Generation of platelet angiostatin mediated by urokinase plasminogen activator: effects on angiogenesis.
BACKGROUND
Angiogenesis, the growth of new capillaries from pre-existing blood vessels, is regulated by a balance between its promoters and inhibitors. Platelets are an important circulating store of angiogenesis regulators. We have previously identified the angiogenesis inhibitor angiostatin in human platelets.


OBJECTIVE
To identify the mechanism of platelet angiostatin generation and its pharmacological regulation.


METHODS
Platelet aggregometry, flow cytometry, Western blot, zymography, immunofluorescence microscopy, matrigel-induced angiogenesis of human umbilical vein endothelial cells (HUVECs), and a panel of selective proteinase inhibitors were used to study the mechanism of angiostatin generation by platelets, its pharmacological regulation, and effects on angiogenesis. Release of pro-MMP-2 by HUVECs was also used to quantify angiogenesis.


RESULTS
Platelet membranes were identified as the site of angiostatin generation from plasminogen. Generation of angiostatin by platelet membranes was not affected by a matrix metalloproteinase (MMP) inhibitor, phenanthroline, but was inhibited by serine proteinase inhibitors aprotinin, leupeptin, plasminogen activator inhibitor-1, and selective inhibitor of urokinase plasminogen activator (uPA), uPA-STOP(TM). Angiostatin generation by intact platelets was inhibited by aprotinin, and the resulting incubate promoted angiogenesis to a greater extent than incubate where angiostatin generation occurred. Furthermore, HUVECs incubated with reaction mixture, where angiostatin generation was inhibited, released more pro-MMP-2 than HUVECs incubated with supernatants, where angiostatin generation occurred.


CONCLUSIONS
We conclude that; (i) platelets constitutively generate angiostatin on their membranes; (ii) this mechanism is dependent on uPA, but not, MMPs; and (iii) inhibition of platelet angiostatin generation can further promote angiogenesis.
Jurasz
P
P
Institute of Molecular Medicine for the Prevention of Human Diseases, Vascular Biology Section, University of Texas Health Sciences Center at Houston, Houston, TX 77030, USA. juraszp@smh.toronto.on.ca
Santos-Martinez
M J
MJ
Radomska
A
A
Radomski
M W
MW
D043168:Angiostatins / Q000096:biosynthesis* / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism; D015153:Blotting, Western; D002460:Cell Line; D005434:Flow Cytometry; D006801:Humans; D008856:Microscopy, Fluorescence; D018919:Neovascularization, Physiologic / Q000187:drug effects*; D010974:Platelet Aggregation; D014568:Urokinase-Type Plasminogen Activator / Q000494:pharmacology*
D043168:Angiostatins; D014568:Urokinase-Type Plasminogen Activator