OBJECTIVE—The rapid-acting insulin analogs aspart and lispro have now been developed in biphasic formulations. This trial compared the postprandial serum glucose control of biphasic insulin aspart 30 (BIAsp 30: 30% aspart, 70% protaminated aspart) with that of biphasic insulin lispro 25 (Mix25: 25% lispro, 75% protaminated lispro) and biphasic human insulin 30 (BHI 30: 30% regular insulin, 70% NPH insulin) in insulin-treated subjects with type 2 diabetes. RESEARCH DESIGN AND METHODS—This was an open-labeled, randomized, single-dose, three-way crossover trial of 61 insulin-treated subjects with type 2 diabetes who had no significant late diabetic complications. BIAsp 30 and Mix25 were injected subcutaneously immediately before a test meal, and BHI 30 was injected 15 min before a test meal. The primary target of analysis was serum glucose excursion 0–5 h after a meal. RESULTS—The postprandial glycemic control with BIAsp 30, as assessed by the 5-h postmeal serum glucose excursion, was superior to that with both BHI 30 and Mix25 (16.6 ± 4.5 vs. 20.1 ± 4.9 and 18.9 ± 6.1 mmol/l per hour, respectively; P < 0.001 and P < 0.05). For BIAsp 30 versus BHI 30, this was supported by a reduced maximum glucose concentration [Cmax(SG)] (−5%; P < 0.05) occurring earlier (−13 min; P < 0.01). Furthermore, BIAsp 30 displayed a higher maximum serum insulin concentration (+101%; P < 0.001) occurring earlier (−55 min; P < 0.001) compared with BHI 30. Compared with Mix25, there was a shorter time to Cmax(SG) (−11 min; P < 0.05) after treatment with BIAsp 30. CONCLUSIONS—This trial demonstrates that BIAsp 30 improves postprandial glycemic control compared with both Mix25 and BHI 30 in subjects with type 2 diabetes.

Coagulation Factor VIIa (FVIIa) lacks the ability to spontaneously complete the conversion to a fully active enzyme after specific cleavage of an internal peptide bond (Arg152-Ile153) in the zymogen. Recently, several variants of FVIIa with enhanced intrinsic activity have been constructed. The in vitro characterization of these variants has shed light on molecular determinants that put restrictions on FVIIa in favour of a zymogen-like conformation and warrants continued efforts. Here we describe a new FVIIa variant with high intrinsic activity containing the mutations Leu305-->Val, Ser314-->Glu, Lys337-->Ala, and Phe374-->Tyr. The variant, called FVIIa(VEAY), processes a tripeptidyl substrate very efficiently because of an unprecedented, 5.5-fold lowering of the K(m) value. Together with a 4-fold higher substrate turnover rate this gives the variant a catalytic efficiency 22 times that of wild-type FVIIa, which is reflected in a considerably enhanced susceptibility to inhibition by antithrombin and other inhibitors. For instance, the affinity of FVIIa(VEAY) for the S1 probe and inhibitor p -aminobenzamidine is represented by an 8-fold lower K(i) value compared with that of FVIIa. Activation of Factor X in solution occurs about 10 times faster with FVIIa(VEAY) than with FVIIa, due virtually exclusively to an increased kcat value. The high activity of FVIIa(VEAY) is not accompanied by an increased burial of the N-terminus of the protease domain. A comparison of the kinetic parameters and molecular properties of FVIIa(VEAY) with those of the previously described mutant V158D/E296V/M298Q-FVIIa (FVIIa(IIa)), and the locations of the substitutions in the two variants, reveals what appear to be two profoundly different structural mechanisms dictating improvements in enzymic performance.

Coagulation factor VIIa belongs to a family of homologous enzymes, including factors IXa and Xa and activated protein C, composed of two epidermal growth factor-like domains located between an N-terminal domain rich in ␥-carboxyglutamic acid residues and a C-terminal serine protease domain. The first epidermal growth factor-like domain in factor VIIa contains a Ca 2؉ binding site, the function of which is largely unknown. Sitedirected mutagenesis of two Ca 2؉ -liganding Asp residues in this domain abolished Ca 2؉ binding and resulted in a 2-3-fold decrease in amidolytic activity at optimal Ca 2؉ concentrations. The lower amidolytic activity persisted in complex with soluble tissue factor, apparently due to a lower k cat of the mutant factor VIIa. Mutant and wild-type factor VIIa bound to lipidated tissue factor were equally efficient activators of factor X. The dissociation constants, derived from amidolytic activity and surface plasmon resonance measurements, were 2-5 nM and 50 -60 nM for the interactions between wild-type and mutant factor VIIa, respectively, and soluble tissue factor. Binding to lipidated tissue factor was characterized by dissociation constants of 7.5 pM for factor VIIa and 160 pM for the factor VIIa mutant. Hence, a functional Ca 2؉ binding site in the first epidermal growth factorlike domain added 7-8 kJ/mol to the total binding energy of the interaction with both lipidated and soluble tissue factor.

To cite this article: Persson E, Østergaard A. Mg 2+ binding to the Gla domain of factor X influences the interaction with tissue factor. J Thromb Haemost 2007; 5: 1977-8. Formation of the factor VIIa (FVIIa)-tissue factor (TF) complex triggers blood coagulation. FVIIa-TF activates FIX and FX, the products of these reactions playing important roles during the initiation and propagation phases [1,2] -induced effect in an attempt to explain it at the molecular level.We confirmed that, in the presence of 1.3 mM Ca 2+, the addition of 0.5 mM Mg 2+ gave approximately a 5-fold faster rate of FX activation by FVIIa-soluble TF (sTF) (Fig. 1A) [6]. The addition of various Mg 2+ concentrations (0.25-1.0 mM) showed that 0.5 mM sufficed to give an optimal rate of FX activation. Similarly, 1.3 mM Ca 2+ (tested at 0.4-1.9 mM) resulted in an optimal rate of activation in a system containing 0. -induced enhancement of FVIIa-TF amidolytic activity previously observed in its absence [7].To locate the Mg 2+ binding site(s) responsible for the enhanced FXa formation we carried out experiments using ccarboxyglutamic acid (Gla)-domainless (des(1-38)) FVIIa as the enzyme and/or Gla-domainless (des(1-44)) FX as the substrate. The activation of FX by des(1-38)-FVIIa bound to sTF was stimulated by Mg 2+ to the same extent as seen with full-length FVIIa (Fig. 1B). As with FVIIa, the activation of FX by des(1-38)-FVIIa alone (500 nM) did not depend on Mg 2+. In sharp contrast, the activation of des(1-44)-FX by FVIIa-sTF (Fig. 1C) or by des(1-38)-FVIIa-sTF (not shown) was insensitive to Mg 2+ , and so were the corresponding activations carried out in the absence of sTF. This strongly suggests that Mg 2+ binding to site(s) in the Gla domain of FX mediates the effect on FX activation. However, although the lack of data to support metal ion binding to TF makes it unlikely, we can not entirely rule out the possibility that the effect is mediated by Mg 2+ binding to sTF (or to sTF and FX).

In a double-blind prospective clinical study patients with at least two verified episodes of urolithiasis, but stone-free at the time of inclusion in the study, were subjected to prophylactic treatment with either bendroflumethiazide (2.5 mg) + 573 mg potassium chloride 3 times a day, or placebo. The material included a total of 22 patients, all males, aged 20-49 years. We found a clear tendency to reduced stone formation in the group treated with the active drug (P less than 0.01). Calcium excretion was reduced by 25-50% in 7 of 12 patients treated with thiazide.

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Forty patients with abnormal radiological findings after blunt renal trauma are presented. A conservative approach to treatment led to only 3 nephrectomies in patients with renal pedicle injury and to surgery in another 4 patients; in only one case was the function of the injured kidney reduced. At follow-up 1 to 5 years later, 4 patients had mild subjective symptoms but the objective findings were few and insignificant. However, since serious sequelae (e.g. hypertension) may follow renal trauma, we suggest that these patients should be seen one year after injury for clinical examination, including renography and measurement of blood pressure.

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