Clinical implications of clotting screens.

INTRODUCTION: Coagulation screens are performed routinely in every hospital with the notion that an abnormal result will pick up low levels of coagulation factors and thus aid in determining patients' bleeding risk. METHODS: We analysed all the clotting factor assays performed for abnormal clotting screen in a tertiary hospital over a 1-year period. RESULTS: Isolated prolongation of prothrombin time (PT) is extremely rarely clinically significant, and there is no correlation with degree of prolongation and factor VII deficiency. One-third cases of isolated prolonged activated partial thromboplastin time (APTT) were clinically insignificant, while in the rest, a factor deficiency which may be deemed as a potential risk for bleeding was noted. Prolonged PT and APTT in combination were noted in 38 cases, but in 29 of these patients all the measured clotting factors and fibrinogen were in the normal range. CONCLUSION: Abnormal clotting screens may not always be associated with clinically significant decrease in coagulation factor.

Identification of catalytically important residues in the active site of Escherichia coli transaldolase.

The roles of invariant residues at the active site of transaldolase B from Escherichia coli have been probed by site-directed mutagenesis. The mutant enzymes D17A, N35A, E96A, T156A, and S176A were purified from a talB-deficient host and analyzed with respect to their 3D structure and kinetic behavior. X-ray analysis showed that side chain replacement did not induce unanticipated structural changes in the mutant enzymes. Three mutations, N35A, E96A, and T156A resulted mainly in an effect on apparent kcat, with little changes in apparent Km values for the substrates. Residues N35 and T156 are involved in the positioning of a catalytic water molecule at the active site and the side chain of E96 participates in concert with this water molecule in proton transfer during catalysis. Substitution of Ser176 by alanine resulted in a mutant enzyme with 2.5% residual activity. The apparent Km value for the donor substrate, fructose 6-phosphate, was increased nearly fivefold while the apparent Km value for the acceptor substrate, erythrose 4-phosphate remained unchanged, consistent with a function for S176 in the binding of the C1 hydroxyl group of the donor substrate. The mutant D17A showed a 300-fold decrease in kcat, and a fivefold increase in the apparent Km value for the acceptor substrate erythrose 4-phosphate, suggesting a role of this residue in carbon-carbon bond cleavage and stabilization of the carbanion/enamine intermediate.

The three-dimensional structure of human transaldolase.

The crystal structure of human transaldolase has been determined to 2.45 A resolution. The enzyme folds into an alpha/beta barrel structure and is thus similar in structure to other class I aldolases. Structure-based sequence alignment of available sequences of the transaldolase subfamily reveals that eight active site residues are invariant in the whole subfamily. Other invariant residues are mainly involved in the formation of the hydrophobic core of the enzyme. Noteworthy is a hydrophobic cluster consisting of five invariant residues. Human transaldolase has been implicated as an autoantigen in multiple sclerosis and four immunodominant peptide segments are located at the surface of the enzyme, accessible to autoantibodies.