An inhibitor of plasminogen activator was purified to apparent homogeneity from human umbilical vein endothelial cell conditioned medium. The purification was achieved by a speedy and simple two-step procedure, without the use of denaturants. The purified protein was a single-chain glycoprotein with apparent molecular mass of 48 kDa.The purified inhibitor had a specific activity of 8500 U/mg protein and the activity could be stimulated about fourteenfold by treatment with denaturants.An antiserum to the purified inhibitor was raised in rabbits. It recognised plasminogen activator inhibitor from platelets and plasma as well as from cultured endothelial cells. The immunoglobulin fraction of the antiserum neutralised the functional activity of the inhibitor from all these sources.The activity of the fibrinolytic system depends on the cleavage of the zymogen plasminogen to form plasmin by plasminogen activators. Two major immunologically distinct types of plasminogen activator, tissue-type (tPA) and urokinase (u-PA) have been identified (reviewed in [l]). t-PA is present in normal resting plasma at a molecular mass of about 110 kDa [2 -41, in contrast to the 65-kDa form isolated from tissues [5]. The high-molecular-mass t-PA in plasma is now known to be an equimolar complex of t-PA with a plasminogen activator inhibitor (PAI) [3, 41. Inhibitors active against both t-PA and u-PA have been discovered in bovine [6] and human [7 -91 endothelial cells, in platelets [lo, 111 and in plasma [3, 12 -141. Immunological cross-reactivity between the bovine endothelial and human platelet inhibitor has been established [15] and this type of PA1 is now designated PAI-1 [16].The bovine endothelial cell PA1 is in an inactive or latent form that is converted to an active form in the presence of denaturants such as SDS and guanidinium hydrochloride [17]. This inhibitor has been purified using SDS/polyacrylamide gel electrophoresis (SDS-PAGE) as the final preparative step; thus the purified product was active [18].In the study reported here, we aimed to purify PA1 from human endothelial cells in the absence of denaturants. T h s Correspondence to
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...
The structural and immunological properties of human thrombospondins isolated from platelets and from endothelial cells were compared. Both thromhospondins were digested with either trypsin or thermolysin, in the presence or absence of calcium, then injected onto a Superose 12 gei filtration column. The isolated the~olysin-generated fragments of throm~s~ndins were identified by radioimmunoassays using either different monoclonal antibodies or a polyclonal antibody directed against platelet thrombospondin. The results show that platelet and endothelial thrombospondins are both partially protected from trypsin digestion in the presence of calcium but have different trypsin and thermolysin fragmentation patterns. The thermolysin-generated fragments from platelet and endothelial thrombospondins are recognized differently by a monoclonal antibody whereas ail of them are identified by a polyclonal antibody.
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