Role of the A chain in thrombin function.
Ontology highlight
ABSTRACT:
SUBMITTER: Papaconstantinou ME
PROVIDER: S-EPMC2667268 | biostudies-literature | 2008 Jun
REPOSITORIES: biostudies-literature
Publications
Role of the A chain in thrombin function.
Papaconstantinou M E ME Bah A A Di Cera E E
Cellular and molecular life sciences : CMLS 20080601 12
The A chain of thrombin is covalently linked to the catalytic B chain but is separate from any known epitope for substrate recognition. In this study we present the results of the Ala replacement of 12 charged residues controlling the stability of the A chain and its interaction with the B chain. Residues Arg4 and Glu8 play a significant role in substrate recognition, even though they are located > 20 A away from residues of the catalytic triad, the primary specificity pocket and the Na+ site. T ...[more]
Similar Datasets
Project description: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.
| S-EPMC3211113 | biostudies-literature
Project description:Thrombin plays an important role in the process of hemostasis and blood coagulation. Studies in thrombin can help us find ways to treat cancer because thrombin is able to reduce the characteristic hypercoagulability of cancer. Thrombin is composed of two chains, the light chain and the heavy chain. The function of the heavy chain has been largely explored, while the function of the light chain was obscured until several disease-associated mutations in the light chain come to light. In this study, we want to explore the dynamic and conformation effects of mutations on the light chain further to determine possible associations between mutation, conformational changes, and disease. The study, which is a follow-up for our studies on apo thrombin and the mutant, ΔK9, mainly focuses on the mutants E8K and R4A. E8K is a disease-associated mutation, and R4A is used to study the role of Arg4, which is suggested experimentally to play a critical role for thrombin's catalytic activities. We performed five all-atom one microsecond-scale molecular dynamics (MD) simulations for both E8K and R4A, and quantified the changes in the conformational ensemble of the mutants. From the root-mean-square fluctuations (RMSF) for the α-carbons, we find that the atomic fluctuations change in the mutants in the 60s loop and γ loop. The correlation coefficients for the α-carbons indicate that the correlation relation for atom-pairs in the protein is also impacted. The clustering analysis and the principal component analysis (PCA) consistently tell us that the catalytic pocket and the regulatory loops are destabilized by the mutations. We also find that there are two binding modes for Na+ by clustering the vector difference between the Na+ ions and the 220s loop. After further analysis, we find that there is a relation between the Na+ binding and the rigidification of the γ loop, which may shed light on the mysterious role of the γ loop in thrombin.
| S-EPMC8990444 | biostudies-literature
Project description:Thrombin and thrombin receptor activation impact cardiomyocyte contraction and ventricular remodeling. However, there is some controversy regarding their effects in cardiac function, especially in cardiac dysfunction after acute myocardial infarction (AMI). A rat AMI model was created by left coronary artery ligation (LCA). Cardiac functional parameters, including the maximum left ventricular (LV) systolic pressure (LVSPmax), LV end-diastolic pressure (LVEDP), and the rise and fall rates in LV pressure (dp/dt max and dp/dt min, respectively), were measured. Hirudin decreased cardiac function within 120 minutes after AMI, whereas treatment with thrombin receptor-activating peptide (TRAP) reversed this hirudin-induced decrease in cardiac function. The mRNA and protein expression levels of inositol 1,4,5-trisphosphate receptor (IP3R) subtypes in infarct area tissues were analyzed by reverse transcription-polymerase chain reaction and immunoreaction. Hirudin decreased the expression levels of IP3R-1, -2, and -3 in the infarct area for up to 40 minutes after AMI, whereas TRAP treatment reversed these hirudin-induced effects. Treatment with the IP3R antagonist 2-aminoethoxydiphenyl borate (2.5 mg/kg) eliminated the effect of TRAP on the hirudin-induced decrease in cardiac function after AMI. Finally, TRAP increased the maximum binding capacity of the three IP3R subtypes, but only enhanced the affinity of IP3R-2. Thrombin and thrombin receptor activation improved cardiac function after AMI by an IP3R-mediated pathway, probably through the IP3R-2 subtype.
| S-EPMC4455342 | biostudies-literature
Project description:IntroductionStudies of thrombin generation (TG) with platelet-rich plasma (PRP) and platelet-poor plasma (PPP) have provided insights on bleeding disorders. We studied TG for a cohort with commonly encountered platelet function disorders (PFD).MethodsParticipants included 40 controls and 31 with PFD due to: nonsyndromic dense granule (DG) deficiency (PFD-DGD, n = 9), RUNX1 haploinsufficiency (n = 6) and aggregation defects from other, uncharacterized causes (n = 16). TG was tested with PRP and PPP samples. As DG store ADP and polyphosphate that enhance platelet-dependent TG, PFD-DGD PRP TG was tested for correction with ADP, polyphosphate and combined additives. Tissue factor pathway inhibitor (TFPI), platelet factor V (FV), and platelet TFPI and ANO6 transcript levels were also evaluated. Findings were tested for associations with TG endpoints and bleeding.ResultsPFD samples had impaired PRP TG, but also impaired PPP TG, with strong associations between their PRP and PPP TG endpoints (P ≤ .005). PFD-DGD PRP TG endpoints showed associations to PPP TG endpoints but not to DG counts, and were improved, but not fully corrected, by adding polyphosphate and agonists. PFD participants had increased plasma TFPI and reduced platelet TFPI (P ≤ .02) but normal levels of platelet FV, and platelet TFPI and ANO6 transcripts levels. PFD plasma TFPI levels showed significant association to several PPP TG endpoints (P ≤ .04). Several PFD PRP TG endpoints showed significant associations to bleeding symptoms, including wound healing problems and prolonged bleeding from minor cuts (P ≤ .04).ConclusionTG is impaired in commonly encountered PFD, with their PRP TG findings showing interesting associations to symptoms.
| S-EPMC8599625 | biostudies-literature
Project description:Background and purposeThrombin and lipocalin-2 (LCN2) contribute to intracerebral hemorrhage-induced brain injury. Thrombin-induced brain damage is partially through a thrombin receptor, protease-activated receptor-1. LCN2 is involved in cellular iron transport and neuroinflammation. This study investigated the role of LCN2 in thrombin-induced brain injury.MethodsThere were 3 parts in this study. First, male adult C57BL/6 wild-type or LCN2 knockout (LCN2 KO) mice had an intracaudate injection of thrombin (0.4 U) or saline. Second, LCN2 KO mice had an injection of thrombin (0.4 U) with recombinant mouse LCN2 protein (1 μg) into the right caudate. Third, protease-activated receptor-1 KO or wild-type mice had an intracaudate injection of thrombin or saline. All mice had T2-weighted magnetic resonance imaging and behavioral tests. Brains were used for histology, immunohistochemistry, and Western blotting.ResultsIntracerebral thrombin injection caused LCN2 upregulation and brain injury in mice. Thrombin-induced brain swelling, blood-brain barrier disruption, neuronal death, and neurological deficits were markedly less in LCN2 KO mice (P<0.05) and were exacerbated by exogenous LCN2 coinjection. In addition, thrombin injection resulted in less LCN2 expression and brain injury in protease-activated receptor-1 KO mice.ConclusionsThrombin upregulates LCN2 through protease-activated receptor-1 activation and causes brain damage.
| S-EPMC4811682 | biostudies-literature
Project description:Thrombin is a key component for chemotherapeutic and antithrombotic therapy development. As the physiologic and pathologic roles of the light chain still remain vague, here, we continue previous efforts to understand the impacts of the disease-associated single deletion of LYS9 in the light chain. By combining supervised and unsupervised machine learning methodologies and more traditional structural analyses on data from 10 μs molecular dynamics simulations, we show that the conformational ensemble of the ΔK9 mutant is significantly perturbed. Our analyses consistently indicate that LYS9 deletion destabilizes both the catalytic cleft and regulatory functional regions and result in some conformational changes that occur in tens to hundreds of nanosecond scaled motions. We also reveal that the two forms of thrombin each prefer a distinct binding mode of a Na+ ion. We expand our understanding of previous experimental observations and shed light on the mechanisms of the LYS9 deletion associated bleeding disorder by providing consistent but more quantitative and detailed structural analyses than early studies in literature. With a novel application of supervised learning, i.e. the decision tree learning on the hydrogen bonding features in the wild-type and ΔK9 mutant forms of thrombin, we predict that seven pairs of critical hydrogen bonding interactions are significant for establishing distinct behaviors of wild-type thrombin and its ΔK9 mutant form. Our calculations indicate the LYS9 in the light chain has both localized and long-range allosteric effects on thrombin, supporting the opinion that light chain has an important role as an allosteric effector.
| S-EPMC6207482 | biostudies-literature
Project description:Diabetic neuropathy is common and disabling despite glycemic control. Novel neuroprotective approaches are needed. Thrombin and hypercoagulability are associated with diabetes and nerve conduction dysfunction. Our aim was to study the role of thrombin in diabetic neuropathy. We measured thrombin activity by a biochemical assay in streptozotocin (STZ)-induced diabetic neuropathy in male Sprague-Dawley rats. Neuropathy severity was assessed by thermal latency and nerve conduction measures. Thermal latencies were longer in diabetic rats, and improved with the non-specific serine-protease inhibitor Tosyl-L-lysine-chloromethyl ketone (TLCK) treatment (p<0.01). The tail nerve of diabetic rats showed slow conduction velocity (p˂0.01), and interestingly, increased thrombin activity was noted in the sciatic nerve (p˂0.001). Sciatic nodes of Ranvier and the thrombin receptor, protease activated receptor 1 (PAR1) reactivity showed abnormal morphology in diabetic animals by immunofluorescence staining (p<0.0001). Treatment of diabetic animals with either the specific thrombin inhibitor, N-alpha 2 naphtalenesulfonylglycyl alpha-4 amidino-phenylalaninepiperidide (NAPAP) or TLCK preserved normal conduction velocity, (p˂0.01 and p = 0.01 respectively), and prevented disruption of morphology (p˂0.05 and p˂0.03). The results establish for the first time an association between diabetic neuropathy and excessive activation of the thrombin pathway. Treatment of diabetic animals with thrombin inhibitors ameliorates both biochemical, structural and electrophysiological deficits. The thrombin pathway inhibition may be a novel neuroprotective therapeutic target in the diabetic neuropathy pathology.
| S-EPMC6611599 | biostudies-literature
Project description:The procofactor, factor VIII, is activated by thrombin or factor Xa-catalyzed cleavage at three P1 residues: Arg-372, Arg-740, and Arg-1689. The catalytic efficiency for thrombin cleavage at Arg-740 is greater than at either Arg-1689 or Arg-372 and influences reaction rates at these sites. Because cleavage at Arg-372 appears rate-limiting and dependent upon initial cleavage at Arg-740, we investigated whether cleavage at Arg-1689 influences catalysis at this step. Recombinant B-domainless factor VIII mutants, R1689H and R1689Q were prepared and stably expressed to slow and eliminate cleavage, respectively. Specific activity values for the His and Gln mutations were approximately 50 and approximately 10%, respectively, that of wild type. Thrombin activation of the R1689H variant showed an approximately 340-fold reduction in the rate of Arg-1689 cleavage, whereas the R1689Q variant was resistant to thrombin cleavage at this site. Examination of heavy chain cleavages showed approximately 4- and 11-fold reductions in A2 subunit generation and approximately 3- and 7-fold reductions in A1 subunit generation for the R1689H and R1689Q mutants, respectively. These results suggest a linkage between light chain cleavage and cleavages in heavy chain. Results obtained evaluating proteolysis of the factor VIII mutants by factor Xa revealed modest rate reductions (<5-fold) in generating A2 and A1 subunits and in cleaving light chain at Arg-1721 from either variant, suggesting little dependence upon prior cleavage at residue 1689 as compared with thrombin. Overall, these results are consistent with a competition between heavy and light chains for thrombin exosite binding and subsequent proteolysis with binding of the former chain preferred.
| S-EPMC2670113 | biostudies-literature
Project description:The expression of the elongated fibrinogen gamma chain, termed gamma', derives from alternative splicing of mRNA and causes an insertion sequence of 20 amino acids. This insertion domain interacts with the anion-binding exosite (ABE)-II of thrombin. This study investigated whether and how gamma' chain binding to ABE-II affects thrombin interaction with its platelet receptors, i.e. glycoprotein Ibalpha (GpIbalpha), protease-activated receptor (PAR) 1, and PAR4. Both synthetic gamma' peptide and fibrinogen fragment D*, containing the elongated gamma' chain, inhibited thrombin-induced platelet aggregation up to 70%, with IC(50) values of 42+/-3.5 and 0.47+/-0.03 microm, respectively. Solid-phase binding and spectrofluorimetric assays showed that both fragment D* and the synthetic gamma' peptide specifically bind to thrombin ABE-II and competitively inhibit the thrombin binding to GpIbalpha with a mean K(i) approximately 0.5 and approximately 35 microm, respectively. Both these gamma' chain-containing ligands allosterically inhibited thrombin cleavage of a synthetic PAR1 peptide, of native PAR1 molecules on intact platelets, and of the synthetic chromogenic peptide D-Phe-pipecolyl-Arg-p-nitroanilide. PAR4 cleavage was unaffected. In summary, fibrinogen gamma' chain binds with high affinity to thrombin and inhibits with combined mechanisms the platelet response to thrombin. Thus, its variations in vivo may affect the hemostatic balance in arterial circulation.
| S-EPMC2662079 | biostudies-literature
Project description:Angiopoietin-1 (Ang1) and Angiopoietin-2 (Ang2) are ligands for Tie2, an endothelial-specific receptor tyrosine kinase that is an essential regulator of angiogenesis. Here we report the identification, via expression cloning, of thrombomodulin (TM) as another receptor for Ang1 and Ang2. Thrombomodulin is an endothelial cell surface molecule that plays an essential role as a coagulation inhibitor via its function as a cofactor in the thrombin-mediated activation of protein C, an anticoagulant protein, as well as thrombin-activatable fibrinolysis inhibitor (TAFI). Ang1 and Ang2 inhibited the thrombin/TM-mediated generation of activated protein C and TAFI in cultured endothelial cells, and inhibited the binding of thrombin to TM in vitro. Ang2 appears to bind TM with higher affinity than Ang1 and is a more potent inhibitor of TM function. Consistent with a potential role for angiopoietins in coagulation, administration of thrombin to mice rapidly increased plasma Ang1 levels, presumably reflecting release from activated platelets (previously shown to contain high levels of Ang1). In addition, Ang1 levels were significantly elevated in plasma prepared from wound blood, suggesting that Ang1 is released from activated platelets at sites of vessel injury. Our results imply a previously undescribed role for angiopoietins in the regulation of hemostasis.
| S-EPMC5765006 | biostudies-literature