Human erythrocyte antigens: II. The In(Lu) gene regulates expression of an antigen on an 80-kilodalton protein of human erythrocytes

We have previously shown that a murine monoclonal antibody (A3D8) identifies a human erythrocyte protein antigen whose expression is regulated by the Lutheran inhibitor [In(Lu)] gene. In the present study, we demonstrated by immunoprecipitation and Western blot techniques that the antigen defined by A3D8 was on an 80-kD erythrocyte membrane protein. A second 170-kD protein was coprecipitated with the 80-kD protein but failed to show antigen activity by Western blot analysis. The 170-kD protein, when analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis in two dimensions, was composed of 50- and 30-kD disulfide-linked subunits. In(Lu) Lu[a-b-) erythrocytes differed from Lu(a+b+) or Lu(a-b+) erythrocytes in that In(Lu) deoxycholate erythrocyte membrane extracts contained trace amounts of immunoprecipitable 80-kD protein compared with detergent-solubilized erythrocyte membrane extracts prepared from Lu(a+b+) or Lu(a-b+) subjects.     

Improved red blood cell preservation correlates with decreased loss of bands 3, 4.1, acetylcholinestrase, and lipids in microvesicles

In earlier studies we have shown that a final concentration of 0.69% glycerol in blood mixed with an experimental additive solution, EAS 25, improves the in vitro quality and in vivo survival of red blood cells (RBCs). The objective of this study was to determine if the better preservation of RBCs in EAS 25 is correlated with the improved maintenance of membrane lipids and proteins and decreased vesiculation. Split units of RBCs were stored in Adsol or EAS 25 (mmol/L: adenine 2/2, dextrose 122/110, mannitol 42/55, glycerol 0/150, NaCl 154/50). After 12 weeks storage, RBC and microvesicle membranes were analyzed for cholesterol, phospholipid, diphenyl hexatriene fluorescence anisotropy, and acetylcholinesterase (AchE) activity. Bands 3 and 4.1 were identified in the microvesicle membranes by immunoblotting. The RBC membrane cholesterol, phospholipids, and AchE remained higher in EAS 25 than in Adsol (P < .001). Vesicle membrane lipids and AchE in EAS 25 were significantly less than in Adsol (P < .001). The fluidity of stored cells in both the solutions was greater than the prestorage samples. Immunoblotting analyses showed that bands 3 and 4.1 were greatly reduced in the microvesicle membranes shed by the RBCs stored in EAS 25 compared with those formed in Adsol.     

Recombinant human megakaryocyte growth and development factor stimulates thrombocytopoiesis in normal nonhuman primates

Megakaryocyte growth and development factor (MGDF) is a novel cytokine that binds to the c-mpl receptor and stimulates megakaryocyte development in vitro and in vivo. This report describes the ability of recombinant human (r-Hu) MGDF to affect megakaryocytopoiesis in normal nonhuman primates. r-HuMGDF was administered subcutaneously to normal, male rhesus monkeys once per day for 10 consecutive days at dosages of 2.5, 25, or 250 micrograms/kg of body weight. Bone marrow and peripheral blood were assayed for clonogenic activity and peripheral blood counts were monitored. Circulating platelet counts increased significantly (P < .05) for all doses within 6 days of r-HuMGDF administration and reached maximal levels between day 12 and day 14 postcytokine administration. The 2.5, 25.0, and 250.0 micrograms/kg/d doses elicited peak mean platelet counts that were 592%, 670%, and 449% of baseline, respectively. Bone marrow-derived clonogenic data showed significant increases in the concentration of megakaryocyte (MEG)- colony-forming unit (CFU) and granulocyte-erythroid-macrophage- megakaryocyte (GEMM)-CFU, whereas that of granulocyte-macrophage (GM)- CFU and burst-forming unit-erythroid (BFU-e) remained unchanged during the administration of r-HuMGDF. These data show that r-HuMGDF is a potent stimulator of thrombocytopoiesis in the normal nonhuman primate.     

Identification of Bakb, a new platelet-specific antigen associated with posttransfusion purpura

Baka is a platelet alloantigen whose putative allele, Bakb, has not been identified previously. By using a serum, "Har," obtained from a patient with posttransfusion purpura, we describe the platelet alloantigen Bakb. The Har serum reacted with an NP-40-extractable platelet membrane protein of 142 kd with mobility similar to platelet glycoprotein IIb alpha. We found that the antigen recognized by the Har serum is inherited in an autosomal dominant mode with an apparent gene frequency of .39. Chi-square analysis of observed and expected phenotype frequencies indicated that serum Har recognizes Bakb, the anticipated allele of Baka. Our findings provide new evidence for polymorphism of glycoprotein IIb and for the association of posttransfusion purpura with alloimmunization to determinants on this glycoprotein.     

Rivaroxaban Plus Aspirin in Obese and Overweight Patients With Vascular Disease in the COMPASS Trial

BackgroundDirect oral anticoagulants are administered in fixed doses irrespective of body weight, but guidelines recommend against their use in patients with extremes of body weight.ObjectivesThis study determined the effects of dual-pathway inhibition antithrombotic regimen (rivaroxaban 2.5 mg twice daily plus aspirin 100 mg/day) compared with aspirin Halone across a range of patient body mass indexes (BMIs) and body weights.MethodsThis was a secondary analysis of the COMPASS (Cardiovascular OutcoMes for People using Anticoagulation StrategieS) trial, which included patients with chronic coronary artery disease or peripheral artery disease. Efficacy and safety outcomes were studied in relation to BMI: (normal 18.5 ≤BMI <25 kg/m², overweight 25 ≤BMI <30 kg/m², obese ≥30 kg/m²) and body weight (≤70 kg, 70 < weight ≤90 kg, and >90 kg; as well as ≤120 kg vs. >120 kg).ResultsAmong 27,395 randomized patients, 6,459 (24%) had normal BMI, 12,047 (44%) were overweight, and 8,701 (32%) were obese. The combination of rivaroxaban and aspirin compared with aspirin produced a consistent reduction in the primary outcome of cardiovascular death, stroke, or myocardial infarction, irrespective of BMI or body weight. For 18.5 ≤BMI <25 kg/m²: 3.5% vs. 5.0%; hazard ratio (HR): 0.73 (95% credible interval [CrI]: 0.58 to 0.90); 25 ≤ BMI <30 kg/m²: 4.3% vs. 5.1%; HR: 0.80 (95% CrI: 0.66 to 0.96); BMI ≥30 kg/m²: 4.2% vs. 6.1%; HR: 0.71 (95% CrI: 0.57 to 0.86). For body weight ≤70 kg: 4.1% vs. 5.3%; HR: 0.75 (95% CrI: 0.62 to 0.91); 70 < weight ≤90 kg: 4.1% vs. 5.3%; HR: 0.76 (95% CrI: 0.65 to 0.89); >90 kg: 4.2% vs. 5.7%; HR: 0.74 (95% CrI: 0.61 to 0.90). Effects on bleeding, mortality, and net clinical benefit were consistent irrespective of BMI or bodyweight.ConclusionsThe effects of dual-pathway antithrombotic therapy are consistent irrespective of BMI or body weight, suggesting no need for dose adjustments in the ranges of weights and BMI of patients enrolled in the COMPASS trial. Further studies need to address this problem in relation to greater extremes of body weight. (Rivaroxaban for the Prevention of Major Cardiovascular Events in Coronary or Peripheral Artery Disease [COMPASS]; NCT01776424)     

Human platelet fibrinogen: purification and hemostatic properties

Conditions were developed in which 80% to 90% of platelet fibrinogen could be routinely purified in nondegraded form from the fluid phase of platelet suspensions stimulated with the calcium ionophore, A23187, in the presence of calcium, leupeptin, and prostaglandin E1. Fibrinogen was separated from other released proteins by chromatography on diethylaminoethanol (DEAE)-cellulose using a continuous pH and ionic strength gradient. Purified platelet fibrinogen, greater than 98% homogeneous by immunoelectrophoresis and sodium-dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE), consisted of intact A alpha, B beta and gamma A chains, but not gamma' chains, and was 95% to 96% clottable. Platelet fibrinogen was shown to compete for the binding of radiolabeled plasma fibrinogen to ADP-activated platelets in a manner identical to that of unlabeled plasma fibrinogen itself. Also, at equivalent protein concentrations, platelet and plasma fibrinogens supported platelet aggregation to an equivalent extent. Based upon these results, we conclude that there is no significant difference between platelet and plasma fibrinogen with respect to their size, their clottability, their affinity for the activated platelet fibrinogen receptor, or their capacity to support subsequent platelet aggregation.     

Molecular cloning, chromosomal location, and tissue-specific expression of the murine cathepsin G gene

We previously have characterized a cluster of genes encoding cathepsin G (CG) and two other CG-like hematopoietic serine proteases, CGL-1 and CGL-2, on human chromosome 14. In this report, we clone and characterize a novel, related murine hematopoietic serine protease gene using human CG (hCG) cDNA as the probe. This murine gene spans approximately 2.5 kb of genomic DNA, is organized into five exons and four introns, and bears a high degree of homology to hCG at both nucleic acid (73%) and deduced amino acid (66%) levels. The predicted cDNA contains an open reading frame of 783 nucleotides that encodes a nascent protein of 261 amino acids. Processing of a putative signal (pre) peptide of 18 residues and an activation (pro) dipeptide would generate a mature enzyme of approximately 27 Kd that has an estimated pI of 12.0. Conserved residues at His44, Asp88, and Ser181 form the characteristic catalytic triad of the serine protease superfamily. The gene is tightly linked to the CTLA-1 locus on murine chromosome 14, where the serine protease genes mCCP1-4 are clustered. Expression of this gene is detected only in the bone marrow and is restricted to a small population of early myeloid cells. These findings are consistent with the identification of the gene encoding murine CG.     

Comparison of Fentanyl and Droperidol Mixture (Neuroleptanalgesia II) with Morphine on Clinical Outcomes in Unstable Angina Patients

The objective of the study was to compare the influence of a fentanyl and droperidol mixture (neuroleptanalgesia) with morphine on the in-hospital instability, development of acute myocardial infarction (AMI), and mortality during a 30-day and 12-month follow-up in unstable angina patients. The study was performed in 112 unstable angina patients. In addition to standard therapy for unstable angina (aspirin, heparin, nitroglycerin, and oxygen), 53 patients (63.2 ± 9.7 years; 32 males) were randomized to receive neuroleptanalgesia (0.025 mg fentanyl and 1.25 mg droperidol in a volume of 1 mL) and 59 patients (58.6 ± 11.5 years; 41 males) to receive morphine. Neuroleptanalgesia was started IV with 2 mL and could be followed by 1 mL every 4 hours. Morphine was started IV with 10 mg and could be followed by 5 mg every 4 hours up to angina resolution during 24 hours of hospitalization. Another 1 mL of neuroleptanalgesia or 5 mg of morphine could be administered on demand if angina lasted or reappeared earlier than the next scheduled dose. Odds ratios with 95% confidence intervals (95% CI) adjusted for the age, sex, smoking, previous myocardial infarction, and hypertension were evaluated for all study outcomes. The odds ratios for clinical in-hospital instability (5.93, 95% CI: 2.49–14.15; P = 0.0001), 12-month AMI development (3.57, 95% CI: 1.51–8.45; P = 0.0038), and 12-month mortality (6.00, 95% CI: 1.63–22.09;P = 0.0070) were significantly increased in the neuroleptanalgesia group compared with the patients on morphine. It is concluded that neuroleptanalgesia negatively influences disease course, AMI development, and total mortality in unstable angina patients.     

Therapeutic efficacy of recombinant human leukemia inhibitory factor in a primate model of radiation-induced marrow aplasia

The therapeutic efficacy of recombinant human leukemia inhibitory factor (LIF) was examined in a nonhuman primate model of radiation- induced marrow aplasia. Rhesus monkeys received 450 cGy of total-body, 1:1 mixed neutron:gamma radiation. For 23 days thereafter, each monkey received a daily subcutaneous injection of LIF or human serum albumin (HSA) at a dose of 15 micrograms/kg body weight. Complete blood counts and white blood cell differentials were monitored for 60 days postirradiation. Administration of LIF significantly decreased (P < or = .05) the duration of thrombocytopenia (platelet count < 30,000 or 20,000/microL), ie, 9.3 days or 6.3 days, respectively, versus the HSA- treated control monkeys, 12.2 days or 10.2 days, respectively. Treatment with LIF did not alter the duration of neutropenia (absolute neutrophil count < 1,000/microL) as compared with the HSA-treated control monkeys. Cytokine administration did not exacerbate the radiation-induced anemia observed in the HSA-treated control monkeys.