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ABSTRACT Under the framework of an project 'Building Asian Climate Change Scenarios by Multi-Regional Climate Models Ensemble', the ability of eight regional climate models and two fine-resolution global climate models to reproduce late 20th century (1981-2000) precipitation climatology is assessed. Future precipitation change (2041-2060) under the A1B scenario is also quantified by applying four different ensemble methods: equal weighting, weighted mean ( WM), reliability ensemble averaging ( REA) and performance-based ensemble averaging, after applying fourfold cross-validation using observation and multi-model-simulated precipitation. The results indicate that the ensemble of simulated precipitation outperforms any single RCM in many aspects. Among the four ensemble approaches, the WM and REA methods show better skill in improving the simulation results, and are used for ensemble prediction of regional climate in Asia. Under the A1B scenario, the WM method estimates future precipitation change of approximately 0.2 mm day−1 with less precipitation in northern and western China and northern India, and more precipitation in most other areas in Asia. The future annual precipitation will decrease by 0.1-0.5 mm day−1 in northern India, Pakistan and the central area of southern China. No significant change is found over eastern Kazakhstan, Mongolia, north-central and western China. [ABSTRACT FROM AUTHOR]

Summary. P-selectin, a member of the selectin family of adhesion molecules, is present in endothelial Weibel–Palade bodies and platelet α-granules, and is rapidly expressed on their surface upon activation, resulting in leucocyte adhesion. LYP20 is a functional monoclonal antibody previously generated in our laboratory that binds with high affinity and specificity directed against P-selectin. This binding is largely imparted by the specific sequence of amino acids present on the hypervariable portions of the IgG chains. We now show that a peptide derived from the heavy chain of mAb LYP20 dose dependently inhibits the adhesion of poly morphonuclear cells to resting and thrombin-activated endothelial cells (EC) and platelets. The scrambled form of this peptide, identical in amino acid composition to the authentic peptide but with altered sequence, was not inhibitory at corresponding concentrations. Binding studies revealed that this peptide also dose dependently bound to both resting and thrombin-activated EC and platelets. Our results may prove useful for the development of new therapeutic inhibitors to modulate leucocyte interactions in inflammatory disorders. [ABSTRACT FROM AUTHOR]

Stimulated human blood platelets release thrombospondin, an α-granule glycoprotein of 450 kDa. The aim of this work was to characterize an anti-thrombospondin monoclonal antibody (P8) in order to study the role of thrombospondin in platelet functions. The presence of thrombospondin receptor sites on resting and thrombin- stimulated platelets of three Glanzmann's thrombasthenia patients and normal donors was investigated using the P8 monoclonal antibody. Monoclonal antibody P8 was extensively characterized using ELISA, immunoprecipitation, immunoadsorbent affinity chromatography combined with tryptic peptide map analysis and crossed immunoelectrophoretic techniques. Labelled P8 bound strongly to thrombin-stimula ted normal platelets (n = 14917 ± 420, mean ± SD) (Kd = 9.2 ± 3.0 nM) and poorly to resting platelets (n = 2697 ± 1278) ((Kd = 24.8 ± 18.6 nM). Moreover, the number of binding sites for P8 on thrombin-stimulated platelets from three Glanzmann's thrombasthenia patients, lacking the IIb-IIIa glycoprotein complex, were found similar to normal samples. F(ab')2 fragments of P8 inhibited aggregation of, and reduced secretion from, washed platelets stimulated by low concentrations of thrombin (0.05 -0.06 U/ml) and collagen (0.5 - 0.6 μg/ml). F(ab')2 fragments of P8 inhibited thrombin-induced platelet aggregation, but did not reduce fibrinogen binding (n) nor affect its dissociation constant ((Kd). Inhibition of platelet aggregation by P8 suggests that thrombospondin plays an active role in promoting platelet aggregation, at low concentrations of thrombin and collagen. Normal binding of P8 to thrombin-stimulated Glanzmann thrombasthenic platelets indicates the presence of a thrombospondin receptor on the platelet surface distinct from the GPIIb-IIIa complex. [ABSTRACT FROM AUTHOR]

Thrombospondin, a 450-kDa glycoprotein composed of three disulphide linked chains, is located in human blood platelet alpha-granules and is released from platelets upon stimulation. This glycoprotein is though to play a major role in platelet aggregation. The aim of this study was to characterize two monoclonal antibodies (P10 and P12) directed against human blood platelet thrombospondin. When the released material obtained after stimulation of platelets with thrombin in the presence of 2 Mm calcium was immediately treated with EDTA, labelled with 125I and incubated with monoclonal antibodies P10 and P12, both immunoprecipitated a major labelled protein band with a molecular mass of 160 kDa and a weaker band at 146 kDa, as analysed on reduced dodecyl sulphate/polyacrylamide gels. The major band corresponds in molecular mass to the thrombospondin subunits. If, however, the released material was left in the presence of Ca2+ for 48 h, then the main band was at 130 kDa and in addition one minor protein band (75 kDa) was immunoprecipitated by P10 whereas P12 recognized two minor protein bands (75 and 60 kDa). When P10 and P12 were incubated with 125I-labelled platelet releasates treated for 48 h at 4°C with 10 mM EDTA, three major protein bands (160, 146 and 130 kDa) were immunoprecipitated in addition to the minor bands mentioned above. These results indicate that thrombospondin is probably degraded by the endogenous platelet calcium-dependent protease. Investigation of tryptic peptide fragments of thrombospondin isolated by fast protein liquid chromatography showed that 125I-labelled antibody P10 bound to 400-kDa and 120-kDa fragments whereas 125I-labelled P12 only recognized a 100-kDa fragment. Competition studies involving solid-phase antibody binding and double antibody sandwich assays showed that P10 and P12 were directed against different determinants of thrombospondin. Purified thrombospondin, isolated in the presence of calcium, either directly or after treatment with EDTA, haemagglutinated trupsinized, formaldehyde-fixed sheep erythrocytes identically. The haemagglutination activity of EDTA-treated thrombospondin was inhibited by P10 and enhanced by P12. On the other hand, P10 and P12, despite their binding to calcium-treated thrombospondin, had no effect on its haemagglutination activity. Monoclonal antibodies P10 and P12 could be useful tools to investigate the role of thrombospondin in platelet aggregation. [ABSTRACT FROM AUTHOR]

Human platelets were surface-labeled by the periodate/NaB3H4 method or by lactoperoxidase-catalysed iodination with 125I. The labeled platelets were treated with chymotrypsin under conditions known to give platelets which aggregate with fibrinogen without stimulation with ADP. Platelets and supernatant were then analysed by various gel electrophoretic techniques including isoelectricfocusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing or non-reducing conditions and two-dimensional non-reduced/reduced sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by fluorography or indirect autoradiography. Chymotrypsin-treatment of surface-labeled platelets degraded the major glycoproteins Ib, IIb and IIIa but also GP1204.9–5.4, GPIc and GPV. The membrane-bound fragments of GPIb, IIb and IIIa could be identified and also the supernatant fragments of GPIb and GPV. GPIIIa was also cleaved within a loop structure formed by disulfide bond(s). The fact that remnants of both GPIIb and IIIa are left on chymotrypsin-treated platelets which aggregate spontaneously with fibrinogen may indicate that a complex formed by these remnants constitutes the fibrinogen-binding site on platelets. [ABSTRACT FROM AUTHOR]

The specificy of live monoclonal antibodies (P1 -P6) against platelet surface components was determined by immunoprecipitation of surface-labelled platelets from normal donors and patients with known platelet glycoprotein defects, followed by analysis by gel electrophoresis. Three (P2, P4 and P6) precipitated glycoproteins lI b and IlI a and, in additions P2 precipitated glycoprotein I a. P1 precipitated normally only glycoprotein lb also Ia when the platelets were pretreated with neuraminidase. P3 precipitated principally glycoprotein Ia but glycoprotein Eb was also weakly precipitated. The effects of the monoclonals on platelet function were tested. P1 and P2 completely inhibited and P3 slightly inhibited thrombin-induced platelet aggregation. P2 also inhibited collagen-induced aggregation and partially inhibited ADP-induced platelet aggregation. P3, P4 and P6 partially inhibited ADP-induced platelet aggregations None had any effect on ristocetin-induced aggregation despite P1 and P3 binding to glycoprotein lb. These results confirm the role of glycoproteins IIb and lIla in aggregation induced by various agents and suggest that the function of glycoprotein lb in thrombin-induced aggregation is more important than previously suspected and that glycoprotein Ia may also be involved in platelet functions. [ABSTRACT FROM AUTHOR]

Platelets from Glanzmann's thrombasthenia patients and from normal donors were surface labelled by techniques specific for sugars (terminal sialic acid, penultimate galactose/N-acetylgalactosamine) and proteins (tyrosine-histidine residues). These labelled platelets were solubilized in sodium dodecyl sulphate and separated on a two-dimensional electrophoretic system [O'Farrell, P. H. (1975) J. Biol. Chem. 250,4007–4021] first according to their isoelectric point (pI) and then according to their molecular weight. In addition, unlabelled sodium- dodecyl-sulphate-solubilized platelets were separated on a two-dimensional polyacrylamide gel and the glyco-proteins were identified by binding of 125I-labelled Lens culinaris lectin (specific for mannose and glucose). In one Glanzmann's thrombasthenia patient glycoproteins IIbA1 and IIIaA1 were absent and in two others lower amounts of two glycoproteins were found in posit ions similar or close to these two membrane glycoproteins. The terminal sialic acid moieties of major glycoproteins (IbA1, IbB1 and IIIbA1) were more intensely labelled in Glanzmann's thrombasthenia than in normals and these glycoproteins had an altered pI. A glycoprotein tentatively designated as Ic/IIa(?) had an altered pI and was labelled more intensely in Glanzmann's thrombasthenia platelets than in normals. A number of low-molecular-weight glycoproteins (IVa, IVb, VII) and one high- molecular weight glycoprotein normally found in platelets of healthy donors were reproducibly not detected in Glanzmann's thrombasthenia platelets. These results obtained by a combination of highly sensitive techniques strongly indicate that in Glanzmann's thrombasthenia the absence or reduction of two major membrane glycoproteins (IIbA1, IIIaA1) is not the only defect but that there appears to be a profound perturbation of the platelet membrane surface. [ABSTRACT FROM AUTHOR]

This study investigated the interaction of apoptotic polymorphonuclear neutrophils (PMN) with thrombospondin (TSP), an important event mediating the clearance of apoptotic neutrophils by macrophages. We developed an in vitro assay to examine this interaction. Based on this assay, we found that apoptotic but not fresh PMN bound specifically to surface-immobilized TSP (33 ± 0.03 × 103 cells/well) compared to fibrinogen, fibronectin or laminin (8.0 ± 0.3 × 103 cells/well). Moreover, the binding was specific for surface bound but not soluble TSP and appeared to be divalent cation dependent, was not significantly inhibited by heparin and was sensitive to cycloheximide (CHX) treatment of senescent PMN (>90% inhibition at 10 μM CHX). In contrast to the binding studies, phagocytosis of senescent PMN by macrophages was not affected by EDTA or cycloheximide. Phosphatidyl-L-serine liposomes, phospho-L-serine, glucosamine, galactosamine, and the acetylated sugars had no effect on phagocytosis. We conclude that: (i) there was specific binding of senescent human PMN to immobilized TSP, which is divalent cation dependent and requires new protein synthesis in the PMN during senescence; (ii) in addition to the recently defined TSP-dependent pathway, there is a TSP-independent pathway mediating phagocytosis of senescent PMN by macrophages. The identity of this pathway remains to be defined. [ABSTRACT FROM AUTHOR]

Extensive evidence is now available to show that the human CD36 antigen is a cellular receptor for thrombospondin, collagen, modified low-density lipoproteins, and long-chain fatty acids. Moreover, CD36 functions as one of the receptors that mediates the adhesion of Plasmodium-falciparum-infected erythrocytes to microvascular endothelium. In an attempt to identify new functional sites of this surface glycoprotein, anti-CD36 monoclonal antibodies were prepared using a vaccinia CD36 recombinant virus as a highly efficient immunization vector. In functional studies, one of these antibodies (clone 10/5) strongly inhibited the adhesion of P. falciparum-infected erythrocytes to purified CD36. This antibody also potentiated ADP-induced platelet activation. In contrast, a second antibody (clone 13/10) did not affect the cytoadherence of infected erythrocytes or platelet functions. Previous structural work pertformed on these antibodies has shown that clone 10/5 is directed against an epitope within the CD36 domain 155-183, whereas clone 13/10 interacts with another antigenic determinant defined by amino acids 30-76 [Daviet, L,, Buckland, R., Puente Navazo, M. D. & McGregor, J. L, (1995) Biochem. J. 305, 221-224]. Taken together; these current studies show that: (a) the methodology of immunization using recombinant vaccinia virus is a powerful tool in the generation of monoclonal antibodies directed against polyimmunogenic membrane glycoproteins such as CD36; (b)the CD36 domain, recognized by clone 10/5 but not by 13/10, is functionnally important regarding the adhesion of P. falciparum-infected erythrocyte and CD36-dependent platelet activation. [ABSTRACT FROM AUTHOR]

The α[Sub]β[SUB3] integrin complex is thought to play an important role in in vivo melanoma tumor growth and metastasis. However, not all human metastatic melanomas, present in lymph node biopsies, express avfi3. In this study, we have investigated the possibility that certain melanoma cell lines can grow aggressively in vivo in the absence of α[Sub]β[SUB3] expression. Established human melanoma cell lines (M[SUB3]Da., M[SUB4]Beu.) were isolated from an achromic skin metastasis or lymph nodes. Two stable variants (7GP, TI P26), derived from a poorly metastatic M[SUB4]Beu. melanoma cell line, were isolated by sequential selection for spontaneous metastasis formation in an immunosuppressed newborn rat model. Flow-cytometry analysis shows an absence of the β[SUB3] integrin subunit (less than 2% of parental levels) in the two variant melanoma cell lines (7GP, T1 P26) compared to M[SUB3]Da. and M[SUB4]Beu. cell lines which express a relatively high number of fl3 subunits. The expression levels of the integrin subunits β[SUB1], β[SUB5], β[SUB6] and α[Sub] were found to be similar for all four melanoma cell lines. Northern blot analysis confirmed the absence of β[SUB3] in 7GP or TIP26 cell lines and its presence in M3Da. and M[SUB4]Beu. Moreover, similar levels of ay transcript were present in the four melanoma cell lines. The functional effect of the absence of fl3 was investigated by subcutaneously implanting the variants and the melanoma cell lines in nude mice. Variant 7GP and TIP26 cell lines yielded tumors which were larger and grew at a faster rate than tumors in M[SUB3]Da. or M[SUB4]Beu. cell lines. The β[SUB3] integrin subunit was not detectable on the surface of cells harvested from tumors after 20 or 35 days. Similarly, subcutaneous innoculation of the two variants into immunosuppressed newborn rats gave rise to extensive spontaneous lung metastases compared to the M[SUB4]Beu. cell line. These results provide evidence that a population of melanoma cells can grow aggressively in vivo and metastasize in the absence of β[SUB3] or α[Sub]β[SUB3] integrin complex. Our results may have clinical relevance and suggest that certain types of melanomas in patients may grow and spread in the absence of the α[Sub]β[SUB3] integrin complex. [ABSTRACT FROM AUTHOR]

Human thrombospondin, a 450-kDa glycoprotein isolated from platelets and endothelial cells, specifically interacts with osteonectin, a protein of 30 kDa isolated from bovine bones and human platelets. Using ELISA, purified osteonectin binds to solid-phase-adsorbed thrombospondin with a dissociation constant (Kd) of 0.7 nM. Binding of thrombospondin to solid-phase-adsorbed osteonectin was also observed {Kd = 0.86 nM), The interaction of thrombospondin with solid-phase-adsorbed osteonectin was significantly decreased (81% inhibition) when using an excess of fluid-phase osteonectin. Thrombospondin-osteonectin complex formation was calcium-dependent as shown by a 50-80% inhibition in the presence of EDTA. None of the proteins known to interact with thrombospondin (fibrinogen, fibronectin, collagen, plasminogen) had a significant inhibitory effect on thrombospondin-osteonectin complex formation. This selective interaction was confirmed by affinity chromatography, lodinated osteonectin, previously incubated with purified thrombospondin, specifically bound to an anti-thrombospondin monoclonal antibody (P10) linked to protein-A--Sepharose 4B. Elution of the antithrombospondin antibody from protein A allowed the recovery of the thrombospondin-osteonectin complex in the eluate, as judged by SDS/polyacrylamide gel electrophoresis and autoradiography. Blotting of purified thrombospondin to osteonectin adsorbed onto nitrocellulose further confirmed complex formation. In addition, when released from thrombin-stimulated platelets, thrombospondin and osteonectin bound to anti-thrombospondin IgG-coated plates indicating that osteonectin was complexed to thrombospondin once the platelet-release reaction has occurred. [ABSTRACT FROM AUTHOR]

Compares the structural and immunological properties of human endothelial, fibroblast and platelet thrombospondins. Indication that under non-denaturing conditions, endothelial and fibroblast thrombospondins are structurally different from plaletet thrombospondin; Observation that these three thrombospondins are immunologically indistinguishable.