PubMed · 12707182
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Vance G Nielsen. 2003. Hemodilution-induced hypercoagulability.. https://doi.org/10.1213/01.ane.0000057764.66559.51
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Endotoxin, also known as lipopolysaccharide (LPS), is the major mediator of septic shock due to Gram-negative bacterial infection. Chemically synthesized S3 peptide, derived from Sushi3 domain of Factor C, which is the endotoxin-sensitive serine protease of the limulus coagulation cascade, was previously shown to bind and neutralize LPS activity. For large-scale production of this peptide and to mimick other pathogen-recognizing molecules, tandem multimers of the S3 gene were constructed and expressed in Escherichia coli. The recombinant tetramer of S3 (rS3-4mer) was purified by anion exchange and digested into monomers (rS3-1mer). Both the rS3-4mer and rS3-1mer were functionally analyzed for their ability to bind LPS by an ELISA-based method and surface plasmon resonance. The LAL inhibition and TNFalpha-release test showed that rS3-1 mer can neutralize the LPS activity as effectively as the synthetic S3 peptide, while rS3-4mer displays an enhanced inhibitory effect on LPS-induced activities. Both recombinant peptides exhibited low cytotoxicity and no haemolytic activity on human cells. This evidence suggests that the recombinant sushi peptides have potential use for the detection, removal of endotoxin and/or anti-endotoxin strategies.
Factor C protein isolated from the horseshoe crab, Carcinoscorpius rotundicauda, has endotoxin binding capability. Synthetic peptides of 34 amino acids based on the sequence of two regions of factor C (Sushi 1 and Sushi 3) as well as their corresponding mutants exhibited activities against 30 clinical isolates of Pseudomonas aeruginosa. Collectively, all four peptides demonstrated exceptionally effective bactericidal activity against P. aeruginosa with 90% minimal bactericidal concentrations (MBC(90)s) in the range of 0.06 to 0.25 microg/ml (16 to 63 nM). Viable bacteria were reduced by 90% after 7 min and were totally eradicated within 40 to 50 min. These peptides are minimally hemolytic against both rabbit and human erythrocytes even at concentrations up to 1,600-fold their MBC(90)s. Both in vitro and in vivo studies indicate that cytotoxic effects are small even at 1,000-fold their MBC(90)s. Furthermore, the Sushi peptides are tolerant of high-salt and adverse pH conditions. These findings demonstrate the promising therapeutic potential of the Sushi peptides.
The binding and assembly of the coagulation proteases on the endothelial cell surface are important steps not only in the generation of thrombin and thrombogenesis, but also in vascular cell signaling. Effector cell protease receptor (EPR-1) was identified as a novel leukocyte cell surface receptor recognizing the coagulation serine protease Factor Xa but not the precursor Factor X. We now demonstrate that EPR-1 is expressed on vascular endothelial cells and smooth muscle cells. Northern blots of endothelial and smooth muscle cells demonstrated three abundant mRNA bands of 3.0, 1.8, and 1.3 kDa. 125I-Labeled Factor Xa bound to endothelial cells in a dose-dependent saturable manner, and the binding was inhibited by antibody to EPR-1. No specific binding was observed with a recombinant mutant Factor X in which the activation site was substituted by Arg196 --> Gln to prevent the proteolytic conversion to Xa. EPR-1 was identified immunohistochemically on microvascular endothelial and smooth muscle cells. Functionally, exposure of smooth muscle cells or endothelial cells to Factor Xa induced a 3-fold and a 2-fold increase in [3H]thymidine uptake, respectively. However, receptor occupancy alone is insufficient for mitogenic signaling because the active site of the enzyme is required for mitogenesis. Thus, EPR-1 represents a site of specific protease-receptor complex assembly, which during local initiation of the coagulation cascade could mediate cellular signaling and responses of the vessel wall.