Bio

Clinical Focus


  • Cancer > Hematology
  • Hematology

Academic Appointments


Administrative Appointments


  • Senior Associate Dean for Veterans Affairs, School of Medicine (2006 - Present)
  • Chief of Staff, VA Palo Alto Health Care System (2006 - Present)

Boards, Advisory Committees, Professional Organizations


  • Editor-in-Chief, Hematology, UpToDate (2008 - Present)
  • Member, Association of American Physicians (2004 - Present)
  • Member, American Society for Clinical Investigation (1987 - Present)

Professional Education


  • Fellowship:New York Hospital (1981) NY
  • Internship:New York Hospital (1976) NY
  • Medical Education:Columbia University (1975) NY
  • Residency:New York Hospital (1978) NY
  • Board Certification: Hematology, American Board of Internal Medicine (1980)
  • Board Certification: Internal Medicine, American Board of Internal Medicine (1978)
  • Board Certification: Medical Oncology, American Board of Internal Medicine (1981)

Research & Scholarship

Current Research and Scholarly Interests


Our long-term interest is to have a better understanding of the natural antithrombotic pathways and the pathophysiology of vascular thrombosis. We have focused on thrombin, the key enzyme in the blood clotting cascade. Using a library of thrombin mutants generated by site-directed mutagenesis, we have mapped the critical functional sites on thrombin for its interactions with its diverse substrates. We are studying a thrombin-activatable carboxypeptidase that plays a key role in the physiologic regulation of thrombin’'s pro-thrombotic and pro-inflammatory properties. Our goal is to develop new antithrombotic agents and devise new diagnostic tests for vascular thrombotic disorders.

Teaching

2018-19 Courses


Publications

All Publications


  • Decoding the Genomics of Abdominal Aortic Aneurysm. Cell Li, J., Pan, C., Zhang, S., Spin, J. M., Deng, A., Leung, L. L., Dalman, R. L., Tsao, P. S., Snyder, M. 2018; 174 (6): 1361

    Abstract

    A key aspect of genomic medicine is to make individualized clinical decisions from personal genomes. We developed a machine-learning framework to integrate personal genomes and electronic health record (EHR) data and used this framework to study abdominal aortic aneurysm (AAA), a prevalent irreversible cardiovascular disease with unclear etiology. Performing whole-genome sequencing on AAA patients and controls, we demonstrated its predictive precision solely from personal genomes. By modeling personal genomes with EHRs, this framework quantitatively assessed the effectiveness of adjusting personal lifestyles given personal genome baselines, demonstrating its utility as a personal health management tool. We showed that this new framework agnostically identified genetic components involved in AAA, which were subsequently validated in human aortic tissues and in murine models. Our study presents a new framework for disease genome analysis, which can be used for both health management and understanding the biological architecture of complex diseases. VIDEO ABSTRACT.

    View details for DOI 10.1016/j.cell.2018.07.021

    View details for PubMedID 30193110

  • Chemerin 156F, generated by chymase cleavage of prochemerin, is elevated in joint fluids of arthritis patients. Arthritis research & therapy Zhao, L., Yamaguchi, Y., Ge, X., Robinson, W. H., Morser, J., Leung, L. L. 2018; 20 (1): 132

    Abstract

    BACKGROUND: Chemerin is a chemoattractant involved in immunity that also functions as an adipokine. Chemerin is secreted as an inactive precursor (chem163S), and its activation requires proteolytic cleavages at its C-terminus, involving proteases in coagulation, fibrinolysis, and inflammation. Previously, we found chem158K was the dominant chemerin form in synovial fluids from patients with arthritis. In this study, we aimed to characterize a distinct cleaved chemerin form, chem156F, in osteoarthritis (OA) and rheumatoid arthritis (RA).METHODS: Purified chem156F was produced in transfected CHO cells. To quantify chem156F in OA and RA samples, we developed a specific ELISA for chem156F using antibody raised against a peptide representing the C-terminus of chem156F.RESULTS: Ca2+ mobilization assays showed that the EC50 values for chem163S, chem156F, and chem157S were 252±141nM, 133±41.5nM, and 5.83±2.48nM, respectively. chem156F was more active than its precursor, chem163S, but very much less potent than chem157S, the most active chemerin form. Chymase was shown to be capable of cleaving chem163S at a relevant rate. Using the chem156F ELISA we found a substantial amount of chem156F present in synovial fluids from patients with OA and RA, 24.06±5.51ng/ml and 20.35±5.19ng/ml (mean±SEM, n=25) respectively, representing 20% of total chemerin in OA and 76.7% of chemerin in RA synovial fluids.CONCLUSIONS: Our data show that chymase cleavage of chem163S to partially active chem156F can be found in synovial fluids where it can play a role in modulation of the inflammation in joints.

    View details for DOI 10.1186/s13075-018-1615-y

    View details for PubMedID 29973268

  • Men and Women Differ in the Biochemical Composition of Platelet-Rich Plasma AMERICAN JOURNAL OF SPORTS MEDICINE Xiong, G., Lingampalli, N., Koltsov, J. B., Leung, L. L., Bhutani, N., Robinson, W. H., Chu, C. R. 2018; 46 (2): 409–19

    Abstract

    Autologous platelet-rich plasma (PRP) is widely used for a variety of clinical applications. However, clinical outcome studies have not consistently shown positive effects. The composition of PRP differs based on many factors. An improved understanding of factors influencing the composition of PRP is important for the optimization of PRP use.Age and sex influence the PRP composition in healthy patients.Controlled laboratory study.Blood from 39 healthy patients was collected at a standardized time and processed into leukocyte-poor PRP within 1 hour of collection using the same laboratory centrifuge protocol and frozen for later analysis. Eleven female and 10 male patients were "young" (aged 18-30 years), while 8 male and 10 female patients were "older" (aged 45-60 years). Thawed PRP samples were assessed for cytokine and growth factor levels using a multiplex assay and enzyme-linked immunosorbent assay. The platelet count and high-sensitivity C-reactive protein levels were measured. Two-way analysis of variance determined age- and sex-based differences.Platelet and high-sensitivity C-reactive protein concentrations were similar in PRP between the groups ( P = .234). Male patients had higher cytokine and growth factor levels in PRP compared with female patients for inflammatory cytokines such as interleukin-1 beta (IL-1β) (9.83 vs 7.71 pg/mL, respectively; P = .008) and tumor necrosis factor-alpha (TNF-α) (131.6 vs 110.5 pg/mL, respectively; P = .048); the anti-inflammatory IL-1 receptor antagonist protein (IRAP) (298.0 vs 218.0 pg/mL, respectively; P < .001); and growth factors such as fibroblast growth factor-basic (FGF-basic) (237.9 vs 194.0 pg/mL, respectively; P = .01), platelet-derived growth factor (PDGF-BB) (3296.2 vs 2579.3 pg/mL, respectively; P = .087), and transforming growth factor-beta 1 (TGF-β1) (118.8 vs 92.8 ng/mL, respectively; P = .002). Age- but not sex-related differences were observed for insulin-like growth factor-1 (IGF-1) ( P < .001). Age and sex interaction terms were not significant. While mean differences were significant, there was also substantial intragroup variability.This study in healthy patients shows differences in the composition of PRP between men and women, with sex being a greater factor than age. There was also proteomic variability within the groups. These data support a personalized approach to PRP treatment and highlight the need for a greater understanding of the relationships between proteomic factors in PRP and clinical outcomes.Variability in the proteomic profile of PRP may affect tissue and clinical responses to treatment. These data suggest that clinical studies should account for the composition of PRP used.

    View details for DOI 10.1177/0363546517740845

    View details for Web of Science ID 000423807500021

    View details for PubMedID 29211968

  • Prochemerin cleavage by factor XIa links coagulation and inflammation BLOOD Ge, X., Yamaguchi, Y., Zhao, L., Bury, L., Gresele, P., Berube, C., Leung, L. L., Morser, J. 2018; 131 (3): 353–64

    Abstract

    Chemerin is a chemoattractant and adipokine that circulates in blood as inactive prochemerin (chem163S). Chem163S is activated by a series of C-terminal proteolytic cleavages resulting in diverse chemerin forms with different levels of activity. We screened a panel of proteases in the coagulation, fibrinolytic, and inflammatory cascades to identify those that process prochemerin in plasma. Factor XIa (FXIa) cleaved chem163S, generating a novel chemerin form, chem162R, as an intermediate product, and chem158K, as the final product. Processing at Arg162 was not required for cleavage at Lys158 or regulation of chemerin bioactivity. Contact phase activation of human platelet-poor plasma by kaolin led to cleavage of chem163S, which was undetectable in FXI-depleted plasma and markedly enhanced in platelet-rich plasma (PRP). Contact phase activation by polyphosphate in PRP resulted in 75% cleavage of chem163S. This cleavage was partially inhibited by hirudin, which blocks thrombin activation of FXI. After activation of plasma, levels of the most potent form of chemerin, chem157S, as well as inactive chem155A, increased. Plasma levels of chem163S in FXI-deficient patients were significantly higher compared with a matched control group (91 ± 10 ng/mL vs 58 ± 3 ng/mL, n = 8; P < .01) and inversely correlated with the plasma FXI levels. Thus FXIa, generated on contact phase activation, cleaves chem163S to generate chem158K, which can be further processed to the most active chemerin form, providing a molecular link between coagulation and inflammation.

    View details for DOI 10.1182/blood-2017-07-792580

    View details for Web of Science ID 000423449900012

    View details for PubMedID 29158361

    View details for PubMedCentralID PMC5774209

  • Dynamic and tissue-specific proteolytic processing of chemerin in obese mice. PloS one Zhao, L., Yamaguchi, Y., Shen, W., Morser, J., Leung, L. L. 2018; 13 (8): e0202780

    Abstract

    Chemerin is a chemoattractant involved in immunity as well as an adipokine, whose activity is regulated by successive proteolytic cleavages at its C-terminus. Chemerin's C-terminal sequence and its proteolytic cleavage sites are highly conserved between human and mouse, as well as in other species. We produced, purified and characterized different mouse chemerin forms. Ca2+ mobilization assay showed that the EC50 values for mchem161T and mchem157R were 135.8 ± 158 nM and 71.2 ± 55.4 nM, respectively, whereas mchem156S and mchem155F had a 20-fold higher potency with an EC50 of 4.6 ± 1.8 nM and 3.6 ± 3.0 nM, respectively, likely representing the two physiologically active forms of chemerin. No agonist activity was found for mchem154A. Similar results were obtained in a chemotaxis assay. To identify and quantify the in vivo mouse chemerin forms in biological samples, we developed specific ELISAs for mchem162K, mchem157R, mchem156S, mchem155F and mchem154A, using antibodies raised against peptides from the C-terminus of the different mouse chemerin forms. The prochemerin form, mchem162K, was the major chemerin form in plasma with its increase matching the increase of total plasma chemerin in obese mice. During the onset of obesity in high-fat diet fed mice, mchem156S was elevated in plasma. In contrast, mchem155F was the dominant form in epididymal fat extracts. Our study provides the first direct evidence that mouse chemerin undergoes extensive, dynamic and tissue-specific proteolytic processing in vivo, similar to human chemerin, underlining the importance of measuring individual chemerin forms in studies of chemerin biology in mouse models.

    View details for DOI 10.1371/journal.pone.0202780

    View details for PubMedID 30161155

  • Chemerin Activation in Human Obesity OBESITY Chang, S., Eisenberg, D., Zhao, L., Adams, C., Leib, R., Morser, J., Leung, L. 2016; 24 (7): 1522-1529

    Abstract

    Chemerin is an inflammatory adipokine, whose activity is regulated by successive proteolytic cleavages at its C-terminus. It is secreted as an inactive precursor (chem163S); cleavage at Lys158 converts it to chem158K with modest activity. Chem157S is the most potent form and chem155A is inactive. The aim of this study was to determine if chemerin was activated in samples from patients with obesity.Using specific ELISAs for different chemerin forms and a pan-chemerin ELISA, chemerin forms in human obesity were characterized.Plasma chemerin from patients with obesity (BMI 44.3 ± 1.3 kg/m(2) , n = 29) was significantly higher than in lean controls (BMI 20.9 ± 0.7 kg/m(2) , n = 10) (160 ± 11 vs. 76.2 ± 5.5 ng/mL, respectively, P < 0.0001). This increase in chemerin was due to increased previously unattributed chemerin, with further C-terminal truncation demonstrated by mass spectrometry, accounting for ∼35% of total plasma chemerin. Chemerin forms in adipose tissue showed a different profile, with minimal chem163S and significant levels of chem157S. Chem155A was present in omental but not in subcutaneous adipose tissue. Unattributed chemerin forms were undetectable in adipose tissue.Chemerin is activated in adipose tissue of subjects with obesity, and further C-terminal processing occurs during the disposition of chemerin from adipose tissue, resulting in substantial levels of novel degraded forms in plasma that correlate with obesity.

    View details for DOI 10.1002/oby.21534

    View details for Web of Science ID 000379303200017

    View details for PubMedID 27222113

  • Plasmin as a complement C5 convertase. EBioMedicine Leung, L. L., Morser, J. 2016; 5: 20-21

    View details for DOI 10.1016/j.ebiom.2016.03.015

    View details for PubMedID 27077104

    View details for PubMedCentralID PMC4816833

  • Thrombin Cleavage of Osteopontin Disrupts a Pro-chemotactic Sequence for Dendritic Cells, Which Is Compensated by the Release of Its Pro-chemotactic C-terminal Fragment JOURNAL OF BIOLOGICAL CHEMISTRY Shao, Z., Morser, J., Leung, L. L. 2014; 289 (39): 27146-27158
  • Open aortic valve replacement in a patient with Glanzmann's thrombasthenia: a multidisciplinary strategy to minimize perioperative bleeding. Transfusion Sheikh, A. Y., Hill, C. C., Goodnough, L. T., Leung, L. L., Fischbein, M. P. 2014; 54 (2): 300-305

    Abstract

    BACKGROUND: Glanzmann thrombasthenia (GT) is an autosomal recessive disorder in which the platelet (PLT) glycoprotein IIb/IIIa complex is either deficient or dysfunctional. In its most severe form, GT may result in spontaneous bleeding, although most cases are first detected in the setting of an invasive procedure. CASE REPORT: A 59-year-old male with Type I GT and a history of transfusion reactions to PLT infusions developed severe aortic stenosis secondary to bicuspid valve disease. He successfully underwent open aortic valve replacement with cardiopulmonary bypass without perioperative bleeding complications. RESULTS: A multidisciplinary team (anesthesia, hematology, cardiac surgery, and transfusion medicine) was established to optimize perioperative hematologic management. Bleeding risk was assessed given the patient's prior history and a dosing timeline for administration of blood products and recombinant clotting factors was established. Successful management was achieved during the operation by prophylactic administration of HLA-matched PLTs and Factor VIIa. Prophylactic PLT administration was continued through the immediate postoperative period and no bleeding complications occurred. Thromboelastograms (TEGs) were used in conjunction with traditional hematologic laboratory analysis to optimize clinical management. CONCLUSION: Patients with GT requiring cardiac surgical procedures are at high risk for perioperative bleeding complications. This case report illustrates the importance of multidisciplinary planning, TEG analysis, and the judicious use of recombinant factors to minimize operative bleeding risk.

    View details for DOI 10.1111/trf.12275

    View details for PubMedID 23710629

  • Brief report: carboxypeptidase B serves as a protective mediator in osteoarthritis. Arthritis & rheumatology (Hoboken, N.J.) Lepus, C. M., Song, J. J., Wang, Q., Wagner, C. A., Lindstrom, T. M., Chu, C. R., Sokolove, J., Leung, L. L., Robinson, W. H. 2014; 66 (1): 101-106

    Abstract

    We previously demonstrated that carboxypeptidase B (CPB) protects against joint erosion in rheumatoid arthritis by inactivating complement component C5a. We also found that levels of CPB are abnormally high in the synovial fluid of individuals with another joint disease, osteoarthritis (OA). We undertook this study to investigate whether CPB plays a role in the pathogenesis of OA.We compared the development of OA in CPB-deficient (Cpb2(-/-) ) mice and wild-type mice by subjecting them to medial meniscectomy and histologically assessing cartilage damage, osteophyte formation, and synovitis in the stifle joints 4 months later. We measured levels of proCPB, proinflammatory cytokines, and complement components in synovial fluid samples from patients with symptomatic and radiographic knee OA. Finally, we used enzyme-linked immunosorbent assay, flow cytometry, and hemolytic assays to assess the effect of CPB on formation of membrane attack complex (MAC)-a complement effector critical to OA pathogenesis.Cpb2(-/-) mice developed dramatically greater cartilage damage than did wild-type mice (P < 0.01) and had a greater number of osteophytes (P < 0.05) and a greater degree of synovitis (P < 0.05). In synovial fluid samples from OA patients, high levels of proCPB were associated with high levels of proinflammatory cytokines and complement components, and levels of proCPB correlated positively with those of MAC. In in vitro complement activation assays, activated CPB suppressed the formation of MAC as well as MAC-induced hemolysis.Our data suggest that CPB protects against inflammatory destruction of the joints in OA, at least in part by inhibiting complement activation.

    View details for DOI 10.1002/art.38213

    View details for PubMedID 24449579

  • Carboxypeptidase B Serves as a Protective Mediator in Osteoarthritis ARTHRITIS & RHEUMATOLOGY Lepus, C. M., Song, J. J., Wang, Q., Wagner, C. A., Lindstrom, T. M., Chu, C. R., Sokolove, J., Leung, L. L., Robinson, W. H. 2014; 66 (1): 101-106

    Abstract

    We previously demonstrated that carboxypeptidase B (CPB) protects against joint erosion in rheumatoid arthritis by inactivating complement component C5a. We also found that levels of CPB are abnormally high in the synovial fluid of individuals with another joint disease, osteoarthritis (OA). We undertook this study to investigate whether CPB plays a role in the pathogenesis of OA.We compared the development of OA in CPB-deficient (Cpb2(-/-) ) mice and wild-type mice by subjecting them to medial meniscectomy and histologically assessing cartilage damage, osteophyte formation, and synovitis in the stifle joints 4 months later. We measured levels of proCPB, proinflammatory cytokines, and complement components in synovial fluid samples from patients with symptomatic and radiographic knee OA. Finally, we used enzyme-linked immunosorbent assay, flow cytometry, and hemolytic assays to assess the effect of CPB on formation of membrane attack complex (MAC)-a complement effector critical to OA pathogenesis.Cpb2(-/-) mice developed dramatically greater cartilage damage than did wild-type mice (P < 0.01) and had a greater number of osteophytes (P < 0.05) and a greater degree of synovitis (P < 0.05). In synovial fluid samples from OA patients, high levels of proCPB were associated with high levels of proinflammatory cytokines and complement components, and levels of proCPB correlated positively with those of MAC. In in vitro complement activation assays, activated CPB suppressed the formation of MAC as well as MAC-induced hemolysis.Our data suggest that CPB protects against inflammatory destruction of the joints in OA, at least in part by inhibiting complement activation.

    View details for DOI 10.1002/art.38213

    View details for Web of Science ID 000337356300014

  • Targeting complement component 5a promotes vascular integrity and limits airway remodeling. Proceedings of the National Academy of Sciences of the United States of America Khan, M. A., Maasch, C., Vater, A., Klussmann, S., Morser, J., Leung, L. L., Atkinson, C., Tomlinson, S., Heeger, P. S., Nicolls, M. R. 2013; 110 (15): 6061-6066

    Abstract

    Increased microvascular dilatation and permeability is observed during allograft rejection. Because vascular integrity is an important indicator of transplant health, we have sought to limit injury to blood vessels by blocking complement activation. Although complement component 3 (C3) inhibition is known to be vasculoprotective in transplantation studies, we recently demonstrated the paradoxical finding that, early in rejection, C3(-/-) transplant recipients actually exhibit worse microvascular injury than controls. In the genetic absence of C3, thrombin-mediated complement component 5 (C5) convertase activity leads to the generation of C5a (anaphylatoxin), a promoter of vasodilatation and permeability. In the current study, we demonstrated that microvessel thrombin deposition is significantly increased in C3(-/-) recipients during acute rejection. Thrombin colocalization with microvessels is closely associated with remarkably elevated plasma levels of C5a, vasodilatation, and increased vascular permeability. Administration of NOX-D19, a specific C5a inhibitor, to C3(-/-) recipients of airway transplants significantly improved tissue oxygenation, limited microvascular leakiness, and prevented airway ischemia, even in the absence of conventional T-cell-directed immunosuppression. As C3 inhibitors enter the clinics, the simultaneous targeting of this thrombin-mediated complement activation pathway and/or C5a itself may confer significant clinical benefit.

    View details for DOI 10.1073/pnas.1217991110

    View details for PubMedID 23530212

    View details for PubMedCentralID PMC3625314

  • Thrombin-cleaved Fragments of Osteopontin Are Overexpressed in Malignant Glial Tumors and Provide a Molecular Niche with Survival Advantage JOURNAL OF BIOLOGICAL CHEMISTRY Yamaguchi, Y., Shao, Z., Sharif, S., Du, X., Myles, T., Merchant, M., Harsh, G., Glantz, M., Recht, L., Morser, J., Leung, L. L. 2013; 288 (5): 3097-3111

    Abstract

    Osteopontin (OPN), which is highly expressed in malignant glioblastoma (GBM), possesses inflammatory activity modulated by proteolytic cleavage by thrombin and plasma carboxypeptidase B2 (CPB2) at a highly conserved cleavage site. Full-length OPN (OPN-FL) was elevated in cerebrospinal fluid (CSF) samples from all cancer patients compared with noncancer patients. However, thrombin-cleaved OPN (OPN-R) and thrombin/CPB2-double-cleaved OPN (OPN-L) levels were markedly increased in GBM and non-GBM gliomas compared with systemic cancer and noncancer patients. Cleaved OPN constituted ∼23 and ∼31% of the total OPN in the GBM and non-GBM CSF samples, respectively. OPN-R was also elevated in GBM tissues. Thrombin-antithrombin levels were highly correlated with cleaved OPN, but not OPN-FL, suggesting that the cleaved OPN fragments resulted from increased thrombin and CPB2 in this extracellular compartment. Levels of VEGF and CCL4 were increased in CSF of GBM and correlated with the levels of cleaved OPN. GBM cell lines were more adherent to OPN-R and OPN-L than OPN-FL. Adhesion to OPN altered gene expression, in particular genes involved with cellular processes, cell cycle regulation, death, and inflammation. OPN and its cleaved forms promoted motility of U-87 MG cells and conferred resistance to apoptosis. Although functional mutation of the RGD motif in OPN largely abolished these functions, OPN(RAA)-R regained significant cell binding and signaling function, suggesting that the SVVYGLR motif in OPN-R may substitute for the RGD motif if the latter becomes inaccessible. OPN cleavage contributes to GBM development by allowing more cells to bind in niches where they acquire anti-apoptotic properties.

    View details for DOI 10.1074/jbc.M112.362954

    View details for Web of Science ID 000314397900019

    View details for PubMedID 23204518

    View details for PubMedCentralID PMC3561533

  • Microfluidic impedance cytometer for platelet analysis LAB ON A CHIP Evander, M., Ricco, A. J., Morser, J., Kovacs, G. T., Leung, L. L., Giovangrandi, L. 2013; 13 (4): 722-729

    Abstract

    We present the design and performance characteristics of a platelet analysis platform based on a microfluidic impedance cytometer. Dielectrophoretic focusing is used to centre cells in a fluid stream, which then forms the core of a two-phase flow (dielectric focusing). This flow then passes between electrodes for analysis by differential impedance spectroscopy at multiple frequencies from 280 kHz to 4 MHz. This approach increases the signal-to-noise ratio relative to a single-phase, unfocused stream, while minimising the shear forces to which the cells are subjected. The percentage of activated platelets before and after passage through the chip was measured using flow cytometry, and no significant change was measured. Measuring the in-phase amplitude at a single frequency is sufficient to distinguish platelets from erythrocytes. Using multi-frequency impedance measurements and discriminant analysis, resting platelets can be discriminated from activated platelets. This multifrequency impedance cytometer therefore allows ready determination of the degree of platelet activation in blood samples.

    View details for DOI 10.1039/c2lc40896a

    View details for Web of Science ID 000313971300028

    View details for PubMedID 23282651

  • Differential Gene Expression in Thrombomodulin (TM; CD141)(+) and TM(-) Dendritic Cell Subsets. PloS one Toda, M., Shao, Z., Yamaguchi, K. D., Takagi, T., D'Alessandro-Gabazza, C. N., Taguchi, O., Salamon, H., Leung, L. L., Gabazza, E. C., Morser, J. 2013; 8 (8)

    Abstract

    Previously we have shown in a mouse model of bronchial asthma that thrombomodulin can convert immunogenic conventional dendritic cells into tolerogenic dendritic cells while inducing its own expression on their cell surface. Thrombomodulin(+) dendritic cells are tolerogenic while thrombomodulin(-) dendritic cells are pro-inflammatory and immunogenic. Here we hypothesized that thrombomodulin treatment of dendritic cells would modulate inflammatory gene expression. Murine bone marrow-derived dendritic cells were treated with soluble thrombomodulin and expression of surface markers was determined. Treatment with thrombomodulin reduces the expression of maturation markers and increases the expression of TM on the DC surface. Thrombomodulin treated and control dendritic cells were sorted into thrombomodulin(+) and thrombomodulin(-) dendritic cells before their mRNA was analyzed by microarray. mRNAs encoding pro-inflammatory genes and dendritic cells maturation markers were reduced while expression of cell cycle genes were increased in thrombomodulin-treated and thrombomodulin(+) dendritic cells compared to control dendritic cells and thrombomodulin(-) dendritic cells. Thrombomodulin-treated and thrombomodulin(+) dendritic cells had higher expression of 15-lipoxygenase suggesting increased synthesis of lipoxins. Thrombomodulin(+) dendritic cells produced more lipoxins than thrombomodulin(-) dendritic cells, as measured by ELISA, confirming that this pathway was upregulated. There was more phosphorylation of several cell cycle kinases in thrombomodulin(+) dendritic cells while phosphorylation of kinases involved with pro-inflammatory cytokine signaling was reduced. Cultures of thrombomodulin(+) dendritic cells contained more cells actively dividing than those of thrombomodulin(-) dendritic cells. Production of IL-10 is increased in thrombomodulin(+) dendritic cells. Antagonism of IL-10 with a neutralizing antibody inhibited the effects of thrombomodulin treatment of dendritic cells suggesting a mechanistic role for IL-10. The surface of thrombomodulin(+) dendritic cells supported activation of protein C and procarboxypeptidase B2 in a thrombomodulin-dependent manner. Thus thrombomodulin treatment increases the number of thrombomodulin(+) dendritic cells, which have significantly altered gene expression compared to thrombomodulin(-) dendritic cells in key immune function pathways.

    View details for DOI 10.1371/journal.pone.0072392

    View details for PubMedID 24009678

    View details for PubMedCentralID PMC3751914

  • Chemerin Bioactivity Is Regulated by Factor XIa: A Novel Interface Linking Between Coagulation, Hemostasis and Immunity 53rd Annual Meeting and Exposition of the American-Society-of-Hematology (ASH) Yamaguchi, Y., Morser, J., Leung, L. L. AMER SOC HEMATOLOGY. 2011: 981–82
  • Chemerin158K Protein Is the Dominant Chemerin Isoform in Synovial and Cerebrospinal Fluids but Not in Plasma JOURNAL OF BIOLOGICAL CHEMISTRY Zhao, L., Yamaguchi, Y., Sharif, S., Du, X., Song, J. J., Lee, D. M., Recht, L. D., Robinson, W. H., Morser, J., Leung, L. L. 2011; 286 (45): 39520-39527

    Abstract

    Chemerin is a chemoattractant involved in immunity that may also function as an adipokine. Chemerin circulates as an inactive precursor (chem163S), and its activation requires proteolytic cleavages at its C terminus, involving proteases involved in coagulation, fibrinolysis, and inflammation. However, the key proteolytic steps in prochemerin activation in vivo remain to be established. Previously, we have shown that C-terminal cleavage of chem163S by plasmin to chem158K, followed by a carboxypeptidase cleavage, leads to the most active isoform, chem157S. To identify and quantify the in vivo chemerin isoforms in biological specimens, we developed specific ELISAs for chem163S, chem158K, and chem157S, using antibodies raised against peptides from the C terminus of the different chemerin isoforms. We found that the mean plasma concentrations of chem163S, chem158K, and chem157S were 40 ± 7.9, 8.1 ± 2.9, and 0.7 ± 0.8 ng/ml, respectively. The total level of cleaved and noncleaved chemerins in cerebrospinal fluids was ∼10% of plasma levels whereas it was elevated ∼2-fold in synovial fluids from patients with arthritis. On the other hand, the fraction of cleaved chemerins was much higher in synovial fluid and cerebrospinal fluid samples than in plasma (∼75%, 50%, and 18% respectively). Chem158K was the dominant chemerin isoform, and it was not generated by ex vivo processing, indicating that cleavage of prochemerin at position Lys-158, whether by plasmin or another serine protease, represents a major step in prochemerin activation in vivo. Our study provides the first direct evidence that chemerin undergoes extensive proteolytic processing in vivo, underlining the importance of measuring individual isoforms.

    View details for DOI 10.1074/jbc.M111.258954

    View details for Web of Science ID 000296759800069

    View details for PubMedID 21930706

    View details for PubMedCentralID PMC3234775

  • Proteolytic Cleavage of Chemerin Protein Is Necessary for Activation to the Active Form, Chem157S, Which Functions as a Signaling Molecule in Glioblastoma JOURNAL OF BIOLOGICAL CHEMISTRY Yamaguchi, Y., Du, X., Zhao, L., Morser, J., Leung, L. L. 2011; 286 (45): 39510-39519

    Abstract

    Chemerin is a chemoattractant involved in innate and adaptive immunity as well as an adipokine implicated in adipocyte differentiation. Chemerin circulates as an inactive precursor in blood whose bioactivity is closely regulated through proteolytic processing at its C terminus. We developed methodology for production of different recombinant chemerin isoforms (chem163S, chem157S, and chem155A) which allowed us to obtain large quantities of these proteins with purity of >95%. Chem158K was generated from chem163S by plasmin cleavage. Characterization by mass spectrometry and Edman degradation demonstrated that both the N and C termini were correct for each isoform. Ca(2+) mobilization assays showed that the EC(50) values for chem163S and chem158K were 54.2 ± 19.9 nm and 65.2 ± 13.2 nm, respectively, whereas chem157S had a ∼50-fold higher potency with an EC(50) of 1.2 ± 0.7 nm. Chem155A had no agonist activity and weak antagonist activity, causing a 50% reduction of chem157S activity at a molar ratio of 100:1. Similar results were obtained in a chemotaxis assay. Because chem158K is the dominant form in cerebrospinal fluid from patients with glioblastoma (GBM), we examined the significance of chemerin in GBM biology. In silico analysis showed chemerin mRNA was significantly increased in tissue from grade III and IV gliomas. Furthermore, U-87 MG cells, a human GBM line, express the chemerin receptors, chemokine-like receptor 1 and chemokine receptor-like 2, and chem157S triggered Ca(2+) flux. This study emphasized the necessity of appropriate C-terminal proteolytic processing to generate the likely physiologic form of active chemerin, chem157S, and suggested a possible role in malignant GBM.

    View details for DOI 10.1074/jbc.M111.258921

    View details for Web of Science ID 000296759800068

    View details for PubMedID 21949124

    View details for PubMedCentralID PMC3234774

  • Plasma carboxypeptidase B downregulates inflammatory responses in autoimmune arthritis JOURNAL OF CLINICAL INVESTIGATION Song, J. J., Hwang, I., Cho, K. H., Garcia, M. A., Kim, A. J., Wang, T. H., Lindstrom, T. M., Lee, A. T., Nishimura, T., Zhao, L., Morser, J., Nesheim, M., Goodman, S. B., Lee, D. M., Bridges, S. L., Gregersen, P. K., Leung, L. L., Robinson, W. H. 2011; 121 (9): 3517-3527

    Abstract

    The immune and coagulation systems are both implicated in the pathogenesis of rheumatoid arthritis (RA). Plasma carboxypeptidase B (CPB), which is activated by the thrombin/thrombomodulin complex, plays a procoagulant role during fibrin clot formation. However, an antiinflammatory role for CPB is suggested by the recent observation that CPB can cleave proinflammatory mediators, such as C5a, bradykinin, and osteopontin. Here, we show that CPB plays a central role in downregulating C5a-mediated inflammatory responses in autoimmune arthritis. CPB deficiency exacerbated inflammatory arthritis in a mouse model of RA, and cleavage of C5a by CPB suppressed the ability of C5a to recruit immune cells in vivo. In human patients with RA, genotyping of nonsynonymous SNPs in the CPB-encoding gene revealed that the allele encoding a CPB variant with longer half-life was associated with a lower risk of developing radiographically severe RA. Functionally, this CPB variant was more effective at abrogating the proinflammatory properties of C5a. Additionally, expression of both CPB and C5a in synovial fluid was higher in patients with RA than in those with osteoarthritis. These findings suggest that CPB plays a critical role in dampening local, C5a-mediated inflammation and represents a molecular link between inflammation and coagulation in autoimmune arthritis.

    View details for DOI 10.1172/JCI46387

    View details for Web of Science ID 000294753700019

    View details for PubMedID 21804193

    View details for PubMedCentralID PMC3163960

  • Enhanced Abdominal Aortic Aneurysm Formation in Thrombin-Activatable Procarboxypeptidase B-Deficient Mice ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Schultz, G., Tedesco, M. M., Sho, E., Nishimura, T., Sharif, S., Du, X., Myles, T., Morser, J., Dalman, R. L., Leung, L. L. 2010; 30 (7): 1363-1370

    Abstract

    To determine whether procarboxypeptidase B (pCPB)(-/-) mice are susceptible to accelerated abdominal aortic aneurysm (AAA) development secondary to unregulated OPN-mediated mural inflammation in the absence of CPB inhibition.Thrombin/thrombomodulin cleaves thrombin-activatable pCPB or thrombin-activatable fibrinolysis inhibitor, activating CPB, which inhibits the generation of plasmin and inactivates proinflammatory mediators (complement C5a and thrombin-cleaved osteopontin [OPN]). Apolipoprotein E(-/-)OPN(-/-) mice are protected from experimental AAA formation. Murine AAAs were created via intra-aortic porcine pancreatic elastase (PPE) infusion. Increased mortality secondary to AAA rupture was observed in pCPB(-/-) mice at the standard PPE dose. At reduced doses of PPE, pCPB(-/-) mice developed larger AAAs than wild-type controls (1.01+/-0.27 versus 0.68+/-0.05 mm; P=0.02 [mean+/-SD]). C5(-/-) and OPN(-/-) mice were not protected against AAA development. Treatment with tranexamic acid inhibited plasmin generation and abrogated enhanced AAA progression in pCPB(-/-) mice.This study establishes the role of CPB in experimental AAA disease, indicating that CPB has a broad anti-inflammatory role in vivo. Enhanced AAA formation in the PPE model is the result of increased plasmin generation, not unregulated C5a- or OPN-mediated mural inflammation.

    View details for DOI 10.1161/ATVBAHA.109.202259

    View details for Web of Science ID 000278856600013

    View details for PubMedID 20431069

  • What has been learnt from the thrombin-activatable fibrinolysis inhibitor-deficient mouse? JOURNAL OF THROMBOSIS AND HAEMOSTASIS Morser, J., Gabazza, E. C., Myles, T., Leung, L. L. 2010; 8 (5): 868-876

    Abstract

    Thrombin-activatable fibrinolysis inhibitor (TAFI) is a circulating zymogen that is activated physiologically by the thrombin/thrombomodulin complex to activated TAFI (TAFIa) which is a basic carboxypeptidase. Substrates include fibrin, leading to a reduction in rate of plasmin generation, and several proinflammatory mediators such as bradykinin, thrombin-cleaved osteopontin and complement factor C5a. TAFI-deficient mice have no phenotype without being challenged and TAFIa appears to play a limited role in physiological fibrinolysis in vivo. In several disease models, the TAFI-deficient mice have different outcomes from the wild type (WT), but whether the difference is beneficial or an exacerbation of the disease depends on the model. The consequences of TAFI deficiency include increased plasmin as a result of enhanced incorporation of plasminogen and tissue plasminogen activator into the fibrin clot, but also loss of its ability to degrade other substrates, with the resultant up-regulation of several proinflammatory mediators, including C5a. Criteria are recommended to demonstrate that a substrate is a physiological substrate of TAFIa.

    View details for DOI 10.1111/j.1538-7836.2010.03787.x

    View details for Web of Science ID 000277328600002

    View details for PubMedID 20128866

  • Thrombospondin Type I Domain Containing 7A (THSD7A) Mediates Endothelial Cell Migration and Tube Formation JOURNAL OF CELLULAR PHYSIOLOGY Wang, C., Su, P., Du, X., Kuo, M., Lin, C., Yang, C., Chan, H., Chang, S., Kuo, C., Seo, K., Leung, L. L., Chuan, Y. 2010; 222 (3): 685-694

    Abstract

    Angiogenesis is a highly organized process controlled by a series of molecular events. While much effort has been devoted to identifying angiogenic factors and their reciprocal receptors, far less information is available on the molecular mechanisms underlying directed endothelial cell migration. To search for novel proteins that participate in this process, we used the serial analysis of gene expression (SAGE) transcript profiling approach to identify genes that are selectively expressed in endothelial cells (ECs). Two EC SAGE libraries were constructed from human umbilical vein and artery ECs to enable data-mining against other non-ECs. A novel endothelial protein, Thrombospondin Type I Domain Containing 7A (THSD7A), with preferential expression in placenta vasculature and in human umbilical vein endothelial cells (HUVECs) was identified and targeted for further characterization. Overexpression of a THSD7A carboxyl-terminal fragment in HUVECs inhibited cell migration and disrupted tube formation, while suppression of THSD7A expression enhanced HUVEC migration and tube formation. Immunohistological analysis revealed that THSD7A was expressed at the leading edge of migrating HUVECs, and it co-localized with alpha(V)beta(3) integrin and paxillin. This distribution was dispersed from focal adhesions after disruption of the actin cytoskeleton, suggesting the involvement of THSD7A in cytoskeletal organization. Our results show that THSD7A is a novel placenta endothelial protein that mediates EC migration and tube formation, and they highlight its potential as a new target for anti-angiogenic therapy.

    View details for DOI 10.1002/jcp.21990

    View details for Web of Science ID 000274273700025

    View details for PubMedID 20020485

  • Heparin-induced thrombosis without thrombocytopenia JOURNAL OF THORACIC AND CARDIOVASCULAR SURGERY Tibayan, F. A., Leung, L. L., Burdon, T. A., Fann, J. I. 2010; 139 (2): E6-E7

    View details for DOI 10.1016/j.jtcvs.2008.07.006

    View details for Web of Science ID 000274014300050

    View details for PubMedID 19660256

  • Prochemerin Is a New Substrate for Thrombin 51st Annual Meeting and Exposition of the American-Society-of-Hematology Du, X., Myles, T., Morser, J., Leung, L. L. AMER SOC HEMATOLOGY. 2009: 1391–91
  • Thrombin-Activatable Carboxypeptidase B Cleavage of Osteopontin Regulates Neutrophil Survival and Synoviocyte Binding in Rheumatoid Arthritis ARTHRITIS AND RHEUMATISM Sharif, S. A., Du, X., Myles, T., Song, J. J., Price, E., Lee, D. M., Goodman, S. B., Nagashima, M., Morser, J., Robinson, W. H., Leung, L. L. 2009; 60 (10): 2902-2912

    Abstract

    Osteopontin (OPN) is a proinflammatory cytokine that plays an important role in the pathogenesis of rheumatoid arthritis (RA). OPN can be cleaved by thrombin, resulting in OPN-R and exposing the cryptic C-terminal alpha4beta1 and alpha9beta1 integrin-binding motif (SVVYGLR). Thrombin-activatable carboxypeptidase B (CPB), also called thrombin-activatable fibrinolysis inhibitor, removes the C-terminal arginine from OPN-R, generating OPN-L and abrogating its enhanced cell binding. We undertook this study to investigate the roles of OPN-R and OPN-L in synoviocyte adhesion, which contributes to the formation of invasive pannus, and in neutrophil survival, which affects inflammatory infiltrates in RA.Using specifically developed enzyme-linked immunosorbent assays, we tested the synovial fluid of patients with RA, osteoarthritis (OA), and psoriatic arthritis (PsA) to determine OPN-R, OPN-L, and full-length OPN (OPN-FL) levels.Elevated levels of OPN-R and OPN-L were found in synovial fluid samples from RA patients, but not in samples from OA or PsA patients. Increased levels of OPN-R and OPN-L correlated with increased levels of multiple inflammatory cytokines, including tumor necrosis factor alpha and interleukin-6. Immunohistochemical analyses revealed robust expression of OPN-FL, but only minimal expression of OPN-R, in RA synovium, suggesting that cleaved OPN is released into synovial fluid. In cellular assays, OPN-FL, and to a lesser extent OPN-R and OPN-L, had an antiapoptotic effect on neutrophils. OPN-R augmented RA fibroblast-like synoviocyte binding mediated by SVVYGLR binding to alpha4beta1, whereas OPN-L did not.Thrombin activation of OPN (resulting in OPN-R) and its subsequent inactivation by thrombin-activatable CPB (generating OPN-L) occurs locally within inflamed joints in RA. Our data suggest that thrombin-activatable CPB plays a central homeostatic role in RA by regulating neutrophil viability and reducing synoviocyte adhesion.

    View details for DOI 10.1002/art.24814

    View details for Web of Science ID 000270696600007

    View details for PubMedID 19790060

    View details for PubMedCentralID PMC3757557

  • Regulation of Chemerin Bioactivity by Plasma Carboxypeptidase N, Carboxypeptidase B (Activated Thrombin-activable Fibrinolysis Inhibitor), and Platelets JOURNAL OF BIOLOGICAL CHEMISTRY Du, X., Zabel, B. A., Myles, T., Allen, S. J., Handel, T. M., Lee, P. P., Butcher, E. C., Leung, L. L. 2009; 284 (2): 751-758

    Abstract

    Chemerin is a potent chemoattractant for cells expressing the serpentine receptor CMKLR1 (chemokine-like receptor 1), such as plasmacytoid dendritic cells and tissue macrophages. The bioactivity of chemerin is post-translationally regulated; the attractant circulates in blood in a relatively inactive form (prochemerin) and is activated by carboxyl-terminal proteolytic cleavage. We discovered that plasma carboxypeptidase N (CPN) and B (CPB or activated thrombin-activable fibrinolysis inhibitor, TAFIa) enhanced the bioactivity of 10-mer chemerin peptide NH(2)-YFPGQFAFSK-COOH by removing the carboxyl-terminal lysine (K). Sequential cleavages of either a prochemerin peptide (NH(2)-YFPGQFAFSKALPRS-COOH) or recombinant full-length prochemerin by plasmin and CPN/CPB substantially increased their chemotactic activities. Endogenous CPN present in circulating plasma enhanced the activity of plasmin-cleaved prochemerin. In addition, we discovered that platelets store chemerin protein and release it upon stimulation. Thus circulating CPN/CPB and platelets may potentially contribute to regulating the bioactivity of leukocyte chemoattractant chemerin, and further extend the molecular link between blood coagulation/fibrinolysis and CMKLR1-mediated immune responses.

    View details for DOI 10.1074/jbc.M805000200

    View details for Web of Science ID 000262122900008

    View details for PubMedID 19010784

    View details for PubMedCentralID PMC2613638

  • Thrombin hydrolysis of human osteopontin is dependent on thrombin anion-binding exosites JOURNAL OF BIOLOGICAL CHEMISTRY Myles, T., Leung, L. L. 2008; 283 (26): 17789-17796

    Abstract

    The cytokine osteopontin (OPN) can be hydrolyzed by thrombin exposing a cryptic alpha(4)beta(1)/alpha(9)beta(1) integrin-binding motif (SVVYGLR), thereby acting as a potent cytokine for cells bearing these activated integrins. We show that purified milk OPN is a substrate for thrombin with a k(cat)/K(m) value of 1.14 x 10(5) m(-1) s(-1). Thrombin cleavage of OPN was inhibited by unsulfated hirugen (IC(50) = 1.2 +/- 0.2 microm), unfractionated heparin (IC(50) = 56.6 +/- 8.4 microg/ml) and low molecular weight (5 kDa) heparin (IC(50) = 31.0 +/- 7.9 microg/ml), indicating the involvement of both anion-binding exosite I (ABE-I) and anion-binding exosite II (ABE-II). Using a thrombin mutant library, we mapped residues important for recognition and cleavage of OPN within ABE-I and ABE-II. A peptide (OPN-(162-197)) was designed spanning the OPN thrombin cleavage site and a hirudin-like C-terminal tail domain. Thrombin cleaved OPN-(162-197) with a specificity constant of k(cat)/K(m) = 1.64 x 10(4) m(-1) s(-1). Representative ABE-I mutants (K65A, H66A, R68A, Y71A, and R73A) showed greatly impaired cleavage, whereas the ABE-II mutants were unaffected, suggesting that ABE-I interacts principally with the hirudin-like OPN domain C-terminal and contiguous to the thrombin cleavage site. Debye-Hückel slopes for milk OPN (-4.1 +/- 1.0) and OPN-(162-197) (-2.4 +/- 0.2) suggest that electrostatic interactions play an important role in thrombin recognition and cleavage of OPN. Thus, OPN is a bona fide substrate for thrombin, and generation of thrombin-cleaved OPN with enhanced pro-inflammatory properties provides another molecular link between coagulation and inflammation.

    View details for DOI 10.1074/jbc.M708629200

    View details for Web of Science ID 000256949200006

    View details for PubMedID 18413297

  • Essential thrombin residues for inhibition by protein C inhibitor with the cofactors heparin and thrombomodulin JOURNAL OF THROMBOSIS AND HAEMOSTASIS Fortenberry, Y. M., Whinna, H. C., Cooper, S. T., Myles, T., Leung, L. L., Church, F. C. 2007; 5 (7): 1486-1492

    Abstract

    Background: Protein C inhibitor (PCI) and antithrombin (AT) are serine protease inhibitors (serpins) that inhibit a wide array of blood coagulation serine proteases including thrombin.Fifty-five Ala-scanned recombinant thrombin mutants were used to determine thrombin residues important for inhibition by PCI with and without the cofactors heparin and thrombomodulin (TM) and compared with the prototypical serpin, AT.Residues around the active site (Tyr50 and Glu202) and the sodium-binding site (Glu229 and Arg233) were required for thrombin inhibition by PCI with and without cofactors. Exosite-2 residues (Arg89, Arg93, Glu94, Arg98, Arg245, Arg248, and Gln251) were critical for heparin-accelerated inhibition of thrombin by PCI. Exosite-1 residues (especially Lys65 and Tyr71) were required for enhanced PCI inhibition of thrombin-TM. Interestingly, we also found that the TM chondroitin sulfate moiety is not required for the approximately 150-fold enhanced rate of thrombin inhibition by PCI. Using the aforementioned thrombin exosite-2 mutants that were essential for heparin-catalyzed PCI-thrombin inhibition reactions we found no change in PCI inhibition rates for thrombin-TM.Collectively, these results show that (i) similar thrombin exosite-2 residues are critical for the heparin-catalyzed inhibition by PCI and AT, (ii) PCI and AT are different in their thrombin-TM inhibition properties, and (iii) PCI has a distinct advantage over AT in the regulation of the activity of thrombin-TM.

    View details for Web of Science ID 000247980000024

    View details for PubMedID 17635698

  • Thrombin-activatable procarboxypeptidase B regulates activated complement C5a in vivo BLOOD Nishimura, T., Myles, T., Piliposky, A. M., Kao, P. N., Berry, G. J., Leung, L. L. 2007; 109 (5): 1992-1997

    Abstract

    Plasma procarboxypeptidase B (proCPB) is activated by the endothelial thrombin-thrombomodulin [corrected] complex. Activated proCPB [corrected] (CPB) functions as a fibrinolysis inhibitor, but it may play a broader role by inactivating inflammatory mediators. To test this hypothesis, C5a-induced alveolitis was studied in wild-type (WT) and proCPB-deficient mice (proCPB-/-). C5a-induced alveolitis, as measured by cell counts and total protein contents in bronchoalveolar lavage fluids, was markedly enhanced in the proCPB-/- mice. E229K thrombin, a thrombin mutant with minimal clotting activity but retaining its ability to activate protein C and proCPB, attenuated C5a-induced alveolitis in WT but not in proCPB-/- mice, indicating that its beneficial effect is mediated primarily by its activation of proCPB. Lung tissue histology confirmed these cellular inflammatory responses. Delayed administration of E229K thrombin after the C5a instillation was ineffective in reducing alveolitis in WT mice, suggesting that the beneficial effect of E229K thrombin is due to the direct inhibition of C5a by CPB. Our studies show that thrombin-activatable proCPB, in addition to its role in fibrinolysis, has intrinsic anti-inflammatory functions. Its activation, along with protein C, by the endothelial thrombin-TM complex represents a homeostatic response to counteract the inflammatory mediators generated at the site of vascular injury.

    View details for DOI 10.1182/blood-2006-03-012567

    View details for Web of Science ID 000244641100036

    View details for PubMedID 17105819

    View details for PubMedCentralID PMC1801069

  • Antiangiogenic antithrombin induces global changes in the gene expression profile of endothelial cells CANCER RESEARCH Zhang, W., Chuang, Y., Jin, T., Swanson, R., Xiong, Y., Leung, L., Olson, S. T. 2006; 66 (10): 5047-5055

    Abstract

    Antithrombin, a serpin family protease inhibitor crucial to hemostasis, acquires antiangiogenic properties on undergoing conformational alterations induced by limited proteolysis or elevated temperature. To better understand the biochemical mechanisms underlying antithrombin antiangiogenic activity, we did genome-wide expression profiling, coupled with quantitative reverse transcription-PCR, Northern blot, and Western blot analyses, to characterize the gene expression patterns that are induced by antiangiogenic antithrombin in cultured primary human umbilical vein endothelial cells. Overall, 35 genes with significantly increased expression and 93 genes with significantly reduced expression (> or =2-fold changes) due to antiangiogenic antithrombin treatment were identified. More than half of the down-regulated genes have well-established proangiogenic functions in endothelial cells, including cell-surface and matrix proteoglycans (e.g., perlecan, biglycan, and syndecans 1 and 3) and mitogenesis-related signaling proteins (e.g., mitogen-activated protein kinase 3, signal transducers and activators of transcription 2, 3, and 6, and early growth response factor 1). In contrast, most up-regulated genes (e.g., caspase-3, p21, tissue inhibitor of metalloproteinases 1, 2, and 3, and adenomatosis polyposis coli) are known for their antiangiogenic functions which include the promotion of cell apoptosis and cell cycle arrest and the inhibition of tumor growth and metastasis. These results show that the antiangiogenic activity of antithrombin is mediated at least in part by a global genetic reprogramming of endothelial cells and strongly implicate an endothelial cell ligand-receptor signaling mechanism in this reprogramming.

    View details for DOI 10.1158/0008-5472.CAN-05-4449

    View details for Web of Science ID 000237679900011

    View details for PubMedID 16707426

  • Perioperative evaluation of bleeding diathesis. Hematology / the Education Program of the American Society of Hematology. American Society of Hematology. Education Program Leung, L. L. 2006: 457-461

    Abstract

    The differential diagnosis of a long APTT with a normal prothrombin time can be due to either a clotting factor deficiency or the presence of an inhibitor, which can be distinguished by using a plasma-mixing study. The various clotting factor deficiency states are reviewed. Clinical bleeding following cardiac bypass surgery due to acquired factor V and thrombin antibodies is also reviewed.

    View details for PubMedID 17124099

  • Thrombin-activatable fibrinolysis inhibitor (TAFI) regulates activated complement C5a in vivo. 46th Annual Meeting of the American-Society-of-Hematology Nishimura, T., Myles, T., Nagashima, M., MORSER, J., Pearl, R. G., Leung, L. L. AMER SOC HEMATOLOGY. 2004: 812A–812A
  • Crystal structure of anticoagulant thrombin variant E217K provides insights into thrombin allostery JOURNAL OF BIOLOGICAL CHEMISTRY Carter, W. J., Myles, T., Gibbs, C. S., Leung, L. L., Huntington, J. A. 2004; 279 (25): 26387-26394

    Abstract

    Thrombin is the ultimate protease of the blood clotting cascade and plays a major role in its own regulation. The ability of thrombin to exhibit both pro- and anti-coagulant properties has spawned efforts to turn thrombin into an anticoagulant for therapeutic purposes. This quest culminated in the identification of the E217K variant through scanning and saturation mutagenesis. The antithrombotic properties of E217K thrombin are derived from its inability to convert fibrinogen to a fibrin clot while maintaining its thrombomodulin-dependent ability to activate the anticoagulant protein C pathway. Here we describe the 2.5-A crystal structure of human E217K thrombin, which displays a dramatic restructuring of the geometry of the active site. Of particular interest is the repositioning of Glu-192, which hydrogen bonds to the catalytic Ser-195 and which results in the complete occlusion of the active site and the destruction of the oxyanion hole. Substrate binding pockets are further blocked by residues previously implicated in thrombin allostery. We have concluded that the E217K mutation causes the allosteric inactivation of thrombin by destabilizing the Na(+) binding site and that the structure thus may represent the Na(+)-free, catalytically inert "slow" form.

    View details for DOI 10.1074/jbc.M402364200

    View details for Web of Science ID 000222003000059

    View details for PubMedID 15075325

  • Molecular cloning of nonsecreted endothelial cell-derived lipase isoforms GENOMICS Ishida, T., Zheng, Z., Dichek, H. L., Wang, H. J., Moreno, I., Yang, E., Kundu, R. K., Talbi, S., Hirata, K. I., Leung, L. L., Quertermous, T. 2004; 83 (1): 24-33

    Abstract

    To expand our knowledge of factors involved in lipid metabolism in the blood vessel wall, we have cloned unique molecular isoforms of endothelial cell-derived lipase (EDL) (HGMW-approved symbol/LIPG). One isoform encoded a truncated protein (EDL2a) lacking the first 80 amino acid residues of the previously characterized EDL1a isoform, including the signal peptide. A similar second clone (EDL2b) was identified that lacked not only the first 80 amino acids, but also a 74-amino-acid region that encodes a portion of the lid domain. RT-PCR analysis confirmed expression of EDL2a/2b isoforms in several human tissues and cultured cells, including endothelial cells. Western blot and immunofluorescence studies using stable transfectants revealed that EDL2a and EDL2b were localized in the cytosol, while, EDL1a was secreted into the culture medium. Cell extracts of EDL2a/2b transfectants did not have triglyceride or phospholipase activity. Thus endothelial cells express three EDL isoforms, two of which remain intracellular and do not function as lipases.

    View details for DOI 10.1016/S0888-7543(03)00181-2

    View details for Web of Science ID 000187775700004

    View details for PubMedID 14667806

  • Thrombin activatable fibrinolysis inhibitor, a potential regulator of vascular inflammation JOURNAL OF BIOLOGICAL CHEMISTRY Myles, T., Nishimura, T., Yun, T. H., Nagashima, M., MORSER, J., Patterson, A. J., Pearl, R. G., Leung, L. L. 2003; 278 (51): 51059-51067

    Abstract

    The latent plasma carboxypeptidase thrombin-activable fibrinolysis inhibitor (TAFI) is activated by thrombin/thrombomodulin on the endothelial cell surface, and functions in dampening fibrinolysis. In this study, we examined the effect of activated TAFI (TAFIa) in modulating the proinflammatory functions of bradykinin, complement C5a, and thrombin-cleaved osteopontin. Hydrolysis of bradykinin and C5a and thrombin-cleaved osteopontin peptides by TAFIa was as efficient as that of plasmin-cleaved fibrin peptides, indicating that these are also good substrates for TAFIa. Plasma carboxypeptidase N, generally regarded as the physiological regulator of kinins, was much less efficient than TAFIa. TAFIa abrogated C5a-induced neutrophil activation in vitro. Jurkat cell adhesion to osteopontin was markedly enhanced by thrombin cleavage of osteopontin. This was abolished by TAFIa treatment due to the removal of the C-terminal Arg168 by TAFIa from the exposed SVVYGLR alpha 4 beta 1 integrin-binding site in thrombin-cleaved osteopontin. Thus, thrombin cleavage of osteopontin followed by TAFIa treatment may sequentially up- and down-modulate the pro-inflammatory properties of osteopontin. An engineered anticoagulant thrombin, E229K, was able to activate endogenous plasma TAFI in mice, and E229K thrombin infusion effectively blocked bradykinin-induced hypotension in wild-type, but not in TAFI-deficient, mice in vivo. Our data suggest that TAFIa may have a broad anti-inflammatory role, and its function is not restricted to fibrinolysis.

    View details for DOI 10.1074/jbc.M306977200

    View details for Web of Science ID 000187206300029

    View details for PubMedID 14525995

  • Thrombin activation of factor XI on activated platelets requires the interaction of factor XI and platelet glycoprotein Ib alpha with thrombin anion-binding exosites I and II, respectively (Retracted Article. See vol 282, pg 29067, 2007) JOURNAL OF BIOLOGICAL CHEMISTRY Yun, T. H., Baglia, F. A., Myles, T., Navaneetham, D., Lopez, J. A., Walsh, P. N., Leung, L. L. 2003; 278 (48): 48112-48119

    Abstract

    Activation of factor XI (FXI) by thrombin on stimulated platelets plays a physiological role in hemostasis, providing additional thrombin generation required in cases of severe hemostatic challenge. Using a collection of 53 thrombin mutants, we identified 16 mutants with <50% of the wild-type thrombin FXI-activating activity in the presence of dextran sulfate. These mutants mapped to anion-binding exosite (ABE) I, ABE-II, the Na+-binding site, and the 50-insertion loop. Only the ABE-II mutants showed reduced binding to dextran sulfate-linked agarose. Selected thrombin mutants in ABE-I (R68A, R70A, and R73A), ABE-II (R98A, R245A, and K248A), the 50-insertion loop (W50A), and the Na+-binding site (E229A and R233A) with <10% of the wild-type activity also showed a markedly reduced ability to activate FXI in the presence of stimulated platelets. The ABE-I, 50-insertion loop, and Na+-binding site mutants had impaired binding to FXI, but normal binding to glycocalicin, the soluble form of glycoprotein Ibalpha (GPIb alpha). In contrast, the ABE-II mutants were defective in binding to glycocalicin, but displayed normal binding to FXI. Our data support a quaternary complex model of thrombin activation of FXI on stimulated platelets. Thrombin bound to one GPIb alpha molecule, via ABE-II on its posterior surface, is properly oriented for its activation of FXI bound to a neighboring GPI alpha molecule, via ABE-I on its anterior surface. GPIb alpha plays a critical role in the co-localization of thrombin and FXI and the resultant efficient activation of FXI.

    View details for DOI 10.1074/jbc.M306925200

    View details for Web of Science ID 000186731400092

  • Structural requirements for the activation of human factor VIII by thrombin BLOOD Myles, T., Yun, T. H., Leung, L. L. 2002; 100 (8): 2820-2826

    Abstract

    The coagulation factors V (FV) and VIII (FVIII) are important at sites of vascular injury for the amplification of the clotting cascade. Natural variants of these factors frequently lead to severe bleeding disorders. To understand the mechanisms of activation of FVIII by thrombin, we used a bank of mutant thrombins to define residues important for its activation. From the initial screening of 53 mutant thrombins for the activation of human recombinant FVIII, we mapped thrombin mutants with 50% or less activity to anion-binding exosite-I (Lys21Ala, His66Ala, Lys65Ala, Arg68Ala, Arg70Ala, and Tyr71Ala) and anion-binding exosite-II (Arg98Ala), the Na(+)-binding site (Glu229Ala, Arg233Ala, Asp234Ala, and Asp193Ala/Lys196Ala), and the 50-insertion loop (Trp50Ala), which were similar to our results for the activation of FV. The role of these residues for cleavage at Arg372 and Arg1689 was investigated using plasma FVIII. Anion-binding exosite-I appears to be important for cleavage at both sites, whereas the anion-binding exosite-II residue Arg98Ala is important for cleavage at Arg372 alone. The Glu229Ala mutant, which contributes to the Na(+)-binding site, and the 50-insertion loop mutant W50A have severely impaired cleavage at Arg372 and Arg1689. This suggests that the integrity of the active site and the Na(+)-bound form of thrombin are important for its procoagulant activity against FVIII. Detailed mutagenic analysis of thrombin can assist in understanding the pathogenesis of bleeding disorders and may lead to the rational design of selective thrombin inhibitors.

    View details for Web of Science ID 000178519100023

    View details for PubMedID 12351390

  • Identification of critical residues on thrombin mediating its interaction with fibrin THROMBOSIS AND HAEMOSTASIS HALL, S. W., Gibbs, C. S., Leung, L. L. 2001; 86 (6): 1466-1474

    Abstract

    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.

    View details for Web of Science ID 000172857400020

    View details for PubMedID 11776315

  • An extensive interaction interface between thrombin and factor V is required for factor V activation JOURNAL OF BIOLOGICAL CHEMISTRY Myles, T., Yun, T. H., HALL, S. W., Leung, L. L. 2001; 276 (27): 25143-25149

    Abstract

    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.

    View details for Web of Science ID 000169800700096

    View details for PubMedID 11312264

  • Thrombin interacts with thrombomodulin, protein C, and thrombin-activatable fibrinolysis inhibitor via specific and distinct domains JOURNAL OF BIOLOGICAL CHEMISTRY Hall, S. W., Nagashima, M., Zhao, L., MORSER, J., Leung, L. L. 1999; 274 (36): 25510-25516

    Abstract

    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.

    View details for Web of Science ID 000082554200046

    View details for PubMedID 10464282

  • Strategies for development of novel antithrombotics: modulating thrombin's procoagulant and anticoagulant properties CELLULAR AND MOLECULAR LIFE SCIENCES HALL, S. W., Gibbs, C. S., Leung, L. L. 1997; 53 (9): 731-736

    Abstract

    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.

    View details for Web of Science ID A1997YB75600002

    View details for PubMedID 9368669

  • Modulation of thrombin's procoagulant and anticoagulant properties THROMBOSIS AND HAEMOSTASIS Leung, L. L., Gibbs, C. S. 1997; 78 (1): 577-580

    Abstract

    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.

    View details for Web of Science ID A1997XE83800102

    View details for PubMedID 9198219

  • SELECTION OF A SUPPRESSOR MUTATION THAT RESTORES AFFINITY OF AN OLIGONUCLEOTIDE INHIBITOR FOR THROMBIN USING IN-VITRO GENETICS JOURNAL OF BIOLOGICAL CHEMISTRY Tsiang, M., Gibbs, C. S., Griffin, L. C., Dunn, K. E., Leung, L. L. 1995; 270 (33): 19370-19376

    Abstract

    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.

    View details for Web of Science ID A1995RP70300030

    View details for PubMedID 7642616

  • FUNCTIONAL MAPPING OF THE SURFACE RESIDUES OF HUMAN THROMBIN JOURNAL OF BIOLOGICAL CHEMISTRY Tsiang, M., Jain, A. K., Dunn, K. E., Rojas, M. E., Leung, L. L., Gibbs, C. S. 1995; 270 (28): 16854-16863

    Abstract

    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.

    View details for Web of Science ID A1995RJ34700062

    View details for PubMedID 7622501

  • Conversion of thrombin into an anticoagulant by protein engineering Nature Leung LLK, Gibbs CS, Coutre SE, Tsiang M, Li WX, Jain AK, Dunn KE, Law VS, Mao CT, Matsumura SY, Mejsa SJ, Paborsky LR 1995