Alpha-1 acid glycoprotein reduces hepatic leukocyte recruitment in murine models of either early endotoxemia or early sepsis.

OBJECTIVE: To characterize the effect of systemically administered AGP on early leukocyte recruitment in the livers of endotoxemic or septic mice and to determine whether this is influenced by LPS sequestration. METHODS: Endotoxemia was induced in C57Bl/6 mice via intraperitoneal injection of LPS. Sepsis was induced in mice by cecal ligation and perforation. AGP (165 mg/kg) or saline (20 mL/kg) or HAS (200 mg/kg) was administered immediately after surgery or LPS injection and the hepatic microcirculation was examined by intravital microscopy at four hour. RESULTS: Leukocyte adhesion in the PSV was reduced by treatment with AGP in mice subjected to either LPS or CLP protocols compared to either saline or HAS treatment. AGP-treated mice also had significantly higher sinusoidal flow in both models. Pre-incubation of LPS with AGP reduced the ability of LPS to recruit leukocytes to the liver microcirculation. CONCLUSIONS: AGP was more effective in limiting hepatic inflammation and maintaining perfusion than saline or HAS, in both endotoxemic and septic mice. AGP sequestration of LPS may contribute to its anti-inflammatory effects.

Recombinant albumins containing additional peptide sequences smaller than barbourin retain the ability of barbourin-albumin to inhibit platelet aggregation.

The previously described fusion protein BLAH(6) (Marques JA et al.,Thromb Haemost 2001; 86: 902-8) is a recombinant protein that combines the small disintegrin barbourin with hexahistidine-tagged rabbit serumalbumin (RSA) produced in Pichia pastoris yeast. We sought to determine: (1) if BLAH(6) was immunogenic; and (2) if its barbourin domain could be productively replaced with smaller peptides. Purified BLAH(6) was injected into rabbits, and anti-barbourin antibodies were universally detected in plasma 28 days later; BLAH(6) was, however, equally effective in reducing platelet aggregation in both naive and pre-treated rabbits. Thrombocytopenia was not observed, and complexing BLAH(6) to alpha(IIb)beta(3) had no effect on antibody detection. The barbourin moiety of BLAH(6) was replaced with each of four sequences: Pep I (VCKGDWPC); PepII (VCRGDWPC); PepIII (bar-bourin 41-54); and PepIV (LPSPGDWR). The corresponding fusion proteins were tested for their ability to inhibit ADP-induced platelet aggregation. PepIII-LAH(6) inhibited neither rabbit nor human platelets. PepI-LAH(6) and PepIV-LAH(6) inhibited rabbit platelet aggregation as effectively as BLAH(6), but PepIV-LAH(6) did not inhibit human platelet aggregation. PepI-LAH(6) and PepIILAH(6) inhibited human platelet aggregation with IC(50)s 10- and 20-fold higher than BLAH(6). Cross-immunoprecipitation assays with human platelet lysates confirmed that all proteins and peptides interacted with the platelet integrin alpha(IIb)beta(3), but with greatly varying affinities. Our results suggest that the antiplatelet activity of BLAH(6) can be retained in albumin fusion proteins in which smaller peptides replace the barbourin domain; these proteins may be less immunogenic than BLAH(6).

A covalently linked recombinant albumin dimer is more rapidly cleared in vivo than are wild-type and mutant C34A albumin.

Mammalian albumins are abundant plasma proteins that exhibit a relatively slow terminal clearance. For this reason they have been fused to potentially therapeutic proteins with rapid terminal clearance to produce fusion proteins with more desirable clearance profiles. A disulfide-linked albumin dimer has been described, but its abundance and stability in plasma are uncertain. To determine whether an obligatory albumin dimer incapable of dissociation would clear less rapidly than monomeric albumin, we expressed 3 recombinant rabbit serum albumin (RSA) polypeptides: H6RSA, RSA modified by the addition of an N-terminal hexahistidinyl tag; H6RSA(C34A), H6RSA with a single cysteine (Cys) 34-to-alanine (Ala) substitution (C34A); and DiRSA, H6RSA(C34A) joined by way of its C-terminus to RSA(C34A) through an intervening hexaglycine spacer. The C34A mutation was introduced to eliminate the possibility of disulfide bond-mediated dimerization. We expressed the proteins with the use of the yeast Pichia pastoris and purified them using nickel-chelate, ion exchange, and gel-filtration chromatography. After radioiodination and injection into rabbits, H6RSA and H6RSA(C34A) exhibited indistinguishable terminal catabolic half-lives (4.9 +/- 0.7 and 4.8 +/- 0.5 days, mean +/- SD), whereas that of DiRSA was reduced to 3.0 +/- 0.3 days (p<.05). The three proteins circulated in intact form, and their distributions in liver, lung, kidney, heart, and spleen did not differ 24 hours after injection. Although more DiRSA than H6RSA(C34A) was present in urine, in both cases it was in acid-soluble form. Ethyl palmitate treatment reduced the relative acceleration of DiRSA clearance compared with that of H6RSA(C34A), suggesting a role for the reticuloendothelial system in the differential clearance of the larger protein. Our results suggest that an albumin fusion protein should include only a single copy of albumin; that if the fusion protein exceeds a certain size, it may not acquire the slow clearance profile of native albumin; and that albumin dimerization through Cys34 probably does not contribute substantially to albumin metabolism in vivo.