Platelets have a crucial role in the maintenance of normal haemostasis, and perturbations of this system can lead to pathological thrombus formation and vascular occlusion, resulting in stroke, myocardial infarction and unstable angina. ADP released from damaged vessels and red blood cells induces platelet aggregation through activation of the integrin GPIIb-IIIa and subsequent binding of fibrinogen. ADP is also secreted from platelets on activation, providing positive feedback that potentiates the actions of many platelet activators. ADP mediates platelet aggregation through its action on two G-protein-coupled receptor subtypes. The P2Y1 receptor couples to Gq and mobilizes intracellular calcium ions to mediate platelet shape change and aggregation. The second ADP receptor required for aggregation (variously called P2Y(ADP), P2Y(AC), P2Ycyc or P2T(AC)) is coupled to the inhibition of adenylyl cyclase through Gi. The molecular identity of the Gi-linked receptor is still elusive, even though it is the target of efficacious antithrombotic agents, such as ticlopidine and clopidogrel and AR-C66096 (ref. 9). Here we describe the cloning of this receptor, designated P2Y12, and provide evidence that a patient with a bleeding disorder has a defect in this gene. Cloning of the P2Y12 receptor should facilitate the development of better antiplatelet agents to treat cardiovascular diseases.

E-cadherin loss is frequently associated with ovarian cancer metastasis. Given that adhesion to the abdominal peritoneum is the first step in ovarian cancer dissemination, we reasoned that down-regulation of E-cadherin would affect expression of cell matrix adhesion receptors. We show here that inhibition of E-cadherin in ovarian cancer cells causes up-regulation of A 5 -integrin protein expression and transcription. When E-cadherin was blocked, RMUG-S ovarian cancer cells were able to attach and invade more efficiently. This greater efficiency could, in turn, be inhibited both in vitro and in vivo with an A 5 B 1 -integrin-blocking antibody. When E-cadherin is silenced, A 5 -integrin is up-regulated through activation of an epidermal growth factor receptor/FAK/Erk1-mitogenactivated protein kinase-dependent signaling pathway and not through the canonical E-cadherin/B-catenin signaling pathway. In SKOV-3ip1 ovarian cancer xenografts, which express high levels of A 5 -integrin, i.p. treatment with an A 5 B 1 -integrin antibody significantly reduced tumor burden, ascites, and number of metastasis and increased survival by an average of 12 days when compared with IgG treatment (P < 0.0005). A 5 -Integrin expression was detected by immunohistochemistry in 107 advanced stage ovarian cancers using a tissue microarray annotated with disease-specific patient follow-up. Ten of 107 tissues (9%) had A 5 -integrin overexpression, and 39% had some level of A 5 -integrin expression. The median survival for patients with high A 5 -integrin levels was 26 months versus 35 months for those with low integrin expression (P < 0.05). Taken together, we have identified A 5 -integrin upregulation as a molecular mechanism by which E-cadherin loss promotes tumor progression, providing an explanation for how E-cadherin loss increases metastasis. Targeting this integrin could be a promising therapy for a subset of ovarian cancer patients.

The proteinase-activated receptor 2 (PAR-2) belongs to the family of seven transmembrane region receptors, and, like the related thrombin receptor, it is activated by specific proteolytic cleavage of its extracellular amino terminus. It is not known which proteinase is the physiological activator of the PAR-2, but candidates can be found among the enzymes involved in the inflammatory cascade systems. Here, we have studied the effects of various mediators on the expression of the PAR-2 and the thrombin receptor in cultured human umbilical vein endothelial cells. Stimulation with the cytokines tumor necrosis factor ␣ or interleukin-1 ␣ as well as bacterial lipopolysaccharide elevated the expression of PAR-2 in a dose-dependent manner. The time course of induction after cytokine stimulation was similar to those published for the adhesion molecules intercellular adhesion molecule-1 and vascular cell adhesion molecule-1. After 20 h of stimulation, PAR-2 mRNA and protein levels were increased to 5-10-fold basal values, and, in the continued presence of tumor necrosis factor ␣, PAR-2 mRNA expression was found to remain elevated for up to 4 days. In contrast, the thrombin receptor gene was not induced by any of these inflammatory mediators. The responses to phorbol ester treatment also differed between the two genes. Thrombin receptor mRNA levels decreased steadily up to 20 h, whereas PAR-2 mRNA levels first rose to about 3-fold basal values at 4 h before decreasing again. Cell surface protein levels of both receptors were decreased after 20 h of phorbol ester stimulation. Elevating intracellular cAMP levels by treatment with forskolin resulted in decreased expression of both receptors, and inhibition of cAMP degradation appeared to blunt the cytokine-induced increase in PAR-2 expression. The induction of the PAR-2 by cytokine treatment supports the concept of PAR-2 involvement in the acute inflammatory response.

Glycoprotein (GP) V is a major substrate cleaved by the protease thrombin during thrombin-induced platelet activation. Previous analysis of platelets from GP V-null mice suggested a role for GP V as a negative modulator of platelet activation by thrombin. We now report the mechanism by which thrombin activates GP V ؊͞؊ platelets. We show that proteolytically inactive forms of thrombin induce robust stimulatory responses in GP V null mouse platelets, via the platelet GP Ib-IX-V complex. Because proteolytically inactive thrombin can activate wild-type mouse and human platelets after treatment with thrombin to cleave GP V, this mechanism is involved in thrombin-induced platelet aggregation. Platelet activation through GP Ib-IX depends on ADP secretion, and specific inhibitors demonstrate that the recently cloned P2Y 12 ADP receptor (Gi-coupled ADP receptor) is involved in this pathway, and that the P2Y1 receptor (G q-coupled ADP receptor) may play a less significant role. Thrombosis was generated in GP V null mice only in response to catalytically inactive thrombin, whereas thrombosis occurred in both genotypes (wild type and GP V null) in response to active thrombin. These data support a thrombin receptor function for the platelet membrane GP Ib-IX-V complex, and describe a novel thrombin signaling mechanism involving an initiating proteolytic event followed by stimulation of the GP Ib-IX via thrombin acting as a ligand, resulting in platelet activation.G lycoprotein (GP) Ib-IX-V is a major complex on the platelet surface, second only to ␣⌱⌱b␤3. This complex consists of several subunits: GP Ib␣, GP Ib␤, GP IX, and GP V in the ratio of 2:2:2:1. Absence of GP Ib-IX-V results in a severe bleeding disorder known as Bernard Soulier syndrome characterized by giant platelets and impaired von Willebrand factor (vWf) binding (1). GP Ib␣ is a receptor for vWf, and the GP Ib-IX-V complex is critical for platelet adhesion under arterial shear conditions (2). A role for GP Ib-IX-V in platelet activation has been proposed on the basis of observations that the signaling molecule 14-3-3 (3, 4) is associated with the complex, and that phosphorylation of pp72 syk occurs upon vWf binding to GP Ib␣ (5). In fact, Zaffran et al. (6) recently showed that in heterologous Chinese hamster ovary (CHO) cells expressing both ␣⌱⌱b␤3 and GP Ib-IX, inside-out activation of ␣⌱⌱b␤3 could occur upon vWf adhesion.The GP Ib␣ subunit also has a thrombin binding site on the extracellular domain that overlaps the vWf binding domain (7). Additionally, the complex has a platelet-specific thrombin substrate, GP V, that is cleaved very early during thrombin-induced platelet aggregation (8). Platelets from Bernard Soulier syndrome patients show an impaired response to thrombin (9), and antibodies that block thrombin binding to GP Ib␣ also partially inhibit platelet responses to thrombin (9). More recently, thrombin binding to GP Ib␣ has been shown to enhance platelet procoagulant activity (10). However, the physiological significance of this interaction ha...

Purpose: Targeted therapeutics have significantly changed the outcome for patients diagnosed with cancer. Still, effective therapeutic intervention does not exist for many cancers and much remains to be done. The objective of this study was to identify novel genes that potentially regulate tumor growth, to target these gene products with monoclonal antibodies, and to examine the therapeutic potential of these antibodies. Experimental Design: Using cDNA microarray analysis, we identified genes overexpressed in several solid malignancies. We generated a mouse monoclonal antibody, 19.2.1, and its humanized counterpart, PDL192, to one such target, TweakR (TWEAK receptor, Fn14, TNFRSF12A, CD266), and characterized the antitumor activities in vitro and in mouse xenograft models. Results: Both 19.2.1 (mouse IgG2a) and PDL192 (human IgG1), like TWEAK, the natural ligand of TweakR, inhibited the growth of several TweakR-expressing cancer cell lines in anchorage-dependent and anchorage-independent assays in vitro. Both antibodies showed significant antitumor activity in multiple mouse xenograft models. PDL192 and 19.2.1 also induced antibody-dependent cellular cytotoxicity (ADCC) of cancer cell lines in vitro. A chimeric version of 19.2.1 containing the mouse IgG1 Fc region (19.2.1×G1) exhibited significantly less ADCC than 19.2.1. However, 19.2.1×G1 showed differential activity in vivo, with activity equivalent to 19.2.1 in one model, but significantly less efficacy than 19.2.1 in a second model. These results indicate that PDL192 and 19.2.1 mediate their antitumor effects by signaling through TweakR, resulting in reduced tumor cell proliferation, and by ADCC. Clin Cancer Res; 16(2); 497–508

Purpose: Trastuzumab-emtansine (T-DM1) is an antibodydrug conjugate (ADC) comprising the cytotoxic agent DM1 conjugated to trastuzumab with a stable linker. Thrombocytopenia was the dose-limiting toxicity in the phase I study, and grade !3 thrombocytopenia occurred in up to 13% of patients receiving T-DM1 in phase III studies. We investigated the mechanism of T-DM1-induced thrombocytopenia.Experimental Design: The effect of T-DM1 on platelet function was measured by aggregometry, and by flow cytometry to detect the markers of activation. The effect of T-DM1 on differentiation and maturation of megakaryocytes (MK) from human hematopoietic stem cells was assessed by flow cytometry and microscopy. Binding, uptake, and catabolism of T-DM1 in MKs, were assessed by various techniques including fluorescence microscopy, scintigraphy to detect T- [H 3 ]-DM1 and 125 I-T-DM1, and mass spectrometry. The role of FcgRIIa was assessed using blocking antibodies and mutant constructs of trastuzumab that do not bind FcgR.Results: T-DM1 had no direct effect on platelet activation and aggregation, but it did markedly inhibit MK differentiation via a cytotoxic effect. Inhibition occurred with DM1-containing ADCs but not with trastuzumab demonstrating a role for DM1. MKs internalized these ADCs in a HER2-independent, FcgRIIa-dependent manner, resulting in intracellular release of DM1. Binding and internalization of T-DM1 diminished as MKs matured; however, prolonged exposure of mature MKs to T-DM1 resulted in a disrupted cytoskeletal structure.Conclusions: These data support the hypothesis that T-DM1-induced thrombocytopenia is mediated in large part by DM1-induced impairment of MK differentiation, with a less pronounced effect on mature MKs.

A role for glycoprotein (GP)V in platelet function has been proposed on the basis of observations that GP V is the major thrombin substrate on intact platelets cleaved during thrombin-induced platelet aggregation, and that GP V promotes GP Ib-IX surface expression in heterologous cells. We tested the hypotheses that GP V is involved in thrombin-induced platelet activation, in GP Ib-IX expression, and in other platelet responses by generating GP V null mice. Contrary to expectations, GP V ؊͞؊ platelets were normal in size and expressed normal amounts of GP Ib-IX that was functional in von Willebrand factor binding, explaining why defects in GP V have not been observed in Bernard-Soulier syndrome, a bleeding disorder caused by a lack of functional GP Ib-IX-V. Moreover, in vitro analysis demonstrated that GP V ؊͞؊ platelets were hyperresponsive to thrombin, resulting in increased fibrinogen binding and an increased aggregation response. Consistent with these findings, GP V ؊͞؊ mice had a shorter bleeding time. These data support a role for GP V as a negative modulator of platelet activation. Furthermore, they suggest a new mechanism by which thrombin enhances platelet responsiveness independent of activation of the classical G-protein-coupled thrombin receptors. P latelet thrombosis and hemostasis are complex reactions that depend on adhesive interactions mediated by specific receptors. A major platelet complex is glycoprotein (GP) Ib-IX-V. The initial adhesion of platelets is primarily mediated by binding of platelet membrane GP Ib-IX-V to von Willebrand factor (vWf) found on damaged vessel walls (1). After adhesion, binding of other agonists such as thrombin, ADP, and collagen induce signaling events that ultimately activate the receptor function of ␣⌱⌱b␤3 for soluble fibrinogen, leading to platelet aggregation (2). Although platelet aggregates are required for normal hemostasis, they can in addition cause arterial thrombosis in atherosclerotic arteries, e.g., acute myocardial infarction and stroke, inducing ischemic complications of cardiovascular disease (3, 4).The importance of the GP Ib-IX-V complex in normal platelet function is underscored by the study of Bernard-Soulier syndrome (BSS), an inherited bleeding disorder characterized by large platelets that are defective in adhesion to damaged vessel walls (1). This genetic disorder is caused by a lack of functional GP Ib-IX-V and has been linked to defects in either GP Ib or GP IX (5). The activities mapped to the GP Ib subunit of the GP Ib-IX-V complex include vWf (6) and thrombin binding (7, 8) on the extracellular domain and actin-binding protein (9-11) and 14-3-3 (12-15) binding on the cytoplasmic domain.Several studies indicate functional activities for the GP V subunit of the GP Ib-IX-V complex. In one example, GP V has been shown to be cleaved by thrombin from the platelet surface during thrombin-mediated platelet stimulation (16), but the role of GP V cleavage in this thrombin-induced platelet response is unresolved (17). In another example, the signa...

Purpose In a recent phase II study of onartuzumab (MetMAb), patients whose non–small cell lung cancer (NSCLC) tissue scored as positive for MET protein by immunohistochemistry (IHC) experienced a significant benefit with onartuzumab plus erlotinib (O+E) versus erlotinib. We describe development and validation of a standardized MET IHC assay and, retrospectively, evaluate multiple biomarkers as predictors of patient benefit. Experimental Design Biomarkers related to MET and/or EGF receptor (EGFR) signaling were measured by IHC, FISH, quantitative reverse transcription PCR, mutation detection techniques, and ELISA. Results A positive correlation between IHC, Western blotting, and MET mRNA expression was observed in NSCLC cell lines/tissues. An IHC scoring system of MET expression taking proportional and intensity-based thresholds into consideration was applied in an analysis of the phase II study and resulted in the best differentiation of outcomes. Further analyses revealed a nonsignificant overall survival (OS) improvement with O+E in patients with high MET copy number (mean ≥5 copies/cell by FISH); however, benefit was maintained in “MET IHC-positive”/MET FISH-negative patients (HR, 0.37; P = 0.01). MET, EGFR, amphiregulin, epiregulin, or HGF mRNA expression did not predict a significant benefit with onartuzumab; a nonsignificant OS improvement was observed in patients with high tumor MET mRNA levels (HR, 0.59; P = 0.23). Patients with low baseline plasma hepatocyte growth factor (HGF) exhibited an HR for OS of 0.519 (P = 0.09) in favor of onartuzumab treatment. Conclusions MET IHC remains the most robust predictor of OS and progression-free survival benefit from O+E relative to all examined exploratory markers.