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Biomedical subjects

R A Allen

Publications and source records attributed to R A Allen.

At least 37 records · Page 2Linked to original sources
PubMed1990

Antibacterial activity of fluoroquinolones in combination with zidovudine.

Since patients with AIDS may receive fluoroquinolones concurrently with zidovudine, the antibacterial interaction of these drugs was investigated. No evidence was found of antagonism between zidovudine and ciprofloxacin, DR-3355, enoxacin, lomefloxacin or ofloxacin against enterobacteria, staphylococci or Pseudomonas aeruginosa. Furthermore, the bactericidal activity of the fluoroquinolones against selected enterobacteria in nutrient broth was not affected by a clinically achievable concentration of zidovudine. It seems unlikely that zidovudine will have an adverse effect on the antibacterial activity of the fluoroquinolones in patients with AIDS.

Anti-Infective Agents↗
PubMed1990

Mechanisms of zidovudine resistance in bacteria.

Unlike their parent strains, zidovudine-resistant derivatives of Escherichia coli KL16 and Salmonella typhimurium NCTC 5710 were found to be incapable of incorporating radiolabelled thymidine into their chromosomal DNA. Since incorporation was still prevented in the presence of EDTA, resistance to zidovudine was not associated with a permeability barrier, but appeared to result from the loss of thymidine kinase activity, required for the phosphorylation of zidovudine. Pseudomonas aeruginosa, which is intrinsically zidovudine-resistant, was also shown to be incapable of incorporating thymidine into its DNA, but Staphylococcus epidermidis SK360 and Staph. aureus E3T, which are also intrinsically zidovudine-resistant, possessed thymidine kinase activity. This suggests that two distinct mechanisms of resistance to zidovudine exist in bacteria. Zidovudine resistance did not appear to confer resistance to other antibacterial agents.

DNA, Bacterial↗
PubMed1989

Reconstitution and characterization of the human neutrophil N-formyl peptide receptor and GTP binding proteins in phospholipid vesicles.

We have developed a unilamellar phospholipid vesicle system which contains the N-formyl peptide receptor and GTP binding proteins. Several detergents were investigated but only two, octyl glucoside (35 mM) and deoxycholate (7.5 mM), were capable of extracting N-formyl peptide receptor from neutrophil membranes in a form which remained functionally active upon reconstitution into phospholipid vesicles. Extracted proteins were reconstituted into phosphatidylcholine vesicles by passage over a Sephadex G-50-80 column. The reconstituted formylpeptide receptor could bind [3H]FMLP (3H-labeled fMet-Leu-Phe) and [125I]FMLPL-SASD (125I-labeled N-formylmethionylleucylphenylalanyl-N epsilon-(2-(p-azidosalicylamido)ethyl- 1,3'-dithiopropionyl)lysine) while the endogenous G protein could bind [35S]GTP gamma S. Furthermore, the functional interaction of the two proteins was preserved. Addition of the nonhydrolyzable guanine nucleotide, GTP gamma S, shifted the N-formyl peptide receptor from a high- to a low-affinity binding state for ligand. The development of this in vitro reconstitution system should provide a basis to study the mechanism of interaction of the N-formyl peptide receptor and the G protein.

Adenosine Diphosphate Ribose↗
PubMed1989

Identification of a human neutrophil protein of Mr 24 000 that binds N-formyl peptides: co-sedimentation with specific granules.

In assaying subcellular fractions of human neutrophils for N-formyl peptide binding sites using the photoaffinity ligand FMLPL-SASD-125I (125I-labelled N-formylmethionylleucylphenylalanyl-N epsilon- (2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionyl)-lysine) several molecular species were observed. We confirmed localization of the N-formyl peptide receptor of Mr 50 000-70 000 in the plasma membrane and specific granule fractions. A species of Mr 33 000-35 000 was detected in the light Golgi/endosomal fraction, whose size is consistent with the deglycosylated form of the receptor. In addition, a major binding species of Mr 24 000 was identified that co-localized on sucrose gradients with specific granule markers. This Mr 24 000 species, which was investigated further, was found to be released upon cell stimulation with phorbol myristate acetate or FMLP in the presence of dihydrocytochalasin B. It had an affinity for FMLPL-SASD of 145 nM (cf. 0.3 nM for the cell surface receptor). The specificity for the formyl group was lost as the nonformylated Met-Leu-Phe was as effective FMLPL in competing with FMLPL-SASD-125I for binding to th Mr 24 000 species. A structurally unrelated peptide, however, did not compete for the binding. The labelling of the Mr 24 000 species was dependent on the presence of Ca2+, as was its apparent Mr, which shifted from 24 000 to 50 000-70 000 in the presence of Ca2+. By incubating photoaffinity-labelled plasma membrane fractions with specific granule fractions, we could generate a receptor fragment of Mr 24 000, although the relationship to this fragment of the specific granule species is unknown at present. The N-terminal sequence of the Mr 24 000 species was determined and it appears to be a novel protein. Further work will allow its relationship to the receptor, if any, to be elucidated and allow assignment of a function to this potentially important molecule.

Affinity Labels↗
PubMed1989

Interaction of amiodarone and desethylamiodarone with the cardiac muscarinic receptor in vitro.

We studied the interaction of amiodarone hydrochloride (Cordarone) and its major metabolite desethylamiodarone with the muscarinic receptor in purified canine cardiac sarcolemmal vesicles by measuring equilibrium binding of the muscarinic antagonist [3H]quinuclidinyl benzilate (QNB) and carbachol displacement of [3H]-QNB. At a [3H]-QNB concentration of 0.02 nM, equilibrium binding was inhibited by amiodarone and desethylamiodarone with an IC50 of 6.86 x 10(-6) M and 2.25 x 10(-6) M, respectively. The presence of increasing concentrations of [3H]-QNB in the incubation medium was able to reverse the inhibition seen with 1 x 10(-6) M amiodarone. Scatchard analysis of [3H]-QNB saturation isotherms (37 degrees C, pH 7.4) in the presence of 1 x 10(-6) M amiodarone showed an apparent increase in equilibrium dissociation constant (Kd) over control from 0.045 +/- 0.002 nM to 0.084 +/- 0.001 nM while maximal binding capacity (Bmax) was unaffected: 10.8 +/- 1.14 and 10.5 +/- 1.48 pmol/mg (means +/- S.E.M., n = 3), respectively. The inhibitory effect of amiodarone on equilibrium binding was highly dependent on the drug:membrane phospholipid mole ratio with effects beginning at a ratio of less than 0.1:1. Hill plot analysis was consistent with the interaction of [3H]-QNB at a single site in the presence or absence of amiodarone. Amiodarone (3 x 10(-6) M) decreased the pseudo-first order forward rate constant of [3H]-QNB (0.02 nM) with the muscarinic receptor (kobs = 4.05 +/- 0.61 x 10(-4)/s under control conditions and 2.36 +/- 0.15 x 10(-4)/s in the presence of amiodarone).(ABSTRACT TRUNCATED AT 250 WORDS)

Amiodarone↗
PubMed1989

Regulation of chemoattractant receptor interaction with transducing proteins by organizational control in the plasma membrane of human neutrophils.

Isolated purified plasma membrane domains from unstimulated human neutrophils were photoaffinity labeled with F-Met-Leu-Phe-N epsilon-(2-(p-azido-[125I]salicylamido)ethyl- 1,3'-dithiopropionyl)-Lys also referred to as FMLPL-SASD[125I]. Most of the photoaffinity-labeled N-formyl peptide receptors were found in light plasma membrane fraction (PM-L) which has been previously shown to be enriched in guanyl nucleotide binding proteins and the plasma membrane marker alkaline phosphatase (Jesaitis, A. J., G. M. Bokoch, J. O. Tolley, and R. A. Allen. 1988. J. Cell Biol. 107:921-928). Furthermore, the heavy plasma membrane fraction (PM-H), which is enriched in actin and fodrin, was depleted in receptors. Solubilization of PM-L and PM-H in divalent cation-free buffer containing octylglucoside and subsequent sedimentation at 180,000 g in detergent-containing sucrose gradients revealed two receptor forms. The major population, found in PM-L sedimented as a globular protein with an apparent sedimentation coefficient of 6-7S, while a minor fraction found in the PM-H fraction sedimented as a 4S particle. In addition, the 6-7S form could be converted to the 4S form by inclusion of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) in the extraction buffer (ED50 = 10-30 nM). ATP was not effective at doses of up to 10 microM. In contrast, isolation and solubilization of receptors from desensitized cells (photoaffinity labeled after a 15 degrees C incubation with FMLPL-SASD[125I]) revealed that the majority of receptors (greater than 60-90%), which are found in PM-H, sedimented as 4S particles. A minor fraction of receptors found in the PM-L sedimented as 6-7S species. The receptors in the PM-H fraction, however, were still capable of interacting with G-proteins, since addition of unlabeled PM-L membrane fraction as a G-protein source reconstituted a more rapidly sedimenting form showing sensitivity to GTP gamma S. These results suggest that receptors in unstimulated human neutrophils have a higher probability of interacting with G-proteins because they are in the light plasma membrane domain. The results also suggest that receptors that have been translocated to the heavy plasma membrane domain during the process of desensitization or response termination have a lower probability of interacting with G-protein. Since the latter receptors are still capable of forming G protein associations, then their lateral segregation would represent a mechanism of controlling of receptor G-protein interactions. This reorganization of the plasma membrane, therefore, may form the molecular basis for response termination or homologous desensitization in human neutrophils.

Affinity Labels↗
PubMed1988

The quaternary structure of the plasma membrane b-type cytochrome of human granulocytes.

Hydrodynamic, crosslinking and immunoprecipitation studies were performed on detergent solubilized cytochrome b to demonstrate that the two copurifying polypeptides of molecular weight 91,000 (glycosylated) and 22,000 [1,2] formed a molecular complex. The hydrodynamic studies indicated that the cytochrome b/detergent complex had a sedimentation coefficient, partial specific volume and Stokes radius of 5.25 S, 0.82 cm3/g and 6.2 nm in Triton X-100 and 6.05 S, 0.80 cm3/g and 5.6 nm in octylglucoside, respectively. These studies also indicated that the detergent-protein complex has a molecular mass of 202 and 188 kDa in Triton X-100 and octylglucoside, respectively, is asymmetric in shape with a frictional coefficient of 1.3-1.4 and binds significant amounts of detergent. The molecular mass of the protein portion of the detergent-cytochrome complex was estimated to be between 100 and 127 kDa. Crosslinking studies with disuccinimidyl suberate and alkaline cleavable bis[2-(succinimidooxy-carbonyloxy)ethyl]sulfone revealed that the Mr = 91,000 and Mr = 22,000 components of purified cytochrome b are closely associated and can be covalently bound to form a polypeptide which, by SDS-polyacrylamide gel electrophoresis, has Mr values of 110,000-120,000 and 120,000-135,000 on 8% and 11% (w/v) SDS-polyacrylamide gels, respectively. Cleavage of the crosslinked species resulted in the reappearance of the Mr = 91,000 and Mr = 22,000 species. Sedimentation profiles of crosslinked cytochrome b in linear sucrose density gradients made up in H2O were identical to those of non-crosslinked controls. A close association of the two protein species was further confirmed by the ability of antibody specific for the smaller subunit to immunoprecipitate the larger one also. Experiments aimed at identifying the heme-carrying subunit(s) were inconclusive, since dissociation of the complex resulted in loss of cytochrome b spectrum. These results, in combination with our SDS-polyacrylamide gel electrophoresis molecular-weight estimates, provide strong evidence for the cytochrome b being an alpha-beta-type heterodimer composed of a glycosylated Mr = 91,000 and non-glycosylated Mr = 22,000 polypeptide.

Cell Membrane↗
PubMed1988

Activation of the neutrophil respiratory burst by chemoattractants: regulation of the N-formyl peptide receptor in the plasma membrane.

The N-formyl peptide receptor mediates a number of host defensive responses of human neutrophils that result in chemotaxis, secretion of hydrolytic enzymes, and superoxide generation. Inappropriate activation or defective regulation of these responses can result in pathogenic states responsible for inflammatory disease. The receptor is a 50 to 70-kD, integral plasma membrane glycoprotein with intracellular and surface localization. Its abundance in the membrane is regulated by membrane flow and recycling processes. Cytoskeletal interactions are believed to control its organization in the plane of the membrane and interaction with other proteins. The receptor's most important interaction is with guanyl nucleotide binding proteins that serve as signal transduction partners ultimately leading to activation of effector responses. Because the interaction of the receptor with G proteins is necessary for transduction, control of this interaction may be at the root of understanding the molecular control of responses in these cells. This review briefly summarizes some of the molecular properties, dynamics, and interactions of this receptor system in human neutrophils and discusses how these characteristics may pertain to the activation and control of superoxide generation.

Cell Membrane↗
PubMed1988

Primary structure and unique expression of the 22-kilodalton light chain of human neutrophil cytochrome b.

Cytochrome b comprising 91-kDa and 22-kDa subunits is a critical component of the membrane-bound oxidase of phagocytes that generates superoxide. This important microbicidal system is impaired in inherited disorders known as chronic granulomatous disease (CGD). Previously we determined the sequence of the larger subunit from the cDNA of the CGD gene, the X chromosome locus affected in "X-linked" CGD. To complete the primary structure of the cytochrome b and to assess expression of the smaller subunit, we isolated cDNA clones for the 22-kDa polypeptide by immunoscreening and confirmed their authenticity by direct N-terminal protein sequencing. Although the deduced amino acid sequence of the 22-kDa subunit is not overtly similar to other known cytochromes, we observed a 31-amino acid stretch of 39% identity with polypeptide I of mitochondrial cytochrome c oxidase centered on a potential heme-coordinating histidine. Similarities in the hydropathy profiles and spacing of histidines of the 22-kDa protein and myoglobin suggest structural motifs in common with other heme-containing proteins that are not readily revealed by primary amino acid sequences. Although RNA for the larger subunit has been found only in cells of the phagocytic lineage, stable RNA encoding the 22-kDa subunit was observed in all cell types. However, the stable 22-kDa protein was detected only in phagocytic cells that were expressing the larger subunit RNA. This observation suggests that the large subunit may play a role in regulating the assembly of the heterodimeric cytochrome b.

Amino Acid Sequence↗
PubMed1988

Lateral segregation of neutrophil chemotactic receptors into actin- and fodrin-rich plasma membrane microdomains depleted in guanyl nucleotide regulatory proteins.

Subcellular fractions were prepared from human neutrophils desensitized at 15 degrees C with stimulatory doses of the photoaffinity derivative F-Met-Leu-Phe-N epsilon-(2-(rho-azido[125I]salicylamido)ethyl-1,3'- dithio-propionyl)-Lys. The covalently labeled receptors were found in a membrane fraction of higher density than those from cells preexposed to ligand at 4 degrees C but not desensitized. The denser fraction (rho approximately equal to 1.155 g/cc) was the cellular locus of the membrane associated cytoskeletal proteins, actin, and fodrin, as detected immunologically on western blots. The light fraction (rho approximately equal to 1.135), cosedimented with neutrophil plasma membrane markers, plasma membrane guanyl nucleotide regulatory proteins, and several characteristic polypeptides identified by SDS-PAGE, including a major 72-kD species. The photoaffinity-labeled species in either case showed the same mobility on SDS-PAGE (Mr = 50,000-70,000) corresponding to previously reported values for N-formyl chemotactic receptors. These labeled receptors were sensitive to proteolysis after exposure of the intact photoaffinity-labeled cells to papain at 4 degrees C. We conclude that (a) the fractions isolated are probably derived from different lateral microdomains of the surface of human neutrophils; (b) the higher density fraction contains occupied N-formyl-chemotactic receptors previously shown to have been converted, to a high affinity, slowly dissociating form coisolating with neutrophil cytoskeleton and implicated in the termination of formyl peptide-induced neutrophil activation; and (c) the translocation of receptors to these microdomains may serve to compartmentalize receptors and perhaps regulate the interaction of the receptor/G-protein transduction pair.

Actins↗
PubMed1988

The chemotactic peptide receptor. A model for future understanding of chemotactic disorders.

Chemotaxis by human phagocytes enables these cells to find sites of injury or inflammation in the host. This function is a complex orchestration of biochemical and morphologic events that begins with the occupancy of specific surface receptors for chemotactic agents. The response is propagated and amplified by a biochemical cascade of transduction reactions. This cascade is mediated by receptor interactions with guanyl nucleotide binding proteins, subsequent activation of phospholipase C, and a resulting increase in phospholipid turnover. These events are spatially and temporarily integrated as elevations in the concentrations of intracellular Ca++ and diacylglycerol combine to activate protein kinase C. At the macromolecular and subcellular level, integration of these functions with as yet undiscovered pathways results in stimulation of actin polymerization, shape change, degranulation, and membrane flow. Final integration of these events results in the morphologic process of directed migration of the cell along concentration gradients of chemotaxins. During this process, chemotactic receptors are continuously regulated and feed back information to the response system that adjusts the biochemistry and morphology accordingly. This article has briefly considered some of the relationships between these transduction events and the control of a specific chemotactic receptor system employing the N-formylated chemotactic peptides. It is hoped that knowledge of this regulation will aid the physician in understanding the origin of chemotactic disorders derived from defects in chemotactic receptor systems of the phagocytic cells.

Calcium↗
PubMed1987

Purified cytochrome b from human granulocyte plasma membrane is comprised of two polypeptides with relative molecular weights of 91,000 and 22,000.

A new method has been developed for purification of cytochrome b from stimulated human granulocytes offering the advantage of high yields from practical quantities of whole blood. Polymorphonuclear leukocytes were treated with diisopropylfluorophosphate, degranulated and disrupted by nitrogen cavitation. Membranes enriched in cytochrome b were prepared by differential centrifugation. Complete solubilization of the cytochrome from the membranes was achieved in octylglucoside after a 1-M salt wash. Wheat germ agglutinin-conjugated Sepharose 4B specifically bound the solubilized cytochrome b and afforded a threefold purification. Eluate from the immobilized wheat germ agglutinin was further enriched by chromatography on immobilized heparin. The final 260-fold purification of the b-type cytochrome with a 20-30% yield was achieved by velocity sedimentation in sucrose density gradients. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified preparation revealed two polypeptides of Mr 91,000 and Mr 22,000. Treatment of the 125I-labeled, purified preparation with peptide:N-glycosidase F, which removes N-linked sugars, decreased relative molecular weight of the larger species to approximately 50,000, whereas beta-elimination, which removes O-linked sugars, had little or no effect on the mobility of the Mr-91,000 polypeptide. Neither of the deglycosylation conditions had any effect on electrophoretic mobility of the Mr-22,000 polypeptide. Disuccinimidyl suberate cross-linked the two polypeptides to a new Mr of 120,000-135,000 by SDS-PAGE. Antibody raised to the purified preparation immunoprecipitated spectral activity and, on Western blots, bound to the Mr-22,000 polypeptide but not the Mr-91,000 polypeptide. Western blot analysis of granulocytes from patients with X-linked chronic granulomatous disease revealed a complete absence of the Mr-22,000 polypeptide. These results (a) suggest that the two polypeptides are in close association and are part of the cytochrome b, (b) provide explanation for the molecular weight discrepancies previously reported for the protein, and (c) further support the involvement of the cytochrome in superoxide production in human neutrophils.

Antibodies↗
PubMed1986

Receptor-cytoskeleton interactions and membrane traffic may regulate chemoattractant-induced superoxide production in human granulocytes.

When dihydrocytochalasin (dhCB) was added either prior to or after CHO-Met-Leu-Phe (fMLP), the rate and duration of superoxide production in human granulocytes stimulated by fMLP was augmented. This effect was maximal when dhCB was added before fMLP, increasing the rate 1.5-3-fold. The effect of dhCB was progressively diminished for later additions and was undetectable after 6-10 min. The effects of dhCB could be blocked by the additional presence of 10 microM t-Boc-Phe-Leu-Phe-Leu-Phe (where t-Boc is t-butoxycarbonyl) indicating a requirement for receptor occupancy. In the presence of dhCB, the reversible binding of fML[3H]P was elevated and the formation of slowly dissociating surface complexes of occupied receptor and cytoskeleton was inhibited. Myeloperoxidase and lactoferrin release from fMLP-stimulated cells was induced by dhCB but was only partially correlated with the potentiating effects of dhCB on superoxide production and receptor expression. To circumvent the complicating effects of degranulation on the analysis of the functional consequences of receptor-cytoskeletal associations, cells were also preincubated with 100 nM fMLP at 15 degrees C. Under these conditions, the majority of the surface receptors became irreversibly occupied and coisolated with the cytoskeletal fraction of the cell. Subsequent exposure of the cells to fMLP at 37 degrees C resulted in no superoxide production. This desensitization was blocked by dhCB which also inhibited coisolation of the ligand and cytoskeletons. Conversion of receptor to a slowly dissociating state may represent its trapping in an inactive form and would provide a role for receptor-cytoskeleton interactions in the termination of the granulocyte response to chemoattractants. The inhibition of such receptor "sequestration" and the induction of new receptor expression could, therefore, partially account for dhCB-induced potentiation of the fMLP response.

Biological Transport↗
PubMed1986

Preparation and properties of an improved photoaffinity ligand for the N-formyl peptide receptor.

A new superior photoaffinity ligand for the N-formyl peptide receptor was prepared by derivatization of N-formyl-Met-Leu-Phe-Lys with a commercially available heterobifunctional crosslinking agent. The product, N-formyl-Met-Leu-Phe-N epsilon-(2-(p-azidosalicylamido)ethyl-1,3'- dithiopropionyl)-Lys was readily synthesized and radiolabelled, and had increased specificity and stability as compared to previously used photoaffinity ligands. The ligand rapidly associated with the receptor with high affinity (Kd = 0.28 nM). Once bound, it was virtually non-dissociable (in the absence of photolysis) in an experimental time-frame (t1/2 (off) = 154 min). The covalent incorporation of the photoaffinity ligand into the receptor upon irradiation was complete within 5 min, minimizing cell damage, and the efficiency of covalent incorporation was approx. 40%. The derivative had enhanced biological activity, having an ED50 for superoxide anion production of 0.23 nM, 27-fold lower than its parent peptide. This derivative of the N-formyl peptide was useful for up to 3 months after radiolabelling, showing a progressive decline in specific activity during storage in the dark at 4 degrees C. The enhanced stability, reproducibility and solubility of the photoaffinity ligand is expected to aid in the purification of the N-formyl peptide receptor and will prove a useful tool for analysing receptor-mediated processes.

Affinity Labels↗
PubMed1986

Physicochemical properties of the N-formyl peptide receptor on human neutrophils.

In order to investigate the physicochemical properties of the N-formyl peptide receptor of human neutrophils, the receptor was specifically and covalently labeled with an iodinated, photoactivatable derivative of the chemotactic hexapeptide, N-formyl-norleucylleucyl- phenylalanyl-norleucyl-[125I]iodotyrosyl-N epsilon-(6- (4'-azido-2'-nitrophenylamino) hexanoyl)-lysine. After labeling isolated neutrophil membranes, the receptor was extracted with Triton X-100, digitonin, or octyl glucoside and subjected to gel filtration on a calibrated Ultrogel AcA 34 column. The Triton X-100- and digitonin-extracted receptor eluted as single molecular species, with Stokes radii of 40 and 33 A, respectively. This material was subjected to further physicochemical analysis. When octyl glucoside-extracted material was gel-filtered, a second peak containing specifically labeled material eluted in the void volume. Subsequent sodium dodecyl sulfate-polyacryl-amide amide gel electrophoresis analysis indicated that this species was the result of disulfide bonded aggregates containing the monomeric species. Sedimentation equilibrium analysis was carried out in H2O and D2O/H2O mixtures, yielding an apparent molecular mass of 63,000 daltons for both Triton X-100- and digitonin-extracted receptor. This agrees closely with the reduced sodium dodecyl sulfate-polyacrylamide gel electrophoretic value of 50,000-60,000 daltons, indicating that the receptor extracted from unstimulated membranes is monomeric in these detergents. From the sedimentation equilibrium data, the partial specific volume (v) and frictional ratio (f/f0) were calculated. The v is high in both Triton X-100 (0.880) and digitonin (0.829), indicating that the receptor may be associated with tightly bound endogenous lipid or that it is a hydrophobic membrane protein. This latter likelihood is further supported by the quantitative extraction of receptor into Triton X-114 by a phase-separation method. The frictional ratio of 1.1-1.3 is consistent with an elongated globular protein having an axial ratio of approximately 3:1. This in conjunction with the Stokes radius of 40 A would indicate that the receptor is capable of spanning the 35-40-A nonpolar center of the lipid bilayer. The state of the receptor in situ is discussed.

Affinity Labels↗
PubMed1984

Acute effect of smoking on fibrinolysis: increase in the activity level of circulating extrinsic (tissue-type) plasminogen activator.

The acute effect of cigarette smoking on the fibrinolytic enzyme system in blood was studied. It was found imperative to have an initial 30 min rest period, after venipuncture, to obtain a stable baseline in the fibrinolytic studies. The average heart rate, in inhaling smokers, increased from 64 to a peak of 79 beats min-1, 5-10 min after commencement of smoking. A peak in fibrinolytic activity was found to occur later, at 22.5 min. Analysis of the increase in fibrinolytic activity revealed no demonstrable activation of intrinsic systems via factor XII, nor changes in plasminogen, prekallikrein and C1-inactivator. No plasmin-alpha 2-antiplasmin complexes were detectable. The increase (P less than 0.01) was found to be due to extrinsic (tissue-type) plasminogen activator, revealed as C1-inactivator-resistant plasminogen activator activity, and further identified by quenching with anti-tissue plasminogen activator IgG. Thus, smoking appears to elicit a significant increase in the level of activity of circulating extrinsic plasminogen activator.

Adult↗