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The Hemostatic System as a Regulator of Angiogenesis

2000; Elsevier BV; Volume: 275; Issue: 3 Linguagem: Inglês

10.1074/jbc.275.3.1521

1083-351X

Timothy Browder, Judah Folkman, Steven Pirie‐Shepherd,

Blood Coagulation and Thrombosis Mechanisms

vascular endothelial growth factor basic fibroblast growth factor hepatocyte growth factor platelet factor 4 antithrombin III thrombospondin transforming growth factor-β1 plasminogen activator inhibitor type 1 urokinase-type plasminogen activator high molecular weight kininogen matrix metalloproteinase Angiogenesis is the process of sprouting and configuring new blood vessels from pre-existing blood vessels, whereas the hemostatic system maintains the liquid flow of blood by regulating platelet adherence and fibrin deposition. Both systems normally appear quiescent, yet both systems remain poised for repair of injury. With vessel injury, a rapid sequence of reactions must occur to occlude the vessel wall defect and prevent hemorrhage. Activated platelets link the margins of the defect and form a provisional barrier that is quickly enmeshed with polymerized fibrin. This clot structure initially requires immobilized vascular endothelial cells to anchor the clot and prevent further bleeding. Thereafter, endothelial cells at the clot margins become mobile, dismantling and invading the cross-linked fibrin structure to rebuild a new vessel wall. Although the positive and negative regulators that control the delicate balance of platelet reactivity and fibrin deposition have been elucidated over the past four decades, analogous proteins that control endothelial cell growth and inhibition have only been discovered within the past decade. Hemostasis and angiogenesis are becoming increasingly inter-related. Proteins generated by the hemostatic system coordinate the spatial localization and temporal sequence of clot/endothelial cell stabilization followed by endothelial cell growth and repair of a damaged blood vessel. We focus here on the regulation of angiogenesis during vessel repair mediated by proteins secreted by platelets and derived as cryptic fragments from the coagulation cascade and fibrinolytic system. At the site of vessel injury, adhered platelets secrete both positive and negative regulators of angiogenesis, mainly from internal α-granules. These positive regulators include: vascular endothelial growth factor-A (VEGF-A)1(1.Mohle R. Green D. Moore M.A.S. Nachman R.L. Rafii S. Constitutive production and thrombin-induced release of vascular endothelial growth factor by human megakaryocytes and platelets..Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 663-668Crossref PubMed Scopus (640) Google Scholar), VEGF-C (2.Wartiovaara U. Salven P. Mikkola H. Lassila R. Kaukonen J. Joukov V. Orpana A. Ristimaki A. Heikinheimo M. Joensuu H. Aitalo K. Palotie A. Peripheral blood platelets express VEGF-C and VEGF which are released during platelet activation..Thromb. Haemostasis. 1998; 80: 171-175Crossref PubMed Scopus (239) Google Scholar), basic fibroblast growth factor (bFGF) (3.Brunner G. Nguyen H. Gabrilove J. Rifkin D.B. Wilson E.L. Basic fibroblast growth factor expression in human bone marrow and peripheral blood cells..Blood. 1993; 81: 631-638Crossref PubMed Google Scholar), hepatocyte growth factor (HGF) (4.Nakamura T. Teramoto H. Ichihara A. Purification and characterization of a growth factor from rat platelets for mature parenchymal hepatocytes in primary culture..Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 6489-6493Crossref PubMed Scopus (620) Google Scholar), angiopoietin-1, 2G. D. Yancopoulos, personal communication. insulin-like growth factor-1 and -2 (6.Karey K.P. Marquardt H. Sirbasku D.A. Human platelet-derived mitogens. I. Identification of insulin-like growth factors I and II by purification and N-terminal amino acid sequence analysis..Blood. 1989; 74: 1084-1092Crossref PubMed Google Scholar, 7.Karey K.P. Sirbasku D.A. Human platelet-derived mitogens. II. Subcellular localization of insulin-like growth factor I to the alpha granule and release in response to thrombin..Blood. 1989; 74: 1093-1100Crossref PubMed Google Scholar), epidermal growth factor (8.Ben-Ezra J. Sheibani K. Hwabg D.L. Lev-Ran A. Megakaryocyte synthesis is the source of epidermal growth factor in human platelets..Am. J. Pathol. 1990; 137: 755-759PubMed Google Scholar, 9.Hwang D.L. Lev-Ran A. Yen C.F. Sniecinski I. Release of different fractions of epidermal growth factor from human platelets in vitro: preferential release of 140 kDa fraction..Regul. Pept. 1992; 37: 95-100Crossref PubMed Scopus (17) Google Scholar), platelet-derived growth factor (10.Bar R.S. Boes M. Booth B.A. Dake B.L. Henley S. Hart M.N. The effects of platelet-derived growth factor in cultured microvessel endothelial cells..Endocrinology. 1989; 124: 1841-1848Crossref PubMed Scopus (111) Google Scholar, 11.Heldin C.-H. Westermark B. Wasteson A. Platelet-derived growth factor: isolation by a large-scale procedure and analysis of subunit composition..Biochem. J. 1981; 193: 907-913Crossref PubMed Scopus (147) Google Scholar), and sphingosine 1-phosphate (12.Lee O.-H. Kim Y.-M. Lee Y.M. Moon E.-J. Lee D.-J. Kim J.-H. Kim K.-W. Kwon Y.-G. Sphingosine 1-phosphate induces angiogenesis: its angiogenic action and signaling mechanism in human umbilical vein endothelial cells..Biochem. Biophys. Res. Commun. 1999; 264: 743-750Crossref PubMed Scopus (337) Google Scholar). Platelets also secrete negative regulators that suppress this inducement of angiogenesis and are discussed below. Moreover, one of the positive angiogenic regulators, hepatocyte growth factor, effects both stimulation and (via the generation of cryptic fragments) suppression of angiogenesis. In contrast to the multiple pro-angiogenic activities of HGF (13.Shima N. Itagaki Y. Nagao M. Yasuda H. Morinaga T. Higashio K. A fibroblast-derived tumor cytotoxic factor/F-TCF (hepatocyte growth factor/HGF) has multiple functions in vitro..Cell Biol. Int. Rep. 1991; 15: 397-408Crossref PubMed Scopus (56) Google Scholar, 14.Rosen E.M. Lamszus K. Laterra J. Polverini P.J. Rubin J.S. Goldberg I.D. Gherardi E. Plasminogen-related Growth Factors. John Wiley & Sons, Chichester, United Kingdom1997: 215-226Google Scholar), alternative processing of the HGF α-chain mRNA generates anti-angiogenic HGF fragments consisting of either the first kringle domain (NK1) or the first two kringle domains (NK2) (14.Rosen E.M. Lamszus K. Laterra J. Polverini P.J. Rubin J.S. Goldberg I.D. Gherardi E. Plasminogen-related Growth Factors. John Wiley & Sons, Chichester, United Kingdom1997: 215-226Google Scholar). These first two kringles contain the HGF binding site for its receptor, c-Met (14.Rosen E.M. Lamszus K. Laterra J. Polverini P.J. Rubin J.S. Goldberg I.D. Gherardi E. Plasminogen-related Growth Factors. John Wiley & Sons, Chichester, United Kingdom1997: 215-226Google Scholar). NK1 and NK2 suppress HGF-induced endothelial cell migration and abrogate HGF-induced angiogenesis in the rat cornea (14.Rosen E.M. Lamszus K. Laterra J. Polverini P.J. Rubin J.S. Goldberg I.D. Gherardi E. Plasminogen-related Growth Factors. John Wiley & Sons, Chichester, United Kingdom1997: 215-226Google Scholar). These observations led to recombinant construction of NK4, which contains all four kringle domains of HGF (15.Date K. Matsumoto K. Kubs K. Shimura H. Tanaka M. Nakamura T. Inhibition of tumor growth and invasion by a four-kringle antagonist (HGF/NK4) for hepatocyte growth factor..Oncogene. 1998; 17: 3045-3054Crossref PubMed Scopus (180) Google Scholar). HGF/NK4 is a more potent antagonist of c-Met activation by HGF (15.Date K. Matsumoto K. Kubs K. Shimura H. Tanaka M. Nakamura T. Inhibition of tumor growth and invasion by a four-kringle antagonist (HGF/NK4) for hepatocyte growth factor..Oncogene. 1998; 17: 3045-3054Crossref PubMed Scopus (180) Google Scholar). HGF/NK4 potently inhibits tumor growthin vivo by increasing tumor cell apoptosis without affecting the proliferation rate of tumor cells (15.Date K. Matsumoto K. Kubs K. Shimura H. Tanaka M. Nakamura T. Inhibition of tumor growth and invasion by a four-kringle antagonist (HGF/NK4) for hepatocyte growth factor..Oncogene. 1998; 17: 3045-3054Crossref PubMed Scopus (180) Google Scholar). A similar pattern of tumor inhibition occurs through angiogenesis inhibition (16.Holmgren L. O'Reilly M.S. Folkman J. Dormancy of micrometastases: balanced proliferation and apoptosis in the presence of angiogenesis suppression..Nat. Med. 1995; 1: 149-153Crossref PubMed Scopus (1706) Google Scholar). Taken together, the anti-tumor activity of HGF/NK4 in vivo is at least partly mediated through an anti-angiogenic activity (15.Date K. Matsumoto K. Kubs K. Shimura H. Tanaka M. Nakamura T. Inhibition of tumor growth and invasion by a four-kringle antagonist (HGF/NK4) for hepatocyte growth factor..Oncogene. 1998; 17: 3045-3054Crossref PubMed Scopus (180) Google Scholar). Thus, expression of NK1 or NK2 or cryptic cleavage of HGF into NK1, NK2, or NK4 could counterbalance HGF-induced angiogenesis in vivo. Unique to platelets, PF4 binds surface heparin-like glycosaminoglycans on endothelial cells, thereby quenching the anti-thrombotic activity of antithrombin III (AT-III) and allowing a clot to form. Nearly two decades ago, PF4 was the first hemostatic protein demonstrated to be an inhibitor of angiogenesisin vivo (17.Taylor S. Folkman J. Protamine is an inhibitor of angiogenesis..Nature. 1982; 297: 307-312Crossref PubMed Scopus (521) Google Scholar, 18.Maione T.E. Gray G.S. Petro J. Hunt A.J. Donner A.L. Bauer S.I. Carson H.F. Sharpe R.J. Inhibition of angiogenesis by recombinant human platelets factor-4 and related peptides..Science. 1990; 247: 77-79Crossref PubMed Scopus (631) Google Scholar). One mechanism for the initial endothelial cell inhibition following platelet secretion is that PF4 blocks heparin-like glycosaminoglycans that function as critical, low affinity receptors for heparin-binding endothelial growth factors on the surface of endothelial cells (18.Maione T.E. Gray G.S. Petro J. Hunt A.J. Donner A.L. Bauer S.I. Carson H.F. Sharpe R.J. Inhibition of angiogenesis by recombinant human platelets factor-4 and related peptides..Science. 1990; 247: 77-79Crossref PubMed Scopus (631) Google Scholar). PF4 also directly neutralizes the heparin-binding region of growth factors (19.Perollet C. Han Z.C. Savona C. Caen J.P. Bikfalvi A. Platelet factor 4 modulates fibroblast growth factor 2 (FGF-2) activity and inhibits FGF-2 dimerization..Blood. 1998; 91: 3289-3299Crossref PubMed Google Scholar). Further, a heparin-independent pathway of PF4 inhibition of endothelial cell growth exists. The endothelial cell stimulatory activity of epidermal growth factor and VEGF-A121, endothelial mitogens that lack heparin affinity, is susceptible to PF4 inhibition (20.Gengrinovitch S. Greenberg S.M. Cohen T. Gitay-Goren H. Rockwell P. Maione T.E. Levi B.-Z. Neufeld G. Platelet factor-4 inhibits the mitogenic activity of VEGF-121 and VEGF-165 using several concurrent mechanisms..J. Biol. Chem. 1995; 270: 15059-15065Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). Moreover, an analogue of PF4 that lacks heparin affinity (rPF4-241) inhibits angiogenesis (21.Maione T.E. Gray G.S. Hunt A.J. Sharpe R.J. Inhibition of tumor growth in mice by an analogue of platelet factor 4 that lacks affinity for heparin and retains potent angiostatic activity..Cancer Res. 1991; 51: 2077-2083PubMed Google Scholar). TSP-1 is the most abundant constituent of platelet α-granules and participates in efficient platelet aggregation (22.Jaffe E. Leung L.L.K. Nachman R.L. Levin R.L. Mosher D.F. Thrombospondin is the endogenous lectin of human platelets..Nature. 1982; 295: 246-248Crossref PubMed Scopus (106) Google Scholar, 23.Rabbi-Sabile S. Thibert V. Legrand C. Thrombospondin peptides inhibit the secretion-dependent phase of platelet aggregation..Blood Coagul. Fibrinolysis. 1996; 7: 237-240Crossref PubMed Scopus (10) Google Scholar). TSP-1 is a large (450 kDa), modular glycoprotein complexed with active transforming growth factor-β1 (TGF-β1) in α-granules and, upon release, can activate latent TGF-β1 secreted by endothelial cells (24.Schultz-Cherry S. Ribeiro S. Gentry L. Murphy-Ullrich J.E. Thrombospondin binds and activates the small and large forms of latent transforming growth factor-beta in a chemically defined system..J. Biol. Chem. 1994; 269: 26775-26782Abstract Full Text PDF PubMed Google Scholar). TSP-1 binds fibrin (25.Panetti T.S. Kudryk B.J. Mosher D.F. Interaction of recombinant procollagen and properdin modules of thrombospondin-1 with heparin and fibrinogen/fibrin..J. Biol. Chem. 1999; 274: 430-437Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), fibronectin (26.Iruela-Arispe M.L. Liska D.J. Sage E.H. Bornstein P. Differential expression of thrombospondin 1, 2, and 3 during murine development..Dev. Dyn. 1993; 197: 40-56Crossref PubMed Scopus (197) Google Scholar), plasminogen (27.Adams J.C. Thrombospondin-1..Int. J. Biochem. Cell Biol. 1997; 29: 861-865Crossref PubMed Scopus (101) Google Scholar), surface heparin-like glycosaminoglycans (26.Iruela-Arispe M.L. Liska D.J. Sage E.H. Bornstein P. Differential expression of thrombospondin 1, 2, and 3 during murine development..Dev. Dyn. 1993; 197: 40-56Crossref PubMed Scopus (197) Google Scholar), CD36 and αvβ3 integrins on activated endothelial cells (27.Adams J.C. Thrombospondin-1..Int. J. Biochem. Cell Biol. 1997; 29: 861-865Crossref PubMed Scopus (101) Google Scholar), and αIIbβ3 integrins on activated platelets (27.Adams J.C. Thrombospondin-1..Int. J. Biochem. Cell Biol. 1997; 29: 861-865Crossref PubMed Scopus (101) Google Scholar). TSP-1 may re-adjust growth factor and integrin signaling pathways between endothelial cells and the fibrin clot (27.Adams J.C. Thrombospondin-1..Int. J. Biochem. Cell Biol. 1997; 29: 861-865Crossref PubMed Scopus (101) Google Scholar) and prevent endothelial cell motility induced by fibrin. TSP-1 stimulates endothelial cell adhesion and spreading but blocks the chemokinetic response of endothelial cells to bFGF (28.Good D.J. Polverini P.J. Rastinejad F. Beau M.M.L. Lemons R.S. Frazier W.A. Bouck N.P. A tumor suppressor-dependent inhibitor of angiogenesis is immunologically and functionally indistinguishable from a fragment of thrombospondin..Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6624-6628Crossref PubMed Scopus (908) Google Scholar). Platelet α-granules are a rich source for active TGF-β1 (29.Assoain R.K. Komoriya A. Meyers C.A. Miller D.M. Sporn M.B. Transforming growth factor-beta in human platelets..J. Biol. Chem. 1983; 258: 7155-7160Abstract Full Text PDF PubMed Google Scholar). TGF-β1 promotes the formation of quiescent capillary tubules in vitro and mediates potent inhibition of endothelial cell proliferation and migration (30.Roberts A.B. Sporn M.B. Regulation of endothelial cell growth, architecture, and matrix synthesis by TGF-beta..Am. Rev. Respir. Dis. 1989; 140: 1126-1128Crossref PubMed Scopus (105) Google Scholar). TGF-β1 blocks the proliferation of endothelial cells to even supramaximal concentrations of bFGF (30.Roberts A.B. Sporn M.B. Regulation of endothelial cell growth, architecture, and matrix synthesis by TGF-beta..Am. Rev. Respir. Dis. 1989; 140: 1126-1128Crossref PubMed Scopus (105) Google Scholar). In vivo, however, TGF-β1 induces angiogenesis (30.Roberts A.B. Sporn M.B. Regulation of endothelial cell growth, architecture, and matrix synthesis by TGF-beta..Am. Rev. Respir. Dis. 1989; 140: 1126-1128Crossref PubMed Scopus (105) Google Scholar, 65.Ucki N. Nakazato M. Ohkawa T. Ikeda T. Amuro Y. Hada T. Higashino K. Excessive production of transforming growth factor beta-1 can play an important role in the development of tumorigenesis by its action for angiogenesis: validity of neutralizing antibodies to block tumor growth..Biochim. Biophys. Acta. 1992; 1137: 189-196Crossref PubMed Scopus (129) Google Scholar) that is thought to reflect recruitment of macrophages, which secrete endothelial cell growth factors (30.Roberts A.B. Sporn M.B. Regulation of endothelial cell growth, architecture, and matrix synthesis by TGF-beta..Am. Rev. Respir. Dis. 1989; 140: 1126-1128Crossref PubMed Scopus (105) Google Scholar). Regulation of plasminogen activation is critical to the sequence of stable fibrin clot formation followed by controlled fibrin digestion. PAI-1 is maintained in an active conformation in complexes with vitronectin within platelet α-granules (31.Hill S.A. Shaughnessy S.G. Joshua P. Ribau J. Austin R.C. Podor T.J. Differential mechanisms targeting type I plasminogen activator inhibitor and vitronectin into the storage granules of a human megakaryocytic cell line..Blood. 1996; 87: 5061-5073Crossref PubMed Google Scholar). Platelet-derived PAI-1 prevents initial fibrinolysis of platelet-rich thrombi (31.Hill S.A. Shaughnessy S.G. Joshua P. Ribau J. Austin R.C. Podor T.J. Differential mechanisms targeting type I plasminogen activator inhibitor and vitronectin into the storage granules of a human megakaryocytic cell line..Blood. 1996; 87: 5061-5073Crossref PubMed Google Scholar) but is less effective in the inhibition of the endothelial cell membrane-associated plasminogen activator (uPA) activity (37.Fukao H. Ueshima S. Okada K. Matsuo O. The role of the pericellular fibrinolytic system in angiogenesis..Jpn. J. Physiol. 1997; 47: 161-171Crossref PubMed Scopus (21) Google Scholar) that is generated by endothelial sprouts (33.Collen A. Koolwijk P. Kroon M. von Hinsbergh V.W.M. Influence of fibrin structure on the formation and maintenance of capillary-like tubules by human microvascular endothelial cells..Angiogenesis. 1998; 2: 153-165Crossref PubMed Google Scholar, 34.Kroon M.E. Koolwijk P. van Goor H. Weidle U.H. Collen A. van der Pluijm G. von Hinsbergh V.W.M. Role and localization of urokinase receptor in the formation of new microvascular structures in fibrin matrices..Am. J. Pathol. 1999; 154: 1731-1742Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar) (Fig.1). By limiting plasmin generation within the clot structure, PAI-1 can suppress angiogenesis (32.Blei F. Wilson E.L. Mignatti P. Rifkin D.B. Mechanism of action of angiostatic steroids: suppression of plasminogen activator activity via stimulation of plasminogen activator inhibitor synthesis..J. Cell. Physiol. 1993; 155: 568-578Crossref PubMed Scopus (145) Google Scholar, 66.Bacharach E. Itin A. Keshet E. In vivo patterns of expression of urokinase and its inhibitor PAI-1 suggest a concerted role in regulating physiological angiogensis..Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10686-10690Crossref PubMed Scopus (159) Google Scholar) (Fig. 1). By scavenging plasmin, platelet-derived and fibrin-bound α2-antiplasmin (35.Plow E.F. Collen D. The presence and release of alpha2-antiplasmin from human platelets..Blood. 1981; 58: 1069-1074Crossref PubMed Google Scholar) and α2-macroglobulin (36.Nachman R.L. Harpel P.C. Platelet alpha2-macroglobulin and alpha1-antitrypsin..J. Biol. Chem. 1976; 251: 4512-4521Abstract Full Text PDF PubMed Google Scholar) may also negatively regulate angiogenesis (37.Fukao H. Ueshima S. Okada K. Matsuo O. The role of the pericellular fibrinolytic system in angiogenesis..Jpn. J. Physiol. 1997; 47: 161-171Crossref PubMed Scopus (21) Google Scholar). HMWK circulates in plasma bound to prekallikrein. Contact activation of this complex can begin the coagulation cascade. Kallikrein-cleaved HMWK (Hka) and vitronectin compete for binding to the endothelial cell urokinase receptor (67.Colman R.W. Pixley R.A. Najamunnisa S. Yan W. Wang J. Mazar A. McCrae K.R. Binding of high molecular weight kininogen to human endothelial cells is mediated via a site within domains 2 and 3 of the urokinase receptor..J. Clin. Invest. 1997; 100: 1481-1487Crossref PubMed Scopus (206) Google Scholar). Cryptic generation of Domain 5 (Lys420–Ser513) from Hka inhibits the migration of endothelial cells to vitronectin and fibronectin, both components of the fibrin clot. Domain 5 of HMWK also inhibits endothelial cell proliferation and is anti-angiogenic on the chicken chorioallantoic membrane (38.Colman R.W. Lin Y. Johnson D. Mousa S.A. Inhibition of angiogenesis by peptides derived from kininogen..Blood. 1998; 92 (suppl.) (abstr.): 174Google Scholar). Activated coagulation factor Xa cleaves factor II (prothrombin) to yield thrombin and a two-kringle amino-terminal domain (fragment 1–2) (39.Rhim T.Y. Park C.-S. Kim E. Kim S.S. Human prothrombin fragment 1 and 2 inhibit bFGF-induced BCE cell growth..Biochem. Biophys. Res. Commun. 1998; 252: 513-516Crossref PubMed Scopus (44) Google Scholar). Thrombin then cleaves fragment 1–2 of prothrombin into single-kringle fragment-1 and fragment-2. Thrombin induces angiogenesis in vivo via cleavage of the tethered ligand of the thrombin receptor on endothelial cells without the requirement for fibrin formation (40.Tsopanoglou N.E. Pipili-Synetos E. Maragoudakis M.E. Thrombin promotes angiogenesis by a mechanism independent of fibrin formation..Am. J. Physiol. 1993; 264: C1302-C1307Crossref PubMed Google Scholar). Simultaneously, these two amino-terminal kringle domains of prothrombin are released. Fragment-1 and fragment-2 of prothrombin inhibit the proliferation of endothelial cells in vitro and angiogenesisin vivo (39.Rhim T.Y. Park C.-S. Kim E. Kim S.S. Human prothrombin fragment 1 and 2 inhibit bFGF-induced BCE cell growth..Biochem. Biophys. Res. Commun. 1998; 252: 513-516Crossref PubMed Scopus (44) Google Scholar). Thus, the stimulatory effects of thrombin on endothelial cells would be antagonized by kringle by-products released upon activation of prothrombin. In the presence of heparin, AT-III avidly inhibits the activated form of factors II (thrombin) and X in plasma. This inactivation is very inefficient when coagulation factors are bound to the anionic phospholipid surface of activated platelets and endothelial cells. AT-III thus serves an important physiologic role in limiting the extent of an evolving clot to the area of vascular injury. Thrombin and neutrophil elastase can cleave the thrombin-binding site of AT-III (41.O'Reilly M.S. Pirie-Shepherd S. Lane W.S. Folkman J. Antiangiogenic activity of the cleaved conformation of the Serpin antithrombin..Science. 1999; 285: 1926-1931Crossref PubMed Scopus (425) Google Scholar). Once generated, cleaved AT-III (anti-angiogenic AT-III) becomes a potent inhibitor of endothelial cell proliferation in vitroand angiogenesis in vivo (41.O'Reilly M.S. Pirie-Shepherd S. Lane W.S. Folkman J. Antiangiogenic activity of the cleaved conformation of the Serpin antithrombin..Science. 1999; 285: 1926-1931Crossref PubMed Scopus (425) Google Scholar). Thus, the angiogenic activity of thrombin generated at the site of clotting may be balanced not only by fragment-1 and -2 of prothrombin but also through production of anti-angiogenic AT-III. Thrombin cleaves small peptides from the amino-terminal ends of the α and β chains of soluble fibrinogen to form insoluble fibrin monomers. Fibrin monomers self-assemble at the site of vessel injury and enmesh the adhered platelets, migration-inhibited endothelial cells, and the exposed subendothelial matrix. Along with binding of latent regulators of plasminogen activation, fibrin also displays high affinity binding for bFGF (42.Sahni A. Odrljin T. Francis C.W. Binding of basic fibroblast growth factor to fibrinogen and fibrin..J. Biol. 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Migration into fibrin gels requires growth factor-stimulated endothelial cell uPA receptor, uPA, and resulting plasminogen activation (33.Collen A. Koolwijk P. Kroon M. von Hinsbergh V.W.M. Influence of fibrin structure on the formation and maintenance of capillary-like tubules by human microvascular endothelial cells..Angiogenesis. 1998; 2: 153-165Crossref PubMed Google Scholar, 34.Kroon M.E. Koolwijk P. van Goor H. Weidle U.H. Collen A. van der Pluijm G. von Hinsbergh V.W.M. Role and localization of urokinase receptor in the formation of new microvascular structures in fibrin matrices..Am. J. Pathol. 1999; 154: 1731-1742Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Localized production of plasmin at the endothelial invasion front lowers the density of the fibrin matrix required for capillary tube formation (45.Vailhe B. Ronot X. Traque P. Usson Y. Tranqup L. In vitro angiogenesis is modulated by the mechanical properties of fibrin gels and is related to alpha-v/beta-3 integrin localization..In Vitro Cell. Dev. Biol. 1997; 33: 763-773Crossref Scopus (95) Google Scholar). As endothelial cells migrate into and align within the more dilute and flexible fibrin gel, residues 15–42 on the β-chain of fibrin interact with vascular endothelial cadherin on endothelial cells and facilitate capillary morphogenesis (46.Bach T.L. Barsigian C. Chalupowicz D.G. Busler D. Yaen C.H. Grant D.S. Martinez J. VE-Cadherin mediates endothelial cell capillary tube formation in fibrin and collagen gels..Exp. Cell Res. 1998; 238: 324-334Crossref PubMed Scopus (184) Google Scholar). Thus, fibrin plays a central role in the sequential events of vascular repair. First, fibrin tightly secures the platelet plug over immobilized endothelial cells to prevent hemorrhage. Second, fibrin serves as a sustained release reservoir for endothelial growth factors (42.Sahni A. Odrljin T. Francis C.W. Binding of basic fibroblast growth factor to fibrinogen and fibrin..J. Biol. Chem. 1998; 273: 7554-7559Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar) and fibrinolytic enzymes (47.Harrison P. Cramer E.M. Platelet alpha-granules..Blood Rev. 1993; 7: 52-62Crossref PubMed Scopus (471) Google Scholar). Third, as the clot is dismantled, partially digested fibrin provides solid-state guidance of endothelial cell migration (44.Thiagarajan P. Rippon A.J. Farrell D.H. Alternative adhesion sites in human fibrinogen for vascular endothelial cells..Biochemistry. 1996; 35: 4169-4175Crossref PubMed Scopus (52) Google Scholar, 48.Henke C.A. Roongta U. Mickelson D.J. Knutson J.R. McCarthy J.B. CD44-related chondroitin sulfate proteoglycan, a cell surface receptor implicated with tumor cell invasion, mediates endothelial cell migration on fibrinogen and invasion into a fibrin matrix..J. Clin. 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Angiostatin, kringles 1–4 of plasminogen, is a circulating inhibitor of angiogenesis originally discovered by its ability to prevent the growth of cancer metastases (50.O'Reilly M.S. Holmgren L. Shing Y. Chen C. Rosenthal R.A. Moses M. Lane W.S. Cao Y. Sage E.H. Folkman J. Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma..Cell. 1994; 79: 315-328Abstract Full Text PDF PubMed Scopus (3197) Google Scholar). Angiostatin potently and specifically inhibits endothelial cell proliferation in vitro and angiogenesis in vivo(50.O'Reilly M.S. Holmgren L. Shing Y. Chen C. Rosenthal R.A. Moses M. Lane W.S. Cao Y. Sage E.H. Folkman J. Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma..Cell. 1994; 79: 315-328Abstract Full Text PDF PubMed Scopus (3197) Google Scholar, 51.O'Reilly M.S. Holmgren L. Chen C. Folkman J. Angiostatin induces and sustains dormancy of human primary tumors in mice..Nat. Med. 1996; 2: 689-692Crossref PubMed Scopus (1155) Google Scholar). Further, portions of all five kringle domains of plasminogen/plasmin possess anti-angiogenic activity (52.Cao Y. Ji R.W. Davidson D. Schaller J. Marti D. Sohndel S. McCance S.G. O'Reilly M.S. Llinas M. Folkman J. Kringle domains of angiostatin: characterization of the anti-proliferative activity on endothelial cells..J. Biol. Chem. 1996; 271: 29461-29467Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar, 53.Cao Y. Chen A. An S.S.A. Ji R.W. Davidson D. Llinas M. Kringle 5 of plasminogen is a novel inhibitor of endothelial cell growth..J. Biol. Chem. 1997; 272: 22924-22928Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). Angiostatin binds to the α/β-subunits of ATP synthase on the surface of endothelial cells, potentially inducing H+cytoplasmic influx into endothelial cells and cytolysis (54.Moser T.L. Stack M.S. Asplin J. Enghild J.J. Hojrup P. Everitt L. Hubchak S. Schnaper H.W. Pizzo S.V. Angiostatin binds ATP synthase on the surface of human endothelial cells..Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2811-2816Crossref PubMed Scopus (470) Google Scholar). Several mechanisms have been demonstrated to generate biologically active angiostatin. These include: (i) cleavage by active matrix metalloproteinase (MMP) -2 (55.O'Reilly M.S. Wiederschain D. Stetler-Stevenson W.G. Folkman J. Moses M.A. Regulation of angiostatin production by matrix metalloproteinase-2 in a model of concomitant resistance..J. Biol. Chem. 1999; 274: 29568-29571Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar), MMP-3 (56.Lijnen H.R. Ugwu F. Bini A. Collen D. Generation of angiostatin-like fragment from plasminogen by stromelysin-1 (MMP-3)..Biochemistry. 1998; 37: 4699-4702Crossref PubMed Scopus (181) Google Scholar), MMP-7 (57.Patterson B.C. Sang Q.A. Angiostatin-converting enzyme activities of human matrilysin (MMP-7) and gelatinase B/type IV collagenase (MMP-9)..J. Biol. Chem. 1997; 272: 28823-28825Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar), and MMP-9 (57.Patterson B.C. Sang Q.A. Angiostatin-converting enzyme activities of human matrilysin (MMP-7) and gelatinase B/type IV collagenase (MMP-9)..J. Biol. Chem. 1997; 272: 28823-28825Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar); (ii) cleavage by a tumor cell-derived plasmin thiolreductase (58.Stathakis P. Fitzgerald M. Matthias L.J. Chestermna C.N. Hogg P.J. Generation of angiostatin by reduction and proteolysis of plasmin: catalysis by a plasmin reductase secreted by cultured cells..J. Biol. Chem. 1997; 272: 20641-20645Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar,59.Stathakis P. Lay A.J. Fitzgerald M. Schlieker C. Matthias L.J. Hogg P.J. Angiostatin formation involves disulfide bond reduction and proteolysis in kringle 5 of plasmin..J. Biol. Chem. 1999; 274: 8910-8916Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar); and (iii) cleavage of plasminogen on the surface of macrophages by granulocyte-macrophage colony-stimulating factor-induced metalloelastase (MMP-12) (60.Dong Z. Kumar K. Yang X. Fidler I.J. Macrophage-derived metalloelastase is responsible for the generation of angiostatin in Lewis lung carcinoma..Cell. 1997; 88: 801-810Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar). Angiostatin also governs the rate of plasminogen activation through non-competitive inhibition of tissue-type plasminogen activator (61.Stack M.S. Gately S. Bafetti L.M. Enghild J.J. Soff G.A. Angiostatin inhibits endothelial and melanoma cellular invasion by blocking matrix-enhanced plasminogen activation..Biochem. J. 1999; 340: 77-84Crossref PubMed Scopus (107) Google Scholar). Thus, generation of angiostatin may regulate the speed of endothelial cell migration and proliferation into the clot both directly and through feedback inhibition of plasminogen activation. During the first days as the nascent clot bridges and stabilizes the vessel defect, any initiation of angiogenesis directed by platelet-derived positive regulators, thrombin, and fibrin must be counteracted. Following clot stabilization, angiogenesis must be tightly regulated to avoid re-bleeding. This regulation is achieved through proteins secreted by platelets and cryptic fragments generated from hemostatic proteins involved in coagulation and fibrinolysis. Although the timing of release of these cryptic fragments is unknown, we can speculate that they may operate when known proteolytic activities develop (5.Hiraoka N. Allen E. Apel I.J. Gyetko M.R. Weiss S.J. 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Platelet secretion is triggered within the first few minutes of hemostasis and results in deposition of both positive and negative regulators of angiogenesis. Platelet-derived PF4, TSP-1, and TGF-β1 may counteract immediate endothelial cell migration and proliferation resulting from platelet-derived positive regulators of angiogenesis and fibrin. In this early context, these positive regulators of angiogenesis derived from platelets may function as anti-apoptotic factors (63.Kwak H.J. So J.-N. Lee S.J. Kim I. Koh G.Y. Angiopoietin-1 is an apoptosis survival factor for endothelial cells..FEBS Lett. 1999; 448: 249-253Crossref PubMed Scopus (207) Google Scholar, 64.Papapetropoulos A. Garcia-Cardena G. Dengler T.J. Maisonpierre P.C. Yancopoulos G.D. Sessa W.C. Direct actions of angiopoietin-1 on human endothelium: evidence for network stabilization, cell survival, and interaction with other angiogenic growth factors..Lab. Invest. 1999; 79: 213-223PubMed Google Scholar). Cleavage of prothrombin to thrombin and fragments 1 and 2 occurs concomitantly with platelet secretion. Further, some of the thrombin that is generated may cleave local AT-III. Thus, fragments 1 and 2 of prothrombin and anti-angiogenic AT-III could also antagonize the initial pro-angiogenic stimulus from platelets, thrombin, and fibrin. PAI-1, α2-antiplasmin, and α2-macroglobulin initially prevent plasmin activity. Thereafter, pericellular fibrinolysis and angiogenesis are initiated by endothelial cell expression of the uPA receptor (34.Kroon M.E. Koolwijk P. van Goor H. Weidle U.H. Collen A. van der Pluijm G. von Hinsbergh V.W.M. Role and localization of urokinase receptor in the formation of new microvascular structures in fibrin matrices..Am. J. Pathol. 1999; 154: 1731-1742Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar) and uPA (34.Kroon M.E. Koolwijk P. van Goor H. Weidle U.H. Collen A. van der Pluijm G. von Hinsbergh V.W.M. Role and localization of urokinase receptor in the formation of new microvascular structures in fibrin matrices..Am. J. Pathol. 1999; 154: 1731-1742Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar) and also membrane type-1 MMP (5.Hiraoka N. Allen E. Apel I.J. Gyetko M.R. Weiss S.J. Matrix metalloproteinases regulate neovascularization by acting as pericellular fibrinolysins..Cell. 1988; 95: 365-377Abstract Full Text Full Text PDF Scopus (649) Google Scholar). Focal generation of angiostatin could then occur via the mechanisms discussed and would regulate both the speed of endothelial repair and rate of plasmin production. Cryptic fragments of HMWK and HGF may also be generated during fibrinolysis. Thus, angiostatin, HMWK domain 5, and HGF/NK1, NK2, or NK4 may limit excessive angiogenesis induced by the unopposed activity of platelet-derived angiogenic growth factors and fibrin during fibrinolysis. Platelet secretory proteins and cryptic fragments generated during clotting and fibrinolysis may sequentially induce endothelial cell immobilization during hemostasis and control the rate of angiogenesis during vessel repair. The capacity of the hemostatic system to store proteins that regulate angiogenesis provides a new conceptual framework to understand how angiogenesis is coordinated by and with hemostasis during vessel repair.