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Biomedical subjects

Perumal Thiagarajan

Publications and source records attributed to Perumal Thiagarajan.

14 recordsLinked to original sources
PubMed2006

Lactadherin binding and phosphatidylserine expression on cell surface-comparison with annexin A5.

BACKGROUND: Transbilayer movement of anionic phospholipids from the inner to the outer leaflet of the plasma membrane occurs during platelet activation, red cell senescence, and apoptosis. The anionic phospholipid-binding protein, annexin A5, has been used to detect the presence of phosphatidylserine on the outer leaflet of the cell membrane. Lactadherin, a glycoprotein secreted by macrophages, binds to phosphatidylserine on apoptotic cells and promote their clearance by macrophages. METHODS: The authors isolated and labeled lactadherin and annexin A5 with FITC and compared their ability to detect phosphatidylserine expression by flow cytometry. RESULTS: FITC-lactadherin induced greater shift in the histogram and a higher mean fluorescence intensity than FITC-annexin A5 when platelets were activated with thrombin (0.1 unit/mL) or Ca(2+) ionophore A23187 (1 microM). Similarly, lactadherin was more sensitive in detecting phosphatidylserine in red cells induced to express phosphatidylserine. Also, in HL 60 cells undergoing apoptosis, lactadherin detected phosphatidylserine expression earlier than annexin A5. In patients with disseminated intravascular coagulation, lactadherin detected phosphatidylserine-expressing platelets in most patients, whereas under similar conditions, FITC-annexin A5 could not. CONCLUSIONS: The authors' studies show that FITC-lactadherin is a better probe than annexin A5 in detecting phosphatidylserine-expressing activated platelets, red cells, and apoptotic cells.

Animals↗
PubMed2006

Leukocyte adhesion and thrombosis.

PURPOSE OF THE REVIEW: The consequences of arterial thrombosis such as myocardial infarction, stroke and peripheral vascular occlusion are the leading causes of morbidity and mortality. A high leukocyte count and an elevation in inflammatory markers are identified as significant risk factors for thrombosis. Leukocytes form the front line in defense against infection and are the first cells arriving at the site of inflammation. This review summarizes the cellular and molecular mechanisms by which adherent leukocytes can induce a prothrombotic state. RECENT FINDINGS: Circulating tissue factor has been recognized as a potential prothrombotic factor initiating thrombosis after vascular injury. The tissue factor is present on microvesicles originated from activated leukocytes. Leukocytes generate tissue factor containing microvesicles following stimulation with cytokines and following platelet adhesion via P-selectin. Additionally, activated leukocytes release several mediators, such as cathepsin G and elastase, which can activate both the coagulation cascade and platelets. Furthermore, new roles for leukocytes have been identified in vascular injury in sickle cell anemia, in vascular occlusion following the rupture of atherosclerotic plaque, and in thrombotic complications of myeloproliferative diseases. SUMMARY: Leukocyte adhesion to endothelium and platelets plays an important role in the activation of the coagulation cascade. An excessive activation of leukocytes during the inflammatory process may induce a systemic procoagulant state. Elucidation of critical steps in activation of coagulation by leukocytes may offer a new therapeutic target for antithrombotic therapy based on blocking leukocyte adhesion.

Animals↗
PubMed2005

Tissue-factor-bearing microvesicles arise from lipid rafts and fuse with activated platelets to initiate coagulation.

Tissue factor (TF) circulates in plasma, largely on monocyte/macrophage-derived microvesicles that can bind activated platelets through a mechanism involving P-selectin glycoprotein ligand-1 (PSGL-1) on the microvesicles and P-selectin on the platelets. We found these microvesicles to be selectively enriched in both TF and PSGL-1, and deficient in CD45, suggesting that they arise from distinct membrane microdomains. We investigated the possibility that microvesicles arise from cholesterol-rich lipid rafts and found that both TF and PSGL-1, but not CD45, localize to lipid rafts in blood monocytes and in the monocytic cell line THP-1. Consistent with a raft origin of TF-bearing microvesicles, their shedding was significantly reduced with depletion of membrane cholesterol. We also evaluated the interaction between TF-bearing microvesicles and platelets. Microvesicles bound only activated platelets, and required PSGL-1 to do so. The microvesicles not only bound the activated platelets, they fused with them, transferring both proteins and lipid to the platelet membrane. Fusion was blocked by either annexin V or an antibody to PSGL-1 and had an important functional consequence: increasing the proteolytic activity of the TF-VIIa complex. These findings suggest a mechanism by which all of the membrane-bound reactions of the coagulation system can be localized to the surface of activated platelets.

Blood Coagulation↗
PubMed2005

Effect of P-selectin on phosphatidylserine exposure and surface-dependent thrombin generation on monocytes.

OBJECTIVE: Stimulation of monocytes with P-selectin induces the synthesis of an array of mediators of inflammation, as well as the expression of tissue factor (TF), the main initiator of coagulation. Because the membrane-bound reactions of coagulation are profoundly influenced by the presence of phosphatidylserine on the membranes of cells, factors that increase its expression may have an impact on coagulation. METHODS AND RESULTS: Using flow cytometry, we studied the effect of P-selectin on phosphatidylserine expression in blood monocytes and in the monocytic cells, THP-1. Soluble P-selectin at biologically relevant concentrations (0.31 to 2.5 microg/mL) induced a time-dependent increase in phosphatidylserine expression, an effect that could be inhibited with an anti-PSGL-1 blocking antibody, and by genistein, a tyrosine kinase inhibitor. Binding of activated platelets to THP-1 cells also resulted in a significant increase in phosphatidylserine expression that was dependent on PSGL-1. Consistent with the role of phosphatidylserine on surface-dependent reactions of coagulation, treatment of monocytic cells with soluble P-selectin led to increased thrombin generation. We excluded P-selectin induced apoptosis of monocyte as a mechanism for the increased phosphatidylserine exposure. CONCLUSIONS: In summary, we show that P-selectin, either soluble or in its membrane-bound form, induces phosphatidylserine exposure in monocytes through a mechanism dependent on PSGL-1.

Apoptosis↗
PubMed2005

Antiphospholipid syndrome with anti-prothrombin autoantibodies in a patient with an axial-flow left ventricular assist device.

Autoantibodies to prothrombin, first described almost 50 years ago, are paradoxically associated with thrombosis. Described is an unusual case of fatal hypercoagulability in a patient with multiple arterial and venous thromboembolic complications despite intense anticoagulation while being bridged to transplantation with a left ventricular assist device. Serum analysis revealed the presence of prothrombin autoantibodies and high titers of anti-nuclear antibodies, and autopsy revealed pulmonary arteriolar vasculitis. These findings suggest an autoimmune basis for the presence of anti-prothrombin antibodies and the hypercoagulable state observed in the present case.

Antibodies, Antiphospholipid↗
PubMed2005

The role of lactadherin in the phagocytosis of phosphatidylserine-expressing sickle red blood cells by macrophages.

Lactadherin is a phosphatidylserine-binding glycoprotein secreted by macrophages. Less than 0.5% of normal circulating red cells showed any binding to lactadherin. However, the red cells from patients with sickle cell disease showed 2 to 10-fold increases in lactadherin binding. Further, lactadherin stimulated the phagocytosis of sickle red blood cells by macrophages suggesting a potential role in sickle red cell clearance.

Adult↗
PubMed2004

Sulfatides activate platelets through P-selectin and enhance platelet and platelet-leukocyte aggregation.

OBJECTIVE: Sulfatides are sulfated glycosphingolipids present on the surface of a variety of cells; however, their exact physiological function is not known. Recently, we have shown that the inhibition of sulfatide-P-selectin interactions leads to disaggregation of platelet aggregates. METHODS AND RESULTS: In this study, we show that sulfatides activated platelets as they increased activation of GPIIb/IIIa (PAC-1 epitope) and expression of P-selectin on the platelet surface. Furthermore, sulfatides aggregated washed platelets in a dose-dependent manner and enhanced platelet aggregation in platelet-rich plasma. Previous activation of platelets was necessary for this effect. Monoclonal anti-P-selectin antibodies inhibited not only sulfatide-induced PAC-1 binding to platelets but also sulfatide-induced platelet aggregation, suggesting that sulfatides activate platelet GPIIb/IIIa via signaling through P-selectin. The proaggegatory effect of sulfatides was also observed in an ex vivo thrombosis model using whole blood and pulsatile flow at 37 degrees C. In this model, sulfatides significantly enhanced platelet aggregation and the formation of platelet-leukocyte aggregates. CONCLUSIONS: We show that sulfatide-P-selectin interactions lead to subsequent platelet activation and P-selectin expression, forming a positive feedback loop that can potentiate formation of stable platelet aggregates. In addition, sulfatides enhance the aggregation of platelet-leukocyte aggregates. These mechanisms may play a significant role in hemostasis and thrombosis.

Adenosine Diphosphate↗
PubMed2004

Characterization of autoantibodies against sulfatide from a V-gene phage-display library derived from patients with systemic lupus erythematosus.

Sulfatide, ceramide galactosyl-3'-sulfate, is mainly present in nervous tissue, kidney, testis, red blood cells, platelets and granulocyte. Antibodies to sulfatide are present in many patients with demyelinating peripheral neuropathy, HIV infection and systemic lupus erythematosus and may account for some of the clinical manifestations. To evaluate the effect of such antibodies, we have constructed a phage-display antibody fragment library from the lymphocytes of patients with systemic lupus erythematosus. Sulfatide-reactive phage were selected by absorption and elution on sulfatide liposomes and soluble single chain variable fragment (ScFv) were isolated from individual colonies and tested in an ELISA assay for binding to bovine brain sulfatide. Five ScFv clones that bound sulfatide were isolated. Two of the clones, PH5 and PA38, bound sulfatide but not phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine, sphingomyelin or ceramide. These two clones also bound sulfatide from human red blood cells. The DNA encoding the fragments was sequenced, revealing predicted polypeptides of 19 kDa for PH5 containing only variable heavy (VH) sequences, and 31 kDa for PA38, with both VH and variable light (VL) sequences. Although they had similar antigen specificities, the VH domains of the two clones were derived from different heavy-chain families. The clustered mutational patterns in the complementarity-determining region (CDR) of the heavy chains in both clones suggest that the V-domains are the products of antigen-driven B cell clonal maturation leading to the development of sulfatide-binding specificity. These results show the presence of sulfatide-specific antibodies in lupus patients, and allow us to test the possibility that the interaction of the antibodies with sulfatide may contribute to some of the symptoms. In addition, the antibodies provide useful reagents to test the role of sulfatide in pathophysiological processes.

Amino Acid Sequence↗
PubMed2004

Lupus-derived antiprothrombin autoantibodies from a V gene phage display library are specific for the kringle 2 domain of prothrombin.

Autoantibodies to prothrombin are common in patients with systemic lupus erythematosus. Although their presence is a risk factor for thrombosis, neither their origin nor their precise role in inducing the procoagulant state is known. We have developed a phage-display antibody library from patients with systemic lupus erythematosus with antiprothrombin antibodies, and we have selected two single-chain Fv antibody fragments (ScFvs) by panning on a prothrombin-coated surface. In prothrombin activation assays using purified components, these antibodies promoted prothrombin activation. These ScFvs, termed AN78 and AN129, bound to immobilized prothrombin in a concentration-dependent specific manner but not to other anionic phospholipid binding proteins such as beta2-glycoprotein I or annexin V. Phosphatidylserine-bound prothrombin, but not soluble prothrombin, inhibited the binding suggesting that the epitope is available only on immobilized prothrombin. To localize the epitope, prothrombin was treated with thrombin or factor Xa and various prothrombin activation fragments were subsequently isolated and tested in ELISA with the ScFvs. Both AN78 and AN129 bound to prethrombin I (the fragment lacking the Gla domain and the first kringle domain), to fragment 1.2 (containing Gla and the two kringle domains only) and to fragment 2 but not to thrombin, thus localizing the cognate epitope to the kringle 2 domain in prothrombin. Analysis of the cDNA sequences of these antibodies show clustered mutational patterns in the complementarity determining region, suggesting that variable domains are the products of antigen-driven B cell clonal maturation.

Amino Acid Sequence↗
PubMed2004

Cross-clade HIV-1 neutralization by an antibody fragment from a lupus phage display library.

A single-chain fragment containing antibody V domains (scFv) isolated from a lupus antibody library displayed the ability to bind gp120 and the conserved gp120 determinant composed of residues 421-436. The scFv neutralized R5 and X4-dependent HIV-1 strains from clades B, C, and D. The lupus repertoire may be useful as a source of neutralizing antibodies to HIV.

Dose-Response Relationship, Immunologic↗
PubMed2002

Polymorphisms beta2-glycoprotein I: phospholipid binding and multimeric structure.

beta2-glycoprotein I is a phospholipid-binding protein of 326 amino acids and is found in plasma at a concentration of approximately 200 microg/ml. It has a sequence of positively charged amino acids located at the carboxy terminus that mediates anionic phospholipid binding. Two polymorphisms (306Cys-->Gly and 316Trp-->Ser) located at the phospholipid-binding site have been described. Homozygous state for either mutation and a compound heterozygous state show no phospholipid binding. Interestingly, heterozygotes for either 306Cys-->Gly or 316Trp-->Ser mutation have normal cardiolipin binding suggesting that beta2-glycoprotein I may circulate as a multimeric structure where wild-type subunits compensate the defective binding of the mutant ones. We investigated the effect of these mutations on quaternary structure of beta2-glycoprotein I and phospholipid binding. As previously reported, under native conditions, beta2-glycoprotein I shows an apparent molecular weight of approximately 320 kDa and it can be dissociated into subunits of lower molecular weight by boiling in 6 M urea. We show that the multimeric structure is not affected by the presence of mutations in the phospholipid-binding domain. beta2-glycoprotein I induces aggregation of anionic phospholipid vesicles suggesting again a multivalent interaction where at least two binding sites are required to bridge adjacent vesicles. beta2-glycoprotein I-induced aggregation does not cause vesicle fusion or damage as demonstrated by fluorescence resonance energy transfer (FRET) or encapsulated calcein release. In conclusion, the normal cardiolipin binding in heterozygous state for mutations at phospholipid-binding domain may be due to the multimeric structure of beta2-glycoprotein I.

Binding Sites↗
PubMed2002

New targets for antithrombotic drugs.

The normal hemostatic process is initiated by disruption in the vascular continuity and exposure of the subendothelial components. Platelets adhere to subendothelium-bound von Willebrand factor via glycoprotein (GP) Ib complex. This initial interaction per se and the release of platelet agonists transduce signals that lead to the rise in intracellular Ca(2+). The rise in Ca(2+) induces shape change, prostaglandin synthesis, release of granular contents and conformational changes in platelet Gp IIb-IIIa. Gp IIb-IIIa in activated platelets becomes competent to bind fibrinogen and other adhesive proteins and mediates platelet cohesion (primary hemostatic plug). Furthermore, the activated platelet surface provides an efficient catalytic surface for the coagulation reactions, ultimately resulting in the formation of fibrin (secondary hemostasis). Normally the hemostatic process plays a delicate balance between keeping the blood in the fluid state to maintain flow and rapidly forming an occluding plug following vessel injury. Thrombosis occurs because of alteration in this delicate balance. Consequences of thrombosis are a major cause of morbidity and mortality in industrialized countries. Arterial thrombosis occurs in the setting of previous vessel wall injury mostly because of atherosclerosis, while venous thrombosis occurs in areas of stasis. The recent advances in our understanding of the hemostatic process have led to a better elucidation of the mechanism of action of many antithrombotic drugs and identification of new targets for drug development. The molecular target of the well known antiplatelet drug ticlopidine has been identified. Large numbers of IIb-IIIa inhibitors have been developed based on the crystal structure of a potent antagonist echistatin. The mechanism of action of heparin has been defined at the molecular level. As a result a synthetic pentasaccharide, based on antithrombin-binding domain of heparin, has been developed and tested successfully in clinical trials. New generation direct thrombin inhibitors are being developed based on the crystal structure of thrombin. Factor Xa has a critical position at the convergence of intrinsic and extrinsic pathway ways. The clinical tolerability and the efficacy of low molecular weight heparins led to the concept that inhibition of further thrombin generation, by blocking factor Xa alone, can be an effective way of preventing thrombus growth without inactivating thrombin. A large number of specific factor Xa inhibitors are under development. Some of these drugs have already undergone preliminary clinical trials and appear to be promising. Future clinical trials will determine whether these new drugs will provide better risk-benefit ratio in treatment of thrombotic disorders.

Anticoagulants↗