The prothrombinase complex, composed of the proteinase, factor Xa, bound to factor Va on membranes, catalyzes thrombin formation by the specific and ordered proteolysis of prothrombin at Arg 323 -Ile 324 , followed by cleavage at Arg 274 -Thr 275 . We have used a fluorescent derivative of meizothrombin des fragment 1 (mIIa⌬F1) as a substrate analog to assess the mechanism of substrate recognition in the second half-reaction of bovine prothrombin activation. Cleavage of mIIa⌬F1 exhibits pseudo-first order kinetics regardless of the substrate concentration relative to K m . This phenomenon arises from competitive product inhibition by thrombin, which binds to prothrombinase with exactly the same affinity as mIIa⌬F1. As thrombin is known to bind to an exosite on prothrombinase, initial interactions at an exosite likely play a role in the enzyme-substrate interaction. Occupation of the active site of prothrombinase by a reversible inhibitor does not exclude the binding of mIIa⌬F1 to the enzyme. Specific recognition of mIIa⌬F1 is achieved through an initial bimolecular reaction with an enzymic exosite, followed by an active site docking step in an intramolecular reaction prior to bond cleavage. By alternate substrate studies, we have resolved the contributions of the individual binding steps to substrate affinity and catalysis. This pathway for substrate binding is identical to that previously determined with a substrate analog for the first half-reaction of prothrombin activation. We show that differences in the observed kinetic constants for the two cleavage reactions arise entirely from differences in the inferred equilibrium constant for the intramolecular binding step that permits elements surrounding the scissile bond to dock at the active site of prothrombinase. Therefore, substrate specificity is achieved by binding interactions with an enzymic exosite that tethers the protein substrate to prothrombinase and directs cleavage at two spatially distinct scissile bonds.Prothrombinase is an archetypal enzyme complex of blood coagulation (2). The enzyme complex assembles through well characterized, reversible, protein-protein and protein-membrane interactions between the serine protease, factor Xa, the cofactor, factor Va, and membranes in the presence of calcium ions (2-4). The resulting complex catalyzes the conversion of prothrombin to thrombin at a greatly enhanced rate, compared with the reaction rate catalyzed by factor Xa alone (2).Prothrombin is activated by proteolytic cleavage at two sites, Arg 274 -Thr 275 and Arg 323 -Ile 324 , which yields the fragment 1.2 activation peptide and thrombin 1 (5, 6). The reaction catalyzed by prothrombinase proceeds almost exclusively via the initial cleavage at Arg 323 -Ile 324 , yielding meizothrombin as an intermediate, followed by cleavage at Arg 274 -Thr 275 to yield the final products of the reaction (7,8). Single turnover kinetic studies indicate that the overall process is likely the sum of two consecutive enzyme-catalyzed reactions (8). Consequently, steady state...

The plasma zymogen prothrombin (II) is converted to the clotting enzyme thrombin (IIa) by two prothrombinase-catalyzed proteolytic cleavages. Thus, two intermediates, meizothrombin (mIIa) and prethrombin-2 (P2), are possible on the reaction pathway. Measurements of the time courses of II, mIIa, P2, and IIa suggested a channeling phenomenon, whereby a portion of the II is converted directly to IIa without free mIIa and P2 as obligatory intermediates. Evidence for this was that the maximum rate of IIa formation preceded the maximum in the level of either intermediate. In addition, analysis of the data according to a model that included two parallel pathways through mIIa and P2 indicated that about 40% of the II consumed did not yield free mIIa or P2. Further studies were carried out in which II was continuously infused in a reactor at a constant rate. Under these conditions II, mIIa, and P2 reached constant steady-state levels, and IIa was produced at a constant rate, equal to that of II infusion. During the steady state, traces of II, mIIa, and P2 were introduced as radiolabels. Time courses of isotope consumption were first order, thus allowing the rates of consumption of II, mIIa, and P2 to be calculated. Under these conditions the rate of II consumption equaled the rate of IIa formation. Rates of consumption of the free intermediates, however, were only 22 (mIIa) and 15% (P2), respectively, of the rate of thrombin formation. Thus, both the time course experiments and the steady-state experiments indicate that an appreciable fraction of II is channeled directly to IIa without proceeding through the free intermediates mIIa and P2.

Preterm neonates exposed to painful NICU procedures exhibit increased pain scores and alterations in oxygenation and heart rate. It is unclear whether these physiologic responses increase the risk of oxidative stress. Using a prospective study design, we examined the relationship between a tissue-damaging procedure (TDP, tape removal during discontinuation of an indwelling central arterial or venous catheter) and oxidative stress in 80 preterm neonates. Oxidative stress was quantified by measuring uric acid (UA) and malondialdehyde (MDA) concentration in plasma before and after neonates experienced a TDP (n=38) compared to those not experiencing any TDP (control group, n=42). Pain was measured before and during the TDP using the Premature Infant Pain Profile(PIPP). We found that pain scores were higher in the TDP group compared to the control group (median scores:11 and 5, respectively, P<0.001). UA significantly decreased over time in control neonates but remained stable in TDP neonates (132.76μM to 123.23μM vs.140.50μM to 138.9μM, P=0.002). MDA levels decreased over time in control neonates but increased in TDP neonates (2.07μM to 1.81μM vs. 2.07μM to 2.21μM, P=0.01). We found significant positive correlations between PIPP scores and MDA. Our data suggest a significant relationship between procedural pain and oxidative stress in preterm neonates.

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The Porphyromonas gingivalis VimA protein has multifunctional properties that can modulate several of its major virulence factors. To further characterize VimA, P. gingivalis FLL406 carrying an additional vimA gene and a vimA-defective mutant in a different P. gingivalis genetic background were evaluated. The vimA-defective mutant (FLL451) in the P. gingivalis ATCC 33277 genetic background showed a phenotype similar to that of the vimA-defective mutant (FLL92) in the P. gingivalis W83 genetic background. In contrast to the wild type, gingipain activity was increased in P. gingivalis FLL406, a vimA chimeric strain. P. gingivalis FLL451 had a five times higher biofilm-forming capacity than the parent strain. HeLa cells incubated with P. gingivalis FLL92 showed a decrease in invasion, in contrast to P. gingivalis FLL451 and FLL406, which showed increases of 30 and 40%, respectively. VimA mediated coenzyme A (CoA) transfer to isoleucine and reduced branched-chain amino acid metabolism. The lipid A content and associated proteins were altered in the vimA-defective mutants. The VimA chimera interacted with several proteins which were found to have an LXXTG motif, similar to the sorting motif of Gram-positive organisms. All the proteins had an N-terminal signal sequence with a putative sorting signal of L(P/T/S)X(T/N/D)G and two unique signatures of EXGXTX and HISXXGXG, in addition to a polar tail. Taken together, these observations further confirm the multifunctional role of VimA in modulating virulence possibly through its involvement in acetyl-CoA transfer and lipid A synthesis and possibly by protein sorting. Porphyromonas gingivalis, a Gram-negative anaerobic bacterium, is one of the main etiological agents of adult periodontitis. While several virulence factors, including fimbriae (28), hemagglutinin (17), capsule (4), and lipopolysaccharide (68), have been implicated in the pathogenicity of P. gingivalis, the strong proteolytic abilities of this organism are considered to be important for its survival and thus play a significant role in virulence (12, 63). The major proteases, called gingipains, consist of arginine-specific (Arg-gingipain [Rgp]) and lysine-specific (Lysgingipain [Kgp]) proteases that are both extracellular and cell membrane associated (32). The activation of these gingipains is associated with several genes, including vimA, vimE, and vimF, that modulate the posttranslational glycosylation of those proteins (44,(62)(63)(64). These genes are part of the 6. locus which has previously been shown to be important to the pathogenic potential of P. gingivalis (1,26,44,(62)(63)(64).We have demonstrated that the bcp and recA genes play the expected role in oxidative stress resistance and DNA repair, respectively (26). The association of these genes with the vim genes on the same transcriptional unit could be considered an important strategy for P. gingivalis to coordinate its oxidative stress and proteolytic activities. A response to oxidative stress will involve binding of oxygen and its toxic derivativ...

Objective: Due to physiological and metabolic immaturity, prematurely born infants are at increased risk because of maternal separation in many neonatal intensive care units (NICUs). The stress induced from maternal–infant separation can lead to well-documented short-term physiologic instability and potentially lifelong neurological, sociological, or psychological sequelae. Based on previous studies of kangaroo mother care (KMC) that demonstrated improvement in physiologic parameters, we examined the impact of KMC on physiologic measures of stress (abdominal temperature, heart rate, oxygen saturation, perfusion index, near-infrared spectrometry), oxidative stress, and energy utilization/conservation in preterm infants. Methods: In this randomized, stratified study of premature neonates, we compared the effects on urinary concentrations of biomarkers of energy utilization and oxidative stress of 1 hr of KMC versus incubator care on Day 3 of life in intervention-group babies ( n = 26) and control-group babies ( n = 25), respectively. On Day 4, both groups received 1 hr of KMC. Urinary samples were collected 3 hr before and 3 hr after intervention/incubator care on both days. Energy utilization was assessed by measures of adenosine triphosphate (ATP) degradation (i.e., hypoxanthine, xanthine, and uric acid). Oxidative stress was assessed using urinary allantoin. Mixed-models analysis was used to assess differences in purine/allantoin. Results: Mean allantoin levels over Days 3 and 4 were significantly lower in the KMC group than in the control group ( p = .026). Conclusions: Results provide preliminary evidence that KMC reduces neonatal oxidative stress processes and that urinary allantoin could serve as an effective noninvasive marker for future studies.

Objective To examine the effects of sucrose on pain and biochemical markers of adenosine trisphosphate(ATP) degradation and oxidative stress in preterm neonates experiencing a clinically required heel lance. Study design Preterm neonates that met study criteria (n=131) were randomized into three groups: (1) control; (2) heel lance treated with placebo and non-nutritive sucking (NNS); and (3) heel lance treated with sucrose and NNS. Plasma markers of ATP degradation (hypoxanthine, xanthine and uric acid) and oxidative stress (allantoin) were measured before and after the heel lance. Pain was measured using the Premature Infant Pain Profile (PIPP). Data were analyzed using repeated measures ANOVA and Spearman rho. Results We found significant increases in plasma hypoxanthine and uric acid over time in neonates who received sucrose. We also found a significant negative correlation between plasma allantoin concentration and PIPP in a subgroup of neonates who received sucrose. Conclusion A single dose of oral sucrose, given before heel lance, significantly increased ATP utilization and oxidative stress in premature neonates. Because neonates are given multiple doses of sucrose per day, randomized trials are needed to examine the effect of repeated sucrose administration on ATP degradation, oxidative stress and cell injury.

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