Presence of active and latent type 1 plasminogen activator inhibitor associated with porcine platelets.
Data from a number of laboratories indicate that human platelets contain type I plasminogen activator inhibitor (PAI-1) primarily in a latent form; however, one report (Biochemistry 28:5773, 1989) indicated that it is predominantly the active form of PAI-1 that is present in and can be purified from an ammonium sulfate precipitate of porcine platelets. To clarify this situation, we investigated and compared the status of PAI-1 in porcine and human platelets. Immunologic analysis of the ability of PAI-1 to form complexes with immobilized t-PA indicated that porcine and human platelets contained 3.7 +/- 0.4 and 1.7 +/- 0.3 U of PAI activity per 10(8) platelets (n = 6; +/- SD), respectively; sodium dodecyl sulfate (SDS)-activation of the lysates increased PAI-1 activity to 10.8 +/- 3.0 and 3.8 +/- 0.5 U per 10(8) platelets. Platelet lysates were also treated with an excess of soluble t-PA, which formed complexes with active PAI-1, whereas the latent form was detected by SDS-polyacrylamide gel electrophoresis and reverse fibrin autography. Furthermore, immobilized t-PA was able to deplete active PAI-1 from the platelet extracts, and the latent form remaining in the absorbed extract could be quantitated by activation with 4 mol/L guanidine. To investigate the differences between our observations and the published data, porcine platelets were extracted, and PAI-1 was partially purified as described in the literature. For quantitative analysis, porcine platelet PAI-1 was also purified to homogeneity using standard chromatographic procedures optimized in our laboratory for endothelial PAI-1, and the purified protein was used to develop an enzyme-linked immunoabsorbent assay for porcine PAI-1 antigen. Our results indicate that: (1) latent PAI-1 in concentrated ammonium sulfate precipitates of porcine platelet lysates cannot be detected unless the precipitates are diluted before treatment with denaturants; and (2) active and latent porcine platelet PAI-1 can be separated by gel filtration over molecular sieving columns. In summary, this report documents that PAI-1 in porcine platelets is present in both an active and a latent form.
Lang
I M
IM
University of California, San Diego.
Marsh
J J
JJ
Moser
K M
KM
Schleef
R R
RR
D000818:Animals; D001792:Blood Platelets / Q000502:physiology*; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D046911:Macromolecular Substances; D008970:Molecular Weight; D017395:Plasminogen Activator Inhibitor 1 / Q000097:blood* / Q000302:isolation & purification / Q000378:metabolism; D013552:Swine; D010959:Tissue Plasminogen Activator / Q000378:metabolism
D046911:Macromolecular Substances; D017395:Plasminogen Activator Inhibitor 1; D010959:Tissue Plasminogen Activator