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. AIM: 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.

Matrix metalloproteinase 2 in tumor cell-induced platelet aggregation: regulation by nitric oxide.

A correlation exists between the ability of tumor cells to aggregate platelets and their tendency to metastasize. Tumor cell-induced platelet aggregation (TCIPA) facilitates the embolization of the vasculature with tumor cells and the formation of metastatic foci. It is well documented that matrix metalloproteinases (MMPs) play an integral part in tumor spread and the metastatic cascade. Therefore, we have examined the role of MMPs during TCIPA and its regulation by nitric oxide (NO) in vitro. Human HT-1080 fibrosarcoma and A549 lung epithelial cancer cells induced TCIPA in a concentration-dependent manner that was monitored by aggregometry. This aggregation resulted in the release of MMIP-2 from platelets and cancer cells, as measured by zymography. HT-1080 cells released significantly more MMP-2 than A549 cells and were more efficacious in inducing TCIPA. Inhibition of MMP-2 with phenanthroline (1-1000 microM), a synthetic inhibitor of MMPs, and by neutralizing anti-MMIP-2 antibody (10 microg/ml) reduced TCIPA induced by HT-1080 cells. TCIPA was abolished by simultaneous inhibition of platelet function with acetylsalicylic acid (100 microM; thromboxane pathway inhibitor), apyrase (250 microg/ml; ADP pathway inhibitor), and phenanthroline. NO donors such as S-nitroso-n-acetylpenicillamine and S-nitrosoglutathione (both at 0.01-100 microM) inhibited TCIPA and MMP-2 release from platelets and tumor cells. The inhibitory actions of S-nitroso-n-acetylpenicillamine and S-nitrosoglutathione were reversed by 1H-[1,2,4]oxadiazole[4,3]quinoxalin-1-one (0.01-30 microM), a selective inhibitor of the soluble guanylyl cyclase. We conclude that (a) human fibrosarcoma cells aggregate platelets via mechanism(s) that are mediated, in part, by MMP-2; (b) NO inhibits TCIPA, in part, by attenuating the release of MMP-2; and (c) these effects of NO are cGMP-dependent.

The potential of enzastaurin to enhance platelet aggregation and growth factor secretion: implications for cancer cell survival.

BACKGROUND: Enzastaurin is a protein kinase C (PKC)ß inhibitor with antiproliferative and proapoptotic effects that was in clinical development for the treatment of a variety of cancers. However, the primary endpoints in several clinical trials of enzastaurin were not met, and thrombosis was reported as an adverse effect in some trials. While investigating the role of PKC in regulating growth factor release from platelets, we found that, unlike other PKC inhibitors, enzastaurin may potentiate platelet aggregation. OBJECTIVE: To investigate the effects of enzastaurin on platelet aggregation, growth factor secretion from α-granules and cancer cell apoptosis in the presence of platelets. METHODS: Prostacyclin-washed platelets and platelet-rich plasma were isolated from the blood of healthy human volunteers. Platelet light-aggregometry was performed in the presence and absence of enzastaurin and acetylsalicylic acid (ASA). P-selectin was measured by flow cytometry, and vascular endothelial growth factor (VEGF) release was measured by ELISA. A549 lung carcinoma cells were treated with releasates from enzastaurin-titrated platelets. A cell death ELISA was performed to measure A549 apoptosis. RESULTS AND CONCLUSIONS: Enzastaurin (10(-8) -10(-6)  m) potentiated aggregation of prostacyclin-washed platelets and caused an increase in VEGF release from α-granules that, in turn, promoted cancer cell survival. In platelet-rich plasma, 10(-6)  m enzastaurin inhibited platelet aggregation, but not 10(-7)  m enzastaurin, which also failed to suppress VEGF secretion. ASA abrogated enzastaurin-potentiated washed-platelet aggregation and VEGF release. These findings indicate that, at high plasma protein-free drug concentrations, enzastaurin potentiates platelet aggregation and growth factor secretion, an effect that may counteract its anticancer activity. ASA nullifies this effect.

Pharmacological characteristics of solid-phase von Willebrand factor in human platelets.

1. The pharmacological characteristics of solid-phase von Willebrand factor (svWF), a novel platelet agonist, were studied. 2. Washed platelet suspensions were obtained from human blood and the effects of svWF on platelets were measured using aggregometry, phase-contrast microscopy, flow cytometry and zymography. 3. Incubation of platelets with svWF (0.2 - 1.2 microg ml(-1)) resulted in their adhesion to the ligand, while co-incubations of svWF with subthreshold concentrations of ADP, collagen and thrombin resulted in aggregation. 4. 6B4 inhibitory anti-glycoprotein (GP)Ib antibodies abolished platelet adhesion stimulated by svWF, while aggregation was reduced in the presence of 6B4 and N-Acetyl-Pen-Arg-Gly-Asp-Cys, an antagonist of GPIIb/IIIa. 5. Platelet adhesion stimulated with svWF was associated with a concentration-dependent increase in expression of GPIb, but not of GPIIb/IIIa. 6. In contrast, collagen (0.5 - 10.0 microg ml(-1)) caused down-regulation of GPIb and up-regulation of GPIIb/IIIa in platelets. 7. Solid-phase vWF (1.2 microg ml(-1)) resulted in the release of MMP-2 from platelets. 8. Inhibition of MMP-2 with phenanthroline (10 microM), but not with aspirin or apyrase, inhibited platelet adhesion stimulated with svWF. 9. In contrast, human recombinant MMP-2 potentiated both the effects of svWF on adhesion and up-regulation of GPIb. 10. Platelet adhesion and aggregation stimulated with svWF were reduced by S-nitroso-n-acetyl-penicillamine, an NO donor, and prostacyclin. 11. Thus, stimulation of human platelets with svWF leads to adhesion and aggregation that are mediated via activation of GPIb and GPIIb/IIIa, respectively. 12. Mechanisms of activation of GPIb by svWF involve the release of MMP-2, and are regulated by NO and prostacyclin.

Role of von Willebrand factor in tumour cell-induced platelet aggregation: differential regulation by NO and prostacyclin.

1. We have studied the effects of a novel agonist, solid-phase von Willebrand Factor (sVWF), on tumour cell-induced platelet aggregation (TCIPA). 2. Washed platelet suspensions were obtained from human blood and the effects of HT-1080 human fibrosarcoma cells and sVWF on platelets were studied using aggregometry, phase-contrast microscopy, and flow cytometry. 3. Incubation of platelets with sVWF (1.2 microg ml(-1)) and HT-1080 cells (5 x 10(3) ml(-1)) resulted in a two-phased reaction characterized first by the adhesion of platelets to sVWF, then by aggregation. 4. TCIPA in the presence of sVWF was inhibited by S-nitroso-glutathione (GSNO, 100 microM) and prostacyclin (PGI(2), 30 nM). 5. Platelet activation in the presence of tumour cells and sVWF resulted in the decreased surface expression of platelet glycoprotein (GP)Ib and up-regulation of GPIIb/IIIa receptors. 6. Pre-incubation of platelets with PGI(2) (30 nM) resulted in inhibition of sVWF-tumour cell-stimulated platelet surface expression of GPIIb/IIIa as measured by flow cytometry using antibodies directed against both non-activated and activated receptor. In contrast, GSNO (100 microM) did not affect sVWF-tumour cell-stimulated platelet surface expression of GPIIb/IIIa. 7. Flow cytometry performed with PAC-1 antibodies that bind only to the activated GPIIb/IIIa revealed that GSNO (100 microM) caused inhibition of activation of GPIIb/IIIa. 8. The inhibitors exerted no significant effects on TCIPA-mediated changes in GPIb. 9. Thus, sVWF potentiates the platelet-aggregatory activity of HT-1080 cells and these effects appear to be mediated via up-regulation of platelet GPIIb/IIIa. 10. Prostacyclin and NO inhibit TCIPA-sVWF-mediated platelet aggregation. The mechanisms of inhibition of this aggregation by PGI(2) differ from those of NO.

Determination of cell surface charge by photometric titration.

A colloid titration method has been frequently used to determine the number of charged residues at the cell surface. Here we present a new version of this technique, based on photometric measurements of a metachromatic shift in the maximum absorption of toluidine blue as it binds to the cell surface. The major improvements are: (1) simplified methodology and (2) increased precision of equivalence point determination. The data are analyzed using Gran's theory, which allows measurements to be taken at regular intervals instead of being concentrated around the equivalence titration point. We used this method to characterize the cell surface charge of three populations of rat mast cells: (1) peritoneal mast cells (PMC), (2) bone marrow-derived mast cells (BMMC) and (3) a rat cultured mast cell line (RCMC). Our results indicate that PMC have (4.23 +/- 0.59) x 10(8), while BMMC (8.58 +/- 0.26) x 10(7) negatively charged residues per cell. The results for RCMC were similar to those for BMMC. Taking into account the size differences between PMC and BMMC, the average charge density of PMC was also significantly higher than that of BMMC. The differences in cell surface charge were analyzed in the light of different sensitivities of mast cells to polycationic secretagogues.