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P C Liaw

Publications and source records attributed to P C Liaw.

10 recordsLinked to original sources
PubMed2001

Molecular basis for the susceptibility of fibrin-bound thrombin to inactivation by heparin cofactor ii in the presence of dermatan sulfate but not heparin.

Although fibrin-bound thrombin is resistant to inactivation by heparin.antithrombin and heparin.heparin cofactor II complexes, indirect studies in plasma systems suggest that the dermatan sulfate.heparin cofactor II complex can inhibit fibrin-bound thrombin. Herein we demonstrate that fibrin monomer produces a 240-fold decrease in the heparin-catalyzed rate of thrombin inhibition by heparin cofactor II but reduces the dermatan sulfate-catalyzed rate only 3-fold. The protection of fibrin-bound thrombin from inhibition by heparin.heparin cofactor II reflects heparin-mediated bridging of thrombin to fibrin that results in the formation of a ternary heparin.thrombin.fibrin complex. This complex, formed as a result of three binary interactions (thrombin.fibrin, thrombin.heparin, and heparin.fibrin), limits accessibility of heparin-catalyzed inhibitors to thrombin and induces conformational changes at the active site of the enzyme. In contrast, dermatan sulfate binds to thrombin but does not bind to fibrin. Although a ternary dermatan sulfate. thrombin.fibrin complex forms, without dermatan sulfate-mediated bridging of thrombin to fibrin, only two binary interactions exist (thrombin.fibrin and thrombin. dermatan sulfate). Consequently, thrombin remains susceptible to inactivation by heparin cofactor II. This study explains why fibrin-bound thrombin is susceptible to inactivation by heparin cofactor II in the presence of dermatan sulfate but not heparin.

Binding Sites↗
PubMed2001

A 23bp insertion in the endothelial protein C receptor (EPCR) gene impairs EPCR function.

EPCR is a type I transmembrane protein, highly expressed on the endothelium of large vessels, that binds protein C and augments its activation. In this study, a 23bp insertion in the EPCR gene was found in 4/198 survivors of myocardial infarction and 3/194 patients with deep vein thrombosis. The EPCR gene with the insertion predicts a protein that lacks part of the extracellular domain, the transmembrane domain and the cytoplasmic tail. Expression studies showed that the truncated protein is not localized on the cell surface, cannot be secreted in the culture medium, and does not bind activated protein C. Since protein C activation depends on the concentration of EPCR, patients with the EPCR insertion could have a diminished protein C activation capacity. Further clinical studies of adequate samples size are necessary to establish whether or not the EPCR insertion predisposes to the development of thrombotic events.

Adult↗
PubMed2000

Identification of the protein C/activated protein C binding sites on the endothelial cell protein C receptor. Implications for a novel mode of ligand recognition by a major histocompatibility complex class 1-type receptor.

The endothelial cell protein C receptor (EPCR) is an endothelial cell-specific transmembrane protein that binds both protein C and activated protein C (APC). EPCR regulates the protein C anticoagulant pathway by binding protein C and augmenting protein C activation by the thrombin-thrombomodulin complex. EPCR is homologous to the MHC class 1/CD1 family, members of which contain two alpha-helices that sit upon an 8-stranded beta-sheet platform. In this study, we identified 10 residues that, when mutated to alanine, result in the loss of protein C/APC binding (Arg-81, Leu-82, Val-83, Glu-86, Arg-87, Phe-146, Tyr-154, Thr-157, Arg-158, and Glu-160). Glutamine substitutions at the four N-linked carbohydrate attachment sites of EPCR have little affect on APC binding, suggesting that the carbohydrate moieties of EPCR are not critical for ligand recognition. We then mapped the epitopes for four anti-human EPCR monoclonal antibodies (mAbs), two of which block EPCR/Fl-APC (APC labeled at the active site with fluorescein) interactions, whereas two do not. These epitopes were localized by generating human-mouse EPCR chimeric proteins, since the mAbs under investigation do not recognize mouse EPCR. We found that 5 of the 10 candidate residues for protein C/APC binding (Arg-81, Leu-82, Val-83, Glu-86, Arg-87) colocalize with the epitope for one of the blocking mAbs. Three-dimensional molecular modeling of EPCR indicates that the 10 protein C/APC binding candidate residues are clustered at the distal end of the two alpha-helical segments. Protein C activation studies on 293 cells that coexpress EPCR variants and thrombomodulin demonstrate that protein C binding to EPCR is necessary for the EPCR-dependent enhancement in protein activation by the thrombin-thrombomodulin complex. These studies indicate that EPCR has exploited the MHC class 1 fold for an alternative and possibly novel mode of ligand recognition. These studies are also the first to identify the protein C/APC binding region of EPCR and may provide useful information about molecular defects in EPCR that could contribute to cardiovascular disease susceptibility.

Amino Acid Sequence↗
PubMed2000

Mechanisms by which soluble endothelial cell protein C receptor modulates protein C and activated protein C function.

The endothelial cell protein C receptor (EPCR) functions as an important regulator of the protein C anticoagulant pathway by binding protein C and enhancing activation by the thrombin-thrombomodulin complex. EPCR binds to both protein C and activated protein C (APC) with high affinity. A soluble form of EPCR (sEPCR) circulates in plasma and inhibits APC anticoagulant activity. In this study, we investigate the mechanisms by which sEPCR modulates APC function. Soluble EPCR inhibited the inactivation of factor Va by APC only in the presence of phospholipid vesicles. By using flow cytometric analysis in the presence of 3 mM CaCl(2) and 0. 6 mM MgCl(2), sEPCR inhibited the binding of protein C and APC to phospholipid vesicles (K(i) = 40 +/- 7 and 33 +/- 4 nM, respectively). Without MgCl(2), the K(i) values increased approximately 4-fold. Double label flow cytometric analysis using fluorescein-APC and Texas Red-sEPCR indicated that the APC.sEPCR complex does not interact with phospholipid vesicles. By using surface plasmon resonance, we found that sEPCR also inhibited binding of protein C to phospholipid in a dose-dependent fashion (K(i) = 32 nM). To explore the possibility that sEPCR evokes structural changes in APC, fluorescence spectroscopy studies were performed to monitor sEPCR/Fl-APC interactions. sEPCR binds saturably to Fl-APC (K(d) = 27 +/- 13 nM) with a maximum decrease in Fl-APC fluorescence of 10.8 +/- 0.6%. sEPCR also stimulated the amidolytic activity of APC toward synthetic substrates. We conclude that sEPCR binding to APC blocks phospholipid interaction and alters the active site of APC.

Binding Sites↗
PubMed1999

Comparison of heparin- and dermatan sulfate-mediated catalysis of thrombin inactivation by heparin cofactor II.

Heparin and dermatan sulfate activate heparin cofactor II (HCII) comparably, presumably by liberating the amino terminus of HCII to bind to exosite I of thrombin. To explore this model of activation, we systematically substituted basic residues in the glycosaminoglycan-binding domain of HCII with neutral amino acids and measured the rates of thrombin inactivation by the mutants. Mutant D, with changes at Arg(184), Lys(185), Arg(189), Arg(192), Arg(193), demonstrated a approximately 130-fold increased rate of thrombin inactivation that was unaffected by the presence of glycosaminoglycans. The increased rate reflects displacement of the amino terminus of mutant D because (a) mutant D inactivates gamma-thrombin at a 65-fold slower rate than alpha-thrombin, (b) hirudin-(54-65) decreases the rate of thrombin inactivation, and (c) deletion of the amino terminus of mutant D reduces the rate of thrombin inactivation approximately 100-fold. We also examined the contribution of glycosaminoglycan-mediated bridging of thrombin to HCII to the inhibitory process. Whereas activation of HCII by heparin was chain-length dependent, stimulation by dermatan sulfate was not, suggesting that dermatan sulfate does not utilize a template mechanism to accelerate the inhibitory process. Fluorescence spectroscopy revealed that dermatan sulfate evokes greater conformational changes in HCII than heparin, suggesting that dermatan sulfate stimulates HCII by producing more effective displacement of the amino terminus.

Amino Acid Sequence↗
PubMed1998

Characterization of the stress-inducing effects of homocysteine.

The mechanism by which homocysteine causes endothelial cell (EC) injury and/or dysfunction is not fully understood. To examine the stress-inducing effects of homocysteine on ECs, mRNA differential display and cDNA microarrays were used to evaluate changes in gene expression in cultured human umbilical-vein endothelial cells (HUVEC) exposed to homocysteine. Here we show that homocysteine increases the expression of GRP78 and GADD153, stress-response genes induced by agents or conditions that adversely affect the function of the endoplasmic reticulum (ER). Induction of GRP78 was specific for homocysteine because other thiol-containing amino acids, heat shock or H2O2 did not appreciably increase GRP78 mRNA levels. Homocysteine failed to elicit an oxidative stress response in HUVEC because it had no effect on the expression of heat shock proteins (HSPs) including HSP70, nor did it activate heat shock transcription factor 1. Furthermore homocysteine blocked the H2O2-induced expression of HSP70. In support of our findings in vitro, steady-state mRNA levels of GRP78, but not HSP70, were elevated in the livers of cystathionine beta-synthase-deficient mice with hyperhomocysteinaemia. These studies indicate that the activation of stress response genes by homocysteine involves reductive stress leading to altered ER function and is in contrast with that of most other EC perturbants. The observation that homocysteine also decreases the expression of the antioxidant enzymes glutathione peroxidase and natural killer-enhancing factor B suggests that homocysteine could potentially enhance the cytotoxic effect of agents or conditions known to cause oxidative stress.

Animals↗
PubMed1998

Localization of the thrombin-binding domain on prothrombin fragment 2.

Co-crystallographic studies have shown that the interaction of human prothrombin fragment 2 (F2) with thrombin involves the formation of salt bridges between the kringle inner loop of F2 and anion-binding exosite II of thrombin. When F2 binds to thrombin, it has been shown to evoke conformational changes at the active site and at exosite I of the enzyme. Using plasma, recombinant, and synthetic F2 peptides (F2, rF2, and sF2, respectively) we have further localized the thrombin-binding domain on F2. F2, rF2-(1-116), rF2-(55-116), and sF2-(63-116), all of which contain the kringle inner loop (residues 64-93) and the acidic COOH-terminal connecting peptide (residues 94-116), bind to thrombin-agarose. In contrast, analogues of the kringle inner loop, sF2-(63-90), or the COOH-terminal connecting peptide, sF2-(92-116), do not bind. Thus, contrary to predictions from the crystal structure, the COOH-terminal connecting peptide as well as the kringle inner loop are involved in the interaction of F2 with thrombin. F2 and sF2-(63-116) bind saturably to fluorescently labeled active site-blocked thrombin with Kd values of 4.1 and 51.3 microM, respectively. The affinity of sF2-(63-116) for thrombin increases about 5-fold (Kd = 10 microM) when Val at position 78 is substituted with Glu. F2 and sF2-(63-116) bind to exosite II on thrombin because both reduce the heparin-catalyzed rate of thrombin inhibition by antithrombin approximately 4-fold. In contrast, only F2 slows the uncatalyzed rate of thrombin inactivation by antithrombin. Like F2, sF2-(63-116) induces allosteric changes in the active site and exosite I of thrombin because it alters the rates of thrombin-mediated hydrolysis of chromogenic substrates and displaces fluorescently labeled hirudin54-65 from active site-blocked thrombin, respectively. Both peptides also prolong the thrombin clotting time of fibrinogen in a concentration-dependent fashion, reflecting their effects on the active site and/or exosite I. These studies provide further insight into the regions of F2 that evoke functional changes in thrombin.

Amino Acid Sequence↗
PubMed1994

Defining the sequence specificity of the Saccharomyces cerevisiae DNA binding protein REB1p by selecting binding sites from random-sequence oligonucleotides.

We have used a random selection protocol to define the consensus and range of binding sites for the Saccharomyces cerevisiae REB1 protein. Thirty-five elements were sequenced which bound specifically to a GST-REB1p fusion protein coupled to glutathione-Sepharose under conditions in which more than 99.9% of the random sequences were not retained. Twenty-two of the elements contained the core sequence CGGGTRR, with all but one of the remaining elements containing only one deviation from the core. Of the core sequence, the only residues that were absolutely conserved were the three consecutive G residues. Statistical analysis of a nucleotide-use matrix suggested that the REB1p binding site also extends into flanking sequences with the optimal sequence for REB1p binding being GNGCCGGGGTAACNC. There was a positive correlation between the ability of the sites to bind in vitro and activate transcription in vivo; however, the presence of non-conformants suggests that the binding site may contribute more to transcriptional activation than simply allowing protein binding. Interestingly, one of the REB1p binding elements had a DNAse 1 footprint appreciably longer than other elements with similar affinity. Analysis of its sequence indicated the potential for a second REB1p binding site on the opposite strand. This suggests that two closely positioned low-affinity sites can function together as a highly active site. In addition, database searches with some of the randomly defined REB1p binding sites suggest that related elements are commonly found within 'TATA-less' promoters.

Base Sequence↗
PubMed1992

TATA-binding protein activates transcription when upstream of a GCN4-binding site in a novel yeast promoter.

In the gal-his3 hybrid promoter, his3-GG1, GCN4 stimulates transcription at the position normally occupied by a TATA element. This expression requires two elements within gal1-10 sequences, a REB1-binding site and a second element, Z, which resides 20 base pairs upstream of the GCN4-binding site. No obvious TATA element is present in this promoter. To characterize the function of Z, we replaced it with short random oligonucleotides and selected for expression in vivo. Fourteen elements were identified and classified into groups based upon sequence and phenotypic similarities. Group 1 elements contained functional TATA sequences that were essential for activity. TATA elements can thus function when positioned upstream of a GCN4-binding site. The Group 2 elements activated transcription poorly when used as conventional TATA elements; however, mutational analyses demonstrated that their activity required TATA-like sequences. These TATA-like sequences bound the yeast TATA-binding protein (TBP) poorly in vitro but function in vivo as TBP interaction sites based upon two criteria. First mutations that improved their TATA character correspondingly improved function and second their activity could be enhanced in the presence of an altered binding specificity mutant of TBP. Furthermore, the Group 2 elements enabled the identification of mutations outside of the TATA-like core that contribute to transcriptional activation without adversely affecting TBP binding. The finding that low affinity TBP-binding sites can be used at unconventional positions suggests that many "TATA-less" promoters contain a cryptic interaction site for TBP.

Alleles↗