Complications of lower extremity arteriovenous grafts in patients with end-stage renal disease.

BACKGROUND: More data are needed to assess lower extremity angioaccess sites for hemodialysis. METHODS: We did a retrospective review of 843 consecutive hospital records of upper and lower extremity arteriovenous (AV) fistulas from 1992 to 1996. RESULTS: Lower extremity grafts accounted for 16% (134/843) of patients in this series. Complications occurred in 58 of 134 patients (43%) and were more prevalent in women, blacks, diabetic, and hypertensive patients, but not of statistical significance. Dialysis was done for a mean duration of 13.3 years, with a mean graft patency rate of 13.8 months. The 12-month survival rate of lower extremity AV grafts was 62% (83/134). Complications in the lower extremity AV graft group (58 patients) included infections in 27 patients (46%), thrombosis within 30 days in 16 (28%), pseudoaneurysm in 9 (16%), and graft hemorrhage in 6 (10%). CONCLUSIONS: There is a decreased patency rate in lower extremity AV grafts.

Electronic absorption, EPR, and resonance raman spectroscopy of CooA, a CO-sensing transcription activator from R. rubrum, reveals a five-coordinate NO-heme.

Electronic absorption, EPR, and resonance Raman spectroscopies revealed that CooA, the CO-sensing transcriptional regulator from Rhodospirillum rubrum, reacts with NO to form a five-coordinate NO-heme. NO must therefore displace both of the heme ligands from six-coordinate, low-spin Fe(II)CooA in forming five-coordinate Fe(II)CooA(NO). CO, in contrast, displaces a single heme ligand from Fe(II)CooA to form six-coordinate Fe(II)CooA(CO). Of a series of common heme-binding ligands, only CO and NO were able to bind to the heme of wild-type CooA; imidazole, azide anion, and cyanide anion had no effect on the heme absorption spectrum. Although NO binds to the heme and displaces the endogenous ligands, NO was not able to induce CooA to bind to its target DNA. The mechanism of CO-dependent activation of CooA is thus more complex than simple displacement of a ligand from the heme iron since NO does not trigger DNA binding. These observations suggest that the CooA heme site discriminates between NO and the biologically relevant signal, CO.

Resonance Raman evidence for a novel charge relay activation mechanism of the CO-dependent heme protein transcription factor CooA.

Resonance Raman spectra of the CO-responsive transcription factor CooA from Rhodospirillum rubrum provides evidence on the nature of heme ligation and its CO activation mechanism. The Fe(III) form gives standard low-spin heme spectrum, while the Fe(II) form is low spin for wild-type (WT) CooA and mixed spin for a CooA variant, H77Y, with an His77Tyr substitution. The Fe(II) porphyrin skeletal mode nu11 is at a value (1541 cm-1) indicative of a neutral donor ligand for the H77Y variant but is at an unusually depressed frequency (1529 cm-1) for the WT protein, indicating a strongly donating ligand. This ligand is proposed to be His77 imidazolate, formed by proton transfer to a nearby acceptor. The WT CO adduct has FeCO and CO stretching frequencies that indicate a neutral trans ligand and negative polarity in the CO binding pocket, while the CO adduct of His77Tyr has both 6- and 5-coordinate signals and a nonpolar CO environment. Photolysis of the WT CO adduct by the Raman laser produced a low-spin product at steady state, indicating fast recombination of the displaced ligand. These data suggest a novel YH- - -His- charge relay mechanism for CooA activation by CO. In this mechanism, His77 is reprotonated upon ligand displacement by CO; CO displaces either His77 or the trans ligand, X. The resulting charge on Y- may induce the protein conformation change required for site-selective DNA binding.

Variable forms of soluble guanylyl cyclase: protein-ligand interactions and the issue of activation by carbon monoxide.

Soluble guanylyl cyclase (sGC) is known to be activated by NO binding to the heme moiety; previous studies have shown that CO does not activate sGC to the same extent as NO. Resonance Raman spectroscopy reveals different heme pocket structures for soluble guanylyl cyclase prepared by alternate methods, all of which display activation by NO. In our preparation, and in the expressed protein sGC1, the resting Fe(II) state is mainly 6-coordinate and low-spin, and the CO adduct has vibrational frequencies characteristic of a histidine-heme-CO complex in a hydrophobic environment. In contrast, the protein sGC2 is 5-coordinate, high-spin in the resting state, and the CO adduct has perturbed vibrational frequencies indicative of a negatively polarizing residue in the binding pocket. The differences may result from the need to reconstitute sGC1 or different isolation procedures for sGC1 versus sGC2. However, both sGC1 and sGC2 are activated by the same mechanism, namely displacement of the proximal histidine ligand upon NO binding, and neither one is activated by CO. If CO is an activator in vivo, some additional molecular component is required.

Erythropoietin therapy improves graft patency with no increased incidence of thrombosis or thrombophlebitis.

BACKGROUND: Recombinant human erythropoietin (rHuEPO) for the treatment of severe anemia in patients with end-stage renal disease (ESRD) is suggested to improve rehabilitation and cognitive function. The criticism is the alleged increase in the failure rate of arteriovenous (AV) access grafts and in the incidence of lower-extremity deep venous thrombophlebitis (DVT). This study addressed the longevity of AV grafts and the incidence of DVT. STUDY DESIGN: We reviewed 481 consecutive patients with ESRD on dialysis with PTFE access grafts, including 173 consecutive patients who were receiving rHuEPO and 308 who were not. rHuEPO was administered during dialysis titrated against the hematocrit to achieve a level of 33% to 38%. The rHuEPO-ESRD group included 173 patients with a mean age of 58 years, including 54% women; 84% of the grafts were in the upper extremity. In the control group of 308 patients, 57% were women. Diabetes and hypertension were controlled in both groups. RESULTS: Forty-five of 173 rHuEPO patients (26%) experienced graft thrombosis within 1 year. Among 88 episodes of thrombosis, 14 patients experienced multiple episodes. Primary patency was 8.9 months; secondary patency was 11.2 months. In the control population, 95 of 308 patients (31%) experienced graft thrombosis; 27 patients had multiple episodes. Primary patency was 7.8 months and secondary patencywas 9.8 months. The hematocrit improved from a mean of 23% in the control group to 34% in the treated rHuEPO group. Two patients in the control group and one patient receiving rHuEPO experienced DVT in the lower extremity. CONCLUSIONS: Primary and secondary AV fistula patency rates were improved by 10% with rHuEPO. rHuEPO did not increase DVT.

De novo heme proteins from designed combinatorial libraries.

We previously reported the design of a library of de novo amino acid sequences targeted to fold into four-helix bundles. The design of these sequences was based on a "binary code" strategy, in which the patterning of polar and nonpolar amino acids is specified explicitly, but the exact identities of the side chains is varied extensively (Kamtekar S, Schiffer JM, Xiong H, Babik JM, Hecht MH, 1993, Science 262:1680-1685). Because of this variability, the resulting collection of amino acid sequences may include de novo proteins capable of binding biologically important cofactors. To probe for such binding, the de novo sequences were screened for their ability to bind the heme cofactor. Among an initial collection of 30 binary code sequences, 15 are shown to bind heme and form bright red complexes. Characterization of several of these de novo heme proteins demonstrated that their absorption spectra and resonance Raman spectra resemble those of natural cytochromes. Because the design of these sequences is based on global features of polar/ nonpolar patterning, the finding that half of them bind heme highlights the power of the binary code strategy, and demonstrates that isolating de novo heme proteins does not require explicit design of the cofactor binding site. Because bound heme plays a key role in the functions of many natural proteins, these results suggest that binary code sequences may serve as initial prototypes for the development of large collections of functionally active de novo proteins.