The seamless web revisited: tissue plasminogen activator in disseminated intravascular coagulation.
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Disseminated Intravascular Coagulation↗
Biomedical subjects
G H Goldsmith
Publications and source records attributed to G H Goldsmith.
Bovine coronary artery endothelium: in vitro culture and production of plasminogen activator.
A new method for isolation and culture of endothelial cells from bovine coronary artery (BCoAEC) is presented. This method involves in situ perfusion and digestion of main coronary arteries with a collagenase solution. The isolated cells were cultured and maintained through many cell passages in Dulbecco's Modified Eagle's Medium supplemented with fetal bovine serum derived from either whole blood or plasma. Confirmation of these cells' endothelial origin was obtained by demonstration of typical morphologic and growth characteristics of endothelium, immunofluorescent staining with antibodies to von Willebrand factor (Factor VIII: vWF), and measurement of plasminogen activator (PA). In addition, production of PA was inhibited by enzymatically active thrombin as has been previously described with bovine aortic endothelial cells in culture.
Animals↗
Studies on a circulating anticoagulant inhibiting factor XI in a patient with congenital deficiency and carcinoma of the prostate.
An inhibitor of plasma thromboplastin antecedent (PTA, factor XI), measured in coagulant and radioimmunoassays, was detected in a 60-year-old man with carcinoma of the prostate who had no evidence of a bleeding tendency. Family studies indicated that the patient was either a homozygote or a heterozygote for hereditary factor XI deficiency. In contrast to earlier described patients with factor XI deficiency in whom inhibitors were detected, the patient was unaware of having been transfused with blood or blood products at any time before the discovery of the inhibitor. The inhibitor of factor XI in the patient's plasma appeared to be predominantly in the IgG4 fraction and to be directed at a locus on the factor XI molecule other than the active site; it did not block the amidolytic properties of activated factor XI (XIa). Rather, it appeared to block adsorption of factor XI to negatively charged surfaces. The inhibitor interfered with measurement of other components of the intrinsic pathway of thrombin formation, perhaps explaining the low titres of other coagulation factors of the intrinsic system reported in patients with strong inhibitors directed against factor XI.
Factor XI↗
Metal and phospholipid binding properties of partially carboxylated human prothrombin variants.
To study the specific role of gamma-carboxyglutamic acid (Gla) residues in prothrombin, we have isolated a series of partially carboxylated prothrombin variants from a patient with a hereditary defect in vitamin K-dependent carboxylation (Goldsmith, G. H., Pence, R. E., Ratnoff, O. D., Adelstein, D. A., and Furie, B. (1982) J. Clin. Invest. 69, 1253-1260). The three variant prothrombins, purified by DEAE-Sephacel, immunoaffinity chromatography, and preparative gel electrophoresis, were indistinguishable from prothrombin in molecular weight, amino acid composition, and NH2-terminal amino acid sequence, with the exception of Gla residues. Variant prothrombin 1, with 8 Gla residues, had 66% of the coagulant activity of prothrombin, one high affinity metal-binding site (Kd = 15 nM), and three lower affinity sites (Kd = 2.7 microM); prothrombin contained two high affinity (36 nM) and four lower affinity sites (Kd = 1 microM). Ca(II) induced a 23% decrease in the intrinsic fluorescence of variant prothrombin 1 fragment 1, compared to a 35% decrease in that of prothrombin fragment 1. The phospholipid binding activity of variant prothrombin 1 was 44% that of prothrombin. Variant prothrombin 2 and variant prothrombin 3, with 4 and 6 Gla residues, respectively, had about 5% of prothrombin coagulant activity and a single high affinity and two lower affinity metal-binding sites and exhibited no phospholipid binding activity. Variant prothrombin 3 fragment 1 and variant prothrombin 2 fragment 1 demonstrated 18 and 13% of Ca(II)-induced fluorescence quenching, respectively. Abnormal prothrombin, with 1 Gla residue, had 8% of prothrombin coagulant activity, a single lower affinity (1 microM) metal-binding site, and 13% Ca(II)-induced fluorescence quenching of the fragment 1 species and did not bind to phospholipid. These results indicate that Gla residues define the metal binding properties of prothrombin. Most, if not all, of the Gla residues are required for complete prothrombin function, and the prothrombin coagulant activity correlates to the phospholipid binding activity of the prothrombin species.
Amino Acid Sequence↗
Inhibitors of plasma thromboplastin antecedent (factor XI): studies on mechanism of inhibition.
Acquired inhibitors of plasma thromboplastin antecedent (PTA) have been described in several recent studies, which collectively emphasize the heterogeneity of PTA functions that are impaired by this class of circulating anticoagulants. To clarify whether acquired inhibitors of PTA exhibit inhibitory properties that vary widely from one patient to the next, IgG inhibitors from four patients were isolated. The effect of these inhibitors on the binding of PTA to glass, the activation of kaolin-bound PTA, the coagulant activity of both kaolin-bound and fluid phase activated PTA (aPTA), and the enzymatic activity of aPTA against a low molecular weight substrate was quantified. The relative inhibition of these individual components of PTA activation and function by the four anticoagulants was quite similar. The results indicate that the principal binding sites for acquired inhibitors of PTA are PTA domains involved with surface binding and conversion of PTA to aPTA, with less effective steric impairment of aPTA enzymatic activity.
Adult↗
A monoclonal antibody that inhibits activation of human Hageman factor (factor XII).
A monoclonal antibody to human Hageman factor (HF, factor XII) was derived from BALB/c mouse spleen cells fused with NS-1 mouse myeloma cells. This antibody, purified from ascites fluid, reacted with HF to inhibit the activation of HF, purified or in normal pooled plasma, as measured by a coagulation assay. The antibody did not inhibit the coagulant activity of activated HF. The antibody also inhibited the generation of amidolytic activity in HF-ellagic acid mixtures, but failed to inhibit the amidolytic properties of the carboxy-terminal fragment of HF (HFf). Amidolytic activity, absent in an HF-monoclonal antibody mixture, was generated upon treatment with insoluble trypsin. Monoclonal antibody, bound to CNBr Sepharose 4B gel (Pharmacia Fine Chemicals, Piscataway, NJ), reversibly bound HF in plasma or in buffer, without activating it. HF was then eluted with 4 mol/L guanidine HCI. The passage of 125I-labeled HF enzymatically cleaved by trypsin through a column of monoclonal antibody-CNBr Sepharose 4B gel resulted in flow-through of HFf with a molecular weight (mol wt) of 30,000 and HF fragments of mol wt 12,000. Elution with 4 mol/L guanidine HCI yielded several HF fragments (mol wt 80,000, 52,000, and 40,000) but not HFf. These data suggest that the single determinant recognized by the murine monoclonal antibody is not on HFf, but rather on the amino-terminal fragment thought to be involved in the binding activity of HF. The monoclonal anti-HF bound to CNBr-activated Sepharose 4B gel could be used to artificially deplete plasma samples of HF.
Animals↗
Bovine aortic endothelial cells elaborate an inhibitor of the generation of lipopolysaccharide-stimulated human blood monocyte procoagulant activity.
We examined the effect of bovine aortic endothelial cell culture supernatants upon the generation of procoagulant activity by human blood monocytes. Confluent endothelial monolayers were cultured for up to 96 h. At timed intervals, culture supernatants were collected and incubated for 5 h with lipopolysaccharide-stimulated human peripheral blood mononuclear cells. The procoagulant activity of mononuclear cell lysates was determined in a one-stage clotting assay. In five experiments, procoagulant activity with culture supernatant (time 0) was 2,294 +/- 761 U/ml (mean +/- SEM). Culture supernatants from endothelial cells incubated for 24-96 h strongly inhibited mononuclear cell generation of procoagulant activity. Indomethacin (10 microM) added to endothelial cells delayed the appearance of procoagulant inhibitor for 72 h. Bovine aortic smooth muscle cell culture supernatants did not inhibit procoagulant activity. The inhibitor was heat stable, effective at 1:50 dilution, soluble, and acid sensitive, with a molecular weight of less than 1,500. Further studies on subpopulations of mononuclear cells demonstrated that endothelial inhibitor selectively decreased the generation of monocyte procoagulant activity and interfered with T lymphocyte amplification of monocyte production of procoagulant activity. Thus, we have demonstrated that endothelial cells elaborate a potent inhibitor of monocyte procoagulant activity.
Animals↗
Myelodysplastic syndromes: is another classification necessary?
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Hematologic Diseases↗
Studies on a family with combined functional deficiencies of vitamin K-dependent coagulation factors.
Two siblings with m ild hemorrhagic symptoms had combined functional deficiencies of vitamin K-dependent clotting factors. Prothrombin (0.18-0.20 U/ml) and Stuart factor (Factor X, 0.18-0.20 U/ml) and Stuart factor (Factor X, 0.18-0.20 U/ml) were most severely affected. Antigenic amounts of affected coagulation factors were normal and normal generation of thrombin activity occurred in the patients' plasmas after treatment with nonophysiologic activators that do not require calcium for prothrombin activation. Hepatobilary disease, malabsorptive disorders, and plasma warfarin were not present. Both parents had normal levels of all coagulation factors. The patients' plasmas contained prothrombin that reacted both with antibody directed against des-gamma-carboxyprothrombin and native prothrombin. Crossed immunoelectrophoresis of patients' plasmas and studies of partially purified patient prothrombin suggested the presence of a relatively homogeneous species of dysfunctional prothrombin, distinct from the heterologous species found in the plasma of warfarin-treated persons. These studies are most consistent with a posttranslational defect in hepatic carboxylation of vitamin K-dependent factors. This kindred uniquely possesses an autosomal recessive disorder of vitamin K-dependent factor formation that causes production of an apparently homogeneous species of dysfunctional prothrombin; the functional deficiencies in clotting factors are totally corrected by oral or parenteral administration of vitamin K1.
Adolescent↗
Interaction of Borrelia spirochetes with human mononuclear leukocytes causes production of leukocytic pyrogen and thromboplastin.
Relapsing fever caused by Borrelia spirochetes is characterized by episodes of spirochetemia, fever, and DIC. We examined the ability of Borrelia hermsii to induce production of leukocytic pyrogen and thromboplastin from human blood leukocytes in vitro. Organisms were found devoid of endotoxin by the Limulus assay. Human peripheral blood leukocytes were separated into MNC and PMN fractions and were incubated with two to five spirochetes per cell in 10% human serum. Supernatant fluids from MNC-spirochete mixtures produced mean increases in the temperature of rabbits of 0.80 degree to 1.35 degrees C, which were significantly higher than those caused by supernatant fluids of MNC or spirochetes alone (p less than 0.05). MNC-spirochete mixtures possessed seven to 15 times the thromboplastic activity of MNC suspensions alone, assayed with a modified one-stage prothrombin time. Supernatant fluids of PMNs and spirochetes, on the other hand, did not contain leukocytic pyrogen, and PMN suspensions did not produce thromboplastin. Cycloheximide (10 micrograms/ml), and inhibitor of protein synthesis, completely suppressed both pyrogen and thromboplastin production. Although intracellular spirochetes were observed within phagosomes of blood monocytes by electron microscopy, the production of leukocytic pyrogen and thromboplastin was not significantly altered by serum opsonins or by the inhibitors of phagocytosis cytochalasin B (5 micrograms/ml) or phenylbutazone (2 mg/ml). These results showed that Borrelia spirochetes stimulated human MNCs to produce increased amounts of leukocytic pyrogen and thromboplastin and that this stimulation required de novo synthesis of protein, was not mediated by endotoxin, and was not prevented by omitting opsonic proteins or by inhibiting phagocytosis.
Borrelia↗
Labile anticoagulant in a patient with lymphoma.
A patient with diffuse lymphocytic lymphoma and monoclonal IgM kappa immunoglobulin was found to have an unstable circulating anticoagulant. The anticoagulant inhibited phospholipid-dependent plasma coagulation reactions. Unlike previously described anticoagulants of this type, this plasma inhibitor was neutralized in vitro by products of platelet lysis but not by exogenous addition of other phospholipids or by intact platelets. Inhibitory activity seemed dependent on the monoclonal immunoglobulin, but isolated immunoglobulin fractions lacked anticoagulant activity, suggesting that the inhibitory function was dependent on an easily disrupted macromolecular aggregate. Recognition and characterization of other similar anticoagulants may provide a means of studying the role of phospholipids in normal hemostasis.
Blood Coagulation↗
Studies on plasminogen activator and other proteases in subcultured human vascular cells.
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Animals↗
Radionuclide scanning and lung cancer.
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Decision Making↗
Chloroform-induced fibrinolysis. Its dependence on Hageman factor, plasma prekallikrein, and high molecular weight kininogen.
Incubation of normal human plasma or its euglobulin fraction with chloroform enhances the generation of proteolytic activity attributable to plasmin. The mechanisms involved remain unclear. Earlier, plasmin formation was found to be impaired in the chloroform-treated euglobulin fraction of Hageman trait plasma. We now demonstrate sharply reduced generation of fibrinolytic and caseinolytic activities in prekallikrein-deficient (Fletcher trait) and HMW kininogen-deficient (Fitzgerald trait) plasmas as well. These defects were corrected by addition of the missing factors. In addition, precipitation of the euglobulin fraction of plasma activated small but measurable amounts of HF. With the use of 125I-HF, cleavage of the native molecule into its amino-terminal and carboxy-terminal fragments occurred pari passu with the generation of plasmin. These experiments suggest that one pathway through which HF can bring about formation of plasmin in chloroform-treated euglobulin fractions involves the participation of plasma prekallikrein and HMW kininogen, presumably through mechanisms similar to those evoked by surface activation of HF.
Animals↗
Contact-activated fibrinolysis: role of surface concentration and high-molecular-weight kininogen.
Activation of plasminogen through contact-activated reactions involves participation of HF, PK, PTA, and HMWK. The concentration of negatively charged surface (kaolin) to which purified factors are exposed critically influences the extent to which the known enzymatic species participate in plasminogen activation as assayed upon a synthetic amide substrate and by fibrinolysis. HMWK is an essential cofactor for activation of plasminogen by PTA and for optimal plasminogen activation by kallikrein in the presence of kaolin. At a given kaolin concentration, HMWK reduces HF binding to kaolin and diminishes plasminogen activation by HF. In the presence of kaolin, HMWK also enhances the activity of plasmin against both synthetic substrates and fibrin.
Factor XII↗
Inhibitory spectrum of alpha 2-plasmin inhibitor.
alpha 2-Plasmin inhibitor (alpha 2PI) has been recently characterized as a fast-reacting inhibitor of plasmin in human plasma and appears to play an important role in the regulation of fibrinolysis in vivo. We have studied the effect of purified alpha 2PI upon various proteases participating in human blood coagulation and kinin generation. At physiological concentration (50 microgram/ml), alpha 2PI inhibited the clot-promoting and prekallikrein-activating activity of Hageman factor fragments, the amidolytic, kininogenase, and clot-promoting activities of plasma kallikrein, and the clot-promoting properties of activated plasma thromboplastin antecedent (PTA, Factor XIa) and thrombin. alpha 2PI had minimal inhibitory effect on surface-bound activated PTA and activated Stuart factor (Factor Xa). alpha 2PI did not inhibit the activity of activated Christmas factor (Factor IXa) or urinary kallikrein. Heparin (1.5-2.0 units/ml) did not enhance the inhibitory function of alpha 2PI. These results suggest that, like other plasma protease inhibitors, alpha 2PI possesses a broad in vitro spectrum of inhibitory properties.
Blood Coagulation Factors↗
Platelets and surface-mediated clotting activity.
In contrast to previously reported studies, no evidence could be adduced for the activation of Hageman factor (factor XII) by platelets, whether or not these cells had been incubated with adenosine diphosphate (ADP).
Adenosine Diphosphate↗
The activation of plasminogen by Hageman factor (Factor XII) and Hageman factor fragments.
Activation of plasminogen through surface-mediated reactions is well recognized. In the presence of kaolin, purified Hageman factor (Factor XII) changed plasminogen to plasmin, as assayed upon a synthetic amide substrate and by fibrinolysis. Kinetic studies suggested an enzymatic action of Hageman factor upon its substrate, plasminogen. Hageman factor fragments, at a protein concentration equivalent to whole Hageman factor, activated plasminogen to a lesser extent. These protein preparations were not contaminated with other agents implicated in surface-mediated fibrinolysis. Diisopropyl fluorophosphate treatment of plasminogen did not inhibit its activation by Hageman factor. These studies indicate that Hageman factor has a hitherto unsuspected function, the direct activation of plasminogen.
Amides↗