A mechanism for NaCl inhibition of Reactive Blue 19 decolorization and ABTS oxidation by laccase.

Laccases produced by white rot fungi have been extensively evaluated for their potential to decolorize textile wastewaters which contain salts like sodium chloride and sodium sulfate. The effect of sodium chloride and sodium sulfate on Trametes versicolor laccase during the decolorization of an anthraquinone dye (Reactive Blue 19) and the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were evaluated by steady-state kinetic analysis. The results showed that, while sodium sulfate did not affect laccase activity, sodium chloride inhibited both ABTS oxidation and dye decolorization. However, the type of inhibition was substrate-dependent: it was hyperbolic, noncompetitive with ABTS and parabolic, noncompetitive with Reactive Blue 19. Furthermore, the results suggested that two chlorides may bind to laccase in the presence of the dye unlike recent inhibition models which suggest that there is only one inhibition site. This investigation is the first to provide evidence for and to propose a two-site model of laccase inhibition, providing new insight into NaCl inhibition of laccase. The proposed model is also useful to predict decolorization rates in the presence of sodium chloride and to determine operating conditions that will minimize inhibition.

Thrombin activatable fibrinolysis inhibitor activation and bleeding in haemophilia A.

Individuals with haemophilia A exhibit bleeding tendencies that are not always predicted by their factor (F)VIII level. It has been suggested that bleeding in haemophilia is due not only to defective prothrombin activation but also aberrant fibrinolysis. Thrombin activatable fibrinolysis inhibitor (TAFI) activation was measured in tissue factor (TF)-initiated blood coagulation in blood samples of 28 haemophiliacs and five controls. Reactions were quenched over time with FPRck and citrate and assayed for TAFIa and thrombin-antithrombin (TAT). The TAFIa potential (TP), TAFI activation rate and the TAFIa level at 20 min (TAFIa(20 min)) was extracted from the TAFI activation progress curve. In general, the time course of TAFI activation follows thrombin generation regardless of FVIII activity and as expected the rate of TAFI activation and TP decreases as FVIII decreases. The magnitude of TP was similar among the control subjects and subjects with <11% FVIII. In severe subjects with <1% FVIII at the time of blood collection, the TAFIa(20 min) was inversely and significantly correlated with haemarthrosis (-0.77, P = 0.03) and total bleeds (-0.75, P = 0.03). In all cases, TAFIa(20 min) was more strongly correlated with bleeding than TAT levels at 20 min. Overall, this study shows that TAFI activation in whole blood can be quantified and related to the clinical bleeding phenotype. Measuring TAFIa along with thrombin generation can potentially be useful to evaluate the differential bleeding phenotype in haemophilia A.

Carboxypeptidase U (TAFIa): a new drug target for fibrinolytic therapy?

Procarboxypeptidase U (TAFI) is a recently discovered plasma procarboxypeptidase that upon activation by thrombin or thrombin-thrombomodulin turns into a potent antifibrinolytic enzyme. Its prominent bridging function between coagulation and fibrinolysis raised the interest of many research groups and of the pharmaceutical industry. The development of carboxypeptidase U (CPU) inhibitors as profibrinolytic agents is an attractive concept and possibilities for rational drug design will become more readily available in the near future as a result of the recently published crystal structure. Numerous studies have been performed and many of them show beneficial effects of CPU inhibitors for the improvement of endogenous fibrinolysis in different animal sepsis and thrombosis models. CPU inhibitors combined with tissue-type plasminogen activator (t-PA) seem to increase the efficiency of pharmacological thrombolysis allowing lower dosing of t-PA and subsequently fewer bleeding complications. This review will focus on recently obtained in vivo data and the benefits/risks of targeting CPU for the treatment of thrombotic disorders.

Soluble thrombomodulin partially corrects the premature lysis defect in FVIII-deficient plasma by stimulating the activation of thrombin activatable fibrinolysis inhibitor.

BACKGROUND: Previous work by others has shown that premature clot lysis occurs in plasmas deficient in components of the intrinsic pathway, due to a failure to activate thrombin activatable fibrinolysis inhibitor (TAFI). This suggests the hypothesis that bleeding in hemophilia is due not only to defective coagulation but also enhanced fibrinolysis. These studies were carried out to quantify the extent of TAFI activation over time in normal plasma (NP) and factor VIII deficient plasma (FVIII-DP) and to determine whether soluble thrombomodulin (sTM) can correct the lysis defect in FVIII-DP. METHODS: The time courses of TAFI activation in both NP and FVIII-DP were monitored after clotting with thrombin, PCPS and Ca(2+), +/- sTM. Clotting and lysis were measured turbidometrically and TAFIa using a functional assay. RESULTS: Premature lysis that occurs in FVIII-DP is corrected by mixing deficient plasma with 10% NP. However, this does not fully correct the defect in TAFI activation. FVIII-DP must be mixed with up to 50% NP to attain the same TAFIa potential as NP. In FVIII-DP, sTM can correct the defect in TAFIa-dependent prolongation of lysis at low tPA concentrations and partially correct this defect at high tPA concentrations. CONCLUSIONS: TAFI activation increases as the concentration of FVIII increases. FVIII at a level of 10% fully corrects the lysis defect in spite of the extent of TAFI activation being only one half that obtained with 100% FVIII. In addition, sTM increases TAFI activation sufficiently to correct the premature lysis defect in FVIII-DP.

A unique function for LRP-1: a component of a two-receptor system mediating specific endocytosis of plasma-derived factor V by megakaryocytes.

BACKGROUND: Factor V is endocytosed by megakaryocytes from plasma via a specific, receptor-mediated, clathrin-dependent mechanism to form the unique platelet-derived FV pool. OBJECTIVE: The role of low-density lipoprotein (LDL) receptor-related protein-1 (LRP-1), or a related family member, in FV endocytosis by megakaryocytes was examined because of its known interactions with other proteins involved in hemostasis. METHODS: LRP-1 expression by megakaryocytes and its functional role in FV endocytosis was confirmed using reverse transcription polymerase chain reaction (RT-PCR) and specific antibodies. FV binding to megakaryocytes was performed under Ca(2+)-free conditions to quantify binding in the absence of endocytosis. RESULTS AND CONCLUSION: Cell surface expression of LRP-1 by CD34+ ex vivo-derived megakaryocytes and the megakaryocyte-like cell line CMK was confirmed using anti-LRP-1 antibodies and was consistent with the detection of LRP-1 message in these cells. All cells capable of endocytosing FV expressed LRP-1. Anti-LRP-1 antibodies and receptor-associated protein (RAP), a known antagonist of LDL receptor family members, displaced only 50% of the [(125)I]FV bound to megakaryocytes. FV binding to megakaryocytes showed positive cooperativity (Hill coefficient = 1.92 +/- 0.18) that was substantially reduced in the presence of RAP (1.47 +/- 0.26). As FV endocytosis is specific to this cofactor, a model is hypothesized where FV binding to a specific receptor facilitates binding and endocytosis of a second FV molecule by LRP-1, or a related family member. These combined observations describe a unique role for LRP-1 in endocytosis of a coagulation protein trafficked to alpha-granules and not destined for lysosomal degradation.

The relative kinetics of clotting and lysis provide a biochemical rationale for the correlation between elevated fibrinogen and cardiovascular disease.

BACKGROUND: Elevated plasma fibrinogen is a well known risk factor for cardiovascular disease. The mechanistic rationale for this is not known. OBJECTIVES: These studies were carried out to determine the fibrinogen concentration dependencies of clotting and lysis times and thereby determine whether these times rationalize the correlation between an increased risk of cardiovascular disease and elevated plasma fibrinogen. METHODS: The time courses of clot formation and lysis were measured by turbidity in systems comprising a) fibrinogen, thrombin and plasmin, or b) fibrinogen, thrombin, plasminogen and t-PA, or c) plasma, thrombin and t-PA. From the lysis times, k(cat) and K(m) values for plasmin action on fibrin were determined. RESULTS: The time to clot increased linearly from 2.9 to 5.6 minutes as the fibrinogen concentration increased from 1 to 9 microM and did not increase further as the fibrinogen concentration was raised to 20 microM. In contrast, the clot lysis time increased linearly over the input fibrinogen concentration range of 2 to 20 microM. A similar linear trend was found in the two systems with t-PA and plasminogen. Apparent K(m) and k(cat) values for plasmin were 1.1 +/- 0.6 microM and 28 +/- 2 min(-1), respectively. K(m) values for plasmin in experiments initiated with t-PA and plasminogen were 1.6 +/- 0.2 microM in the purified system and 2.1 +/- 0.9 microM in plasma. CONCLUSION: As the concentration of fibrinogen increases, especially above physiologic level, the balance between fibrinolysis and clotting shifts toward the latter, providing a rationale for the increased risk of cardiovascular disease associated with elevated fibrinogen.

Role of mRNA transcript stability in modulation of expression of the gene encoding thrombin activable fibrinolysis inhibitor.

Regulation of mRNA stability has emerged as a major control point in eukaryotic gene expression. The abundance of a particular mRNA can be rapidly regulated in response to a stimulus by altering the stability of existing translatable transcripts rather than by altering the rate of transcription initiation. Alternative polyadenylation of transcripts during mRNA processing can be important in determining transcript abundance if the different forms of mRNA possess different stabilities or translatability. The mRNA transcript encoding thrombin activable fibrinolysis inhibitor (TAFI) is an attractive candidate for regulation of mRNA stability because of the relatively long length of its 3'-untranslated region and because the transcript can be polyadenylated at three different sites. As well, we have previously reported that treatment of HepG2 cells with interleukins (IL) - 1beta and - 6 destabilizes the endogenous TAFI mRNA expressed in this cell line. In the current study, we report that the TAFI 3'-untranslated region contains cis-acting instability element(s) and that these elements in fact determine the intrinsic stability of the TAFI transcript. Moreover, we found that the three different polyadenylated mRNA forms have different intrinsic stabilities, with the mRNA half-life increasing from the longest to the shortest transcript. Interestingly, treatment with IL-1beta plus IL-6 not only resulted in a 2-fold decrease in stability of the transcript produced using the 3'-most polyadenylation site but also resulted in profound shifts in the relative abundances of the respective polyadenylated forms through changes in the frequency of utilization of the three polyadenylation sites. As such, in the presence of IL-1beta and IL-6, the longest transcript is over a thousand times more abundant than the two shorter transcripts whereas in the absence of the stimulus it comprises only 1% of the total TAFI transcripts.

Temporal changes in factors associated with neutrophil elastase and coagulation in intensive care patients with a biphasic waveform and disseminated intravascular coagulation.

Summary. The biphasic waveform is an early marker of disseminated intravascular coagulation (DIC). Neutrophil elastase (NE) cleaves coagulation factors; thus, elevated elastase levels or its dysregulation by alpha-1-protease inhibitor (Alpha1PI) may be linked to DIC. Time courses over a period were determined for factors associated with NE and coagulation in 14 Intensive Care Unit patients with a biphasic waveform who developed DIC. The data were analyzed using a random coefficient linear regression model to predict the variables' mean values on day 0 and their mean rates of change over the period in which the biphasic waveform appeared. The biphasic waveform was normal on day 0, maximized on day 1, and approached normal again by day 4. Alpha1PI/NE complex levels were 2.5-fold greater than normal for the entire period. The A1PI activity, antigen, and specific activity levels were normal on day 0 and increased thereafter by 21.0, 10.5, and 8.9% of normal per day, respectively. Factor II, V, VII, IX, and X activity levels were, respectively, 57, 46, 46, 77, and 46% of normal on day 0, whereas factor VIII and fibrinogen levels were normal. All coagulation factor levels trended upward with time but not significantly. The prothrombin time, but not the activated partial thromboplastin time, was prolonged, and the platelet counts and hematocrits were below normal on day 0 and remained so thereafter. We conclude that events associated with neutrophil activation, elastase release, and perturbations of coagulation precede both the appearance of the biphasic waveform and the diagnosis of DIC in these patients.

Reversible inhibitors of TAFIa can both promote and inhibit fibrinolysis.

The plasma carboxypeptidase activated thrombin-activable fibrinolysis inhibitor (TAFIa), is thermally unstable at 37 degrees C, with a half-life of 8 or 15 min depending on the isoform. The arginine analog, 2-guanidinoethylmercaptosuccinate (GEMSA), not only inhibits TAFIa but also slows the spontaneous inactivation of the enzyme, thereby reducing the activity of TAFIa, while extending its apparent half-life. Because, as shown in previous work, the ability of TAFIa to prolong clot lysis can be more dependent on its half-life than its concentration, in this study we determined whether reversible inhibitors of TAFIa could paradoxically prolong clot lysis. Potato tuber carboxypeptidase inhibitor (PTCI) or GEMSA were titrated into normal pooled human plasma, in the presence of soluble thrombomodulin. Both inhibitors mediate a biphasic antifibrinolytic effect, prolonging clot lysis at lower concentrations and enhancing clot lysis at higher concentrations. The antifibrinolytic effect of GEMSA is maximized at 1 mmol L-1, increasing clot lysis time from 100 min to 350 min. The antifibrinolytic effect of PTCI is maximized at 100 nmol L-1, increasing clot lysis time from 100 min to 240 min. To further characterize the nature of this biphasic effect, TAFI at various concentrations was added to TAFI-immunodepleted human plasma in the presence of PTCI or GEMSA. The magnitude of the effect depends on the concentration of TAFIa, the concentration of inhibitor, and the potency of the inhibitor. We propose that the biphasic antifibrinolytic effect is mediated by the dynamic equilibrium of free TAFIa that inactivates quickly, and TAFIa bound to inhibitor that inactivates slowly. TAFIa inhibitors used as therapeutic agents might not only enhance lysis at higher concentrations, but also stabilize fibrin clots at intermediate concentrations.

Factor Xa is highly protected from antithrombin-fondaparinux and antithrombin-enoxaparin when incorporated into the prothrombinase complex.

Antithrombin and its cofactor, heparin, target both the product of prothrombin activation by prothrombinase, thrombin, as well as the enzyme responsible for the reaction, factor (F)Xa. These studies were carried out to quantify the effects of each of the prothrombinase components on the half-life of FXa in the presence of antithrombin and the low-molecular-weight heparins (enoxaparin, Aventis, Laval, Quebec, Canada) or the heparin pentasaccharide (fondaparinux, Organon Sanofi-Synthelabo, Cypress, TX, USA). Experiments were carried out using a recombinant form of prothrombin in which the active site serine has been mutated to cysteine and subsequently labeled with fluorescein. This mutant allowed calculation of the second order rate constant for inhibition of FXa by antithrombin in such a way that competition for antithrombin by thrombin is eliminated and competition for FXa by prothrombin is accounted for. Intrinsic rate constants for the inhibition of FXa by antithrombin-enoxaparin and antithrombin-fondaparinux, in the presence of the various prothrombinase components, were calculated. Addition of phospholipid had no significant effect on the second order rate constant for inhibition of FXa by antithrombin, while addition of FVa appeared to be mildly protective. Further addition of prothrombin however, caused profound protection of FXa, increasing its half-life from 1.1 to 353 s in the case of fondaparinux, and from 0.4 to 42 s in the case of enoxaparin. Similar results were reported for unfractionated heparin previously [1]. Therefore, in the presence of unfractionated heparin, fondaparinux, or enoxaparin, prothrombinase is profoundly protected from antithrombin.

Nutritional impact of intestinal helminthiasis during the human life cycle.

Poor people in developing countries endure the burden of disease caused by four common species of soil-transmitted nematode that inhabit the gastrointestinal tract. Disease accompanying these infections is manifested mainly as nutritional disturbance, with the differing infections having their deleterious effects at different phases during the human life cycle. Reduced food intake, impaired digestion, malabsorption, and poor growth rate are frequently observed in children suffering from ascariasis and trichuriasis. Poor iron status and iron deficiency anemia are the hallmarks of hookworm disease. The course and outcome of pregnancy, growth, and development during childhood and the extent of worker productivity are diminished during hookworm disease. Less is known about the impact of these infections in children under 2 years of age. The severity of disease caused by soil-transmitted nematodes has consistently been found to depend on the number of worms present per person. Cost-effective measures based on highly efficacious anthelminthic drugs are now available to reduce and control disease caused by these infections.

Myocardial infarction and the balance between fibrin deposition and removal.

When fibrin deposition and removal are properly balanced, the organism is protected from both a catastrophic loss of blood at the site of injury and the inappropriate loss of fluidity within the vascular system. When these activities are not properly balanced, however, severe bleeding or thromboses can occur. Myocardial infarction is a common and morbid consequence of the latter. The thrombin/thrombomodulin complex plays an essential role in regulating this balance because it generates both an anticoagulant substance, activated protein C, and an antifibrinolytic substance, activated TAFI (thrombin activatable fibrinolysis inhibitor, also known as plasma carboxypeptidase B or carboxypeptidase U). Thus, the coagulation and fibrinolytic cascades are explicitly linked by virtue of thrombin catalyzed activation of TAFI, either by the thrombin/thrombomodulin complex or, in the absence of thrombomodulin, by the massive amounts of thrombin generated through the factor XI-dependent pathway after clotting. Some potential targets for diagnosis, prognosis and therapy related to the balance between fibrin formation and removal include: development of a convenient global assay for plasma fibrinolytic potential; an assay for plasma or urine thrombomodulin that had been oxidized at methionine 388 and thereby has lost its capacity to stimulate activation of protein C but not TAFI; an assay for activated TAFI; discovery of a means for tapping the tremendous potential of the vasculature to acutely release tissue-type plasminogen activator; and an assessment of the potential role of polymorphisms in the TAFI gene which might influence TAFI levels or the properties TAFIa. In addition, a much fuller and quantitative understanding of the properties of the coagulation and tibrinolytic cascades is needed in order to optimize diagnosis, prognosis and therapy in disorders such as myocardial infarction that are related to the balance between fibrin formation and removal.

Modulation of fibrin cofactor activity in plasminogen activation.

Fibrin is a cofactor for the formation of plasmin from plasminogen as catalyzed by tissue plasminogen activator. Initial cleavages of fibrin by plasmin upregulates the cofactor activity of fibrin by exposing carboxyl terminal lysine residues. This effect is eliminated by a carboxypeptidase B-like enzyme generated from the precursor, thrombin activatable fibrinolysis inhibitor (TAFI) that is generated by thrombin during the formation of fibrin. Thus, TAFI and its activation to TAFIa create a link between the coagulation and fibrinolytic cascade, such that activation of the former suppresses the latter. Complete solubilization of fibrin results in a family of very large fibrin degradation products. These also have very substantial tissue plasminogen activator cofactor activity that is very highly downregulated by TAFIa.

Channeling during prothrombin activation.

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.