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L L Leung

Publications and source records attributed to L L Leung.

At least 19 recordsLinked to original sources
PubMed2001

An extensive interaction interface between thrombin and factor V is required for factor V activation.

The interaction interface between human thrombin and human factor V (FV), necessary for complex formation and cleavage to generate factor Va, was investigated using a site-directed mutagenesis strategy. Fifty-three recombinant thrombins, with a total of 78 solvent-exposed basic and polar residues substituted with alanine, were used in a two-stage clotting assay with human FV. Seventeen mutants with less than 50% of wild-type (WT) thrombin FV activation were identified and mapped to anion-binding exosite I (ABE-I), anion-binding exosite II (ABE-II), the Leu(45)-Asn(57) insertion loop, and the Na(+) binding loop of thrombin. Three ABE-I mutants (R68A, R70A, and Y71A) and the ABE-II mutant R98A had less than 30% of WT activity. The thrombin Na(+) binding loop mutants, E229A and R233A, and the Leu(45)-Asn(57) insertion loop mutant, W50A, had a major effect on FV activation with 5, 15, and 29% of WT activity, respectively. The K52A mutant, which maps to the S' specificity pocket, had 29% of WT activity. SDS-polyacrylamide gel electrophoresis analysis of cleavage reactions using the thrombin ABE mutants R68A, Y71A, and R98A, the Na(+) binding loop mutant E229A, and the Leu(45)-Asn(57) insertion loop mutant W50A showed a requirement for both ABEs and the Na(+)-bound form of thrombin for efficient cleavage at the FV residue Arg(709). Several basic residues in both ABEs have moderate decreases in FV activation (40-60% of WT activity), indicating a role for the positive electrostatic fields generated by both ABEs in enhancing complex formation with complementary negative electrostatic fields generated by FV. The data show that thrombin activation of FV requires an extensive interaction interface with thrombin. Both ABE-I and ABE-II and the S' subsite are required for optimal cleavage, and the Na(+)-bound form of thrombin is important for its procoagulant activity.

Amino Acid Substitution↗
PubMed2001

A thrombin receptor function for platelet glycoprotein Ib-IX unmasked by cleavage of glycoprotein V.

Glycoprotein (GP) V is a major substrate cleaved by the protease thrombin during thrombin-induced platelet activation. Previous analysis of platelets from GP V-null mice suggested a role for GP V as a negative modulator of platelet activation by thrombin. We now report the mechanism by which thrombin activates GP V -/- platelets. We show that proteolytically inactive forms of thrombin induce robust stimulatory responses in GP V null mouse platelets, via the platelet GP Ib--IX--V complex. Because proteolytically inactive thrombin can activate wild-type mouse and human platelets after treatment with thrombin to cleave GP V, this mechanism is involved in thrombin-induced platelet aggregation. Platelet activation through GP Ib-IX depends on ADP secretion, and specific inhibitors demonstrate that the recently cloned P2Y(12) ADP receptor (G(i)-coupled ADP receptor) is involved in this pathway, and that the P2Y(1) receptor (G(q)-coupled ADP receptor) may play a less significant role. Thrombosis was generated in GP V null mice only in response to catalytically inactive thrombin, whereas thrombosis occurred in both genotypes (wild type and GP V null) in response to active thrombin. These data support a thrombin receptor function for the platelet membrane GP Ib--IX--V complex, and describe a novel thrombin signaling mechanism involving an initiating proteolytic event followed by stimulation of the GP Ib--IX via thrombin acting as a ligand, resulting in platelet activation.

Animals↗
PubMed2001

Identification of critical residues on thrombin mediating its interaction with fibrin.

Thrombin binding to fibrin may be important in localizing thrombin to the site of vascular injury. However, fibrin-bound thrombin retains its catalytic activity toward fibrinogen, and may be prothrombotic under certain conditions. A collection of 52 purified thrombin mutants was used to identify those residues mediating the thrombin-fibrin interaction. Comparison of fibrinogen clotting activity with fibrin binding activity identified twenty residues involved in fibrinogen recognition with four of these residues important in fibrin binding (Lys65, His66, Tyr71, Arg73). No mutant was identified with normal clotting activity and deficient fibrin binding, suggesting that these two properties are not readily dissociable. A DNA thrombin aptamer that binds to these residues was able to inhibit the thrombin-fibrin interaction, and displace thrombin that was already bound. Mapping of these fibrin-binding residues on thrombin revealed that they are localized within exosite I, and comprise a subset of the residues important in fibrinogen recognition.

Allosteric Regulation↗
PubMed2000

Dissociation of thrombin's substrate interactions using site-directed mutagenesis.

Thrombin is an allosteric enzyme that interacts with multiple procoagulant substrates such as specific clotting factors and cell surface thrombin receptors, as well as the anticoagulant substrate protein C. Functional mapping of thrombin's interactions with its various substrates has been carried out using a collection of thrombin mutants generated by systematic alanine scanning mutagenesis. A thrombin mutant, E229K, has been identified that has essentially lost all of its procoagulant properties while retaining its ability to activate protein C, thus functioning as an anticoagulant in vitro and in vivo. It is also found that specific and distinct domains are involved in thrombin's interaction with thrombomodulin (TM) and the subsequent activation by the thrombin/TM complex of protein C and the thrombin-activatable fibrinolysis inhibitor (TAFI).

Animals↗
PubMed1999

Thrombin interacts with thrombomodulin, protein C, and thrombin-activatable fibrinolysis inhibitor via specific and distinct domains.

A collection of 56 purified thrombin mutants, in which 76 charged or polar surface residues on thrombin were mutated to alanine, was used to identify key residues mediating the interactions of thrombin with thrombomodulin (TM), protein C, and thrombin-activatable fibrinolysis inhibitor (TAFI). Comparison of protein C activation in the presence and absence of TM identified 11 residues mediating the thrombin-TM interaction (Lys(21), Gln(24), Arg(62), Lys(65), His(66), Arg(68), Thr(69), Tyr(71), Arg(73), Lys(77), Lys(106)). Three mutants (E25A, D51A, R89A/R93A/E94A) were found to have decreased ability to activate TAFI yet retained normal protein C activation, whereas three other mutants (R178A/R180A/D183A, E229A, R233A) had decreased ability to activate protein C but maintained normal TAFI activation. One mutant (W50A) displayed decreased activation of both substrates. Mapping of these functional residues on thrombin revealed that the 11 residues mediating the thrombin-TM interaction are all located in exosite I. Residues important in TAFI activation are located above the active-site cleft, whereas residues involved in protein C are located below the active-site cleft. In contrast to the extensive overlap of residues mediating TM binding and fibrinogen clotting, these data show that distinct domains in thrombin mediate its interactions with TM, protein C, and TAFI. These studies demonstrate that selective enzymatic properties of thrombin can be dissociated by site-directed mutagenesis.

Animals↗
PubMed1998

Senescent human neutrophil binding to thrombospondin (TSP): evidence for a TSP-independent pathway of phagocytosis by macrophages.

This study investigated the interaction of apoptotic polymorphonuclear neutrophils (PMN) with thrombospondin (TSP), an important event mediating the clearance of apoptotic neutrophils by macrophages. We developed an in vitro assay to examine this interaction. Based on this assay, we found that apoptotic but not fresh PMN bound specifically to surface-immobilized TSP (33 +/- 0.03 x 10(3) cells/well) compared to fibrinogen, fibronectin or laminin (8.0 +/- 0.3 x 10(3) cells/well). Moreover, the binding was specific for surface bound but not soluble TSP and appeared to be divalent cation dependent, was not significantly inhibited by heparin and was sensitive to cycloheximide (CHX) treatment of senescent PMN (>90%) inhibition at 10 microM CHX). In contrast to the binding studies, phagocytosis of senescent PMN by macrophages was not affected by EDTA or cycloheximide. Phosphatidyl-L-serine liposomes, phospho-L-serine, glucosamine, galactosamine, and the acetylated sugars had no effect on phagocytosis. We conclude that: (i) there was specific binding of senescent human PMN to immobilized TSP, which is divalent cation dependent and requires new protein synthesis in the PMN during senescence; (ii) in addition to the recently defined TSP-dependent pathway, there is a TSP-independent pathway mediating phagocytosis of senescent PMN by macrophages. The identity of this pathway remains to be defined.

Carbohydrates↗
PubMed1997

Strategies for development of novel antithrombotics: modulating thrombin's procoagulant and anticoagulant properties.

Thrombin is a serine proteinase that can interact with a large number of diverse macromolecular substrates, which results in either a procoagulant or anticoagulant effect. These divergent properties are physiologically regulated by the endogenous protein thrombomodulin. This review summarizes recent work on a variety of methods used to exploit the allosteric nature of the enzyme. The procoagulant and anticoagulant functions of thrombin can be modulated by sodium binding, site-directed mutagenesis, and a small synthetic molecule. Modulation of thrombin's intrinsic properties represents a novel approach to the development of unique antithrombotic agents.

Allosteric Regulation↗
PubMed1997

Modulation of thrombin's procoagulant and anticoagulant properties.

The procoagulant and anticoagulant functions of thrombin are controlled physiologically by allosteric changes induced by Na+ and vascular cell-surface TM. Key residues that mediate Na+ interaction with thrombin have been identified. Based on a site-directed mutagenesis approach, E229K thrombin is found to be the most optimal and potent PC activator with a marked shift in substrate specificity for PC over fibrinogen. E229K thrombin demonstrates significant anticoagulant and antithrombotic efficacy in animal models in vivo. Alternatively, a synthetic organic molecule (LY254603) has been discovered which interacts with thrombin and effectively modulates its functions in vitro. This new class of antithrombotic agents exploits the powerful natural PC anticoagulant pathway and may have a superior therapeutic profile than direct thrombin inhibitors.

Allosteric Regulation↗
PubMed1996

Protein engineering thrombin for optimal specificity and potency of anticoagulant activity in vivo.

Previous alanine scanning mutagenesis of thrombin revealed that substitution of residues W50, K52, E229, and R233 (W60d, K60f, E217, and R221 in chymotrypsinogen numbering) with alanine altered the substrate specificity of thrombin to favor the anticoagulant substrate protein C. Saturation mutagenesis, in which residues W50, K52, E229, and R233 were each substituted with all 19 naturally occurring amino acids, resulted in the identification of a single mutation, E229K, that shifted the substrate specificity of thrombin by 130-fold to favor the activation of the anticoagulant substrate protein C over the procoagulant substrate fibrinogen. E229K thrombin was also less effective in activating platelets (18-fold), was resistant to inhibition by antithrombin III (33-fold and 22-fold in the presence and absence of heparin), and displayed a prolonged half-life in plasma in vitro (26-fold). Thus E229K thrombin displayed an optimal phenotype to function as a potent and specific activator of endogenous protein C and as an anticoagulant in vivo. Upon infusion in Cynomolgus monkeys E229K thrombin caused an anticoagulant effect through the activation of endogenous protein C without coincidentally stimulating fibrinogen clotting and platelet activation as observed with wild-type thrombin. In addition, E229K thrombin displayed enhanced potency in vivo relative to the prototype protein C activator E229A thrombin. This enhanced potency may be attributable to decreased clearance by antithrombin III, the principal physiological inhibitor of thrombin.

Animals↗
PubMed1996

Identification of a domain (155-183) on CD36 implicated in the phagocytosis of apoptotic neutrophils.

Clearance of apoptotic neutrophils by macrophages is a crucial event following the resolution of acute inflammation. CD36, together with alphavbeta3, has been identified as one of the adhesion molecules on the surface of macrophages implicated in the clearance of polymorphonuclear leukocytes. The domain on CD36 implicated in the phagocytosis of aged neutrophils remains to be elucidated. In this study, COS cells transfected with human CD36 cDNA had a significantly higher capacity to phagocytose human apoptotic neutrophils compared with murine CD36 cDNA. Moreover, monoclonal antibodies 10/5 or OKM5 (epitopes identified on amino acids 155-183) but not monoclonal antibody 13/10 (epitope identified on amino acids 30-76) inhibited phagocytosis of apoptotic neutrophils by COS cells transfected by human CD36. Swapping the human CD36 155-183 domain from human to murine CD36 (human-murine CD36 chimera) imparted to murine CD36-transfected COS cells an increased capacity to phagocytose apoptotic neutrophils. Conversely, when the murine domain 155-183 was inserted in human CD36, a decreased phagocytic capacity was observed. In addition, a synthetic peptide(155-169) but not its scrambled form significantly inhibited phagocytosis. These results identify for the first time a functional domain encompassing amino acids 155-183 on human CD36 implicated in the recognition and phagocytosis of apoptotic neutrophils.

Animals↗
PubMed1995

A peptide derived from a tissue factor loop region functions as a tissue factor--factor VIIa antagonist.

Tissue factor (TF) is a transmembrane protein that functions in the initiation of blood coagulation in vivo. At sites of vascular injury, TF serves as a cell-surface receptor for the serine protease factor VIIa (FVIIa), forming an enzyme--cofactor complex and enhancing the catalytic activity of FVIIa. Tissue factor, along with the receptors for alpha- and gamma-interferons, is a member of the class 2 cytokine receptor superfamily. Crystallographic analysis demonstrated that the extracellular domain of TF consists of two immunoglobulin-like domains joined by a linker region. Each domain is comprised of two antiparallel beta-sheets containing seven conserved beta-strands separated by more variable loop regions. Extensive mutagenesis has been performed in order to map the FVIIa binding site on TF. Results indicated that the discontinuous binding site for FVIIa lies at the domain--domain interface and includes residues from extended loops and beta-strands within both the N- and C-terminal domains. Our previous study provided evidence that three consecutive residues (D44, W45, K46) within the TF loop region between beta-strands C and C' of the N-terminal domain were important for interactions with FVIIa. We have presently extended our alanine-scanning mutagenesis to include the residues within the flanking beta-strands. Thirteen sTF mutants were screened for their ability to enhance FVIIa activity. Three residues within strand C (Y34, Q37, I38) and two residues within C' (K48, Y51) were shown to be important for TF cofactor function.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗
PubMed1995

Selection of a suppressor mutation that restores affinity of an oligonucleotide inhibitor for thrombin using in vitro genetics.

The thrombin aptamer is a single-stranded DNA of 15 nucleotides that was identified by the selection of thrombin-binding molecules from a large combinatorial library of oligonucleotides. This prototype aptamer of thrombin has a unique double G-tetrad structure capable of inhibiting thrombin at nanomolar concentrations through binding to a specific region within thrombin exosite I. Substitution of arginine 70 in thrombin exosite I with glutamic acid effectively eliminated binding of the prototype thrombin aptamer. In contrast, aptamers selected against R70E thrombin were able to bind and inhibit both wild-type and R70E thrombins, and displayed potassium-independent inhibition. Aptamers selected against R70-E thrombin bound to sites identical or overlapping with that of the prototype thrombin aptamer. These aptamers retained the potential to form double G-tetrad structures; however, these structures would be destabilized by a T-->A substitution, disrupting the T4-T13 base pairing found in the prototype. This destabilization appeared to be partially compensated by newly recruited structural elements. Thus, selection against R70E thrombin did not lead to aptamers that bound to alternative sites, but instead to ssDNA structures with a suppressor mutation that accommodated the mutation in thrombin within a double G-tetrad context. These results provide insight into the aptamer-thrombin interaction and suggest that the binding site for the prototype is the dominant aptamorigenic site on thrombin.

Amino Acid Sequence↗
PubMed1995

Functional mapping of the surface residues of human thrombin.

Utilizing site-directed mutagenesis, 77 charged and polar residues that are highly exposed on the surface of human thrombin were systematically substituted with alanine. Functional assays using thrombin mutants identified residues that were required for the recognition and cleavage of the procoagulant substrate fibrinogen (Lys21, Trp50, Lys52, Asn53 + Thr55, Lys65, His66, Arg68, Tyr71, Arg73, Lys77, Lys106 + Lys107, Asp193 + Lys196, Glu202, Glu229, Arg233, Asp234) and the anticoagulant substrate protein C (Lys21, Trp50, Lys65, His66, Arg68, Tyr71, Arg73, Lys77, Lys106 + Lys107, Glu229, Arg233), interactions with the cofactor thrombomodulin (Gln24, Arg70) and inhibition by the thrombin aptamer, an oligonucleotide-based thrombin inhibitor (Lys65, His66, Arg70, Tyr71, Arg73). Although there is considerable overlap between the functional epitopes, distinct and specific residues with unique functions were identified. When the functional residues were mapped on the surface of thrombin, they were located on a single hemisphere of thrombin that included both the active site cleft and the highly basic exosite 1. No functional residues were located on the opposite face of thrombin. Residues with procoagulant or anticoagulant functions were not spatially separated but interdigitated with residues of opposite or shared function. Thus thrombin utilizes the same general surface for substrate recognition regardless of substrate function although the critical contact residues may vary.

Amino Acid Sequence↗
PubMed1995

Involvement of CD31 in lymphocyte-mediated immune responses: importance of the membrane-proximal immunoglobulin domain and identification of an inhibiting CD31 peptide.

CD31 (PECAM-1) is an immunoglobulin gene superfamily cell adhesion molecule found on vascular endothelium, platelets, and leukocytes. Lymphocyte expression of CD31 is most closely associated with the CD45RA+CD8+ naive T phenotype. CD31 has recently been shown to play a role in leukocyte egress to inflammatory sites. The mechanism of CD31 adhesion remains under investigation. Several investigators have reported evidence for a heterotypic ligand. We have previously shown that CD31 is phosphorylated with cell activation, which suggests a possible role for CD31 in cell activation events. We therefore studied the effects of CD31 antibodies on in vitro assays of lymphocyte activation. One CD31 antibody, LYP21, inhibited the mixed lymphocyte reaction (MLR) in a specific and dose-dependent fashion. An LYP21 epitope was localized to the sixth Ig domain of CD31. This peptide and a scrambled control peptide were synthesized and used to study effects of this epitope on lymphocyte activation. The CD31 peptide strongly inhibited the MLR. Because CD31 is expressed on both stimulator and responder populations, stimulator peripheral blood leukocytes and responder lymphocyte populations were separately incubated with CD31 peptide or control peptide and then washed before mixing. The CD31 peptide inhibited the MLR equally when either stimulator or responder cells were preincubated with the CD31 peptide. We further sorted responder cells into CD31-high and CD31-low populations and separately incubated these subsets with peptides. The CD31 peptide strongly inhibited MLRs, regardless of level of responder-cell CD31 expression. Examination of MLR reactions involving the CD31 peptide showed dispersed small aggregates of cells, rather than the single large aggregate observed in control MLRs. The CD31 peptide did not affect activation of lymphocytes by phorbol myristate acetate (PMA) and ionomycin. These results suggest that a surface CD31-ligand interaction may have a functional role in alloimmune lymphocyte activation and identify a functionally important domain of CD31.

Amino Acid Sequence↗
PubMed1995

Application of combinatorial libraries and protein engineering to the discovery of novel anti-thrombotic drugs.

Combinatorial libraries and protein engineering represent two new powerful tools in drug discovery and development. The application of a combinatorial ssDNA library to thrombin led to the discovery of a sequence-specific nucleotide-based thrombin inhibitor (thrombin aptamer). The thrombin aptamer has a novel tertiary structure revealed by NMR and shows potent rapid anticoagulation with a short half-life in vivo. It has been used successfully to replace heparin in a canine cardiopulmonary bypass model. Functional mapping of the surface residues of thrombin led to the generation of a modified thrombin with markedly diminished procoagulant properties while retaining its ability to activate protein C. This engineered thrombin functions as a protein C activator and demonstrates potent anticoagulation in vivo without prolongation of the bleeding time.

Angioplasty, Balloon↗