Biomedical subjects

R Lottenberg

Publications and source records attributed to R Lottenberg.

At least 37 records · Page 2Linked to original sources

PubMed1994

Characterization of a novel streptokinase produced by Streptococcus equisimilis of non-human origin.

Streptokinases are proteins with plasminogen activator activity produced by certain hemolytic streptococci. We previously identified equine streptococcal isolates which produced streptokinases (ESKs) that bound both human and equine plasminogen but only readily activated equine plasminogen (14). This property was exploited to purify a representative ESK produced by Streptococcus equisimilis strain 87-542-W. Affinity chromatography with human plasminogen resulted in the isolation of a M(r) approximately 49,000 molecule with two isoforms. This ESK was subsequently compared to well characterized streptokinases (HSKs) that efficiently activate human plasminogen. Differences in streptokinases were identified in the highly conserved amino-terminal amino acid sequence, peptide maps, and antigenic properties, and these differences were supported by DNA hybridization studies. These results indicate that the family of proteins identified as streptokinases has much greater diversity than previously appreciated.

Amino Acid Sequence↗

PubMed1992

Identification of two type IIa IgG-binding proteins expressed by a single group A streptococcus.

Functional heterogeneity associated with Ig-binding proteins expressed by group A streptococci is well documented. In this study we have demonstrated that treatment of group A streptococcal isolate 64/14 with CNBr resulted in the solubilization of two different sized proteins that displayed identical functional reactivity with human IgG1, IgG2, and IgG4 (characteristics of a type IIa binding protein). Monospecific polyclonal antibodies to each form of type IIa molecule were prepared and no antigenic cross-reactivity between the two m.w. forms of type IIa binding protein could be detected. The smaller m.w. protein was shown to be identical or closely related to the recombinant type IIa protein cloned from strain CS110. These studies provide further evidence for the heterogeneity of type II Ig-binding proteins expressed by pathogenic group A streptococci.

Bacterial Proteins↗

PubMed1992

Streptokinase-producing streptococci grown in human plasma acquire unregulated cell-associated plasmin activity.

Group A streptococci grown in the presence of human plasma generated plasmin from plasminogen and captured the functional enzyme to a specific cell-surface receptor. Bacteria-bound plasmin was not regulated by alpha 2-antiplasmin present in the medium. The ability of the bacteria to acquire cell-associated plasmin activity was dependent on both the presence of plasminogen in the culture medium and the production of a bacterial plasminogen activator, streptokinase. The ability of group A streptococci to produce a plasminogen activator and capture resulting plasmin in an unregulatable form could provide the organism with a mechanism for invasion of normal tissue barriers.

Blotting, Western↗

PubMed1992

Cloning, sequence analysis, and expression in Escherichia coli of a streptococcal plasmin receptor.

Plasmin(ogen) receptors are expressed by many gram-positive and gram-negative bacteria. We previously isolated a plasmin receptor from a pathogenic group A streptococcal strain (C. C. Broder, R. Lottenberg, G. O. von Mering, K. H. Johnston, and M. D. P. Boyle, J. Biol. Chem. 266:4922-4928, 1991). The gene encoding this plasmin receptor, plr, was isolated from a lambda gt11 library of chromosomal DNA from group A streptococcal strain 64/14 by screening plaques with antibodies raised against the purified streptococcal plasmin receptor protein. The gene was subcloned by using a low-copy-number plasmid and stably expressed in Escherichia coli, resulting in the production of an immunoreactive and functional receptor protein. The DNA sequence of the gene contained an open reading frame encoding 335 amino acids with a predicted molecular weight of 35,787. Upstream of the open reading frame, putative promoter and ribosomal binding site sequences were identified. The experimentally derived amino acid sequences of the N terminus and three cyanogen bromide fragments of the purified streptococcal plasmin receptor protein corresponded to the predicted sequence encoded by plr. The deduced amino acid sequence for the plasmin receptor protein revealed significant similarity (39 to 54% identical amino acid residues) to glyceraldehyde 3-phosphate dehydrogenases.

Amino Acid Sequence↗

PubMed1991

Isolation of a prokaryotic plasmin receptor. Relationship to a plasminogen activator produced by the same micro-organism.

Plasminogen receptors have been identified on the surface of a number of prokaryotic and eukaryotic cells. A receptor demonstrating high affinity for plasmin with minimal reactivity with the native zymogen Glu-plasminogen has been identified on the surface of certain group A streptococci. In this study the group A streptococcal plasmin receptor has been solubilized and purified to homogeneity. The isolated protein was an Mr approximately 41,000 molecule which retained its ability to bind plasmin following solubilization and affinity purification on a column of enzymatically inactivated human plasmin. The isolated plasmin receptor was compared functionally, antigenically, and physicochemically to the secreted plasminogen activator, streptokinase, produced by the same organism. The Mr approximately 41,000 surface plasmin receptor was shown to be functionally and antigenically distinct from the Mr approximately 48,000 streptokinase molecule produced by the same strain and lacked any plasminogen activator activity. The streptokinase molecule produced by this strain was shown to be closely related to the plasminogen activator protein secreted by other group A and C streptococci. This study represents the first report of the isolation of a plasmin receptor, either prokaryotic or eukaryotic, with functional activity.

Antigens, Bacterial↗

PubMed1991

Streptokinases produced by pathogenic group C streptococci demonstrate species-specific plasminogen activation.

The species specificities of plasminogen activation and binding of plasmin by pathogenic group C streptococci isolated from humans, horses, and pigs were examined. Of 56 streptococcal isolates, 52 elaborated plasminogen activator activity and 49 of these had specificity for plasminogen of the homologous host. Analysis of supernatants from 13 isolates indicated that the plasminogen activator activity resulted from secreted streptokinases. These 13 streptokinases were antigenically related and bound all three plasminogens, indicating that the binding recognition sites were conserved despite the observed species-specific activation. In addition, all group C isolates tested demonstrated surface receptors that bound human, equine, and porcine plasmin. Species-specific plasminogen activation may be an early step in events resulting in infection and may account for the species preference of certain streptococci.

Animals↗

PubMed1991

Immunological and molecular characterization of plasminogen activator inhibitors 1 and 2 in baboon (Papio anubis) placental tissues.

Two major plasminogen activator inhibitors (PAI-1 and PAI-2) increase in the peripheral circulation during pregnancy in humans. PAI-1 is of vascular endothelial origin whereas PAI-2 is produced primarily by human placental tissues. This study was undertaken to determine a) if PAI-1 and PAI-2 are also present in the baboon and b) their association with pregnancy. Citrated plasma was obtained from pregnant baboons sequentially at 15 +/- 3-day intervals between Days 30 and 140 of pregnancy. PAI activity increased significantly (p less than 0.05) at Day 120 (15.3 IU/ml) and 140 (21.8 IU/ml) of gestation and returned to baseline (2.6 IU/ml) 48 h post cesarean section. Placental tissues obtained at cesarean section during the third trimester were either placed in explant culture, fixed for immunocytochemistry, or frozen for RNA extraction. Western blot analysis of tissue culture media (TCM) indicated that the polyclonal antibody to PAI-1 reacted with a major band (Mr 47 000) in TCM from placental tissues while the PAI-2 antibody reacted primarily with a doublet (Mr 67 000 and 69 000) in these same media. PAI-1 was immunocytochemically localized primarily in the chorioamniotic tissue (CAM-D) and PAI-2 was found predominantly in placental villi. Slot blot hybridization with cDNAs to PAI-1 and PAI-2 indicated that the mRNA for PAI-2 was found primarily in placental villi, whereas the mRNA for PAI-1 was present in all three tissue compartments.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Group A streptococci bind human plasmin but not other structurally related proteins.

Certain group A streptococci are known to possess a receptor for the human enzyme plasmin. Plasmin is a member of a super gene family that includes other serine proteases and kringle containing proteins. In this study we have examined the interaction of a group A streptococcus with structurally related proteins, including plasmin, glu-plasminogen, tissue plasminogen activator, kallikrein, factor XII, prothrombin, thrombin, trypsin, and urokinase. Our studies indicate that only the key fibrinolytic enzyme, plasmin, demonstrates significant binding activity to the group A streptococcus.

Animals↗

PubMed1989

Fibrinolytic capacity following stimulation with desmopressin acetate in patients with diabetes mellitus.

Tissue plasminogen activator (t-PA), tissue plasminogen activator inhibitor, (PAI), and von Willebrand factor (vWF) were measured in 30 diabetics and 17 control subjects. These studies were performed to clarify the role of obesity in causing abnormalities of the fibrinolytic system in diabetics. The t-PA antigen response measured after the infusion of desmopressin acetate (DDAVP) was similar in all groups. Peak responses to DDAVP for controls, type I diabetics, and type II diabetics were 21.2 +/- 9.5 ng/mL, 27.5 +/- 9.0 ng/mL, and 28.8 +/- 11.0 ng/mL (NS), respectively. These responses did not correlate with the body mass index (BMI) or any other of the indices examined. A significant decrease of t-PA activity as contrasted with t-PA antigen following DDAVP occurred in type II diabetics only. The decrease of t-PA activity strongly correlated with greater basal levels of plasminogen activator inhibitor in these same subjects. The plasma level of plasminogen activator inhibitor correlated with BMI but with no other index examined. In contrast to t-PA activity and PAI, vWF responses to DDAVP inversely correlated to basal vWF concentration in all groups. Basal concentrations of vWF were increased in both type I and II diabetics and showed no relationship to degree of obesity. In summary, these results suggest that type II diabetic subjects have decreased t-PA activity, which is best explained by increased levels of PAI. The increased PAI appears related to obesity and not diabetes per se.

Adolescent↗

PubMed1989

Mapping of the human plasmin domain recognized by the unique plasmin receptor of group A streptococci.

A high-affinity surface receptor for human plasmin has been reported on certain group A streptococci. To map the region of the plasmin molecule that binds to the bacterial receptor, isolated domains of plasmin were tested for their ability to inhibit the binding of intact radiolabeled plasmin to receptor-positive bacteria. Complete inhibition of binding of labeled plasmin to bacteria by isolated heavy chains was achieved, but this inhibition was not as efficient on a molar basis when compared with that of unlabeled plasmin. By contrast, a conformationally altered form of native plasminogen was found to bind to bacteria and was as efficient a competitive inhibitor as intact plasmin was. The results of this study indicate that the selective binding of human plasmin to a group A streptococcus is dependent on structures present in the conformationally altered form of native plasminogen or plasmin that are not found on the native zymogen, the plasminogen with NH2-terminal glutamic acid.

Amino Acid Sequence↗

PubMed1989

Familial protein S deficiency with a variant protein S molecule in plasma and platelets.

A protein S deficient family presenting a variant protein S molecule in plasma and platelets is described. The propositus, age 20, and two brothers suffered from venous thrombotic disease. The propositus, the only family member studied while taking oral anticoagulants, had a protein S antigen (ag) level of 17% and undetectable activity. As demonstrated by immunoblotting both the propositus and one clinically affected brother (42% ag, 7% activity) presented variant protein S molecules of 65,000 molecular weight (mol wt) while the other clinically affected brother (64% ag, 11% activity) had only protein S with normal electrophoretic mobility of 70,000 mol wt. The mother had normal protein S levels (93% ag, 100% activity) but had both normal and variant protein S molecules and based on her functional protein S data a normal anticoagulant activity of the variant molecule is suggested. One asymptomatic but protein S deficient sister (68% ag, 9% activity) as well as the asymptomatic protein S deficient father (59% ag, 10% activity) had only protein S molecules of 70,000 mol wt. The variant protein S bound to C4b-binding protein in plasma, and differed from normal protein S in carbohydrate content. Platelets of each family member contained the same immunoblotting pattern of normal and variant protein S forms as found in plasma, consistent with the hypothesis that protein S gene expression involves codominant expression of two alleles that is similar in cells that control the synthesis of both platelet and plasma forms of protein S.

Blood Coagulation Factors↗

PubMed1988

Desmopressin stimulates parallel norepinephrine and tissue plasminogen activator release in normal subjects and patients with diabetes mellitus.

Desmopressin acetate administration markedly stimulates release of tissue plasminogen activator (t-PA) from vascular endothelial cells. The mechanism for this effect is unknown. Because infusion of epinephrine has been shown to increase t-PA levels, we examined the role of endogenous catecholamine mediation of t-PA release by desmopressin. Intravenous desmopressin acetate (0.3 micrograms/kg) was infused over 30 min in 9 controls and 11 subjects with diabetes mellitus, a condition associated with abnormalities of the fibrinolytic system. Plasma was collected in the supine, overnight fasted state at 15 min intervals (0-60 min) for measurement of t-PA activity, t-PA antigen and fractionated catecholamines. t-PA activity peaked at 30-45 min and subsequently decreased. The norepinephrine levels paralleled the t-PA activity. t-PA activity increased 10-fold from 0.14 +/- .12 to 1.49 +/- 0.79 IU/ml (Mean +/- SD) and plasma norepinephrine increased 2-fold from 426 +/- 90 to 780 +/- 292 pg/ml. However, epinephrine and dopamine levels did not change significantly. The response to desmopressin of control and diabetic subjects was not shown to differ and their data were combined. We conclude that desmopressin increases plasma norepinephrine in addition to t-PA and that the parallel time course of change suggests a possible role for norepinephrine in mediating endothelial cell t-PA release.

Antigens↗

PubMed1988

Congenital deficiency of plasminogen and its relationship to venous thrombosis.

In a patient with deep venous thrombosis, plasma concentrations of coagulant and inhibitor proteins were normal except for moderate deficiency of plasminogen. Family studies revealed a similar deficiency in the mother and sister of the propositus. Evaluation of purified plasminogen demonstrated that it functioned normally. The patient represents our only example of plasminogen deficiency in 435 German individuals evaluated with a history of thromboembolism.

Blood Coagulation Disorders↗

PubMed1988

Enzyme-labeled type III bacterial Fc receptors. A versatile tracer for immunoassay.

The type III bacterial Fc receptor isolated form a group C streptococcus has been conjugated to alkaline phosphatase and used as a tracer in a variety of direct and indirect immunoassays. These immunoassays have utilized specific antibodies prepared in species whose immunoglobulins are poorly reactive with the type I Fc receptor, staphylococcal protein A. The value of the type III Fc receptor as a tracer for immunoassays utilizing antibodies produced in sheep and goats is documented.

Alkaline Phosphatase↗

PubMed1988

Aprotinin inhibits urokinase but not tissue-type plasminogen activator.

The protease inhibitor, aprotinin, has been examined for its ability to inhibit urokinase and tissue-type plasminogen activators at pH 7.4 in assays utilizing pyroGlu-Gly-Arg-p-nitroanilide and H-D-Ile-Pro-Arg-p-nitroanilide as substrates, respectively. Aprotinin inhibited both two-chain low molecular weight urokinase and the high molecular weight form of the enzyme in a competitive manner with a similar Ki (27 microM). There was no observable inhibition of tissue-type plasminogen activators at aprotinin concentrations up to 500 microM. These findings suggest that sensitivity to inhibition by aprotinin could be used to distinguish tissue-type and urokinase-type plasminogen activators.

Aprotinin↗

PubMed1988

Procoagulant synthesis by exudate and bone marrow-derived murine macrophages.

Murine exudate macrophages elicited by different stimuli and bone marrow-derived macrophages were studied for their capacity to synthesize factor VII and tissue factor in a basal state and on stimulation with endotoxin (LPS). Cells elicited by different stimuli varied in their production of both factors. Thioglycollate-elicited cells generally made more, but not significantly more, tissue factor in response to endotoxin than cells elicited with periodate or streptococci. Cells elicited with proteose-peptone, fetal calf serum (FCS), or LPS produced less or very little tissue factor. Thioglycollate-elicited cells and cells elicited with streptococci or proteose-peptone consistently made more factor VII than cells elicited with periodate, FCS, and LPS. Bone marrow-derived macrophages were responsive to LPS by the production of tissue factor by the fifth day of culture, and this rose to a maximum by day 10. The maximal production of factor VII occurred on day 5 of culture and declined with longer cultivation. Factor VII production was not enhanced by LPS, and prolonged cultivation in the presence of LPS turned off the synthesis of both tissue factor and factor VII. We conclude that exudate cells are heterogeneous in the production of coagulant factors and that the production of these factors varies with the maturity of the cells. In addition, the production of the tissue factor and the factor VII were not necessarily expressed in a coordinate fashion.

Animals↗

PubMed1988

Characterization of the interaction of human plasmin with its specific receptor on a group A streptococcus.

Certain Group A beta-hemolytic streptococci express a receptor that is capable of specifically binding the human plasma protease plasmin. Once bound, plasmin remains enzymatically active and is unregulated by its naturally occurring inhibitor alpha-2-antiplasmin (Lottenberg, R., C. C. Broder and M. D. P. Boyle, 1987. Infect. Immun. 55: 1914-1918). In this study certain characteristics of the interaction between plasmin and the receptor expressed on a group A beta-hemolytic streptococcus, strain 64/14, were examined. Binding occurred optimally at physiologic pH and ionic strength. The KD was 5 x 10(-11) M and there were approximately 800 receptors per bacterium. Mouse passage of strain 64 had no significant effect on the KD of the receptor. Binding of plasmin to the bacteria was inhibited by lysine and epsilon-aminocaproic acid in a concentration dependent manner. Similarly these amino acids would displace pre-bound plasmin from the bacteria. These findings suggest a role for plasmin's high affinity lysine binding site in the interaction of plasmin with the bacteria.

Aminocaproic Acid↗

PubMed1987

Acquired hemophilia. A natural history study of 16 patients with factor VIII inhibitors receiving little or no therapy.

Rarely, a patient develops an antibody against factor VIII coagulant activity. The resultant hemorrhagic diathesis is clinically distinct from inherited hemophilia, being characterized by few hemarthroses but frequent skin and other soft-tissue hematomas. Hematuria may be troublesome. These patients represent therapeutic challenges. This study is one institution's results with 16 such patients followed up over an average of 31 months (range, four to 120 months; median, 19 months). It describes the largest group from a single institution receiving essentially no immunosuppressive agents, yet has one of the better overall results. Two patients experienced fatal hemorrhage and five patients underwent spontaneous remission. Long-term survival is not incompatible with persistence of the inhibitor. We conclude that this hemorrhagic diathesis is clinically distinct, less fatal than usually perceived, and may undergo spontaneous remission. Clearly, there is no mandate for any particular therapeutic regimen, such as immunosuppression, in the attempt to rid the patient of the antibody.

Adult↗