Proteolysis of human plasma reveals the presence of complexes formed by endogenous heparin and peptides that stimulate angiogenesis.
Human endogenous plasma heparin associates with proteins that mask its anticoagulant activity. This association persists after exhaustive proteolysis of plasma, and resulting peptide/heparin complexes have no anticoagulant activity. Looking for functions other than inhibition of coagulation, we considered that commercial preparations of heparin from bovine or porcine sources show alternative effects on angiogenesis, either stimulating or inhibiting the process. However, the effects of endogenous human heparin on angiogenesis are unknown. In this study, the fraction of plasma containing endogenous heparin was prepared by means of exhaustive proteolysis, either in the presence or in the absence of 35S-labeled heparin. Plasma from healthy donors was digested and the supernatant was precipitated with 66% ethanol, dialyzed, and submitted to basic and acidic ion-exchange chromatography. 35S-Labeled heparin as well as endogenous heparin bound plasmatic peptides, forming acidic, basic, and neutral complexes. Binding of peptides, eluting from both resins, impaired migration of heparin on cellulose acetate electrophoresis. Endogenous neutral complexes (i.e., those formed by human endogenous plasma heparin and peptides) were tested for angiogenic activity in chick embryo chorioallantoic membrane assay. Bovine heparin induced a moderate angiogenic response. Neutral complexes of human endogenous plasma heparin and basic plasma peptides induced a very strong angiogenic response. Treatment of neutral complexes with nitrous acid, which degrades heparin, abolished the angiogenic effect, thus demonstrating that it was due to the presence of heparin. These results demonstrate that proteolysis of human plasma generates angiogenic peptide/heparin complexes.
Pacini
Stefania
S
Department of Anatomy, Histology and Forensic Medicine, University of Firenze, Italy.
Ruggiero
Marco
M
Cecchi
Fabiola
F
Peruzzi
Benedetta
B
Vannucchi
Simonetta
S
D000085:Acetates / Q000737:chemistry; D000818:Animals; D000925:Anticoagulants / Q000494:pharmacology; D001777:Blood Coagulation; D002417:Cattle; D002482:Cellulose / Q000031:analogs & derivatives* / Q000737:chemistry; D002642:Chick Embryo; D049033:Chorioallantoic Membrane / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D000431:Ethanol / Q000494:pharmacology; D006493:Heparin / Q000097:blood* / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008954:Models, Biological; D009389:Neovascularization, Pathologic; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D013552:Swine; D013997:Time Factors
D000085:Acetates; D000925:Anticoagulants; D010455:Peptides; C005062:acetylcellulose; D000431:Ethanol; D002482:Cellulose; D006493:Heparin