Heparin-induced thrombocytopenia: recent experience in a large teaching hospital.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is a potentially serious adverse reaction caused by platelet-activating antibodies. AIM: To describe experience with HIT. METHODS: Twenty-two patients identified by laboratory records of heparin-associated antibodies with a 50% or greater decrease in platelet count were reviewed in our 600-bed metropolitan teaching hospital from 1999 to April 2005. RESULTS: There was an increase in the frequency of HIT diagnosed during the review period, which was associated with a rise in the number of requests for HIT antibodies. Thrombotic complications were identified in 14 of 22 patients with HIT. Mean age was 65 years, and 11 patients were men. Seven patients died and HIT was considered contributory in four. One patient required mid-forearm amputation. Unfractionated heparin was used in all cases and five patients also received enoxaparin. Mean time to HIT screen, reflecting when the diagnosis was first suspected, was 14 days. Platelet nadir ranged from 6 x 10(9)/L to 88 x 10(9)/L, with a percentage drop in platelet count of 67-96%. Alternative anticoagulation (danaparoid) was not used in three patients, two of whom died. CONCLUSIONS: HIT is a potentially life-threatening complication of heparin therapy, associated with a fall in platelet count and a high incidence of thromboembolic complications. It is most frequently seen using unfractionated heparin therapy. The increase in frequency of HIT diagnosed in our hospital appears to be associated with a greater awareness of the entity, although detection is often delayed. Platelet count should be monitored in patients on heparin and the presence of antiplatelet antibodies determined if HIT is suspected. Treatment involves both discontinuation of heparin and the use of an alternative anticoagulant such as danaparoid because of the persisting risk of thrombosis.

Evaluation of dosage adjustment in patients with renal impairment.

AIMS: To determine whether appropriate dosage adjustments are made in patients with significant renal impairment for drugs with a high fractional renal clearance. METHODS: Evaluation of dosage adjustment was performed in patients who were admitted to a 480-bed metropolitan hospital (Princess Alexandra Hospital, Brisbane, Australia) with an estimated creatinine clearance of < or =40 mL/min. All drugs had a high fractional renal excretion. A prescribed dose within 30% of the calculated dose was considered appropriate. RESULTS: Doses were found to be inappropriately high in 111 (44.8%) of 248 admission prescriptions of the targeted drugs. Doses were appropriately reduced in hospital in 26 patients (23.4%). Seventy-three (29.3%) prescriptions were continued with excessive doses. Only 34 prescriptions for the target drugs were initiated in hospital, of which 88.2% were appropriately dosed. CONCLUSIONS: A significant percentage of patients with renal impairment are admitted to hospital on inappropriately high doses of drugs, with a high fractional renal excretion and low therapeutic index. Doses are appropriately reduced in hospital in some patients but there is still room for improvement [corrected].

Sidechain interactions in parallel beta sheets: the energetics of cross-strand pairings.

BACKGROUND: Both backbone hydrogen bonding and interactions between sidechains stabilize beta sheets. Cross-strand interactions are the closest contacts between the sidechains of a beta sheet. Here we investigate the energetics of cross-strand interactions using a variant of the B1 domain of immunoglobulin G (IgG) binding protein G (beta1) as our model system. RESULTS: Pairwise mutations of polar and nonpolar residues were made at a solvent-exposed site between the two central parallel beta strands of beta1. Both stabilizing and destabilizing interactions were measured. The greatest stabilizations were observed for charge-charge interactions. Our experimental study of sidechain interactions correlates with statistical preferences: residue pairs for which we measure stabilizing interaction energies occur together frequently, whereas destabilizing pairs are rarely observed together. CONCLUSIONS: Sidechain interactions modulate the stability of beta sheets. We propose that cross-strand sidechain interactions specify correct strand register and ordering through the energetic benefit of optimally arranged pairings.

Structure of the capsid of Pf3 filamentous phage determined from X-ray fibre diffraction data at 3.1 A resolution.

We have recorded X-ray diffraction patterns at 3.1 A resolution from magnetically aligned fibres of the Pf3 strain of filamentous bacteriophage (Inovirus). The patterns are similar to patterns from the higher-temperature form of the Pf1 strain, indicating that the Pf3 and Pf1 virions have the same helix symmetry and similar protein subunit shape. This is of particular interest, given that the primary structures of the two protein subunits are quite different; and the nucleotide/protein subunit ratio in the Pf3 virion is more than twice that in Pf1, indicating important differences in DNA packaging. We have built a molecular model of the Pf3 protein capsid based on the model of Pf1, and refined it against the diffraction data using simulated annealing. The refinement confirms that the two structures are similar, which may reflect a fundamental motif of alpha-helix packing. However, there are some differences between the structures: the Pf3 subunit appears to be completely alpha-helical, beginning at the N terminus, whereas the first few residues of the Pf1 subunit are not helical; and the structure of the C-terminal region of the Pf3 subunit at the inner surface of the tubular capsid indicates that DNA/protein interactions in this virion may involve both aromatic side-chains and positively charged side-chains, whereas those in the Pf1 virion involve predominantly only the latter. In the course of this work, we have developed new approaches to refinement and validation of helical structures with respect to continuous transform fibre diffraction data.

A differential scanning calorimetric study of the thermal unfolding of apo- and holo-cytochrome b562.

Cytochrome b562 is a four-helix-bundle protein containing a non-covalently bound b-type heme prosthetic group. In the absence of heme, cytochrome b562 remains highly structured under native conditions. Here we report thermodynamic data for the thermal denaturation of the holo- and apoproteins as determined by differential scanning calorimetry. Thermal denaturation of holocytochrome b562 is a highly reversible process, and unexpectedly does not involve dissociation of the heme prosthetic group. Thermal denaturation of the corresponding apoprotein, with the heme group chemically removed, remains a cooperative, reversible process. Apocytochrome b562 is substantially destabilized relative to the holoprotein: the t1/2 is more than ten degrees lower, and enthalpy and heat capacity changes are about one-half of the holoprotein values. However, the energetic parameters of apocytochrome b562 denaturation are within the range of observed values for small proteins.

A thermodynamic study of the binding of linear and cyclic oligosaccharides to the maltodextrin-binding protein of Escherichia coli.

Isothermal titration calorimetric (ITC) studies over a range of temperatures of the binding of maltose, maltotriose, maltotetraose and beta-cyclodextrin to the maltodextrin-binding protein (MBP) of Escherichia coli are reported. The binding constants of maltose, maltotriose and beta-cyclodextrin are not very different, namely 8.7 x 10(5), 13.0 x 10(5) and 2.55 x 10(5) M-1, respectively at 25 degrees C. The calorimetric data obtained with maltotetraose cannot be interpreted in terms of a definite binding constant. The binding of maltose and maltotriose is endothermic with a large entropy increase while that of beta-cyclodextrin is exothermic, with a smaller entropy increase. The binding of maltotetraose was endothermic or exothermic depending on the temperature.

The adaptability of Escherichia coli thioredoxin to non-conservative amino acid substitutions.

The adaptability of Escherichia coli thioredoxin to the substitution of a series of non-natural amino acids has been investigated. Different thiosulfonated alkyl groups were inserted into the hydrophobic core of the protein in position 78 via disulfide bonding with a buried cysteine residue as previously described (Wynn R, Richards FM. 1993. Unnatural amino acid packing mutants of Escherichia coli thioredoxin produced by combined mutagenesis/chemical modification techniques. Protein Sci 2:395-403). The side chains added to the cysteine included methyl, ethyl, n-propyl, n-butyl, n-pentyl, and cyclo-pentyl derivatives. The side chains appear to exploit the presence of the large cavities to incorporate these variant forms, enabling the protein to fold and have some activity. Solution structural and kinetic data suggested that these substitutions had little effect on the overall fold of the protein. Thermodynamic data revealed that the entropic effect of restricting the side chains in the folded protein has an effect on the stability. The variant forms of thioredoxin have different propensities to form dimers despite the limited structural perturbations. Molecular modeling studies allow the conformation of the side chains to be assessed.

Significant discrepancies between van't Hoff and calorimetric enthalpies. III.

The study of a wide variety of reversible reactions in solution indicates that the enthalpy, DeltaH(vH), which controls the temperature variation of the equilibrium constant for a reaction, can seldom, if ever, be taken to be independent of the temperature. It is also found in most cases that the values for DeltaH(vH), properly evaluated as varying with the temperature, differ significantly from the values for the enthalpy, DeltaH(cal), determined by direct calorimetry under the same experimental conditions. In a continuing search for reactions which show agreement between DeltaH(vH) and DeltaH(cal), we have studied by isothermal titration calorimetry the reactions of heptylamine with heptanoic acid in dodecane solution and of alpha-cyclodextrin with sodium heptanoate in aqueous solution.

Energetics of heme binding to native and denatured states of cytochrome b562.

Cytochrome b562 is a four-helix bundle protein containing a noncovalently bound b-type heme prosthetic group. For the first time, energetics of heme binding to an apocytochrome were measured by isothermal titration calorimetry. The heme is tightly bound to native apocytochrome b562, with a dissociation constant (Kd) of approximately 9 nM (DeltaG degrees = 11 kcal mol-1) at 25 degrees C. Unexpectedly, the thermally denatured apoprotein is also capable of specifically binding heme with modest affinity (Kd = 3 microM, DeltaG degrees = 7.6 kcal mol-1). This interaction results in the dependence of holocytochrome b562 stability on protein concentration in the submicromolar range.

Surface point mutations that significantly alter the structure and stability of a protein's denatured state.

Significantly different m values (1.9-2.7 kcal mol-1 M-1) were observed for point mutations at a single, solvent-exposed site (T53) in a variant of the B1 domain of streptococcal Protein G using guanidine hydrochloride (GuHCl) as a denaturant. This report focuses on elucidating the energetic and structural implications of these m-value differences in two Protein G mutants, containing Ala and Thr at position 53. These two proteins are representative of the high (m+) and low (m-) m-value mutants studied. Differential scanning calorimetry revealed no evidence of equilibrium intermediates. A comparison of GuHCl denaturation monitored by fluorescence and circular dichroism showed that secondary and tertiary structure denatured concomitantly. The rates of folding (286 S-1 for the m+ mutant and 952 S-1 for the m- mutant) and the rates of unfolding (11 S-1 for m+ mutant and 3 S-1 for the m- mutant) were significantly different, as determined by stopped-flow fluorescence. The relative solvation free energies of the transition states were identical for the two proteins (alpha ++ = 0.3). Small-angle X-ray scattering showed that the radius of gyration of the denatured state (Rgd) of the m+ mutant did not change with increasing denaturant concentrations (Rgd approximately 23 A); whereas, the Rgd of the m- mutant increased from approximately 17 A to 23 A with increasing denaturant concentration. The results indicate that the mutations exert significant effects in both the native and GuHCl-induced denatured state of these two proteins.

What makes a protein a protein? Hydrophobic core designs that specify stability and structural properties.

Here we describe how the systematic redesign of a protein's hydrophobic core alters its structure and stability. We have repacked the hydrophobic core of the four-helix-bundle protein, Rop, with altered packing patterns and various side chain shapes and sizes. Several designs reproduce the structure and native-like properties of the wild-type, while increasing the thermal stability. Other designs, either with similar sizes but different shapes, or with decreased sizes of the packing residues, destabilize the protein. Finally, overpacking the core with the larger side chains causes a loss of native-like structure. These results allow us to further define the roles of tight residue packing and the burial of hydrophobic surface area in the construction of native-like proteins.

A scanning calorimetric study of unfolding equilibria in homodimeric chicken gizzard tropomyosins.

Using both circular dichroism (CD) and differential scanning calorimetry (DSC), several laboratories find that the thermal unfolding transitions of alpha alpha and beta beta homodimeric coiled coils of rabbit tropomyosin are multistate and display an overall unfolding enthalpy of near 300 kcal (mol dimer)(-1). In contrast, an extant CD study of beta beta and gamma gamma species of chicken gizzard tropomyosin concludes that their unfolding transitions are simple two-state transitions, with much smaller overall enthalpies (98 kcal mol(-1) for beta beta and 162 kcal mol(-1) for gamma gamma). However, these smaller enthalpies have been questioned, because they imply a concentration dependence of the melting temperatures that is far larger than observed by CD. We report here DSC studies of the unfolding of both beta beta and gamma gamma chicken gizzard homodimers. The results show that these transitions are very similar to those in rabbit tropomyosins in that 1) the overall unfolding enthalpy is near 300 kcal mol(-1); 2) the overall delta C(rho) values are significantly positive; 3) the various transitions are multistate, requiring at least two and as many as four domains to fit the DSC data. DSC studies are also reported on these homodimeric species of chicken gizzard tropomyosin with a single interchain disulfide cross-link. These results are also generally similar to those for the correspondingly cross-linked rabbit tropomyosins.

The observed change in heat capacity accompanying the thermal unfolding of proteins depends on the composition of the solution and on the method employed to change the temperature of unfolding.

The apparent change in heat capacity, delta C(p), accompanying the thermally induced unfolding of lysozyme and of ribonuclease A was determined by means of differential scanning calorimetry in dilute aqueous buffer containing one of the following added solutes: 0.5 M or 1.0 M sucrose, 1.0 M glycine, 0.5 M, 1.0 M, or 2.0 M guanidinium chloride, 10% glycerol, or 0.5 M NaCl over a pH range. In each system the temperature of half-completion, t1/2, of the unfolding transition was varied by varying the pH. The resulting enthalpies of denaturation were linearly dependent on t1/2 for each solvent system. The resulting values of delta C(p) for each protein showed variation of almost 2-fold. Such large variations in the sensitivity of the proteins to temperature changes are not readily interpreted.

Thermodynamic studies of SHC phosphotyrosine interaction domain recognition of the NPXpY motif.

The N-terminal 200 amino acids of SHC constitute a unique phosphotyrosine (Tyr(P)) interaction (PI) domain that shows no significant sequence similarity to the other Tyr(P)-recognizing module, the SH2 domain. We describe the thermodynamic parameters characterizing PI domain binding to various tyrosyl phosphopeptides, using isothermal titration calorimetry. The PI domain forms 1:1 complexes of similar affinity with a 12-mer peptide (ISLDNPDpYQQDF) derived from Tyr-1148 of the epidermal growth factor receptor (EGFR) (KD = 28 nm) and an 18-mer (LQGHIIENPQpYFSDACVH) derived from Tyr-490 of Trk (KD = 42 nM). Binding of the EGFR-derived peptide was largely enthalpy-driven at 25 degrees C, while Trk490 peptide binding was entropy-driven. Based on the change in heat capacity upon binding, approximately 700 A2 of nonpolar surface was estimated to be buried upon interaction. Alteration of the Asn or Pro to Ala in the NPXpY motif of the EGFR Tyr-1148 peptide increased the KD of PI domain interactions to 238 and 370 nM, respectively. Alteration of a Leu at position -5 (with respect to Tyr(P)) in the EGFR peptide to Gly also reduced the binding affinity (KD = 580 nM). It is proposed that the PI domain recognizes the beta1 turn that is found in NPXpY-containing peptides and also interacts with a larger segment of the peptide than seen for SH2 domains.

Significant discrepancies between van't Hoff and calorimetric enthalpies. II.

Isothermal calorimetric titration of 18-crown-6 ether with BaCl2 in pure aqueous solution over the temperature range 7-40 degrees C gives precise binding constants and enthalpy changes. Nonlinear least-squares fitting of the binding constants to the integrated van't Hoff equation, including a temperature-independent change in heat capacity, leads to van't Hoff enthalpies that differ significantly from the observed calorimetric enthalpies. This perplexing discrepancy appears at present to be very widely occurring.

Thermodynamics of the thermal unfolding of eglin c in the presence and absence of guanidinium chloride.

The thermal unfolding of eglin c, a small proteinase inhibitor of molecular weight 8.1 kDa, is studied by means of high sensitivity scanning calorimetry over a wide pH range in dilute buffer solutions, and in the presence of varying concentrations of guanidinium chloride at pH 7.00 and 10.55. The temperature of half-completion of the unfolding transition, t1/2, in dilute buffer varies from 41 degrees C at pH 1.1 to 86 degrees C at pH 7.0 to 10.55, with corresponding enthalpy changes of approximately 40 kcal mol-1 and 71 kcal mol-1. This latter enthalpy change, amounting to 8.7 cal g-1, is unusually large for a protein, especially for one of unusually small molecular weight. Addition of 3.3 M guanidinium chloride at pH 10.55 lowered t1/2 from 86 degrees C to 40 degrees C and decreased the enthalpy change from approximately 71 kcal mol-1 to 25 kcal mol-1.

Significant discrepancies between van't Hoff and calorimetric enthalpies.

In this paper we show that the usual assumption in studies of the temperature variation of equilibrium constants for equilibria of the form A+B <-->AB that a plot of ln K vs. 1/T (K = equilibrium constant, T = temperature in degrees kelvin) is a straight line with slope equal to -delta HvH/R (delta HvH = van't Hoff or apparent enthalpy, R = gas constant) is not valid in many cases. In all the cases considered here, delta HvH is temperature dependent and is significantly different from the true or calorimetrically measured enthalpy, and the respective values for delta Cp are also significantly different.

A thermodynamic study of mutant forms of Streptomyces subtilisin inhibitor. I. Hydrophobic replacements at the position of Met103.

The thermodynamic effects of replacing the Met residue at amino acid position 103 of Streptomyces subtilisin inhibitor with other non-polar aliphatic residues were studied by means of differential scanning calorimetry. All but the Leu mutant, which is as stable as the wild-type but has different cooperative units in the course of unfolding, showed destabilization in terms of free energy. Similar losses in free energy, however, were caused by different reasons, i.e. by increased entropy for the Ala mutant and by decreased enthalpy for the Ile mutant, with a tendency that increases in entropy are accompanied by increases in enthalpy. The gain in entropy that caused the largest loss in free energy for the Gly mutant was unexpectedly smaller than that for the Ala mutant. The changes in enthalpy and entropy induced by the mutations exhibited some correlations with hydrophobicity, while no clear correlation was found between the changes in free energy and hydrophobicity.