Cell Surface-Dependent Generation of Angiostatin4.5

Abstract

Angiostatin4.5 (AS4.5) is a naturally occurring human angiostatin isoform, consisting of plasminogen kringles 1-4 plus 85% of kringle 5 (amino acids Lys78 to Arg529). Prior studies indicate that plasminogen is converted to AS4.5 in a two-step reaction. First, plasminogen is activated to plasmin. Then plasmin undergoes autoproteolysis within the inner loop of kringle 5, which can be induced by a free sulfhydryl donor or an alkaline pH. We now demonstrate that plasminogen can be converted to AS4.5 in a cell membrane-dependent reaction. Actin was shown previously to be a surface receptor for plasmin(ogen). We now show that ␤-actin is present on the extracellular membranes of cancer cells (PC-3, HT1080, and MDA-MB231), and ␤-actin can mediate plasmin binding to the cell surface and autoproteolysis to AS4.5. In the presence of ␤-actin, no small moleculefree sulfhydryl donor is needed for generation of AS4.5. Antibodies to actin reduced membrane-dependent generation of AS4.5 by 70%. In a cell-free system, addition of actin to in vitro-generated plasmin resulted in stoichiometric conversion to AS4.5. Annexin II and ␣-enolase have been reported to be plasminogen receptors, but we did not demonstrate a role for these proteins in conversion of plasminogen to AS4.5. Our data indicate that membrane-associated ␤-actin, documented previously as a plasminogen receptor, is a key cell membrane receptor capable of mediating conversion of plasmin to AS4.5. This conversion may serve an important role in regulating tumor angiogenesis, invasion, and metastasis, and surface ␤-actin may also serve as a prognostic marker to predict tumor behavior.

Aldehyde dehydrogenase 1A1 and gelsolin identified as novel invasion-modulating factors in conditioned medium of pancreatic cancer cells

“…Although membrane-bound and secreted proteins are more likely to be cleaved and found in the circulation [12], our GO STAT analysis determined that most up-regulated and down-regulated proteins were involved in glycolysis, cytoplasm, cytosol and intracellular part. Proteins, such as heat shock proteins and actins, generally viewed as cytoplasmic, have been increasingly implicated in extracellular functions [13,14]. Eukaryotic protein secretion normally routes through the endoplasmatic reticulum (ER) and Golgi, ending up in a secretory vesicle fusing to the cell membrane.…”

Antiangiogenic plasma activity in patients with systemic sclerosis

“…Individual plasminogen kringles and various subsets of kringle domains have angiostatin activity, and many are more potent angiogenesis inhibitors than the 38-kd fragment containing K 1-4 , which was originally identified as angiostatin (51)(52)(53). Thus, the K 1-3 , K 1-5 , and K 1-4.5 (kringles 1-4 plus 85% of kringle 5) fragments exhibit more antiangiogenic activity than K 1-4 (12,54,55). Remarkably, we detected the presence of several "angiostatins" in the SSc patient plasma, including the presence of a 25-kd form (lysine K 1-3 ).…”

Upregulation of Vitamin D binding protein (Gc-globulin) binding sites during neutrophil activation from a latent reservoir in azurophil granules

“…A well-known high affinity ligand for DBP is the monomeric form of actin (Gactin), and plasma DBP serves as an extracellular scavenger for actin released from necrotic tissue (White and Cooke, 2000). Actin is the most prevalent intracellular protein, however, several investigators have reported actin on the extracellular face of the plasma membrane in lymphocytes and endothelial cells (Dudani et al, 2005;Petrini et al, 1983;Petrini et al, 1985;Wang et al, 2006;Wang et al, 2004). Reports have also shown that other cytoskeletal proteins are expressed on the cell surface (Moisan and Girard, 2006).…”