Characterization of an anti‐thrombospondin monoclonal antibody (P8) that inhibits human blood platelet functions

Abstract

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‐stimulated 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.

Selective inhibition of platelet macroaggregate formation by a recombinant heparin-binding domain of human thrombospondin.

“…This resulted in the formation of smaller aggregates, as we previously observed with mAb MAIL 11 In agreement with previous work that demonstrated the role of endogenous or exogenously added TSP in platelet aggregation, 10 - 43 we found that higher concentrations of ADP (10 /miol/L) or thrombin (0.2 U/mL) could overcome the inhibitory effect of the recombinant proteins. These results underline the important role played by TSP in promoting platelet aggregation at low concentrations of stimulating agents.…”

A Model of Platelet Aggregation Involving Multiple Interactions of Thrombospondin-1, Fibrinogen, and GPIIbIIIa Receptor

“…12D). This latter model may appear controversial because platelets from patients with Glanzmann's thrombastenia, which lack GPIIbIIIa, have been shown to express normal levels of TSP at the cell surface upon thrombin activation, with no aggregation detected in an aggregometer (23,47). However, this apparent contradiction with our model may rather arise from the facts that (i) micro-aggregates of Ͻ10 platelets observed in one Glanzmann's thrombastenia patient by phase contrast microscopy may not be detected by aggregometry (48) and (ii) electron microscopy studies revealed that upon thrombin activation, TSP was not distributed normally on the platelet surface of one Glanzmann's thrombastenia patient, with decreased size and number of TSP clusters (49).…”

mda -9/Syntenin promotes metastasis in human melanoma cells by activating c-Src

“…Cell extracts in RIPA buffer were prepared, and equal amounts of proteins were resolved in SDS/PAGE, transferred, and evaluated for c-Src and FAK expression and activity with the indicated antibodies as previously described (4). For coimmunoprecipitations, equivalent amounts of cell lysates were incubated for 2 h at 4°C with antibodies to c-Src coupled to protein G-Sepharose as described previously (32). Immunoprecipitates were extensively washed, and the eluted precipitates were resolved by SDS/7% PAGE, transferred, and probed with the appropriate antibodies.…”