Differential binding of plasminogen, plasmin, and angiostatin4.5 to cell surface beta-actin: implications for cancer-mediated angiogenesis.

Angiostatin4.5 (AS4.5) is the product of plasmin autoproteolysis and consists of kringles 1 to 4 and approximately 85% of kringle 5. In culture, cancer cell surface globular beta-actin mediates plasmin autoproteolysis to AS4.5. We now show that plasminogen binds to prostate cancer cells and that the binding colocalizes with surface beta-actin, but AS4.5 does not bind to the cell surface. Plasminogen and plasmin bind to immobilized beta-actin similarly, with a Kd of approximately 140 nmol/L. The binding is inhibited by epsilon-aminocaproic acid (epsilonACA), indicating the requirement for a lysine-kringle domain interaction. Using a series of peptides derived from beta-actin in competitive binding studies, we show that the domain necessary for plasminogen binding is within amino acids 55 to 69 (GDEAQSKRGILTLKY). Substitution of Lys61 or Lys68 with arginine results in the loss of the ability of the peptide to block plasminogen binding, indicating that Lys61 and Lys68 are essential for plasminogen binding. Other actin peptides, including peptides with lysine, did not inhibit the plasminogen-actin interaction. AS4.5 did not bind actin at concentrations up to 40 micromol/L. Plasminogen, plasmin, and AS4.5 all contain kringles 1 to 4; however, kringle 5 is truncated in AS4.5. Isolated kringle 5 binds to actin, suggesting intact kringle 5 is necessary for plasminogen and plasmin to bind to cell surface beta-actin, and the truncated kringle 5 in AS4.5 results in its release from beta-actin. These data may explain the mechanism by which AS4.5 is formed locally on cancer cell surfaces and yet acts on distant sites.

Alternatively spliced human tissue factor promotes tumor growth and angiogenesis in a pancreatic cancer tumor model.

INTRODUCTION: Tissue Factor (TF) expression is observed in many types of cancer, associated with more aggressive disease, and thrombosis. Alternatively-spliced human tissue factor (asHTF) has recently been identified in which exon 5 is deleted. asHTF is soluble due to the substitution of the transmembrane and cytoplasmic domains of exon 6 with a unique COOH-terminal domain. MATERIALS AND METHODS: We examine the expression and function of asHTF and full-length Tissue Factor ((FL)TF) in six human pancreatic cancer cells. Further, we transfected asHTF, (FL)TF, and control expression vectors into a non-expressing, human pancreatic cancer line (MiaPaCa-2). We studied the procoagulant activity of asHTF and (FL)TF and the effect on tumor growth in mice. RESULTS: asHTF is expressed in 5 of 6 human pancreatic cancer cell lines, but not in normal human fibroblasts, nor the MiaPaCa-2 line. (FL)TF conferred procoagulant activity, but asHTF did not. Transfected cells were injected subcutaneously in athymic mice. Interestingly, compared with control transfection, (FL)TF expression was associated with reduced tumor growth (mean 7 mg vs 85 mg), while asHTF-expression was associated with enhanced tumor growth (mean 389 mg vs. 85 mg). asHTF expression resulted in increased mitotic index and microvascular density. CONCLUSIONS: These data suggests that asHTF expression promotes tumor growth, and is associated with increased tumor cell proliferation and angiogenesis in vivo. Our results raise a new perspective on the understanding of the relationship between TF expression and cancer growth, by showing a dissociation of the procoagulant activity of (FL)TF and the cancer-promoting activity of asHTF.

In vivo generation of angiostatin isoforms by administration of a plasminogen activator and a free sulfhydryl donor: a phase I study of an angiostatic cocktail of tissue plasminogen activator and mesna.

PURPOSE: Angiostatin4.5 (AS4.5), the endogenous human angiostatin, is derived from plasminogen in a two-step process. A plasminogen activator converts plasminogen to plasmin, then plasmin undergoes autoproteolysis to AS4.5. A free sulfhydryl donor can mediate plasmin autoproteolysis. To translate this process to human cancer therapy, we conducted a phase I trial of administration of a tissue plasminogen activator (tPA) with a free sulfhydryl donor (mesna). PATIENTS AND METHODS: Fifteen patients with advanced solid tumors were treated. The dose of tPA was escalated (cohorts; 1, 2, 3, 5, and 7.5 mg/h for 6 hours). Mesna was administered as a 240 mg/m2 bolus followed by an infusion of 50 mg/h, concurrent with tPA. Both tPA and mesna were administered 3 consecutive days every 14 days. RESULTS: No dose-limiting toxicity was observed. Two AS4.5 isoforms were generated, Lys-AS4.5 and Glu-AS4.5. Mean baseline Lys-AS4.5 level was 20.4 nmol/L (SE, 2.9). In the 5 mg/h tPA cohort, Lys-AS4.5 levels increased by an average of 143% or 24 nmol/L (SE, 4.9) above baseline. Glu-AS4.5 (M(r) approximately 62,000) was also generated (additional 77 amino acids at amino terminus compared with Lys-AS4.5). Glu-AS4.5 level at baseline was undetectable in four of five patients in the 5 mg/h tPA cohort, but at end of infusion, was approximately 67 nmol/L (SE, 20). Two patients in the 5 mg/h tPA cohort experienced decreases in tumor markers with treatment, although no clinical objective responses were observed. CONCLUSION: This study shows that in vivo generation of AS4.5 is safe in humans and may provide a practical approach to achieve antiangiogenic therapy.

Cell surface-dependent generation of angiostatin4.5.

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 beta-actin is present on the extracellular membranes of cancer cells (PC-3, HT1080, and MDA-MB231), and beta-actin can mediate plasmin binding to the cell surface and autoproteolysis to AS4.5. In the presence of beta-actin, no small molecule-free 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 alpha-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 beta-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 beta-actin may also serve as a prognostic marker to predict tumor behavior.

Angiostatin(4.5)-mediated apoptosis of vascular endothelial cells.

Angiostatin, a proteolytic cleavage product of plasminogen, acts via a selective, yet poorly understood mechanism to potently inhibit angiogenesis (M. S. O'Reilly et al., Cell, 79: 315-328, 1994). Vascular endothelial cell proliferation assays revealed that angiostatin(4.5), a naturally occurring human isoform consisting of plasminogen kringle domains 1-4 and most of kringle domain 5 (G. A. Soff, Cancer Metastasis Rev., 19: 97-107, 2000), dose dependently reduces cell number despite the presence of a potent stimulus of proliferation. Flow cytometry using the vital dyes Hoechst 33342 and Pyronin Y revealed that approximately 40% of both control and angiostatin(4.5)-treated cells were in the proliferative phase, indicating that cell cycle progression is not impaired by exposure to angiostatin(4.5). Both bovine aortic endothelial cells and human umbilical endothelial cells were shown to undergo apoptosis in response to angiostatin(4.5). Caspases-3, -8, and -9 activation, specified by cleavage of fluorophore-conjugated specific peptide substrates, revealed a cascade of caspase activation that peaks at 36 h of angiostatin(4.5) treatment. Angiostatin(4.5) exposure induced release of cytochrome c from mitochondria in a caspase-dependent manner, but a pan-caspase inhibitor, zVAD-fmk, blocked cytochrome c release. Overall, these data indicate that human angiostatin(4.5) may function in vivo to block blood vessel formation by specifically inducing vascular endothelial cells to apoptose in a process likely involving both the intrinsic and extrinsic apoptosis pathways.

Venous thromboembolic disease in users of low-estrogen combined estrogen-progestin oral contraceptives.

OBJECTIVE: To assess the relationship between venous thromboembolic disease (VTE) and use of low-estrogen dose (<50 microg) combined estrogen-progestin oral contraceptives (OC) and three thrombosis-related gene mutations in a United States population. DESIGN: This case-control study was conducted in 1998-2000 among women ages 15-44 years who were members of the Kaiser Permanente Medical Care Program [KPMCP] (Northern and Southern California). Cases were women with incident VTE; about three times as many women frequency matched for age were randomly selected as controls from the KPMCP membership in the same years. Data were collected in a 1 h face-to-face interview; blood was drawn to extract DNA to test for gene polymorphisms. The analysis data set comprised 196 cases (mean age 35.3 years) and 746 controls (mean age 36.2 years). RESULTS: The adjusted odds ratio (OR) for VTE associated with current OC use was 4.07 (95% confidence interval [CI]: 2.77-6.00). The OR associated with OC use was higher for women who were obese than in the nonobese (p = 0.01 for likelihood test for interaction) and in women without predisposing medical conditions (p = 0.02 for interaction). The adjusted OR for VTE was 7.10 (95% CI: 2.33-21.61) in women with factor V Leiden (G1691A) mutation, 2.83 (95% CI: 0.70-11.63) in women with prothrombin G20210A mutation and 0.26 (95% CI: 0.10-0.65) in women with the MTHFR C677T mutation. The OR for VTE in OC users with factor V Leiden mutation (11.32) was elevated more than in OC users without the mutation (3.20) and women with the mutation who were non-OC users (8.42), but confidence intervals overlapped. CONCLUSIONS: The risk of VTE is increased in users of low-estrogen OC formulations. Obese women appear to be at greater risk of VTE when using OCs.