Thrombin A-Chain: Activation Remnant or Allosteric Effector?

Although prothrombin is one of the most widely studied enzymes in biology, the role of the thrombin A-chain has been neglected in comparison to the other domains. This paper summarizes the current data on the prothrombin catalytic domain A-chain region and the subsequent thrombin A-chain. Attention is given to biochemical characterization of naturally occurring prothrombin A-chain mutations and alanine scanning mutants in this region. While originally considered to be simply an activation remnant with little physiologic function, the thrombin A-chain is now thought to play a role as an allosteric effector in enzymatic reactions and may also be a structural scaffold to stabilize the protease domain

Granzyme B Cleaves Decorin, Biglycan and Soluble Betaglycan, Releasing Active Transforming Growth Factor-β1

Objective: Granzyme B (GrB) is a pro-apoptotic serine protease that contributes to immune-mediated target cell apoptosis. However, during inflammation, GrB accumulates in the extracellular space, retains its activity, and is capable of cleaving extracellular matrix (ECM) proteins. Recent studies have implicated a pathogenic extracellular role for GrB in cardiovascular disease, yet the pathophysiological consequences of extracellular GrB activity remain largely unknown. The objective of this study was to identify proteoglycan (PG) substrates of GrB and examine the ability of GrB to release PG-sequestered TGF-b1 into the extracellular milieu. Methods/Results: Three extracellular GrB PG substrates were identified; decorin, biglycan and betaglycan. As all of these PGs sequester active TGF-b1, cytokine release assays were conducted to establish if GrB-mediated PG cleavage induced TGF-b1 release. Our data confirmed that GrB liberated TGF-b1 from all three substrates as well as from endogenous ECM and this process was inhibited by the GrB inhibitor 3,4-dichloroisocoumarin. The released TGF-b1 retained its activity as indicated by the induction of SMAD-3 phosphorylation in human coronary artery smooth muscle cells. Conclusion: In addition to contributing to ECM degradation and the loss of tissue structural integrity in vivo, increase

Novel function of coagulation factor Xa : conversion into a clot-dissolving cofactor

PLASMIN-MEDIATED CONVERSION OF FXa INTO A CLOT-DISSOLVING COFACTOR. Factor Xa (FXa) is an essential blood clotting enzyme. A previously identified FXa derivative, Xa33/13, is generated by two distinct cleavages by the clot-dissolving (fibrinolytic) enzyme, plasmin. FXa is first converted to FXaβ by excision of a small C-terminal peptide and then proteolyzed at Lys330 in the autolysis loop to yield Xa33/13, which cannot participate in clotting. Instead, these cleavages confer novel fibrinolytic function to Xa33/13 as a tissue plasminogen activator (tPA) cofactor, thereby accelerating plasmin generation. To understand the importance of each cleavage and the role of individual residues in this functional conversion of FXa, five mutants were generated by mutation of basic residues to glutamine: Lys330 and four residues in the β-peptide region. Mutation at Lys330 prevented autolysis loop cleavage, and this mutant dissolved purified fibrin clots faster than plasma-derived FXa derivatives. Additionally, no basic residue within the β-peptide was uniquely targeted by plasmin and no single-point mutation in this region prevented subsequent autolysis loop cleavage. FX-DEFICIENT PATIENT. Factor X (FX) can be activated by two separate protein complexes, known as the initiating (extrinsic) and amplifying (intrinsic) tenases, which are assembled during coagulation. I describe a FX-deficient patient with a novel compound heterozygous mutation associated with differential clotting pathway function. Quantification of plasma FX antigen revealed 15 % of normal, which was consistent with extrinsic pathway activity. Intrinsic pathway activity was reduced to 5 % of normal, suggesting an activatable specific activity 3-fold lower than expected for this branch of the clotting pathway. DNA sequence analysis identified two heterozygous mutations: (1) a previously reported mutation that disrupts the splice site between exons I and II; (2) a novel mutation resulting in an Arg386Cys substitution in the protease domain. I propose that alternate disulfide bond formation and protein folding may reduce circulating FX antigen levels. Additionally, Arg386 may be involved in substrate recognition by the intrinsic tenase complex, providing a possible explanation for the differential effect on the two branches of the coagulation cascade. Recombinant FX mutant studies confirmed our findings in patient plasma and provided further support for these hypotheses.Medicine, Faculty ofPathology and Laboratory Medicine, Department ofGraduat

GrB-mediated PG cleavage is inhibited by DCI and cleavage site identification.

<p>GrB was incubated with decorin (a), biglycan (b) and betaglycan (c), +/− DCI and the solvent control DMSO, for 4 h and 24 h. Cleavage sites in biglycan and betaglycan were identified by N-terminal Edman degradation. * denotes full length protein, arrows indicate cleavage fragments, and cleavage sites are displayed on the right.</p

TGF-β1 released by GrB is active and induces SMAD-3 activation in HCASMCs.

<p>GrB+/−DCI was incubated on betaglycan/TGF-β1 complexes for 24 h. Supernatants (containing released TGF-β1) were added to HCASMC for 20 m and phosphorylated SMAD-2 and SMAD-3 levels were examined. TGF-β1 released by GrB is active and induces SMAD-3 signalling in HCASMCs (P<0.05). The result shown is representative of at least 5 experiments.</p

GrB-mediated cleavage of decorin, biglycan and betaglycan.

<p>Increasing concentrations of GrB (25, 50, 100 and 200 nM) were incubated with decorin (a), biglycan (b), and betaglycan (c) for 24 h at RT. * denotes full length protein, arrows indicate cleavage fragments and ∧ indicates GrB.</p

HPLC Analysis of Phenolics Compounds and Antioxidant Capacity of Leaves of Vitex megapotamica (Sprengel) Moldenke

Vitex megapotamica (Sprengel) Moldenke belongs to the Verbenaceae family and is popularly known as “tarumã”. The antioxidant capacity of fractions and crude extract from the leaves of V. megapotamica were determined in this study through the capacity to remove reactive species and phenolic compounds were quantified in the various fractions. The IC50 (DPPH) ranged from 14.17 ± 0.76 to 37.63 ± 0.98 µg/mL. The ethyl acetate fraction might contain the strongest lipid peroxidation inhibitory compounds with an IC50 of 16.36 ± 5.09 µg/mL, being also the one with the highest content of polyphenols (522.4 ± 1.12 mg/g), flavonoids (220.48 ± 0.30 mg/g) and condensed tannins (3.86 ± 0.53 mg/g). Compounds quantified by HPLC/DAD in the crude extract and fractions were chlorogenic and rosmarinic acids. Higher dosages of the extracts were more effective in reducing levels of plasma protein carbonyls and were also shown to be able to remove reactive species by a 2&#039;,7&#039;-dichlorofluorescein diacetate assay, reducing oxidative stress in all tested fractions. Results obtained indicated that V. megapotamica exhibits good potential to prevent diseases caused by the overproduction of free radicals and it might also be used as a potential source of natural antioxidant agents