Prognostic value of protein C concentrations in neutropenic patients at high risk of severe septic complications.

OBJECTIVE: To assess the prognostic value of protein C, endogenous activated protein C, and D-dimer concentrations in patients at high risk of developing severe septic complications secondary to cytostatic chemotherapy. DESIGN: Prospective, comparative, single-center study. SETTING: Specialized ward for treating patients with acute leukemia and associated intensive care unit at a university hospital. SUBJECTS: Twenty-six consecutive patients who developed either severe sepsis (n = 13) or septic shock (n = 13) during chemotherapy-induced neutropenia (leukocytes <1,000/microL). INTERVENTION: None, other than standard care. MEASUREMENTS AND MAIN RESULTS: Baseline blood samples were obtained from 97 adult patients treated with intensive cytostatic chemotherapy. Serial blood sampling was performed in 62 of 97 patients who developed fever (>38.3 degrees C). Thirteen patients progressed to severe sepsis and 13 patients to septic shock. Protein C, endogenous activated protein C, and D-dimer were measured in these 26 patients. At fever onset, protein C concentrations decreased from normal baseline concentrations and were significantly lower in the group of patients who progressed to septic shock compared with those who developed severe sepsis (medians for protein C activity: 23.1% vs. 69.5%; p = .0003). The median elapsed time between detection of fever and the diagnosis of severe sepsis or septic shock was 16 hrs and 12 hrs, respectively. All septic shock patients died, whereas patients who progressed only to severe sepsis survived. CONCLUSIONS: Septic shock in neutropenic patients is associated with increased protein C consumption. The data demonstrate that the coagulation cascade is activated and produces a hypercoagulable state before the onset of clinical symptoms of severe sepsis and septic shock. Low protein C concentrations at the onset of fever and before the onset of clinical symptoms of severe sepsis or septic shock may have prognostic value in predicting an unfavorable outcome. Protein C measurements may help identify patients at risk in an early phase of neutropenic sepsis. It is also attractive to speculate that because low protein C concentrations were seen in these patients, protein C replacement may be beneficial in sepsis.

Isolation and characterization of mouse coagulation factor X -- biophysical and enzymological properties.

Mouse factor X was highly purified from plasma using barium ion precipitation and chromatography on anion-exchange and heparin-agarose affinity chromatography columns. Intact and reduced patterns of mouse factor X in SDS-PAGE were similar to those of human factor X. The specific absorption E 1%/1 cm at 280 nm of mouse factor X was found to be 11.2. Content of carbohydrate moieties of mouse factor X, determined to be 10% by weight, differs both quantitatively and quantitatively from that of human factor X, while the gamma-carboxyglutamic acid (Gla) and beta-hydroxy-aspartic acid (beta-OH-Asp) content were essentially the same as for human factor X. The amino-terminal amino acid sequences of the light and heavy chains of mouse factor X separated by SDS-PAGE were ANSFF--FKK and SVALXTSDSE, respectively. Underlined residues are non-identical with those of human factor X. Clotting time-based assays using human factor X-deficient plasma as substrate exhibited the following apparent extents of activation of factor X in mouse plasma, using human plasma as the standard: 195% (intrinsic); 200% (extrinsic); and 190% (RVV-X). Using the purified proteins in the same assay systems, the following apparent activation of mouse factor X was demonstrated, compared with human factorX: 195% (intrinsic); 27% (extrinsic); and 41% (RVV-X). These activity profiles suggest that the human extrinsic coagulation pathway functions less efficiently than the corresponding mouse pathway in the activation of mouse factor X. Furthermore, mouse brain thromboplastin satisfactorily replaced rabbit brain thromboplastin in extrinsic activation of factor X in mouse plasma, but not of human plasma or purified mouse or human factor X, in line with studies by others suggesting that human factor VIIa poorly activates factor X in the presence of mouse tissue factor. While fully RVV-X-activated mouse factor X activated human prothrombin at a rate equal to about 117% of that for human factor X, it hydrolyzed the synthetic substrate, S-2222, at a rate of only about 18% of that for human factor X. These results are expected to be useful in making the mouse suitable for study of the mammalian blood coagulation pathways.

High mobility group protein, HMG-1, contains insignificant glycosyl modification.

High mobility group protein-1 (HMG-1) is a ubiquitous, highly conserved, and abundant nuclear protein. Recent findings suggest that HMG-1 may serve as a DNA chaperone protein and play a role in the regulation of transcription. There is a mounting interest in elucidating the mechanism by which HMG-1 protein takes part in these activities. HMG-1 has been reported to undergo an extensive array of posttranslational modifications, including glycosylation. We extend the earlier findings on the glycosylation of HMG-1 by quantitating the amount of carbohydrate on HMG-1 from calf thymus and chicken erythrocytes isolated by 2 different purification procedures. In addition, 2 different developmental stages (embryonic and adult) were examined in the chicken erythrocytes. The glycosyl composition was quantitated using the Dionex HPAE-PAD II system. Furthermore, the presence of O-linked GlcNAc on HMG-1 was determined by the enzymatic incorporation of 3H-galactose into HMG-1 protein. Contrary to earlier reports, less than 0.5 mol of total monosaccharides (Fuc, Man, GalNH2, GlcNH2, Gal) were detected per mole of HMG-1 protein, regardless of the source of the protein or the method of isolation. In addition, less than 0.002 mol of O-linked GlcNAc per mole of HMG-1 protein was detected. Thus, insignificant amount of glycosylation was found on HMG-1 protein. Because O-linked GlcNAc modification of proteins is believed to be a reversible posttranslational event, more definitive studies will need to be conducted before ruling out that the function of HMG-1 protein is not regulated by glycosylation.

Novel Asn-linked oligosaccharides terminating in GalNAc beta (1-->4)[Fuc alpha (1-->3)]GlcNAc beta (1-->.) are present in recombinant human protein C expressed in human kidney 293 cells.

Recombinant human Protein C (rHPC), expressed in human kidney 293 cells, has a higher anticoagulant activity than plasma HPC, while its in vivo circulatory half-life is essentially unaltered compared to that of the natural protein. In seeking to elucidate the molecular basis for the improved efficacy of the recombinant antithrombotic drug, we focused on the carbohydrate moiety of rHPC. Protein C is a heavily post-translationally modified serine protease with four N-glycosylation sites. Glycosyl composition analysis of rHPC revealed a 5-fold higher fucose content and a 2-fold lower sialic acid content compared to plasma HPC. In addition, we found that rHPC contains N-acetylgalactosamine (2.6 mol GalNAc/mol rHPC) in its Asn-linked oligosaccharides, while plasma HPC is devoid of GalNAc. The Asn-linked oligosaccharides of rHPC were released by N-glycanase and separated into 25 fractions by high-pH anion-exchange chromatography. The most abundant oligosaccharides were structurally characterized by glycosyl composition and linkage analysis, in conjunction with 1H-NMR spectroscopy at 600 MHz. The structure of the major neutral oligosaccharide in rHPC was determined to be: [formula: see text] Two representatives of the sialylated oligosaccharides in rHPC are: [formula: see text] and [formula: see text] Thus, many of the Asn-linked oligosaccharides in rHPC were found to terminate in GalNAc beta (1-->4)GlcNAc beta (1-->.), in NeuAc alpha (2-->6)GalNAc beta (1-->4)GlcNAc beta (1-->.), and/or in GalNAc beta (1-->4)[Fuc alpha (1-->3)]GlcNAc beta (1-->.). Since the latter trisaccharide was first [Yan, S.B., Chao, B.Y. and Van Halbeek,H. (1992) J. Cell. Biochem., 16D, 151] observed in the Asn-linked oligosaccharides of rHPC derived from human kidney 293 cells, we propose to label the GalNAc beta-(1-->4)[Fuc alpha (1-->3)]GlcNAc beta (1-->.) terminal trisaccharide the PC-293 determinant. The PC-293-containing oligosaccharides may contribute to the higher anticoagulant activity of rHPC as compared to plasma HPC.

Characterization and novel purification of recombinant human protein C from three mammalian cell lines.

Human Protein C (HPC), an antithrombotic factor with potential clinical utility, is a vitamin K-dependent protein that has several complex post-translational modifications. In an effort to define the functional roles of these modifications, recombinant HPC (rHPC) was expressed in and characterized from 3 adenovirus-transformed cell lines. The rHPC in crude culture medium from the 3 cell lines displayed anticoagulant activities that were either higher, slightly lower or much lower than that of plasma HPC. The rHPC from each cell line was purified and characterized using a novel, but simple chromatographic method, termed "pseudo-affinity", capable of resolving molecules differing by only very slight modifications. We demonstrate the critical dependence of full gamma-carboxylation on the function of this protein. In addition, our data indicate that both the gamma-carboxyglutamate and glycosyl contents affect the functional activities of rHPC.