SP-B is a protein in pulmonary surfactant that is, in greatest part, responsible for resistance to surface tension and prevention of collapse of pulmonary alveoli. Peptides of 21 residues, synthesized following the sequence of SP-B or resembling the hydrophobic and hydrophilic domains of SP-B (such as RLLLLRLLLLRLLLLRLLLLR, R, Arg, and L, Leu), enhanced the abilities of phospholipids to reduce surface tension both in vitro and in vivo. Intermittent positively charged residues were essential for this activity. The SP-B-like peptides were found by tryptophan fluorescence to partition within the phospholipid layer in contact with both polar head groups and acyl side chains. These data, together with findings that the SP-B-related peptides increase inter- and intramolecular order of the phospholipid layer, suggest that SP-B resists surface tension by increasing lateral stability of the phospholipid layer.
The present study was undertaken to determine if a synthetic peptide, KLLLLKLLLLKLLLLKLLLLK (KL4), in which K = lysine and L = leucine, in an aqueous dispersion of phospholipids (DPPC and POPG), would expand pulmonary alveoli and improve gas exchange in premature human infants with respiratory distress syndrome (RDS). The KL4 peptide was synthesized to resemble the amino acid pattern of surfactant protein B (SP-B). Forty-seven infants with RDS were treated within 4 h of birth with the KL4-peptide/phospholipid mixture, called KL4-Surfactant. The average arterial-to-alveolar oxygen tension ratios (a/A O2) of 39 patients included in efficacy analyses rose from pretreatment values of 0.14 +/- 0.02 (mean +/- SEM) to 0.40 +/- 0.04 (normal value > or = 0.40) by 12 h of age. Mean airway pressures and oxygenation index values fell concomitantly, and expansion of the lungs was observed on radiographs. The median duration of mechanical ventilation was 5.0 d. Of the 39 included infants, 29 required only a single dose. Radiographic data indicate that those patients requiring a second instillation of KL4-Surfactant but not showing a sustained rise in a/A O2 ratios did, in fact, exhibit expansion of alveoli in the lung. There were no RDS-related deaths; the incidence of complications was no higher than found in other comparable published studies. The data demonstrate that the synthetic peptide, KL4, which mimics the hydrophobic and hydrophilic pattern of SP-B, when formulated in an aqueous dispersion with the phospholipids DPPC and POPG, creates a strong and durable surfactant activity as judged by expansion of pulmonary alveoli and improvement of gas exchange in infants with RDS.
ABSTRACf. Synthetic pulmonary surfactants consisting of mixtures of phospholipids with synthetic peptides based on the amino acid sequence of human surfactant apoprotein SP-B were prepared. These surfactants were analyzed for their ability to lower surface tension on a pulsating bubble surfactometer and for their capacity to improve lung compliance and increase alveolar expansion in a fetal rabbit model of surfactant deficiency. The data demonstrate that several peptides, ranging from 17 to 45 residues in length, matching the carboxy-terminal sequence of the SP-B protein, when appropriately recombined with the phospholipids dipalmitoylphosphatidylcholine and phosphatidylglycerol (3:1), are capable of producing a synthetic surfactant with biophysical and biologicactivity approaching that of human surfactant derived from amniotic fluid. (Pediatr Res 29: 460-465, 1991) Abbreviations DPPC, dipalmitoylphosphatidylcholine lA, iodoacetic acid PG, phosphatidylglycerol PL, phospholipid SP, surfactant protein or swine, or synthetic lipids, generally in combination with SPBand SP-C (7, 10, 11, 13-15, 17-19, 21-23, 27), are being evaluated for their potential use in surfactant replacement therapies.Our previous studies (10) and those of Curstedt et al. (2), and Yu and Possmayer (19), suggested that when recombined with PL, SP-B resulted in greater surface tension lowering and biologic activity than did SP-C. For this reason, we decided to focus our attention on this protein. SP-B, also called SP-18 (4, 10), SPL(Phe) (3), PSP-B (5), and SAP-6-14 (7,16,17,23), has been shown to be a disulfide-linked dimer of two identical polypeptides of approximately 9000 D having an amino terminal sequence of phe-pro-ile-pro-leu-pro-tyr-(human) (3, 5, 10). Data based on amino acid compositions (10) and fast atom bombardment mass spectroscopy (unpublished observations) have led us to conclude that human SP-B is 80-81 residues in length. The sequence is shown in Figure I.To investigate the mechanisms by which SP-B protein augments the surfactant capacity of PL, peptides were synthesized that conform to portions of the native sequence of this protein. These peptides could be combined with PL to reconstitute the biophysical and biologic activities of natural surfactant. We report here the results of studies, using the pulsating bubble surfactometer to measure the capacity of these synthetic surfactants to lower surface tension at an air/liquid interface and the fetal rabbit model to determine their ability to increase lung compliance and alveolar expansion. MATERIALS AND METHODS Preparation ofpurified SP-B monomer, dimer, and oligomers.SP-B was isolated from human amniotic fluid surfactant ad escribed previously (10). One hundred J.Lg of SP-B, in a volume of 206 J.LL methanol, were incubated with 5.14 mg DTT (Sigma Chemical Co, St. Louis, MO) in 17 J.LL methanol, for 1 h at 37°C. Analysis of 2 J.LL on SDS-PAGE showed the SP-B to be approximately 70% reduced as noted by a shift in band position from 18000 D to approximately 9000 D. The SP-B/...
Two low molecular weight (LMW) apoproteins were isolated from human pulmonary surfactant. SDS polyacrylamide gel analysis showed one protein (SP 18) to have an apparent molecular weight of 18,000 when unreduced and 9,000 D after reduction. The second protein (SP 9) migrated at -9,000 D in the presence or absence of reducing agents. Both proteins contain a high number of hydrophobic amino acids. The NH2-terminal sequence of SP 18 was determined to be: NH2-phe-proile-pro-leu-pro-tyr-. A cDNA clone isolated from a human adult lung cDNA library contained a long open reading frame encoding at an internal position the human SP 18 amino-terminal sequence.Mixtures of phospholipids (PL) and SP 9 and SP 18 were assessed for their capacity to reduce surface tensions on a pulsating bubble surfactometer. The addition of 1% apoprotein resulted in a reduction of surface tension after 15 s from 42.9 dyn/cm for PL alone to 16.7 and 6.3 dyn/cm for preparations containing SP 9 and SP 18, respectively. In vivo assessment of reconstituted surfactant activity was performed in fetal rabbits. Reconstituted surfactant consisting of PL + 0.5% SP 18 instilled intratracheally at delivery resulted in a marked increase in lung compliance, while the incorporation of 0.5% SP 9 yielded a moderate increase. These data show the ability to produce biologically active surfactant by the addition of isolated LMW apoproteins to defined PL.
A large number of negatively charged macromolecules, including DNA, glycosaminoglycans, and proteoglycans, were tested as possible activators of the contact (Hageman factor) system in vitro. Activation was assessed by conversion of prekallikrein to kallikrein, as determined by amidolytic assay and by cleavage of 125I-Hageman factor into 52,000- and 28,000-dalton fragments. Of particular interest to these studies, heparin proteoglycan and glycosaminoglycan from rat peritoneal mast cells, and squid chondroitin sulfate E, which is representative of the glycosaminoglycan from cultured mouse bone marrow derived mast cells, induced the reciprocal activation between Hageman factor and prekallikrein. In addition, naturally occurring heparin glycosaminoglycans from pig mucosa, bovine lung, and rat mast cells also induced activation. In contrast, native connective tissue matrix glycosaminoglycans and proteoglycans from several sources were inactive, although when one such chondroitin sulfate was further sulfated in vitro, it gained activity. When the negative charge of the activating agents was blocked by the addition of hexadimethrine bromide, the cleavage of 125I-Hageman factor in the presence of prekallikrein was prevented. The active negatively charged macromolecules induced cleavage of 125I-high molecular weight kininogen in normal plasma but not in Hageman factor-deficient or prekallikrein- deficient plasmas. Reconstitution of prekallikrein-deficient plasma with purified prekallikrein restored the kininogen cleavage upon addition of the active proteoglycans. These results suggest that both heparin from connective tissue mast cells and highly sulfated chondroitin sulfate E from cultured mouse bone marrow derived mast cells (which are considered synonomous with mucosal mast cells) could activate the contact system of plasma subsequent to an activation secretion response.
Proteolytic cleavage and activation of isolated, single chain, zymogen Hageman factor was observed in the presence of kaolin alone. The rate of cleavage of kaolin-bound Hageman factor was enhanced 50-fold by the presence of prekallikrein and high molecular weight kininogen. The two-chain 82,000 dalton form of activated Hageman factor (α-HF(a)) also cleaved kaolin- bound single-chain Hageman factor in a dose-dependent manner, yielding fragments of 28,000 and, 50,000 dahons under reducing conditions. Cleavage of kaolin-bound single-chain Hageman factor was not inhibited by preincubation with diisopropylfluorophosphate (12 mM) for 10 min, but long-term incubation of Hageman factor with diisopropylfluorophosphate (up to 48 h) resulted in inhibition of cleavage of kaolin-bound Hageman factor to an extent proportional to the inhibition of procoagulant Hageman factor activity. Hageman factor cleavage was maximal when the kaolin concentration was {approximately} 10-fold greater than the Hageman factor concentration (wt:wt), and was partially inhibited by high molecular weight kininogen. Kaolin-bound Hageman factor cleaved clotting factor XI in an amount which correlated with the extent of cleavage of the Hageman factor. These findings are compatible with the concept that single-chain Hageman factor and α- HF(a), are both capable of cleaving and activating kaolin-bound Hageman factor and that a close molecular association of kaolin-bound Hageman factor molecules is required for this reaction.
Studies were conducted to assess the efficacy and safety of a synthetic peptide-containing surfactant in the treatment of respiratory distress syndrome (RDS) in preterm (approximately 80% of normal gestation) infant rhesus monkeys. Surfactant was prepared consisting of the phospholipids dipalmitoylphosphatidyl choline and palmitoyl-oleoyl phosphatidyl glycerol and a synthetic peptide modeled after surfactant protein B (SP-B), "KL4-Surfactant" contained a peptide having the sequence KLLLLKLLLLKLLLLKLLLLK, where "K" is lysine and "L" is leucine. The peptide was selected because it mimics the repeating stretches of hydrophobic residues with intermittent basic hydrophilic residues seen in SP-B. KL4-Surfactant was shown to have biophysical activity assessed as the ability to lower surface tension at an air-liquid interface in a pulsating bubble surfactometer. Thirty premature rhesus monkeys were treated shortly after birth with one dose of KL4-Surfactant. The arterial to alveolar oxygen partial pressure ratio (a/A) was found to rise from a pretreatment level of 0.11 +/- 0.01 (mean +/- SEM), indicative of severe RDS, to 0.40 +/- 0.02 at 12-13 h post-treatment. The improvement in oxygenation persisted throughout the study period, with a mean a/A at 22-23 h of 0.45 +/- 0.07. Chest radiographs and gross and microscopic examination of the lungs all confirmed the reversal of the atelectasis seen before treatment. Animals treated with a dose of 200 mg/kg showed a faster, more consistent, and greater response than did a group treated with an average dose of 127 mg/kg. There was no evidence of toxicity after treatment with the higher dose as demonstrated by physiologic, hematologic, biochemical, and pathologic data. The importance of the peptide in the synthetic surfactant was apparent from the results obtained with a control group of nine premature monkeys treated with a non-peptide-containing surfactant; the a/A of this group was 0.15 +/- 0.03 at nine hours of age as compared with a value of 0.38 +/- 0.02 for 30 comparable animals receiving KL4-Surfactant.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.