombosis (DVT) had approximately twice the amount of proteolysed factor VII found in plasmas of patients in whom prophylaxis with heparin or low M(r) heparin was successful. These and other studies also reported higher concentrations of thrombin-antithrombin III in pre- and post-operative plasmas of patients who developed post-operative thrombosis than in plasmas of patients in whom prophylaxis was successful. Whether the extent of factor VII proteolysis seen in the patients who developed post-operative DVT is related to the severity of their disease or age is not known. This report investigated age-related changes in the concentrations of total factor VII protein, factor VII zymogen, factor VIIa, tissue factor pathway inhibitor, thrombin-antithrombin III, and prothrombin fragment 1 + 2 in normal plasmas and the relationships between these parameters. With the exception of thrombin-antithrombin III, statistically significant increases in the concentrations of these parameters with age were found. Additionally, the differences between the concentrations of total factor VII protein and factor VII zymogen, an index factor VII proteolysis in vivo, were statistically significant only for individuals over age 40. Using linear regression analysis, a significant correlation was found to exist between the concentrations of plasma factor VIIa and prothrombin fragment 1 + 2. Since factor VIIa-tissue factor probably initiates coagulation in vivo, we hypothesize that the elevated plasma factor VIIa (reflecting a less tightly regulated tissue factor activity and therefore increased thrombin production in vivo) accounts for the high risk for post-operative thrombosis seen in individuals over the age of 40.

Patients with acute myocardial infarction who undergo thrombolytic therapy may shortly thereafter present evidence for increased platelet activation and thrombin activity, and recurrent thrombosis. This study investigated whether plasmin activates platelets and prothrombin in recalcified platelet-rich plasma (RPRP) to cause (at least in part) these side-effects of thrombolytic therapy. Plasmin (0.1 and 1.0 CU/ml) addition to RPRP with microM r-tick anticoagulant peptide (the latter a factor Xa inhibitor which abrogates prothrombin activation by prothrombinase at the concentration used) resulted in no change in the concentration of prothrombin fragment 1 + 2, or in the expression of GMP-140, the resting and activated GP IIb-IIIa conformers, and GPIb on platelets. Thus, plasmin neither activates platelets nor prothrombin in RPRP. However, plasmin accelerated platelet activation and secretion, and prothrombin fragment 1 + 2 production in RPRP. When combined with 1 microM r-tick anticoagulant peptide and 1 or 10 mM alpha-thrombin to RPRP, plasmin also increased the number of GMP-140 molecules expressed/platelet without enhancing alpha-thrombin binding to the platelets. Additionally, plasmin accelerated prothrombin activation when it was added to washed platelets resuspended in factor V depleted plasma simultaneously with 10 mM CaCl2, 10 nM alpha-thrombin for 10 s (to activate platelets and platelet factor V), followed by 4 microM hirudin and 1 nM factor Xa. Thus, plasmin potentiates the platelet release reaction in response to alpha-thrombin (probably by increasing the availability of factor V on the platelets) to enhance prothrombin activation in RPRP. These actions of plasmin may contribute to the increased platelet activation and thrombotic side-effects that can occur after thrombolytic therapy.

PCR amplification and analysis of short tandem repeats (STR) have provided a useful tool for genetic linkage studies and for the diagnosis of genetic disorders. We have recently identified a novel trinucleotide STR, (ATT).(TAA), in the fifth intron of the human antithrombin gene (AT3) located on chromosome 1q23. PCR amplification, cloning, and sequence analysis revealed this AT3-STR to be highly polymorphic with repeat units ranging in size from (ATT)5 to (ATT)18. Ten distinct alleles were found in 81 unrelated Caucasian individuals (162 alleles) with an observed heterozygosity of 81%. Genetic linkage studies using the AT3-STR in two previously described antithrombin (AT)-deficient kindreds, AT-Hamilton (Ala 382 Thr) and AT-Amiens (Arg 47 Cys), demonstrate, in a given kindred, that a specific AT3-STR polymorphism is strongly associated with a particular AT mutation. Thus, this highly polymorphic AT3-STR should be very useful in performing linkage studies in AT-deficient kindreds as well as in investigating other chromosome 1-related genetic disorders.

The prevalence of antithrombin (AT) deficiency in the general population has been variously estimated to be between 0.05 and 5 per 1,000 in the population; 2,491 blood donors were screened in an attempt to clarify this issue using plasma samples taken from the blood donor units. From this initial population, 122 individuals were identified as having plasma AT levels lower than 2 standard deviations below the normal mean. Twenty-two samples had evidence that thrombin had been generated during blood collection and the remaining cohort of 100 blood donors were asked to return but only 59 complied. The data obtained from these 59 were compared with that from 51 age- and sex-matched control blood donors. Both groups of subjects were assessed for previous evidence, or family history, of thrombotic events, as well as exposure to risk factors associated with the development of deep vein thrombosis (DVT). All had venous blood samples taken from which the supernatant plasma was immediately removed and quick frozen for later assaying. Only 6 of the 59 subjects with initial low AT levels had repeat AT-Xa levels below 0.80 units/ml (normal range 0.94 +/- 0.14). Upon repeating the AT-Xa determinations on new samples from these six individuals, only three were found again to be low. One was found to have a type 3 AT deficiency (an Arg47Cys substitution). The other two with a low AT level had mean functional AT-Xa levels of 0.61 and 0.71 units/ml, respectively, with correspondingly low AT:Ag levels consistent with a type 1 AT deficiency. Two of these three subjects has been in high risk situations without evidence of having developed DVT and none had evidence of venous reflux on Doppler venography. In addition, none had personal or family histories of previous thrombotic events. These present data indicate that the prevalence of AT deficiency in our blood donor population is 2 per 1,000 (95% confidence intervals: 0.7-6/1,000).

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.

Patients over 40 years of age who undergo elective orthopaedic surgery have a relatively high risk for developing post-surgical deep vein thrombosis (DVT). Prophylactic use of heparin or low molecular weight heparins can reduce the incidence of post-operative DVT by up to 80%. It is not known whether prophylaxis is achieved by inhibition of prothrombin activation or catalysis of thrombin inhibition in vivo. We determined the changes in concentrations of factor VII zymogen and thrombin-antithrombin III (the latter as an index of prothrombin activation) in the plasmas of 129 patients randomized to receive two daily subcutaneous injections of placebo or 30 mg of Enoxaparin after elective knee surgery. Enoxaparin reduced the frequency of post-surgical DVT by 70%. The concentration of factor VII zymogen had decreased by approximately 50% within 24 h after the knee surgery, followed by a gradual increase to near presurgical values. Additionally, post-Enoxaparin plasmas had statistically significant higher concentrations of factor VII zymogen than post-placebo plasmas. Post-Enoxaparin plasmas had significantly lower concentrations of endogenous thrombin-antithrombin III than comparable post-placebo plasmas. Finally, post-Enoxaparin plasmas inactivated exogenous factor Xa and thrombin more effectively than comparable post-placebo plasmas. As Enoxaparin moderated the generation of endogenous thrombin-antithrombin III after elective knee surgery, inhibition of prothrombin activation in vivo by Enoxaparin may be important for its prophylactic antithrombotic effect.

This study compared how Enoxaparin and unfractionated (UF) heparin influenced in vivo coagulation in patients randomized to receive, by twice daily subcutaneous injections, either 30 mg of Enoxaparin or 7500 I.U. of UF heparin after elective hip surgery. These two regimens were equally effective in reducing the incidence of post-operative deep vein thrombosis DVT. We compared the concentrations of endogenous thrombin-antithrombin III in pre- and post-surgical plasmas to determine how each prophylactic regimen influenced prothrombinase activity in vivo, and found the same concentrations of endogenous thrombin-antithrombin III in post-heparin and post-Enoxaparin plasmas. However, significantly higher concentrations of endogenous thrombin-antithrombin III were found in pre- and post-surgical plasmas of patients who developed post-operative DVT than the levels found in comparable plasmas of patients who remained DVT-negative, regardless of the drug received for prophylaxis. Human factor Xa was added to an equal volume of each patient's plasmas and the amount of added enzyme inactivated by antithrombin III measured using an enzyme-linked immunosorbent assay for factor Xa-antithrombin III. Post-heparin and post-Enoxaparin plasmas inactivated approximately 4 times more factor Xa than the pre-surgical plasmas, regardless of the clinical outcome. Thus, before and after surgery, a higher than normal in vivo prothrombinase activity may be a significant risk factor for developing post-operative DVT.

A family with an antithrombin III variant (AT-III-Amiens) demonstrating abnormal heparin cofactor activity is described. Amplification and direct sequencing of genomic DNA by the polymerase chain reaction procedure permitted the identification of an Arg47----Cys mutation in exon 2 of the variant antithrombin III gene.

The effects of standard heparin, three low molecular weight derivatives of heparin, dermatan sulphate and pentosan polysulphate on the intrinsic coagulation pathway were compared in order to evaluate the contributions of the anti-factor Xa and anti-thrombin activities to their anticoagulant activities. The anticoagulant potency was measured by the ability of each sulphated polysaccharide to inhibit the generation of thrombin activity in plasma. Similarly, the ability of the six sulphated polysaccharides to enhance the rates of inactivation either factor Xa or thrombin in defibrinated plasma containing calcium chloride and cephalin were also determined. Standard heparin was the only sulphated polysaccharide that could equally inhibit thrombin generation and enhance the inactivation of factor Xa and thrombin by plasma. Dermatan sulphate and pentosan polysulphate were more effective as inhibitors of thrombin generation than potentiators of factor Xa inactivation. The two smallest derivatives of heparin, which had high anti-factor Xa (but low antithrombin) activity, were the poorest inhibitors of thrombin generation. Our results therefore suggest that only sulphated polysaccharides that enhance the inactivation of thrombin by plasma and/or inhibit the generation of thrombin activity in plasma are good anticoagulants. These two activities of sulphated polysaccharides appear to be good predictors of the relative antithrombotic potency in vivo.

A murine hybridoma cell line which secretes monoclonal antibody to factor VII has been prepared to facilitate the immunodepletion of this clotting factor from plasma. Specific monoclonal antibody was purified from mouse ascites tumours by protein A-Sepharose chromatography and shown to be of the IgG1 immunoglobulin subclass. On immunoblotting, this antibody reacted with a single protein band identical to purified factor VII. The purified monoclonal antibody was coupled to Sepharose 4B and was used to immuno-deplete factor VII from pooled normal human plasma. The prothrombin time of plasma immunodepleted in this way was 35 s compared to 12 s for the starting plasma. Specific factor assays of the immunodepleted plasma showed factor VII activity to be less than 1% while the levels of the other clotting factors were unchanged. The immunodepleted plasma was equivalent to severe congenital factor VII deficient plasma as a substrate for factor VII assays. Bound factor VII could be eluted from the immunoaffinity column with citrate buffer, pH 6.0, with good recovery.

Platelet prothrombin-converting activity and factor Xa binding were studied after exposure of human platelet rich plasma (PRP) to various conditions leading to platelet activation. Zymosan resulted in increased platelet-bound C3, enhanced prothrombin-converting activity and increased factor Xa binding. Similar findings were observed with normal platelets resuspended in factor XII-deficient plasma. The combined use of zymosan and thrombin to activate platelets resulted in synergistic prothrombin-converting activity and factor Xa binding. In contrast, no synergism was obtained with the concomitant use of zymosan and collagen, suggesting that collagen and zymosan share the same pathway for platelet activation. Heterologous antibody to factor V completely inhibited the platelet prothrombin-converting activity for all modes of platelet activation, indicating that this activity is mediated by factor V.

Platelets and phospholipids have been shown to protect factor Xa from inhibition by the heparin--antithrombin III complex. The studies reported herein investigated the effects of gel filtered platelets, activated platelets, brain phospholipids (cephalin), and brain tissue factor on the inactivation of thrombin and factor Xa by the heparin--antithrombin III complex. In addition, the relative anticoagulant effects of heparin on the extrinsic and intrinsic coagulation pathways were investigated. Our results suggest that gel filtered platelets, activated platelets, cephalin and tissue factor protect thrombin, as well as factor Xa, from inactivation by the heparin--antithrombin III complex. Tissue factor had the greatest anti-heparin activity. Activated platelets, gel filtered platelets, cephalin and tissue factor did not alter the protease--antithrombin III reaction rates measured in the absence of heparin. These observations are consistent with the hypothesis that platelets, brain phospholipids, and tissue factor, in the presence of calcium, partition heparin from antithrombin III, and thus prevent full expression of the antithrombin III-dependent anticoagulant activity of heparin.

Erythrocyte-associated IgG was measured on washed red cells from 26 children with P. falciparum parasitaemia using an immunoradiometric assay. The values obtained were compared with 15 age-matched controls living in the same area who did not have malaria. The amount of red cell-associated IgG was related to the haemoglobin concentration, degree of parasitaemia and serum immunoglobulin level. The mean red cell-associated immunoglobulin for patients with malaria was 629 molecules per red cell (range 215-1770) which was significantly higher than the control group, who had a mean of 395 molecules per red cell (range 190-930). A statistically significant correlation was established between red cell-associated IgG and the degree of anaemia in the infected patients. In contrast, there was no relationship between the red cell-associated IgG and the degree of parasitaemia or the serum immunoglobulin level. These observations support the hypothesis that an immunological mechanism could be involved in the anaemia associated with malarial infections.

Five children with 'paroxysmal cold haemoglobinuria' are described. In all of them an upper respiratory tract infection preceded the episode of acute intravascular haemolysis and potentially pathogenic organisms were recovered from four. The haematological features are presented and the importance of technique in the detection of the pathognomonic antibody is stressed and discussed in detail. Although the clinical manifestations are usually self-limiting and of short duration, it may be necessary, because of the rapidity and severity of anaemia, to provide interim support with appropriate blood transfusion. Since the disease is not usually recurrent and not clearly related to exposure to cold, it is suggested that it be re-named Donath-Landsteiner haemolytic anaemia.