Disseminated intravascular coagulation in prostatic carcinoma reversed by ketoconazole.
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Aged↗
Biomedical subjects
W R Bell
Publications and source records attributed to W R Bell.
Deglycosylation of a native, protease-sensitive glycoprotein by peptide N-glycosidase F without protease inhibitors.
The glycoprotein fibrinogen was deglycosylated in its native state and in the absence of protease inhibitors by peptide N-glycosidase F following removal of protease contaminants from the enzyme by HPLC. Fibrinogen is sensitive both to proteolysis by contaminants which may constitute as little as 0.2% of the enzyme protein and to denaturation by 1,10-o-phenanthroline, the only substance known to inhibit the proteolysis. Thus removal of protease contaminants from the enzyme is a prerequisite for the deglycosylation of native fibrinogen. The starting material for the present method is the final material obtained from the purification described by A. L. Tarentino, C. M. Gomez, and T. H. Plummer (1985, Biochemistry 24, 4565). Three sequential passages over a PolyCAT A (20 X .46 cm) cation-exchange column and elutions with a linear gradient of NaCl from 0 to 0.4 M were necessary to completely overcome the tenacious but noncovalent association of peptide N-glycosidase F with contaminants that proteolyze fibrinogen. All three chromatographic runs could be completed in 1 day. Using this "protease-free" enzyme at up to a 1:20 molar ratio, fibrinogen that is completely deglycosylated and native has been generated in order to determine the role of the carbohydrate moieties in its function.
Amidohydrolases↗
A kinetic analysis of fibrinogenolysis during plasminogen activator therapy.
A mathematic model of the systemic fibrinogenolysis that accompanies coronary thrombolysis with recombinant tissue plasminogen activator (rt-PA) has been devised. The kinetic parameters of the model were estimated by nonlinear regression analysis with data from a clinical trial of rt-PA. The plasma elimination rate constant for rt-PA was estimated to be 10.0 hr-1, the second-order catalytic rate constant for the in vivo rt-PA-mediated conversion of plasminogen to plasmin 0.0078 (microgram rt-PA/kg body weight)-1 hr-1, the plasma elimination rate constant for plasmin 2.33 hr-1, and the second-order catalytic rate constant for the in vivo plasmin-catalyzed degradation of fibrinogen 0.466 (mg plasmin/dl)-1 hr-1. Computer simulation studies based on these parameter estimates show that the magnitude of fibrinogenolysis induced by rt-PA is related to the dose in a curvilinear fashion, showing diminishing increments in the effect at increasing doses. The degree of fibrinogenolysis induced by rt-PA administration is nearly independent of the dosing schedule.
Clinical Trials as Topic↗
Platelet-collagen adhesion enhances platelet aggregation induced by binding of VWF to platelets.
Ristocetin-induced platelet aggregation (RIPA) was evaluated in the presence of platelet-collagen adhesion. RIPA of normal donor platelet-rich plasma (PRP) demonstrated a primary wave of aggregation mediated by the binding of von Willebrand factor (VWF) to platelets and a secondary aggregation wave, due to a platelet-release reaction, initiated by VWF-platelet binding and inhibitable by acetylsalicylic acid (ASA). An enhanced RIPA was observed in PRP samples to which collagen had been previously added. These subthreshold concentrations of collagen, which by themselves were insufficient to induce aggregation, caused measurable platelet-collagen adhesion. Subthreshold collagen did not cause microplatelet aggregation, platelet release of [3H]serotonin, or alter the dose-responsive binding of 125I-labeled VWF to platelets, which occurred with increasing ristocetin concentrations. However, ASA inhibition of the platelet release reaction prevented collagen-enhanced RIPA. These results demonstrate that platelet-collagen adhesion altered the platelet-release reaction induced by the binding of VWF to platelets causing a platelet-release reaction at a level of VWF-platelet binding not normally initiating a secondary aggregation. These findings suggest that platelet-collagen adhesion enhances platelet function mediated by VWF.
Blood Platelets↗
Cold agglutinin hemolytic anemia: management with an environmental suit.
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Adult↗
Extensive thrombus formation with heparin resistance during extracorporeal circulation. A new presentation of familial antithrombin III deficiency.
Extensive thrombus formation during extracorporeal circulation despite the administration of heparin sodium prompted investigation of a 15-year-old boy with a calcified right ventricular thrombus and a history of subacute bacterial endocarditis. In vitro studies confirmed the failure of heparin in standard doses to have an anticoagulant effect. Antithrombin III concentrations were low. The patient's mother, who had no history of thromboembolic disease, was also antithrombin III deficient. Resistance to heparin is a theoretical, but inconsistently documented, feature of antithrombin III deficiency. This deficiency state should be considered whenever heparin resistance is encountered, even in the absence of a personal and family history of thromboses.
Adolescent↗
Application of a nitrocellulose immunoassay for quantitation of proteins secreted in culture media.
A macro-dot immunoassay was developed to quantitate proteins (antigens) secreted in the culture media of primary rat hepatocytes. Dilutions of protein standards and undiluted spent culture media were applied to numbered sheets of nitrocellulose (NC) paper by vacuum filtration (in volumes up to 1 ml) through a specially designed macrofiltration apparatus constructed of plexiglass. Sequential incubation of the NC with bovine serum albumin blocking buffer, monospecific antibody, and 125I Protein A enabled quantitation of protein concentration by determination of NC bound radioactivity. Linear and reproducible standard curves were obtained with fibrinogen, albumin, transferrin, and haptoglobin. A high degree of coefficient of correlation between radioactivity (cpm) and protein concentration was found. Intra- and interest reproducibility was excellent (C.V.'s less than 7%). By using monospecific antibodies, single proteins (i.e., fibrinogen), as low as 32 ng/ml, could be quantified in heterogeneous protein mixtures and in spent culture media. The assay was sensitive to the difference of fibrinogen secretion under nonstimulatory (serum-free hormonally defined medium, SFHD) and stimulatory (SFHD plus hydrocortisone) culture conditions. The procedure and techniques described are applicable to the quantitation of any protein in a suitable buffer.
Albumins↗
Fibrinogen Seattle II: congenital dysfibrinogenemia with an Arg (A alpha 16)----his substitution.
Incubation of fibrinogen Seattle II with thrombin (17 mu/ml) resulted in the release of two forms of fibrinopeptide A (FpA) which were resolved by high-performance liquid chromatography. Amino acid analysis disclosed that the abnormal FpA contained histidine in place of arginine. At lower, approximately physiologic thrombin concentrations only half the normal amount of FpA was released, and fibrinopeptide B (FpB) release was delayed. The effect of this substitution on the time course of fibrinopeptide release is consistent with conclusions drawn from other studies on the kinetics of fibrinopeptide release, viz., that prior removal of FpA is not required before FpB hydrolysis by thrombin, and that optimal rates of FpB release occur after formation of fibrin I polymer.
Afibrinogenemia↗
Post-transfusion purpura: a report of five patients and a review of the pathogenesis and management.
We report five cases of post-transfusion purpura. Despite having profound thrombocytopenia, their management consisted only of steroid administration if serious bleeding was present. In one patient without bleeding, no specific therapy was given. This patient recovered uneventfully. Two patients died. One of these died from complicating illnesses rather than bleeding. The other suffered brain death, the result of a ruptured cerebral aneurysm. Four patients were PLA1 negative. The fifth patient had an antibody to an unidentified platelet-specific antigen.
Adult↗
Histidyl-tRNA synthetase, the myositis Jo-1 antigen, is cytoplasmic and unassociated with the cytoskeletal framework.
The myositis-specific anti-Jo-1 autoantibody, which is directed against histidyl-tRNA-synthetase, is found in 30% of polymyositis patients. The Jo-1 antigen has been reported to be a nuclear antigen by some authors. On the contrary we show that less than 2% of the total histidyl-tRNA and lysyl-tRNA synthetase activities are associated with purified rat liver nuclei or the hepatocyte intermediate filament-nuclear fraction. In the presence of polyethylene glycol, in which the high Mr multi-enzyme complex containing lysyl-tRNA synthetase is insoluble, 65% of the lysyl-tRNA synthetase and only 15% of histidyl-tRNA synthetase activities remained associated with the cytoskeletal framework. The Jo-1 antigen exhibited a diffuse granular cytoplasmic distribution in cultured rat hepatocytes as determined by indirect immunofluorescent microscopy. Hence, the Jo-1 antigen is cytoplasmic and unassociated with the cytoskeletal framework or high Mr synthetase complex in situ.
Amino Acyl-tRNA Synthetases↗
Analysis of red cell survival data.
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Erythrocyte Aging↗
Plasma clearance rates of coagulation factors VIII and IX in factor-deficient individuals.
The plasma clearance rates of factors IX and VIII were determined in patients with hemophilia A and B who had received factor replacement by prolonged, continuous infusion of factor concentrates. The clearance rates were calculated by dividing the factor infusion rates by the steady-state plasma factor activities corrected for base-line factor activities. The mean factor IX clearance rate in eight factor IX-deficient patients was 233 mL/h (range 159 to 340 mL/h). The mean normalized clearance rate was 3.4 mL/h/kg. The mean factor VIII clearance rate in eight factor VIII-deficient patients was 294 mL/h (range 229 to 361 mL/h) and the mean normalized rate was 5.0 mL/h/kg. Both factors show linear relationships between the factor infusion rates and the steady-state plasma factor activities achieved.
Factor IX↗
Platelet-collagen interaction: inhibition by ristocetin and enhancement by von Willebrand factor-platelet binding.
The contribution of von Willebrand factor (vWF)-platelet binding to platelet-collagen interaction was examined in vitro. The binding of vWF to platelets was mediated and regulated by ristocetin. Subthreshold concentrations of ristocetin (less than or equal to 1 mg/mL), insufficient to cause ristocetin-induced platelet aggregation (RIPA), were added to platelet-rich plasma (PRP) prior to the addition of collagen. The collagen-induced platelet aggregation (CIPA) was modified by ristocetin and the degree of alteration was dependent on the ristocetin concentration. Response as a function of ristocetin concentration was designated the Collagen-Platelet Aggregation Response (CoI-PAR). In normal PRP the CoI-PAR was a progressive inhibition followed by decreasing inhibition and then an enhanced response. The enhanced response occurred over a narrow range of ristocetin concentrations (0.8 to 1.0 mg/mL). In the absence of vWF (severe von Willebrand's disease, Type I, vWF less than 1%) the CoI-PAR was a progressive, eventually complete inhibition with no enhanced response (with ristocetin concentrations up to 3.0 mg/mL). With addition of vWF to this PRP an enhanced response was observed at a ristocetin concentration inversely proportional to the vWF level. PRP from a patient with severe Hemophilia A showed a response within the normal range. Subthreshold ristocetin did not cause plasma protein precipitation or platelet release of 3H-serotonin, nor induce micro platelet aggregate formation. Digestion of platelet membrane glycoproteins (GP(s] with chymotrypsin demonstrated that upon removal of GPI, RIPA was absent, CIPA retained and the CoI-PAR was progressive inhibition, with no enhancement. With removal of GPs I, II, and III, RIPA, CIPA, and the CoI-PAR were absent. A dose-response 125I-vWF-platelet binding occurred with increasing ristocetin concentrations which was unchanged by the addition of collagen. These results demonstrated that ristocetin-platelet association inhibited CIPA, and vWF-platelet binding enhanced platelet-collagen adhesion and platelet aggregation. The in vitro-enhanced CIPA represents a vWF-dependent aggregation of sufficient magnitude to overcome the inhibitory effect of ristocetin. These studies demonstrate an influential interaction of ristocetin, vWF, and collagen with the platelet membrane and imply an important hemostatic contribution of vWF-platelet binding in platelet-collagen interaction.
Blood Platelets↗
Studies on the pathophysiology of posttransfusion purpura.
Posttransfusion purpura typically occurs in PLA1 negative blood recipients who have been previously immunized to the PLA1 antigen. Following transfusion, severe thrombocytopenia develops with the formation of anti-PLA1. Since the patients' platelets lack the PLA1 antigen, one would not expect this antibody to destroy autologous platelets. In this study we show that PLA1 antigen exists in stored blood and can absorb to PLA1 negative platelets making them PLA1 reactive. Incubating PLA1 (-) platelets with ultracentrifuged plasma from PLA1 (+) blood donors allowed anti-PLA1 to bind to PLA1 (-) platelets. Control plasma from PLA1 (-) blood donors did not lead to anti-PLA1 binding. Using an inhibition assay, we showed that stored blood contains PLA1 material that was not removed by ultracentrifugation. The material absorbing to PLA1 (-) platelets represented the PLA1 antigen, which was confirmed by Western blotting. After incubating plasma containing PLA1 antigen with PLA1 (-) platelets, reactivity at 95,000 D was observed. Native PLA1 (+) platelets showed a similar band. When PLA1 (-) platelets were incubated with plasma from a PLA1 (-) donor, this band was not present. These studies show that a soluble form of PLA1 antigen exists in stored blood that can absorb to PLA1 (-) platelets. Consequently, anti-PLA1 can bind to these platelets leading to thrombocytopenia. These observations may explain the autologous destruction of platelets in posttransfusion purpura.
Antigens, Human Platelet↗
Reduction in the plasma clearance rate of warfarin induced by cimetidine.
The interaction between the histamine H-2 receptor antagonist, cimetidine, and warfarin sodium was prospectively studied in 14 patients who were previously anticoagulated for five years. The patients received warfarin and cimetidine concomitantly for a minimum of ten days. Seven of the patients experienced increases in plasma warfarin concentrations. This correspondingly resulted in abnormal prolongation of their prothrombin times. In these patients the elevation in the steady-state warfarin concentration demonstrates that coadministration of cimetidine significantly reduces the plasma clearance rate of warfarin. The serum and urine metabolite levels of warfarin were not qualitatively different in the absence in contrast to the presence of cimetidine. It is apparent that cimetidine can act as an inhibitory influence on the catabolic degradation of warfarin.
Aged↗
Localization of a fibrinogen calcium binding site between gamma-subunit positions 311 and 336 by terbium fluorescence.
Calcium is required for effective fibrin polymerization. The high affinity Ca2+ binding capacity of fibrinogen was directly localized to the gamma-chain by autoradiography of nitrocellulose membrane blots of fibrinogen subunits incubated with 45Ca2+. Terbium (Tb3+) competitively inhibited 45Ca2+ binding to fibrinogen during equilibrium dialysis, accelerated fibrin polymerization, and limited fibrinogen fragment D digestion by plasmin. The intrinsic fluorescence of Ca2+-depleted fibrinogen was maximally enhanced by Ca2+ and Tb3+, but not by Mg2+, at about 3 mol of cation/mol of fibrinogen. Protein-bound Tb3+ fluorescence at 545 nm was maximally enhanced by resonance energy transfer from tryptophan (excitation at 290 nm) at about 2 mol of Tb3+mol of fibrinogen and about 1 mol of Tb3+/mol of plasmic fragment D94 (Mr 94,000). Fibrinogen fragments D78 (Mr 78,000) and E did not show effective enhancement of Tb3+ fluorescence, suggesting that the Ca2+ site is located within gamma 303 to gamma 411, the peptide which is absent in fragment D78 but present in D94. When CNBr fragments of the carboxyamidated gamma-subunit were assayed for enhancement of Tb3+ fluorescence, peptide CBi (gamma 311-336) bound 1 mol of Tb3+/mol of CBi. Thus, the Ca2+ site is located within this peptide. The sequence between gamma 315 and gamma 329 is homologous to the calmodulin and parvalbumin Ca2+ binding sites.
Amino Acid Sequence↗
Protective effect of divalent cations in the plasmin degradation of fibrinogen.
Calcium limits the plasmic proteolysis of fibrinogen fragment D by binding to a specific site on the carboxy-terminal segment of the D gamma chain. Employing sodium dodecyl sulfate-polyacrylamide gel electrophoresis to visualize plasmic fragments, Sr2+, Ba2+, and Mn2+ were found to have an equivalent capacity to limit the degradation of fibrinogen fragment D (Mr 94,000). Mg2+, Fe2+, Co2+, and Zn2+ did not comparably limit the digestion of fragment D. Equilibrium dialysis demonstrated that Ba2+ competitively inhibited Ca2+ binding to fibrinogen, suggesting that the ions occupied the Ca2+ binding site of fibrinogen and thereby limited the plasmic digestion of fragment D. The results suggest that Ca2+, Sr2+, Ba2+, and Mn2+ limit plasmin digestion of fragment D by interacting with a Ca2+ binding site in the D domain of the fibrinogen molecule.
Binding Sites↗
Fibrinogen Baltimore IV: congenital dysfibrinogenemia with delayed fibrin monomer polymerization.
A congenital dysfibrinogenemia, fibrinogen Baltimore IV, has been found in a 56-year-old Caucasian man. Clinical laboratory studies disclosed a slightly prolonged prothrombin time, but were otherwise unremarkable. Release of fibrinopeptides by thrombin occurs normally, as does ligation of the fibrin polymer by Factor XIII. Approximately half of the isolated fibrin monomers polymerize normally, but the remainder polymerize at about 2% of the initial rate. The functional defect is thus limited to a decrease in the rate of fibrin monomer polymerization.
Afibrinogenemia↗