Non-Hodgkin's lymphoma as an unexpected diagnosis in a shoulder arthroplasty.

Shoulder arthroplasty (SA) is commonly performed in patients with rheumatoid arthritis (RA) who have been treated with long-term immunosuppressive medication. RA is associated with an increased risk of neoplasms of the immune system. A case of non-Hodgkin's lymphoma as an unexpected diagnosis after the routine pathologic examination of the soft tissues after SA was detected in a 54-year-old woman with long-standing RA and prolonged immunosuppressive therapy. Although this case does not support the cost-effectiveness of routine specimen evaluation during SA, we suggest that histological analysis of the surgical tissues is appropriate and should be performed in all patients who have been treated with prolonged immunosuppressive medication, especially RA patients as well as patients who have suspicious surgical findings.

Inhibition of platelet aggregation by an irreversible local anesthetic.

Three known local anesthetic agents were examined for their ability to inhibit platelet aggregation induced by adenosine diphosphate or epinephrine. Drug-treated platelet-rich plasma samples were filtered through agarose gel to remove free drug, and the ability of the platelets to aggregate was again determined. One of the local anesthetics contained a "one-armed" nitrogen mustard structure and appeared to produce an irreversible inhibition of aggregation. The other two agents gave a block of aggregation that was reversed upon gel filtration.

The impact of delayed fibrinopeptide-A release on fibrin structure. Studies of an abnormal fibrinogen.

The impact of delayed fibrinopeptide-A release on polymerization and structure of fibrin gels was studied utilizing a heterozygously transmitted variant fibrinogen. An arginine to histidine substitution at position 16 of the alpha chain of the abnormal fibrinogen delayed release of an abnormal fibrinopeptide-A (A) by thrombin and completely blocked release of A by reptilase. When clotted with thrombin, patient fibrin formed more slowly than normal fibrin, but clottability was normal and gel fiber mass/length ratios were decreased less than 10%. Gels formed with reptilase clotted slowly, demonstrated reduced clottability, but had normal fiber mass/length ratios. Reptilase clotted the normal but not the variant component of the patient fibrinogen. Thrombin-induced cleavage of fibrinopeptide-B prior to A occurred in these experiments, but polymerization of this species beyond trimers has been reported to be minimal under the conditions used. With time, A is removed by thrombin resulting in the slow production of normal fibrin monomer from the abnormal component. These monomers subsequently polymerize. The minimal change in gel fiber size caused by slow A release implies that fibrin fiber size is primarily a function of ionic environment and not of the sequence of peptide release.

Effect of 5-HT2-selective agonists on cat platelet aggregation.

The effects of the 5-HT2-selective agonists 1-(4-bromo-2,5-dimethoxyphenyl)-2-aminopropane (DOB) and 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) on cat platelet aggregation were investigated and compared with those produced by serotonin (5-HT) and a positional isomer of DOB (i.e., isoDOB). Serotonin, DOB, and DOI enhanced the aggregation of platelets induced by a suboptimal concentration of ADP. This effect was completely inhibited by pre-incubation of the platelet suspension with the 5-HT2-selective antagonist ketanserin. IsoDOB, an isomer of DOB with a very low affinity for central 5-HT2 binding sites, was inactive in the platelet aggregation assay. The present results are consistent with the proposed role of 5-HT2 receptors in serotonin-induced platelet aggregation.

Colchicine does not provide a sustained blockage of collagen and plasma protein secretion by rat hepatocytes.

Colchicine has been reported to disrupt microtubules and thereby inhibit collagen secretion. Because of this "anti-collagen" activity, colchicine has been suggested for use in the treatment of hepatic fibrosis. Using biochemical and immunohistochemical techniques, our laboratory has identified the hepatocyte as one possible source of collagen in the liver. The present study examined the direct effect of colchicine on collagen secretion by hepatocytes in culture. Parenchymal cells were isolated from the livers of adult rats four days following a two-thirds hepatectomy. Total collagen and the fraction secreted into the medium were quantitated as incorporation of [3H]-proline into bacterial collagenase-sensitive protein. Treatment of the hepatocytes with 100 microM colchicine (2-3 hours) resulted in a substantial inhibition of collagen secretion. However, upon longer exposure of the hepatocytes to the drug (24 hours and 8 days), the inhibitory effect on collagen secretion was abolished. The anti-protein secretion activity of the colchicine in the conditioned medium removed from the hepatocytes was still present as verified by a 2.5 hour fibroblast-collagen secretion bioassay. The secretion of the plasma proteins albumin, fibrinogen and the third component of complement was not altered by the presence of colchicine. We conclude that the hepatocyte is a highly efficient secretory cell, and is not entirely dependent upon microtubules as organelles for protein secretion. Therefore, to the extent that hepatocytes may contribute to the hepatic fibrosis, the therapeutic use of colchicine to block collagen secretion might be expected to have only limited effectiveness.

The in vitro and in vivo effects of isoflurane and nitrous oxide on platelet aggregation.

In view of the possible antiaggregation effects of newer general anesthetics we investigated the in vitro and in vivo effects of isoflurane and nitrous oxide on platelet aggregation. Platelets obtained from 18 healthy volunteers, were exposed in vitro for 30 min in a closed chamber to oxygen-carbon dioxide (90%, 5%) (control), oxygen-carbon dioxide-nitrous oxide (80%), or oxygen-carbon dioxide-isoflurane (1.5%) with or without nitrous oxide (80%). Samples were tested in ADP- and collagen-induced aggregation tests. Both nitrous oxide and isoflurane produced statistically significant inhibition of ADP-induced aggregation. Inhibition of collagen-induced aggregation was not statistically significant. In 18 patients who received nitrous oxide (3 L/min) and isoflurane (1-2%) during anesthesia, platelet aggregation was reduced significantly. We conclude that both nitrous oxide and isoflurane cause moderate but statistically significant inhibition of platelet aggregation that may be clinically important in some patients.

In vitro comparison of antiplatelet effects of beta-lactam penicillins.

beta-Lactam antibiotics have been shown to cause platelet dysfunction and bleeding in some patients. However, relative antiplatelet activity of various beta-lactams has remained controversial. Results of clinical studies have been variable because of the presence of underlying disease in the study patients, in addition to inherent difficulties of in vivo experimentation such as individual variations of drug metabolism and drug kinetics. Thus, we designed in vitro experiments to study the direct effect of penicillin G, carbenicillin, ticarcillin, mezlocillin, piperacillin, nafcillin, and azlocillin on platelets. Platelets obtained from normal volunteers were exposed in vitro for 15 minutes to increasing concentrations of the test penicillins (10.0, 12.5, 15.0, and 20.0 mmol/L), and the platelet aggregation response determined after the additional of adenosine diphosphate (2.5 to 5.0 mumol/L), epinephrine (0.1 X 10(-3) mol/L), thrombin (0.01 to 0.02 U/ml), and collagen (11.62 micrograms/ml). All tested penicillins inhibited platelet aggregation in a saturable dose-dependent manner that was reversible by platelet washing. Biostatistical comparison of inhibition of platelet aggregation demonstrated nafcillin to cause significantly more inhibition, followed by azlocillin, mezlocillin, and piperacillin as a group. Penicillin G, carbenicillin, and ticarcillin were the least inhibitory. The mean percent inhibition (epinephrine) at 20 mmol/L concentration was nafcillin 86.4%, mezlocillin 83.2%, piperacillin 80.3%, azlocillin 76.4%, ticarcillin 73.2%, carbenicillin 66.4%, and penicillin G 58.4% (overall P less than 0.001). We conclude that all penicillins tested in vitro inhibit platelet aggregation in normal individuals, but to varying degrees. The inhibitory response, which is most likely a membrane-related phenomenon, is dose dependent and reversible.

In vitro and in vivo studies of the effect of aztreonam on platelet function and coagulation in normal volunteers.

The in vitro effects of aztreonam on platelet aggregation were compared with those of cefotaxime, moxalactam, piperacillin, and carbenicillin. In addition, the in vivo effects of intravenously administered aztreonam on blood coagulation and platelet function were examined in 10 normal male volunteers in a randomized crossover study. In vitro, at concentrations of greater than 6.25 mM (2.7 mg/ml), aztreonam inhibited ADP-induced platelet aggregation in a dose-dependent manner. The effect was less than that produced by equimolar concentrations of cefotaxime, moxalactam, piperacillin, or carbenicillin. At all concentrations tested, aztreonam and cefotaxime inhibited epinephrine-induced aggregation least. All antibiotics inhibited collagen-induced aggregation, but only at inordinately high concentrations (25 mM). In vivo studies in 10 male subjects, randomly infused intravenously with 2 g of aztreonam or saline placebo every 6 h for 21 consecutive doses in a single-blind crossover study, revealed no evidence of bleeding or visible adverse side effects. Although plasma coagulation and platelet adhesion remained within normal limits in all subjects throughout the study, inhibition of ADP-induced platelet aggregation significantly (P less than 0.0001) increased on days 3 and 6, but still was below 40%. With the exception of one subject who had a mean template bleeding time of 7.3 min (normal, 2 to 7 min at 95% confidence limits) on day 6 of aztreonam administration, all volunteers exhibited bleeding times within the normal range. No abnormalities in platelet morphology were observed. Mean peak serum aztreonam concentrations on days 1 and 6 were 90.1 +/- 16.7 and 95.9 +/- 13.7 micrograms/ml, respectively; accumulation did not occur. Thus, in normal volunteers, aztreonam produced no significant recognizable abnormalities of hemostasis after 6 days of maximal recommended doses.

Protein C levels in nephrotic syndrome: use of a new enzyme-linked immunoadsorbent assay for protein C antigen.

Activated protein C is a potent, physiologic anticoagulant that inactivates the activated forms of factors V and VIII as well as facilitates in vivo fibrinolysis. We developed a competitive protein-binding enzyme-linked immunoadsorbent assay (ELISA) for protein C that was utilized to investigate if the hypercoagulability of the nephrotic syndrome is related to a deficiency of circulating plasma protein C. Protein C was measured in plasma of 11 patients with nephrotic syndrome (24 hr protein 8.4 +/- 1.6 g, SEM; serum creatinine 4.2 +/- .74 mg/dl, SEM). Ten azotemic nonnephrotic patients were employed as controls (serum creatinine 6.0 +/- 1.25 mg/dl, SEM). No significant reduction of protein C values was observed (mean 108%, range 65-200%) in nephrotic patients when compared with the controls (mean 127%, range 100-200%) even though protein C antigen was present in all nephrotic urine samples tested. Also, no correlation existed between blood levels of urea nitrogen, creatinine, albumin, total protein, or 24-hr urine protein excretion and the observed protein C values. These results suggest that in patients with the nephrotic syndrome, a hypercoagulable state may not be related to a deficiency of protein C and that the level of this zymogen in nephrotic syndrome reflects a dynamic balance between urinary losses and systemic production.

Stimulation of fibrinogen synthesis in cultured rat hepatocytes by fibrinogen fragment E.

Because of the inherent difficulties of experimentation in intact animals, we used primary monolayer cultures of non-proliferating adult rat hepatocytes to study the effects of fibrinogen degradation products on fibrinogen biosynthesis. The freshly isolated hepatocytes obtained by collagenase perfusion of the liver in situ were cultured in a chemically defined serum-free medium. The rate of fibrinogen synthesis in control cultures was 40-50 pmol/2.5 X 10(6) cells per 24 h. Additions of 20, 60 or 100 micrograms of homologous stage I fibrinogen degradation products had no effect on fibrinogen synthesis. In contrast, addition of the same amounts of homologous or heterologous (human) stage III fibrinogen degradation products resulted in a concentration-dependent increase in fibrinogen biosynthesis without affecting the rate of synthesis of albumin. When purified stage III fibrinogen degradation products D and E (human) were tested in 10, 30 or 50 micrograms/3 ml medium only fragment E showed a significant increase in fibrinogen biosynthesis (1.9-, 2.8- and 5.6-fold, respectively, over the control cultures). The presence of excess fibrinogen had no effect. These results suggest that fibrinogen fragment E may be a specific stimulator of fibrinogen biosynthesis which may play an important role in maintaining normal levels of plasma fibrinogen.

Comparison of indium 111-labeled platelets v leukocytes in a pyogenic abscess.

This study sought to determine which blood component, WBCs or platelets, is the more specific indicator of an abscess and where each localizes. An abscess was created using stool in the hind limb of dogs. After 24 hours, one group was given autologous indium 111-labeled platelets and another group was given autologous indium 111-labeled WBCs. Blood, abscess fluid, infected operative control muscle tissue, and nonoperative control muscle tissue were counted for radioactivity 24 hours after administration of the labeled cells. There was significantly (P less than .001) less WBC radioactivity in blood and more within abscess fluid compared with platelets. The highest platelet activity occurred in muscle tissue adjacent to the abscess (P less than .002) compared with platelet activity in abscess fluid or control muscle tissue. The unwanted high platelet blood background activity and the desirable high concentration of WBC radioactivity within the abscess fluid makes the latter the preferential radionuclide imaging agent.

Warfarin resistance with nafcillin therapy.

Drug-induced warfarin resistance may be mediated by the direct effect of a drug on warfarin's absorption, excretion, distribution, or metabolism. A 29-year-old man on long-term stable anticoagulation therapy with warfarin sodium developed resistance to warfarin while receiving nafcillin. His prothrombin time ranged between 14 and 17 s (control, 12 s) despite an increase in his warfarin dosage to 25 mg/d. Pharmacokinetic studies showed that the decreased hypoprothrombinemic effect of warfarin was most likely due to rapid metabolism of the anticoagulant induced by nafcillin. Warfarin's half-life was 11 hours when the patient was on nafcillin therapy and 44 hours when he was off nafcillin therapy. This interaction may be clinically important in patients requiring concomitant administration of nafcillin and warfarin.

Gamma scintigraphic localization of platelets labeled with indium 111 in a focus of infection.

To determine if autologous platelets would localize in a focus of infection, a pyogenic abscess was created in the left hind limb of dogs, using previously processed human stool, while an identical surgical procedure without bacterial inoculation was performed on the right hind limb. Autologous platelets labeled with indium 111 (500 microCi) were administered intravenously to five control dogs that had not undergone surgery, to eight dogs two hours following stool inoculation, and to five dogs 24 hours after stool inoculation. A statistically significant scintigraphic increase in tracer activity was apparent within 24 hours in each animal at the site of abscess creation. Tissue samples, obtained at 48 hours after the administration of labeled platelets, revealed a significant increase in percent dose of 111In per gram of infected muscle compared with control muscle. These studies show that platelets localize at the site of bacterial infection.

Acquired warfarin resistance and weight-reducing diet.

A frequent cause of acquired warfarin resistance is drug interaction; however, ingestion of large amounts of vitamin K in food may also be an etiologic factor. A 31-year-old woman on a weight-reducing diet showed evidence of resistance to warfarin sodium therapy. On a regimen of 35 mg of oral warfarin sodium daily, prothrombin time was 14 s (control, 12 s). Pharmacokinetic studies did not reveal any evidence of impaired adsorption or increased catabolism of the drug. The half-life of her oral warfarin was 26 hours (normal, 15 to 56 hours). Although end-organ resistance was not studied fully, a change of her vegetable-rich, weight-reducing diet (vitamin K content, 1,277 microgram) to a regular diet (vitamin K content, 360 microgram) resulted in substantial reduction in her warfarin resistance. We conclude that in patients on vegetable-rich, weight-reducing diets, a relative resistance to warfarin may be secondary to their increased dietary intake of vitamin K.

Collagen synthesis by the hepatocyte: studies in primary cultures of parenchymal cells from adult rat liver.

Based on the finding that prolyl hydroxylase, a key enzyme in collagen biosynthesis, is a constituent of the hepatic parenchymal cell, we have suggested that the hepatocyte may synthesize collagen (Exp. Cell Res. 123: 269-279, 1979). We now report that, consistent with this idea, collagen formation has been detected in primary nonproliferating cultures of isolated rat hepatocytes prepared from either normal liver or regenerated liver four days after partial hepatectomy. The characteristics of the radiolabeled collagen formed in two-day old cultures incubated for 24 hours in the presence of either [3H]-proline or [35S]-cystine were its resistance to pepsin and its susceptibility to degradation by highly purified, protease-free bacterial collagenase. The presence of fibroblasts in the hepatocyte cultures was excluded as an explanation for these results because we detected no type I collagen, a universal product of the cultured fibroblast. The initial low rates of synthesis of collagen relative to total cellular protein (0.1-0.4 percent) increased dramatically upon continued incubation of the cells reaching 0.31 and 0.81 percent in nine-day old cultures of normal or regenerated hepatocytes, respectively. This change was accompanied by the synthesis of an additional 100,000 molecular weight from of collagen, possibly type I or A, B. Morphologically, the hepatocytes progressively flattened and overlapped adjacent cells with time in culture. However, their identify as hepatocytes was confirmed by the fact that synthesis of fibrinogen, a liver-specific function, was maintained above initial levels throughout the experiment. We conclude that synthesis of collagen is a constitutive function of the hepatocyte. This function is linked to hepatocyte replication, is subject to phenotypic change in culture, and may be important in the pathogenesis of hepatic fibrosis or cirrhosis.

Isolation of anticoagulant proteins from cobra venom (Naja nigricollis). Identity with phospholipases A2.

Three anticoagulant proteins were isolated from the venom of Naja nigricollis (spitting cobra). The peaks of anticoagulant activity co-chromatographed with phospholipase A2 activities. The three proteins were homogeneous by the criteria of electrophoresis in two acidic polyacrylamide gel systems. Electrophoresis in sodium dodecyl sulfate also yielded single protein bands with molecular weights of about 15,000. The amino acid compositions of the three anticoagulant proteins are reported. All three proteins have only one amino acid replacement in the first 25 to 30 amino acids of their NH2-terminal sequences, compared to the sequence of the basic phospholipase from N. nigricollis venom. Removal of Ca2+ from the crude venom caused loss of both anticoagulant and phospholipase activities, and restoration of Ca2+ caused partial recovery of both activities. Both activities were lost in parallel when the venom was heated between pH 6.0 and 8.5. The association of the anticoagulant effect with phospholipase activity contradicts the previous conclusion that phospholipase is not responsible for the anticoagulant action of cobra venom. The previous results might be explained by independence of the anticoagulant and phospholipase effects within the same protein molecule, or by different activity levels of monomer and dimer forms of the enzyme.

In vitro stability of human fibrinopeptide B.

In anticipation of a future clinical application of plasma fibrinopeptide B (FPB) measurement, we studied the stability of FPB in an ultrafiltrate of normal plasma, normal urine and alkaline buffer by measuring the immunoreactivity of the peptide by FPB radioimmunoassay using anti FPB serum (R-29). FPB was unstable in an ultrafiltrate of plasma and urine and demonstrated a temperature dependent loss of activity. In plasma ultrafiltrate the loss of immunoreactivity was not significant during the first 24 hours, however, 92% of the peptide activity was lost at the end of seven days at 25 degrees C and 37 degrees C. The rate of FPB degradation in urine was comparable. The peptide was stable in an alkaline buffer (pH 8.5) at temperatures ranging from -10 degrees C to 37 degrees C or in plasma ultrafiltrate or urine when incubated at -10 degrees C. Treatment with carboxypeptidase B or leucine aminopeptidase for two hours at 37 degrees C (enzyme/substrate molar ratio of up to 1:100) did not cause a loss of FPB immunoreactivity. EDTA (1.0 mM) and Trasylol (500 units/ml) completely stabilized the peptide in a plasma ultrafiltrate.