Structural and immunological comparison of human thrombospondins isolated from platelets and from culture supernatants of endothelial cells and fibroblasts

Abstract

Thrombospondin is a 450-kDa glycoprotein secreted by a variety of cells including endothelial cells, fibroblasts and platelets. The aim of this study was to compare the structural and immunological properties of human endothelial, fibroblast and platelet thrombospondins. All three thrombospondins were purified, digested with thermolysin, and the subsequent thermolysin-generated fragments isolated on a Superose 12 gel-permeation column using non-denaturating conditions. Each isolated proteolytic fragment of thrombospondins was then detected using either a radioimmunoassay with a polyclonal antibody or an enzyme-linked immunosorbent assay with three monoclonal antibodies (P10, MA-I, MA-11) directed against different epitopes of whole platelet thrombospondin. The fragmentation pattern of human endothelial thrombospondin consists of six major thermolysin-generated fragments (1 35 -110, 98 -82, 54 -47, 25 -20, 18 -15 and 10 kDa) having molecular masses very similar to those observed with human fibroblast thrombospondin (1 15 -100, 92 -80, 54 -49, 27 -21, 17-13 and 12-10 kDa). Treatment of platelet thrombospondin with thermolysin only generated four proteolytic fragments having molecular masses of 110, 50, 25 and 12/10 kDa respectively. All these proteolytic fragments of endothelial, fibroblast and platelet thrombospondins were recognized by a 'polyclonal antibody. Monoclonal antibodies MA-I and P10 essentially recognized two proteolytic fragments (135 -110;98 -82 kDa) of endothelial and fibroblast (1 15 -100, 92-80 kDa) thrombospondins, and the 110-kDa fragment of platelet thrombospondin. Monoclonal antibody MA-I1 recognized three proteolytic fragments (54 -47, 25 -20, 18 -15 kDa) of endothelial and fibroblast (54-49, 27-21, 17-13 kDa) thrombospondins, and two fragments (50, 25 kDa) of platelet thrombospondin, different from those detected by P10 an MA-I. The results clearly demonstrate that, under non-denaturating conditions, endothelial and fibroblast thrombospondins are structurally different from platelet thrombospondin since two fragments of endothelial thrombospondin (98 -82, 18 -15 kDa), equivalent to those of fibroblast thrombospondin (92-80, 17-13 kDa), are not released from platelet thrombospondin after thermolysin treatment. These three forms of thrombospondin are, however, immunologically indistinguishable. To investigate further the structural differences observed between platelet and the two other forms of thrombospondin, their degree of polymerization was compared. Prior to thermolysin treatment, the three forms of thrombospondin were separated into several oligomers ranging from 450 kDa to 3300 kDa when injected onto a Superose 6 gel-permeation column. In the presence of thermolysin, platelet thrombospondin oligomers were all digested whereas the 3300 kDa oligomer of endothelial or fibroblast thrombospondin was unaffected. All these oligomers were recognized by a polyclonal antibody and three monoclonal antibodies directed against platelet thrombospondin. Such a resistance to proteolysis of endothelia...

Production, Characterization, and Use of Monoclonal Antibodies Directed Against Human Blood Platelet Thrombospondin: Immunologic Comparison with Human Endothelial and Fibroblast Thrombospondins

“…Human endothelial cells and fibroblasts were cultured using methods and materials previously described. 16 Cell supernatants were harvested at confluence.…”

“…Under such experimental conditions, no proteolytic degradation of thrombospondin occurred, as previously reported. 16…”

“…The immunologic compari son of platelet, endothelial, and fibroblast thrombo spondins was performed using a polyclonal antibody 15 and three monoclonal antibodies (P10, MA-I, MA-II). 16,17 Two of these monoclonal antibodies (P10, MA-II) are, respectively, directed against the hemagglutinating activity domain and the heparinbinding domain of platelet thrombospondin, 6 ' 18 which are supposed to be involved in the binding of thrombospondin to stimulated platelets. 2 The results clearly indicate that when using a polyclonal antibody and the aforementioned monoclonal antibodies, thrombospondin originating from platelets, endothelial cells, and fibroblasts are immunologically indistinguishable.…”

Cell attachment and fibrinogen binding properties of platelet and endothelial cell thrombospondin are not affected by structural differences in the 70 and 18 kDa protease‐resistant domains

“…Endothelial cells from human umbilical cord veins and smooth muscle cells from human aorta were cultured using methods and materials previously described [5,8]. Human endothelial cell and platelet thrombospondin (TBSP) were purified as outlined in [S] .…”

“…We reported recently [5] that human endothelial cell TBSP is more resistant to proteolysis than platelet TBSP. However, the molecular basis for this increased resistance to proteolysis of endothelial cell TBSP is unknown.…”