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Biomedical subjects

H L Nossel

Publications and source records attributed to H L Nossel.

At least 19 recordsLinked to original sources
PubMed1986

Immunologic identification of the cleavage products from the A alpha- and B beta-chains in the early stages of plasmin digestion of fibrinogen.

Fragment X components (Mr 225,000 to 333,000) were distinguished on sodium dodecyl sulfate polyacrylamide gels. Western blotting with monoclonal antibodies to A alpha-chain segments demonstrated that the A alpha-chains of fibrinogen and the largest fragment X components (Mr 285,000-340,000) contained both A alpha 259-276 and A alpha 540-554. Fragment X components of Mr 270,000-285,000 contained A alpha 259-276 but lacked A alpha 540-554, whereas the smallest fragment X components (Mr 225,000-270,000) contained neither A alpha 540-554 nor A alpha 259-276. Studies of the small peptides generated during fragment X formation complemented the studies of the large molecules, by demonstrating peptides containing both A alpha 259-276 and A alpha 540-554 (Mr 41,600-41,800 and Mr 38,700-38,900), peptides containing A alpha 540-554 but not A alpha 259-276 (Mr 20,500-21,000 and Mr 17,300-17,500) and peptides containing only A alpha 259-276 (Mr 23,600-24,000 and Mr 20,500-21,000). Cleavage of B beta 1-42 from the amino terminal ends of the B beta-chains, measured with a specific radioimmunoassay, was linear until 1.6 moles per mole of fibrinogen had been released, and coincided with loss of the central and carboxy terminal A alpha-chain regions, i. e. A alpha 259-276 and A alpha 540-554. Based on present and previously reported data, a model is proposed for the evolution of the heterogeneous group of fragment X derivatives from fibrinogen with the simultaneous release of small peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1984

Activation of factor IX bound to cultured bovine aortic endothelial cells.

Previous studies have shown that factor IX and its activated form, factor IXa, bind to cultured vascular endothelial cells and that cell-bound factor IXa retains its procoagulant activity. The present studies provide evidence that factor IX bound to cultured bovine aortic endothelial cells can be activated. Factor IX activation was assessed by finding cleavage of the factor IX molecule on NaDodSO4/polyacrylamide gel electrophoresis and by the generation of procoagulant activity as assessed by thrombin-treated factor VIII-dependent generation of factor Xa activity. Cell-bound factor IX (0.8 micrograms per 4 X 10(8) cells per ml) could be activated by factor XIa (5 micrograms/ml) or by factor VIIa (0.1 micrograms/ml) without exogenous tissue factor when endothelial cells were treated with phorbol ester and acquired tissue factor-like procoagulant activity. Regardless of how factor IX was activated, the cell-bound factor IXa required thrombin-treated factor VIII and calcium, but not exogenous phospholipid, to activate factor X. In further experiments, factor X bound to endothelial cells specifically and reversibly with a dependence on calcium and with a lower affinity (half-maximal at 480 nM) than factor IX. At saturation, 9.1 X 10(6) factor X molecules were bound per cell. After activation of factor X by factor IXa, approximately 50% of the factor Xa formed could be eluted from the cells by 10 mM EDTA, suggesting that the factor Xa was cell associated. These observations indicate that endothelial cells can bind and promote the activation of factors IX and X in the absence of platelets or exogenous phospholipid.

Animals↗
PubMed1984

Fibrinogen proteolysis and platelet alpha-granule release in preeclampsia/eclampsia.

Serial measurements of the plasma concentration of fibrinopeptide A, thrombin-increasable fibrinopeptide B (reflecting B beta 1-42), desarginyl fibrinopeptide B, beta thromboglobulin, and platelet factor 4 were made before, during, and after delivery in patients with preeclampsia/eclampsia. The data were correlated with routine coagulation studies, hematologic and renal status, as well as with the clinical manifestations. In 11 patients with mild preeclampsia, there were small increases in the fibrinopeptides at the time of delivery, but no other hematologic changes. In 5 patients with severe preeclampsia/eclampsia, there were marked increases in plasma levels of fibrinopeptides and platelet alpha granule proteins, which correlated in time with the clinical manifestations. When the changes in these patients were compared with those occurring in patients undergoing intraamniotic hypertonic saline infusion, it was noted that: (1) patients with severe preeclampsia/eclampsia usually presented when plasmin action on fibrinogen exceeded that of thrombin; (2) in patients with preeclampsia/eclampsia the increase in fibrinopeptides lasted from 3 to 7 days, rather than for several hours as occurred after the infusion of hypertonic saline, indicating a more persistent stimulus to intravascular coagulation in preeclampsia/eclampsia; (3) severe thrombocytopenia and increased platelet protein levels were seen in these patients and were disproportionate to the degree of increase in the fibrinopeptide A level, suggesting that a mechanism other than thrombin must have contributed to the platelet changes; and (4) in two patients with severe preeclampsia/eclampsia, high desarginyl fibrinopeptide B levels preceded renal insufficiency, possibly reflecting fibrin II formation in renal vessels.

Adolescent↗
PubMed1984

Prostacyclin production by perturbed bovine aortic endothelial cells in culture.

This study reports that endotoxin (Escherichia coli serotype 026:B6) and 12-O-tetradecanoyl-phorbol-13-acetate stimulate cultured bovine aortic endothelial cells to generate prostacyclin. The prostacyclin concentration of the culture medium was measured indirectly by radioimmunoassay for 6-keto-PGF1 alpha. The amount of prostacyclin generated depended on the concentration of endotoxin or phorbol diester. Prostacyclin generation was not immediate, but occurred slowly after a six-hour lag period. The perturbed cells contracted and showed marked shape changes that correlated temporally with the start of enhanced prostacyclin production. Cytochalasins B and D, vinblastine, and colchicine inhibited prostacyclin production, indicating involvement of the cytoskeleton in the cellular response to endotoxin and phorbol diester. The increase in prostacyclin production was prevented by trifluoperazine, an inhibitor of the Ca++-calmodulin system, which is known to be involved in cytoskeletal function. Generation of prostacyclin was inhibited by cycloheximide and actinomycin D, indicating dependence on protein and ribonucleic acid synthesis. It is postulated that exposure to endotoxin or phorbol diester leads, via a series of reactions that involve RNA and protein synthesis and require intact cytoskeletal function, to the generation of toxic active intermediate(s) that stimulate the enzymes necessary for prostacyclin production.

Animals↗
PubMed1983

The release of B beta 1-42 from fibrinogen and fibrin by plasmin.

The balance between thrombin and plasmin action has been postulated to be an important determinant of thrombosis. Measurement of plasma concentrations of fibrinopeptide A (FPA), which reflect thrombin action on the NH2-terminal end of the A alpha chain, and of B beta 1-42 (thrombin-increasable fibrinopeptide B immunoreactivity-TIFPB) which reflect plasmin action on the NH2-terminal end of the B beta chain have shown systematic changes in the relative concentrations of the two peptides in thrombotic states. This paper reports kinetic data for TIFPB release by plasmin using fibrinogen, fibrin I monomer, and fibrin I polymer as substrates. For fibrinogen and fibrin I monomer the data fit the Michaelis-Menten equation. Experiments were performed with human proteins in 0.15M Tris-buffered saline at pH 7.4 and at 37 degrees C. With fibrinogen as substrate the Km was calculated to be 0.87 microM and the Vmax 3.75 X 10(-5) M/min/unit of plasmin. With fibrin I monomer as the substrate the Km was calculated to be 1.25 microM and the Vmax 5.5 X 10(-5) M/min/unit of plasmin. With fibrin I polymer as substrate the data did not fit the Michaelis-Menten equation but there appeared to be no dramatic differences in rates from those obtained with the other two substrates. The influence of factor XIIIa-induced cross-linking of fibrin was not examined. It is concluded from these findings that fibrinogen and non-cross-linked fibrin I are equally good substrates for plasmin cleavage of the NH2-terminal end of the B beta chain.

Batroxobin↗
PubMed1983

Sequence of plasmin proteolysis at the NH2-terminus of the b beta-chain of human fibrinogen.

Employing high-performance liquid chromatography (HPLC), we have isolated and quantified the peptides that are released from the NH2-terminus of human fibrinogen B beta-chains by plasmin proteolysis. The peptides were identified by amino acid composition and by a radioimmunoassay developed for fibrinopeptide B detection. B beta 1-42 was the earliest fragment released during limited plasmin proteolysis. The level of this peptide reached a maximum and then began to decline during the course of the digestion. In addition, increasing levels of B beta 1-21 and of FPB followed the production of B beta 1-42. Using purified B beta 1-42 as a substrate, preferential cleavage was shown to occur at the 21-22 bond, with a minor cleavage at the 14-15 bond. Exhaustive digestion yielded two major components which were separated by HPLC: B beta 1-14 (FPB) and beta 22-42. The rate of cleavage at the 14-15 bond, which is the customary site of thrombin proteolysis, was not affected by the addition of hirudin indicating that this was not the result of trace contamination with thrombin. We have also examined plasmin proteolysis at the NH2-terminal region of the B beta-chains of a variety of fibrinogen derivatives and have found similar patterns of B beta 1-42 release. Using HPLC data, we have estimated the Km for plasmic cleavage of the beta 21-22 bond to be 1.8 X 10(-5) M and of the beta 14-15 bond to be 2.8 X 10(-5) M.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1983

Binding of factors IX and IXa to cultured vascular endothelial cells.

Factor IX and its activated form IXa have been found to bind to confluent cultured bovine aortic and human umbilical vein endothelial cells. Binding of bovine factors IX and IXa to the bovine endothelial cells was saturable and specific and reached a plateau in 75 min at 4 degrees C and 30 min at 37 degrees C. Binding was half-maximal at a total factor IX or IXa concentration of 2.3 +/- 0.2 nM. At 4 degrees C, a maximum of 42 fmol of tritiated factor IX or IXa bound to 10(6) cells (an average of 20,000 molecules per cell). The binding of tritiated factor IX or IXa was inhibited by excess unlabeled factor IX or IXa but not by factor X, prothrombin, or thrombin. Competition studies indicated that factors IX and IXa interacted with the same site. Binding was reversible, with 50% of the specifically bound factor IX or IXa eluted in 40 min by a 400-fold excess of unlabeled protein. Specific binding required Ca2+ with half-maximal binding at 1.2 mM CaCl2. Factor IXa bound to the cells was tested for procoagulant activity in a clotting assay with factor IX-deficient plasma, cephalin, and CaCl2. Cell-bound factor IXa was at least 3-fold more active than was factor IXa in solution. The retention of procoagulant activity by cell surface-bound factor IXa provides a mechanism for the localization of clot-promoting activity.

Animals↗
PubMed1983

Wasp sting anaphylaxis.

Profuse hemostatic defects were demonstrable 14 hr after wasp sting anaphylaxis. The patient's plasma contained an agent or agents that interfered with the action of thrombin, impeding the release of fibrinopeptide A from fibrinogen and the hydrolysis of the synthetic amide H-D-prolyl-L-phenylalanyl-L-arginine p-nitroanilide. This inhibitor could not be equated with known plasma inhibitors of thrombin nor with heparin. Additionally, the titers of nearly all other known clotting factors were reduced as compared to levels obtained after the patient's recovery. Of particular interest were profound reductions in the titers of proaccelerin (factor V) and high molecular weight kininogen. A normal titer of Hageman factor (factor XII) argued against participation of contact-activated mechanisms in the induction of the multiple abnormalities observed. Attempts to demonstrate the release of procoagulant or anticoagulant substances from the patient's convalescent blood, plasma, serum, or leukocytes upon challenge with wasp venom were unsuccessful. The observations reported confirm and extend information concerning hemostatic abnormalities in anaphylaxis, and point out the need to examine further this puzzling association.

Adult↗
PubMed1982

Acquired antibody to factor XI in a patient with congenital factor XI deficiency.

The results of studies in a patient with congenital deficiency of Factor XI who developed an inhibitor are presented. The patient presented with a severe, apparently spontaneous bleed into the thigh, which progressed despite infusion of fresh frozen plasma, but which responded promptly to activated prothrombin complex. During therapy with plasma his clotting time and Factor XI level were unresponsive and a Factor XI inhibitor titer of 6,000 U/ml was attained. The inhibitor was isolated and found to be polyclonal immunoglobulin G (IgG), predominantly of subclass 4. The specificity of the antibodies for Factor XI was shown by the ability of isolated inhibitor bound to polyacrylamide beads to remove Factor XI selectively from normal plasma. The binding of (125)I-labeled factor XI to the inhibitor was studied and an affinity constant of 1.65 x 10(10) liter/mol was found. Complexing of the antibodies with Factor XI was shown to block multiple activities of the clotting factor. Factor XI complexed with antibody did not bind to high molecular weight kininogen or undergo activation and cleavage by two-chain Factor XII. The complex of activated Factor XI with inhibitor prevented the cleavage and activation of Factor IX. Hence the inhibitor appears to act by binding to multiple sites on the Factor XI molecule and preventing its interaction with other molecules. Clinically these interactions of the inhibitor with Factor XI result in a state of severe Factor XI deficiency. The clinical circumstances of the case, with severe hemorrhage refractory to plasma infusion but readily responsive to an alternate clot-promoting agent, suggest that a defect of intrinsic system activation was critical, supporting the inference that Factor XI does participate in normal hemostasis. The clinical course of this patient, who has only had two documented hemorrhages in the presence of the inhibitor, is not as severe as that of patients with severe Factor VIII or IX deficiency. This suggests that physiologic activation of Factors XI and IX does not occur exclusively in series because deficiency of factors XII, XI, VIII, and IX should then have similar hemostatic consequences. We propose that independent mechanisms for bypass of Factors XII and XI are important in physiologic activation of coagulation.

Animals↗
PubMed1982

Immunochemical studies of antisera to human fibrinopeptide-B.

The immunochemical specificity of rabbit antisera to human fibrinopeptide-B (FPB) has been studied by comparing the relative abilities of FPB and of various proteins and peptides containing the NH2-terminal segment of the B beta-chain of human fibrinogen to inhibit the binding of a radioiodinated FPB derivative by each of seven anti-FPB sera. Anti-FBP sera varied in the extent to which they cross-reacted with fibrinogen, the NH2-terminal disulfide knot of fibrinogen (N-DSK), B beta 1(Pyr)-118(Met), B beta 1(Pyr)-42(Arg), and desarginyl-FPB. Anti-FPB sera have been identified that discriminate effectively between FPB and larger FBP-containing peptides; such antisera can be used to measure FPB in the absence of the larger peptides or to demonstrate the presence of larger peptides such as B beta 1(Pyr)-42(Arg) in extracts of clinical plasma samples by means of an increase in FPB immunoreactivity following thrombin treatment. One anti-FPB serum has been identified that is capable of detecting desarginyl-FPB, and this antiserum has been used in the development of a radioimmunoassay for desarginyl-FPB. Thus, by precisely defining the specificity of anti-FPB sera, it has been possible to identify antisera that are useful, not only in the measurement of FPB, but also in the detection of other important related molecules, such as B beta 1(Pyr)-42(Arg) and desarginyl-FPB. The immunochemical detection of these FPB-related peptides should provide useful information concerning the action of proteolytic enzymes, such as plasmin on the NH2-terminal segment of the B beta-chain of fibrinogen, and of carboxypeptidase-B on free FPB, in human plasma.

Cross Reactions↗
PubMed1982

Fibrinopeptide A, platelet factor 4, and beta-thromboglobulin levels in coronary heart disease.

In vivo platelet alpha-granule release and fibrin I formation were measured in 82 patients with ischemic heart disease by radioimmunoassay of platelet factor 4, beta-thromboglobulin, and fibrinopeptide A. The presence and extent of coronary artery disease were determined by coronary arteriography, and the extent of left ventricular regional dysfunction was assessed by contrast left ventriculography. In patients with abnormal coronary arteriograms without previous myocardial infarction, mean levels of platelet factor 4, beta-thromboglobulin, and fibrinopeptide A were not elevated. In patients in whom myocardial infarction had occurred more than 6 mo previously, platelet factor 4 (8.3 ng/ml; p less than 0.01) and beta-thromboglobulin (33.2 ng/ml; p less than 0.001) levels were significantly elevated, but fibrinopeptide A levels were normal. Levels of platelet factor 4 and beta-thromboglobulin were unrelated to the extent of coronary artery disease. In the patients with prior infarction, beta-thromboglobulin correlated directly with extent of left ventricular regional dysfunction (r = 0.53; p less than 0.01) and inversely with ejection fraction (r = -056; p less than 0.05). In a small group of patients with left ventricular aneurysm, mean fibrinopeptide A levels were also elevated. We interpret these findings as indicating that platelet release in patients with ischemic heart disease results from platelet reaction with previously infarcted myocardium rather than with the atherosclerotic coronary arteries.

Adult↗