Dissolution of arterial platelet thrombi in vivo with a bifunctional platelet GPIIIa49-66 ligand which specifically targets the platelet thrombus.

Patients with HIV-1 immune-related thrombocytopenia have a unique antibody (Ab) against integrin GPIIIa49-66 capable of inducing oxidative platelet fragmentation via Ab activation of platelet nicotinamide adenine dinucleotide phosphate oxidase and 12-lipoxygenase releasing reactive oxygen species. Using a phage display single-chain antibody (scFv) library, we developed a novel human monoclonal scFv Ab against GPIIIa49-66 (named A11) capable of inducing fragmentation of activated platelets. In this study, we investigated the in vivo use of A11.

Role of thrombin as a tumor growth factor.

The clinical observation that thrombosis in some patients heralds the onset of malignancy has been recognized for over a century. Thrombin the key terminal enzyme of coagulation also promotes angiogenesis and stimulates tumor-platelet adhesion, adhesion to endothelium, tumor implantation, tumor cell growth and metastasis. The thrombin receptor, a member of the protease-activated receptor family, is expressed on many tumor cell lines and on breast tumor biopsy specimens.

Thrombin induces tumor cell cycle activation and spontaneous growth by down-regulation of p27Kip1, in association with the up-regulation of Skp2 and MiR-222.

The effect of thrombin on tumor cell cycle activation and spontaneous growth was examined in synchronized serum-starved tumor cell lines and a model of spontaneous prostate cancer development in TRAMP mice. BrdUrd incorporation and propidium iodide staining of prostate LNCaP cells arrested in G(0) and treated with thrombin or serum revealed a 48- and 29-fold increase in S phase cells, respectively, at 8 hours. Similar results were obtained with TRAMP cells and a glioblastoma cell line, T98G.

C-terminal ADAMTS-18 fragment induces oxidative platelet fragmentation, dissolves platelet aggregates, and protects against carotid artery occlusion and cerebral stroke.

Anti-platelet integrin GPIIIa49-66 antibody (Ab) induces complement-independent platelet oxidative fragmentation and death by generation of platelet peroxide following NADPH oxidase activation. A C-terminal 385-amino acid fragment of ADAMTS-18 (a disintegrin metalloproteinase with thrombospondin motifs produced in endothelial cells) induces oxidative platelet fragmentation in an identical kinetic fashion as anti-GPIIIa49-66 Ab. Endothelial cell ADAMTS-18 secretion is enhanced by thrombin and activated by thrombin cleavage to fragment platelets.

Role of molecular mimicry of hepatitis C virus protein with platelet GPIIIa in hepatitis C-related immunologic thrombocytopenia.

Patients with HIV-1 immune-related thrombocytopenia (HIV-1-ITP) have a unique Ab against platelet GPIIIa49-66 capable of inducing oxidative platelet fragmentation in the absence of complement. HIV-1-seropositive drug abusers are more prone to develop immune thrombocytopenia than non-drug abusers and have a higher coinfection with hepatitis C virus (HCV) than non-drug abusers (90% vs 30%). Molecular mimicry was sought by screening a phage peptide library with anti-GPIIIa49-66 antibody as bait for peptides sharing homology sequences with HCV.

Thrombin up-regulates cathepsin D which enhances angiogenesis, growth, and metastasis.

Cathepsin D (CD) up-regulation has been associated with human malignancy and poor prognosis. Thrombin up-regulated CD mRNA and protein in eight tumor cell lines as well as in human umbilical vascular endothelial cells (HUVEC). Thrombin increased the secretion of CD by 3- to 8-fold and enhanced chemotaxis ( approximately 2-fold) in 4T1 murine mammary CA cells, which was completely inhibited with the knockdown of CD.

Twist is required for thrombin-induced tumor angiogenesis and growth.

Twist, a master regulator of embryonic morphogenesis, induces functions that are also required for tumor invasion and metastasis. Because thrombin contributes to the malignant phenotype by up-regulating tumor metastasis, we examined its effect on Twist in five different tumor cell lines and two different endothelial cell lines. Thrombin up-regulated Twist mRNA and protein in all seven cell lines.

Platelet fragmentation requires a specific structural conformation of human monoclonal antibody against beta3 integrin.

We have described an autoantibody against beta3 (GPIIIa49-66), a region of platelet integrin alphaIIbbeta3 that is unique. It induces platelet fragmentation in the absence of complement via antibody activation of platelet NADPH oxidase and 12-lipoxygenase to release reactive oxygen species, which destroy platelets. To study the mechanism of anti-GPIIIa antibody-induced platelet fragmentation, we screened a human single chain Fv antibody library with the GPIIIa49-66 peptide.

Platelet particle formation by anti GPIIIa49-66 Ab, Ca2+ ionophore A23187, and phorbol myristate acetate is induced by reactive oxygen species and inhibited by dexamethasone blockade of platelet phospholipase A2, 12-lipoxygenase, and NADPH oxidase.

An HIV antibody (Ab) against platelet integrin GPIIIa49-66 induces complement-independent platelet particle formation by the elaboration of reactive oxygen species (ROS) downstream of the activation of the platelet NADPH oxidase by the 12-lipoxygenase (12-LO) product 12(S)-HETE. To determine whether other inducers of platelet particle formation also function via the induction of ROS, we examined the effects of the Ca(2+) ionophore A23187 and phorbol myristate acetate (PMA). Both agents induced oxidative platelet particle formation in an identical fashion as Ab, requiring Ca(2+) flux and 12(S)-HETE production as well as intact NADPH oxidase and 12-LO pathways.

Thrombin induces tumor growth, metastasis, and angiogenesis: Evidence for a thrombin-regulated dormant tumor phenotype.

The association of idiopathic venous thrombosis with occult cancer is generally recognized. However, it has not been fully appreciated that thrombin generated during thrombosis can augment the malignant phenotype. Thrombin activates tumor cell adhesion to platelets, endothelial cells, and subendothelial matrix proteins; enhances tumor cell growth; increases tumor cell seeding and spontaneous metastasis; and stimulates tumor cell angiogenesis.

Growth-regulated oncogene is pivotal in thrombin-induced angiogenesis.

The mechanism of thrombin-induced angiogenesis is poorly understood. Using a gene chip array to investigate the pro-malignant phenotype of thrombin-stimulated cells, we observed that thrombin markedly up-regulates growth-regulated oncogene-alpha (GRO-alpha) in several tumor cell lines as well as endothelial cells by mRNA and protein analysis. Thrombin enhanced the secretion of GRO-alpha from tumor cells 25- to 64-fold.

Hematopoiesis and stem cell renewal in long-term bone marrow cultures containing catalase.

Culturing mouse bone marrow in the presence of catalase dramatically alters hematopoiesis. Granulocyte output is initially increased 4- to 5-fold. This increase is transient and granulocyte production declines as immature (Sca-1+/LIN-) cells accumulate.

Role of molecular mimicry to HIV-1 peptides in HIV-1-related immunologic thrombocytopenia.

Patients with early HIV-1 infection develop an autoimmune thrombocytopenia in which antibody is directed against an immunodominant epitope of the beta3 (glycoprotein IIIa [GPIIIa]) integrin, GPIIIa49-66. This antibody induces thrombocytopenia by a novel complement-independent mechanism in which platelets are fragmented by antibody-induced generation of H2O2 derived from the interaction of platelet nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and 12-lipoxygenase. To examine whether sharing of epitope between host and parasite may be responsible for this immunodominant epitope, we screened for antibody-reactive peptides capable of inhibiting platelet lysis and oxidation in vitro, using a filamentous phage display 7-mer peptide library.

Role of endogenous thrombin in tumor implantation, seeding, and spontaneous metastasis.

Tumor/host-generated thrombin (endogenous thrombin) was investigated with tumor growth and metastasis experiments in mice by the use of hirudin, a highly potent specific inhibitor of thrombin. Pretreatment with hirudin inhibited tumor implantation in nude or syngeneic mice, following subcutaneous injection of 2 human and 2 murine tumors. Hirudin induced a considerable lag period in the appearance of tumor growth, compared with phosphate-buffered saline (PBS) treatment, but had no effect on established tumor nodule growth in vivo or on tumor growth in vitro.

Complement-independent Ab-induced peroxide lysis of platelets requires 12-lipoxygenase and a platelet NADPH oxidase pathway.

Antiplatelet GPIIIa49-66 Ab of HIV-related thrombocytopenic patients induces thrombocytopenia and platelet fragmentation by the generation of peroxide and other reactive oxygen species (ROS). Here we report the presence of a functional platelet NADPH oxidase pathway that requires activation by the platelet 12-lipoxygenase (12-LO) pathway to fragment platelets. A new Ab-mediated mechanism is described in which the platelet 12-LO product, 12(S)-HETE activates the NADPH oxidase pathway to generate ROS.

Thrombin induces neoangiogenesis in the chick chorioallantoic membrane.

Most tumors have constitutively active tissue factor on their surface, capable of generating thrombin in the surrounding environment, and thrombosis is associated with cancer. Thrombin is known to induce a malignant phenotype by enhancing tissue adhesion and cell growth in vitro and in vivo in mice. Because tumors require angiogenesis for growth, we examined whether thrombin induces neoangiogenesis in a physiologically intact in vivo model.

Thrombin induces increased expression and secretion of VEGF from human FS4 fibroblasts, DU145 prostate cells and CHRF megakaryocytes.

Angiogenesis is required for tumor growth and metastasis. It has recently been suggested that thrombin is a potent promoter of angiogenesis. We therefore examined the possibility that thrombin could be inducing the expression of vascular endothelial growth factor (VEGF), which promotes endothelial growth.