Since recent studies demonstrate that vascular smooth muscle cells synthesize two distinct guanylate cyclase–stimulatory gases, NO and CO, we examined possible regulatory interactions between these two signaling molecules. Treatment of rat aortic smooth muscle cells with the NO donors, sodium nitroprusside, S -nitroso- N -acetyl-penicillamine, or 3-morpholinosydnonimine, increased heme oxygenase-1 (HO-1) mRNA and protein levels in a concentration- and time-dependent manner. Both actinomycin D and cycloheximide blocked NO-stimulated HO-1 mRNA and protein expression. Nuclear run-on experiments demonstrated that NO donors increased HO-1 gene transcription between 3- and 6-fold. In contrast, NO donors had no effect on the stability of HO-1 mRNA. Incubation of vascular smooth muscle cells with the membrane-permeable cGMP analogues, dibutyryl cGMP and 8-bromo-cGMP, failed to induce HO-1 gene expression. Treatment of vascular smooth muscle cells with NO donors also stimulated the production and release of CO, as demonstrated by the CO-dependent increase in intracellular cGMP levels in coincubated platelets. Finally, incubating vascular smooth muscle cells with interleukin-1β and tumor necrosis factor-α induced NO synthesis and also significantly increased the level of HO-1 protein. The cytokine-stimulated production of both NO and HO-1 protein in smooth muscle cells was blocked by the NO synthase inhibitor methyl- l -arginine. These results demonstrate that exogenously administered or endogenously released NO stimulates HO-1 gene expression and CO production in vascular smooth muscle cells. The ability of NO to induce HO-catalyzed CO release from vascular smooth muscle cells provides a novel mechanism by which NO might modulate soluble guanylate cyclase and, thereby, vascular smooth muscle cell and platelet function.

IntroductionThe association between malignant tumors and elevated platelet counts raises the possibility of a pathophysiologic interaction between platelets and cancer cells. Cancer cells activate and aggregate platelets. 1-3 Conversely, platelets promote metastasis by protecting cancer cells against natural killer (NK) cells in the blood, 4 by enhancing attachment of cancer cells to the blood vessel wall, 5 by disrupting endothelial junctions, 6 and by promoting angiogenesis through selective sequestering, transporting, and releasing of several growth factors. [7][8][9][10][11] In murine models of metastasis, a deficiency in platelet adhesion molecules, such as P-selectin, glycoprotein Ib␣ (GPIb␣), or GPIIIa, reduces the number of metastatic lesions. [12][13][14][15] Recently, Labelle et al showed that TGF-␤1 secreted from platelets enhances an epithelial to mesenchymal transition in cancer cells promoting metastasis via activation of TGF-␤/Smad and NF-B pathways. 16 We have recently shown that reducing platelet counts decreased the size and number of tumor nodules in a murine model of orthotopic ovarian cancer. 17 In the same study, we detected extravasation of platelets from tumor vasculatures and direct contact between platelets and cancer cells. In the current study, we investigated whether platelets directly affect tumor growth. Methods Coincubation of platelets with ovarian cancer cellsGel-filtered murine or human platelets were isolated from whole blood. 17 Twenty million resting or lysed platelets (prepared by 3 cycles of freeze-and-thaw) were incubated with cancer cells. In some experiments, platelets were isolated from C57BL/6 mice with syngeneic tumors induced 3-4 weeks after intraperitoneal injection of 1 ϫ 10 6 ID8 murine ovarian cancer cells. 17 A total of 50 000 murine ID8 or 2C6 and human SKOV3 or OVCA5 ovarian cancer cells were incubated with serum-free media overnight. In some experiments, cancer cells were transfected with TGF-␤R1 siRNA using 2 g siRNA and 3 L of lipofectamine reagent (Invitrogen). Approximately 20 ϫ 10 6 platelets were added to each well and incubated for 24 hours at 37°C. Murine cancer cells were incubated with murine platelets and human cancer cells with human platelets. In some experiments, different concentrations of blocking antibodies to GPIb␣ (Xia-B2, Emfret Analytics), P-selectin (RB40.34, BD Biosciences), TGF-␤1 (N1C2, Gene Tex Inc); or eptifibatide (0.5M, Merck); or aspirin (15 g/mL, SigmaAldrich) were added to platelets before incubation with cancer cells. In control samples, appropriate buffer was added to the cells instead of platelets. To differentiate between the effect of direct contact between platelets and cells from an indirect paracrine effect, we seeded platelets either on cancer cells or on porous membranes (0.4 m, PET membrane, BD Biosciences) separated from cells in a coculture system. To determine cell proliferation, we measured incorporation of fluorescence-conjugated EdU (5-ethynil-2Ј-deoxyuridine) to newly synthesized DNA according to the manufact...

The hypothesis that von Willebrand factor (vWF) binding to platelet membrane glycoprotein lb (GpIb) initiates intracellular pathways of platelet activation was studied. We measured the biochemical responses of intact human platelets treated with ristocetin plus vWF multimers purified from human cryoprecipitate. vWF plus ristocetin causes the breakdown of phosphatidylinositol 4,5-bisphosphate, the production of phosphatidic acid (PA), the activation of protein kinase C (PKC), increase of ionized cytoplasmic calcium (ICa2`J), and the synthesis of thromboxane A2. PA production, PKC activation, and the rise of [Ca211 stimulated by the ristocetin-induced binding of vWF multimers to platelets are inhibited by an anti-GpIb monoclonal antibody, but are unaffected by anti-GpIIb-IIIa monoclonal antibodies. Indomethacin also inhibits these responses without impairing platelet aggregation induced by vWF plus ristocetin. These results indicate that vWF binding to platelets initiates specific intraplatelet signaling pathways. The mechanism by which this occurs involves an arachidonic acid metabolite-dependent activation of phospholipase C after vWF binding to platelet membrane GpIb. This signal then causes PKC activation and increases of [Ca2+'], which promote platelet secretion and potentiate aggregation. (J. Clin. Invest.

These results indicate that vascular SMCs have both constitutive and inducible HO activity, and they respond to specific stimuli to generate guanylyl cyclase-stimulatory CO in the same SMCs and in coincubated platelets.

Binding of the multimeric adhesive glycoprotein, von Willebrand Factor (vWF), to the platelet membrane glycoprotein (GP) Ib-IX-V complex mediates platelet adhesion and initiates signal transduction leading to platelet activation. Recently described viper venom proteins that bind to the GP Ib alpha-chain and inhibit vWF binding provide novel probes for studying receptor function. We have purified a 50-kDa form of alboaggregin from the white-lipped tree viper (Trimeresurus albolabris) and two 25-kDa proteins, CHH-A and CHH-B, from the timber rattlesnake (Crotalus horridus horridus) in addition to a previously described 25-kDa alboaggregin and echicetin. Complete or partial amino acid sequencing of CHH-A, CHH-B, and 50-kDa alboaggregin and cross-reactivity of these proteins with an anti-botrocetin antiserum confirmed that they were disulfide-linked heterodimers or higher multimers of the C-type lectin protein family. These proteins, together with 25-kDa alboaggregin and echicetin, specifically bound to GP Ib alpha within the N-terminal peptide domain, His-1-Glu-282, and inhibited vWF binding with comparable IC50 values (approximately 0.2 microgram/mL). However, cross-blocking studies between these structurally related proteins and anti-GP Ib alpha monoclonal antibodies demonstrated that the venom protein binding sites were not congruent. Further, the 50-kDa alboaggregin, but not the other venom proteins, potently induced platelet activation as assessed by dense granule serotonin release or elevation of cytosolic ionized calcium. Treatment of platelets with the 50-kDa alboaggregin was associated with activation of protein kinase C and tyrosine kinase(s), resulting in a platelet protein phosphorylation profile similar to that seen on shear-stress-induced vWF binding to platelets. These results suggest that the 50-kDa alboaggregin induces cytoplasmic signaling coincident with its binding to the GP Ib-IX-V complex and provides a potentially useful probe for studying the mechanism of vWF-dependent platelet activation.