Duvelisib treatment is associated with altered expression of apoptotic regulators that helps in sensitization of chronic lymphocytic leukemia cells to venetoclax (ABT-199).

Duvelisib, an oral dual inhibitor of PI3K-δ and PI3K-γ, is in phase III trials for the treatment of chronic lymphocytic leukemia (CLL) and indolent non-Hodgkin's lymphoma. In CLL, duvelisib monotherapy is associated with high iwCLL (International Workshop on Chronic Lymphocytic Leukemia) and nodal response rates, but complete remissions are rare. To characterize the molecular effect of duvelisib, we obtained samples from CLL patients on the duvelisib phase I trial. Gene expression studies (RNAseq, Nanostring, Affymetrix array and real-time RT-PCR) demonstrated increased expression of BCL2 along with several BH3-only pro-apoptotic genes. In concert with induction of transcript levels, reverse phase protein arrays and immunoblots confirmed increase at the protein level. The BCL2 inhibitor venetoclax induced greater apoptosis in ex vivo-cultured CLL cells obtained from patients on duvelisib compared with pre-treatment CLL cells from the same patients. In vitro combination of duvelisib and venetoclax resulted in enhanced apoptosis even in CLL cells cultured under conditions that simulate the tumor microenvironment. These data provide a mechanistic rationale for testing the combination of duvelisib and venetoclax in the clinic. Such combination regimen (NCT02640833) is being evaluated for patients with B-cell malignancies including CLL.

Polymorphisms of ABCC5 and NOS3 genes influence doxorubicin cardiotoxicity in survivors of childhood acute lymphoblastic leukemia.

Anthracyclines are efficient chemotherapy agents. However, their use is limited by anthracycline-induced cardiotoxicity (CT). We investigated the influence of polymorphisms in doxorubicin metabolic and functional pathways on late-onset CT as estimated by echocardiography in 251 childhood acute lymphoblastic leukemia (cALL) patients. Association analyses revealed a modulating effect of two variants: A-1629 T in ABCC5, an ATP-binding cassette transporter, and G894T in the NOS3 endothelial nitric oxide synthase gene. Individuals with the ABCC5 TT-1629 genotype had an average of 8-12% reduction of ejection (EF) and shortening fractions (SF; EF: P<0.0001, and SF: P=0.001, respectively). A protective effect of the NOS3 TT894 genotype on EF was seen in high-risk patients (P=0.02), especially in those who did not receive dexrazoxane (P=0.002). Analysis of an additional cohort of 44 cALL patients replicated the ABCC5 association but was underpowered for NOS3. In summary, we identified two biomarkers that may contribute to cALL anthracycline CT risk stratification.

The phosphoinositide-3-kinase (PI3K)-delta and gamma inhibitor, IPI-145 (Duvelisib), overcomes signals from the PI3K/AKT/S6 pathway and promotes apoptosis in CLL.

The functional relevance of the B-cell receptor (BCR) and the evolution of protein kinases as therapeutic targets have recently shifted the paradigm for treatment of B-cell malignancies. Inhibition of p110δ with idelalisib has shown clinical activity in chronic lymphocytic leukemia (CLL). The dynamic interplay of isoforms p110δ and p110γ in leukocytes support the hypothesis that dual blockade may provide a therapeutic benefit. IPI-145, an oral inhibitor of p110δ and p110γ isoforms, sensitizes BCR-stimulated and/or stromal co-cultured primary CLL cells to apoptosis (median 20%, n=57; P<0.0001) including samples with poor prognostic markers, unmutated IgVH (n=28) and prior treatment (n=15; P<0.0001). IPI-145 potently inhibits the CD40L/IL-2/IL-10 induced proliferation of CLL cells with an IC50 in sub-nanomolar range. A corresponding dose-responsive inhibition of pAKT(Ser473) is observed with an IC50 of 0.36 nM. IPI-145 diminishes the BCR-induced chemokines CCL3 and CCL4 secretion to 17% and 37%, respectively. Pre-treatment with 1 µM IPI-145 inhibits the chemotaxis toward CXCL12; reduces pseudoemperipolesis to median 50%, inferring its ability to interfere with homing capabilities of CLL cells. BCR-activated signaling proteins AKT(Ser473), BAD(Ser112), ERK(Thr202/Tyr204) and S6(Ser235/236) are mitigated by IPI-145. Importantly, for clinical development in hematological malignancies, IPI-145 is selective to CLL B cells, sparing normal B- and T-lymphocytes.

Cisplatin benefit is predicted by immunohistochemical analysis of DNA repair proteins in squamous cell carcinoma but not adenocarcinoma: theranostic modeling by NSCLC constituent histological subclasses.

BACKGROUND: Most non-small-cell lung cancer (NSCLC) patients receive cisplatin-based chemotherapy though clinical response is restricted to a subset of patients. DNA repair protein levels are possible surrogates for cisplatin-induced DNA adduct (and subsequent cell death) repair efficiency and thus molecular determinants of therapeutic efficacy. The International Adjuvant Lung Trial (IALT)-Bio study previously suggested ERCC1 and MSH2 as predictive of cisplatin-based therapeutic benefit. PATIENTS AND METHODS: DNA repair protein expression (XPF, BRCA1, ERCC1, MSH2, p53, PARP1, and ATM) was assessed by immunohistochemistry on a large subset of patients (N = 769) from the IALT trial. Tissue Microarray slides were digitally scanned and signal quantified by user-defined macros. Statistical analyses (univariate and multivariate) of 5-year disease-free survival (DFS) and 5-year overall survival used binary cut-offs (H score low/high expression). RESULTS: In patients with squamous cell carcinoma (SCC), ATM, p53, PARP1, ERCC1, and MSH2 displayed significant (borderline) predictive values, mainly on DFS with chemotherapy efficacy limited to low marker levels. Adenocarcinoma (ADC) results were not significant. BRCA1 and XPF were not significant for predictive modeling in either SCC or ADCs. CONCLUSION: Here predictive utility of DNA repair enzymes co-segregates with SCC histology, focusing their predictive value to this histological subclass of NSCLC. Distinct mechanisms of chemotherapeutic response or resistance might exist among histological subclasses of solid tumors.

BRD-NUT oncoproteins: a family of closely related nuclear proteins that block epithelial differentiation and maintain the growth of carcinoma cells.

An unusual group of carcinomas, here termed nuclear protein in testis (NUT) midline carcinomas (NMC), are characterized by translocations that involve NUT, a novel gene on chromosome 15. In about 2/3rds of cases, NUT is fused to BRD4 on chromosome 19. Using a candidate gene approach, we identified two NMCs harboring novel rearrangements that result in the fusion of NUT to BRD3 on chromosome 9. The BRD3-NUT fusion gene encodes a protein composed of two tandem chromatin-binding bromodomains, an extra-terminal domain, a bipartite nuclear localization sequence, and almost the entirety of NUT that is highly homologous to BRD4-NUT. The function of NUT is unknown, but here we show that NUT contains nuclear localization and export sequences that promote nuclear-cytoplasmic shuttling via a leptomycin-sensitive pathway. In contrast, BRD3-NUT and BRD4-NUT are strictly nuclear, implying that the BRD moiety retains NUT in the nucleus via interactions with chromatin. Consistent with this idea, FRAP studies show that BRD4, BRD4-NUT and BRD3-NUT have significantly slower rates of lateral nuclear diffusion than that of NUT. To investigate the functional role of BRD-NUT fusion proteins in NMCs, we investigated the effects of siRNA-induced BRD3-NUT and BRD4-NUT withdrawal. Silencing of these proteins in NMC cell lines resulted in squamous differentiation and cell cycle arrest. Together, these data suggest that BRD-NUT fusion proteins contribute to carcinogenesis by associating with chromatin and interfering with epithelial differentiation.

Focused antibody response in plasma cell-infiltrated non-medullary (NOS) breast cancers.

Breast tumors with prominent plasma cell (PC) infiltrates often have a more favorable natural course that may plausibly be mediated by anti-tumor activity of the elicited antibodies. These breast tumor-associated PCs are typically IgG dominant in contrast to normal breast PCs, which are mainly IgA. It is our hypothesis that this PC infiltration represents a host immune response that is driven by one or more tumor antigens. Previously, we and others showed that medullary carcinoma (MC) had a focused repertoire and features suggestive of a protein antigen driven response. Infrequently, non-MC, not otherwise specified (NOS) breast tumors may exhibit heavy PC infiltrations, also of IgG isotype. In this first characterization of this favorable prognosis NOS subgroup, IgG heavy chain (Hc) and light chain (Lc) variable (V) regions from three PC-infiltrated NOS tumors were randomly cloned and sequenced. We found biased (V) gene usage by the infiltrating PCs and somatic hypermutation in the rearranged Ig Hc and Lc V regions that were compatible with antigenic selection of the progenitor B cells. The antibody response of NOS infiltrated breast cancer is repertoire-focused, with 13-68% of isolates being clonally reiterated in the samples. Each NOS patient used distinct Hc V-D-J and Lc V-J rearrangements, with her own immune response "footprint," but the overall pattern of gene usage followed that typical of exogenous antigen-induced immune responses. The data are consistent with the hypothesis that PC infiltrates infrequently arising in NOS tumors, as previously inferred for MC, are in response to one or more breast cancer-associated protein tumor antigens.

Spectrum of Epstein-Barr virus-associated diseases.

The association between Epstein-Barr virus (EBV) and a large number of benign and malignant diseases is unique among DNA viruses. Within infected tissues, proteins that are expressed during the normal lytic and latent viral life cycle lead to cellular alterations that contribute to these EBV-associated diseases. Although the early events of EBV infection are poorly understood, increasing knowledge of the viral processes that govern viral latency has shed light upon the potential mechanisms by which EBV infection can lead to cellular transformation. Our current understanding of the role of EBV in the development of Burkitt lymphoma, Hodgkin lymphoma, nasopharyngeal carcinoma, and other EBV-associated diseases is discussed.

Inflammatory pseudotumor of lymph node and spleen: an entity biologically distinct from inflammatory myofibroblastic tumor.

Inflammatory pseudotumors (IPTs) of the lymph node and spleen are an uncommon, benign cause of lymphadenopathy and/or splenomegaly that often bear striking clinicopathologic similarities to the inflammatory myofibroblastic tumors (IMTs) found in soft tissues. These tumors have classically been grouped together under the umbrella category of "inflammatory pseudotumor." Recent evidence shows that IMTs are in fact neoplastic processes that often harbor balanced chromosomal translocations involving the ALK kinase gene. These translocations result in expression of ALK kinase in IMTs as assessed by immunohistochemical studies. However, the relationship between IMT and IPT of the lymph node and spleen is uncertain. To determine if ALK tyrosine kinase expression is also present in IPT, 13 cases of IPT (9 involving lymph nodes, 4 splenic lesions) were examined for the presence of ALK tyrosine kinase by immunohistochemical staining on paraffin-embedded tissue. In addition, in situ hybridization studies for Epstein-Barr virus--encoded RNAs (EBER) and immunoperoxidase studies for human herpesvirus-8 (HHV8)--specific proteins were performed. All cases had clinical, morphologic, and immunophenotypic findings typical of IPT and had varying proportions of fibroblastic and inflammatory components. Age ranged from 11 to 75 (median, 40) years; 8 subjects were male, and 5 were female. None of the cases (0 of 13) had positive staining for ALK kinase or HHV8, and in 1 a lymph node (1 of 13) was focally positive for EBV (EBER) by in situ hybridization. The absence of ALK kinase as detected by immunohistochemical studies in IPT of the lymph node and spleen suggests that this entity is biologically distinct from the histologically similar IMT.

A novel, rapid, multiparametric approach for flow cytometric analysis of intranuclear terminal deoxynucleotidyl transferase.

This report describes a novel, reliable, and simplified approach for determination of intranuclear terminal deoxynucleotidyl transferase (TdT) expression. This approach utilizes standard permeabilization/fixation solutions to reliably detect intracellular antigens with minimal alterations in the light scatter properties of the analyzed cells. In contrast to other described methods, fluorescein isothiocyanate-conjugated anti-TdT antibody is added to previously analyzed and permeabilized cells after a leukemic cell population has been identified using characteristic surface staining patterns. The method eliminates the need for additional sample preparation or cumbersome permeablization steps and can easily be incorporated into any clinical laboratory's existing flow cytometry panels. Sixty-eight cases were analyzed with this method, including 31 acute myelogenous leukemias, 30 acute lymphoblastic leukemias, and 7 chronic lymphoproliferative disorders. To confirm the validity of the method, parallel immunoperoxidase staining and microscopic evaluation of cytocentrifuge test sample preparations were performed. Statistical analysis of the results reveals the method to be highly sensitive and specific, demonstrating exact correlation to the microscopic method. The ease and expeditiousness of this new procedure allows TdT testing to be routinely incorporated into the immunophenotyping repertoire of a busy clinical laboratory. In addition, the method should be readily adaptable to analyze a variety of other clinically relevant intranuclear and intracytoplasmic antigens.

Four-color flow cytometric immunophenotypic determination of peripheral blood CD4+ T-lymphocyte counts: a comparison of validity and cost-effectiveness with a two-color method.

The clinical usefulness of monitoring CD4+ T-lymphocyte counts in patients infected with HIV is now well established. The need for accurate, rapid, and cost-effective methods for making these determinations is evident. Recent technologic advances have allowed for the development of a system for the determination of CD4+ T-lymphocyte counts by simultaneous 4-color flow cytometry. A new 4-color 2-tube flow cytometric method for analyzing CD4+ T-lymphocyte subsets in whole blood was compared with a standard 2-color 5-tube method. The new method provides results almost identical to those of the well-established 2-color method used in our clinical laboratory. Statistical analyses indicate very low variability in CD4+ counts between the 2 methods, strongly supporting the usefulness of this new procedure. In addition, the 4-color procedure provides a 15% reduction in the materials cost per test compared with the 2-color method, as well as a marked reduction in the time expenditure of flow cytometry technologists.

Partial inhibition of platelet aggregation and fibrinogen binding by a murine monoclonal antibody to GPIIIa: requirement for antibody bivalency.

We produced a murine monoclonal antibody, 7H2, and localized its epitope to one or more small regions on platelet glycoprotein (GP) IIIa. 7H2-IgG and 7H2-F(ab')2 completely inhibit platelet aggregation and fibrinogen binding at low agonist concentrations, but only partially inhibit aggregation and fibrinogen binding at high agonist concentrations; 7H2-Fab has no effect on aggregation or fibrinogen binding at any agonist concentration. 7H2-IgG binds to the entire platelet population as judged by flow cytometry. At near saturating concentrations, approximately 40,000 7H2-IgG antibody molecules bind per platelet. In contrast, approximately 80,000 7H2 Fab molecules bind per platelet, suggesting that 7H2-IgG binding is bivalent. 7H2 was unable to inhibit fibrinogen binding to purified, immobilized GPIIb/IIIa. These data indicate that the bivalent binding of 7H2 to GPIIIa is required for its partial inhibition of fibrinogen binding to platelets, perhaps through dimerization of GPIIb/IIIa surface receptors (or more complex GPIIb/IIIa redistribution triggered by 7H2 binding) resulting in limited accessibility of fibrinogen to its binding site(s).

Identification of a novel thrombin receptor sequence required for activation-dependent responses.

Thrombin receptor (TR) activation by alpha-thrombin requires proteolytic cleavage, although synthetic peptides modeled after the new N-terminus directly effect receptor activation without cleavage, presumably by interacting with an unidentified region of the receptor. To further define critical residues responsible for receptor activation, we performed epitope mapping of anti-TR1-160, a previously described polyclonal antibody that inhibits peptide ligand-induced receptor activation in various cell types expressing a functional TR. An enzyme-linked immunosorbent assay (ELISA) using overlapping decapeptides derived from the TR extracellular domains identified four immunodominant peaks within the long N-terminal extension centered between amino acids 34-44, 48-67, 65-79, and 87-94. Soluble peptides derived from regions 83-94, but not those from other regions of the receptor, neutralized the ability of anti-TR1-160 to inhibit peptide ligand-induced platelet aggregation, suggesting that antibodies directed against this region of the TR are important in ligand-mediated activation. Thrombin receptor mutants lacking discrete regions of the TR were subsequently evaluated using microinjected Xenopus oocytes. Whereas a TR mutant lacking amino acid residues Thr67-Lys82 (TR delta 67-82) showed normal to exaggerated responses to either alpha-thrombin or synthetic peptide ligands, only TR mutants with limited deletions spanning the residues Gln83-Ser93 exhibited dysfunctional responses to either agonist (200 nmol/L alpha-thrombin or 200 mumol/L TR42-47). These data provide a model for receptor activation that implicates a discrete and previously uncharacterized sequence within the TR N-terminal extension that is necessary for initiation of signal transduction events independent of the initiating agonist.

Studies of activated GPIIb/IIIa receptors on the luminal surface of adherent platelets. Paradoxical loss of luminal receptors when platelets adhere to high density fibrinogen.

The accessibility of activated GPIIb/IIIa receptors on the luminal surface of platelets adherent to damaged blood vessels or atherosclerotic plaques is likely to play a crucial role in subsequent platelet recruitment. To define better the factors involved in this process, we developed a functional assay to assess the presence of activated, luminal GPIIb/IIIa receptors, based on their ability to bind erythrocytes containing a high density of covalently coupled RGD-containing peptides (thromboerythrocytes). Platelets readily adhered to wells coated with purified type I rat skin collagen and the adherent platelets bound a dense lawn of thromboerythrocytes. With fibrinogen-coated wells, platelet adhesion increased as the fibrinogen-coating concentration increased, reaching a plateau at about 11 micrograms/ml. Thromboerythrocyte binding to the platelets adherent to fibrinogen showed a paradoxical response, increasing at fibrinogen coating concentrations up to approximately 4-6 micrograms/ml and then dramatically decreasing at higher fibrinogen-coating concentrations. Scanning electron microscopy demonstrated that the morphology of platelets adherent to collagen was similar to that of platelets adherent to low density fibrinogen, with extensive filopodia formation and ruffling. In contrast, platelets adherent to high density fibrinogen showed a bland, flattened appearance. Immunogold staining of GPIIb/IIIa receptors demonstrated concentration of the receptors on the filopodia, and depletion of receptors on the flattened portion of the platelets. Thus, there is a paradoxical loss of accessible, activated GPIIb/IIIa receptors on the luminal surface of platelets adherent to high density fibrinogen. Two factors may contribute to this result: engagement of GPIIb/IIIa receptors with fibrinogen on the abluminal surface leading to the loss of luminal receptors, and loss of luminal filopodia that interact with thromboerythrocytes. These data provide insight into the differences in thrombogenicity between surfaces, and may provide a mechanism for purposefully passivating platelet-reactive artificial surfaces.

Immunologic analysis of the cloned platelet thrombin receptor activation mechanism: evidence supporting receptor cleavage, release of the N-terminal peptide, and insertion of the tethered ligand into a protected environment.

The recently cloned functional thrombin receptor is thought to be activated by thrombin cleavage of the bond between R41 and S42, followed by the insertion of the new N-terminal region ("tethered ligand") into an unknown site in the receptor. Antibodies to peptides at or near the cleavage site have been reported to inhibit thrombin-induced platelet activation to varying extents, but the precise mechanism(s) of their inhibition is unknown. We have produced: (1) a polyclonal antibody in rabbits to a peptide containing amino acids 34 to 52 (anti-TR34-52); enzyme-linked immunosorbent assays (ELISA) indicate that anti-TR34-52 contains antibodies to regions on both sides of the thrombin cleavage site; (2) two murine monoclonal antibodies (MoAbs) to a peptide containing amino acids 29 to 68; one antibody reacts primarily with residues N-terminal to the thrombin cleavage site, and the other reacts primarily with residues C-terminal to the cleavage site; and (3) a polyclonal rabbit antibody to a peptide containing amino acids 83 to 94 (anti-TR83-94). Anti-TR34-52 binds to platelets as judged by flow cytometry, and pretreating platelets with a thrombin receptor peptide ligand does not lead to loss of antibody reactivity, suggesting that platelet activation does not initiate redistribution or internalization of surface thrombin receptors. In contrast, pretreating platelets with thrombin leads to complete loss of anti-TR34-52 binding. Similarly, the binding of both MoAbs to platelets is dramatically reduced by pretreatment with thrombin. However, the binding of anti-TR83-94 is not decreased by thrombin activation, confirming that the receptor is not internalized. Anti-TR34-52 profoundly inhibits low dose thrombin-induced platelet shape change and aggregation, but the inhibition can be overcome with higher thrombin doses. However, anti-TR34-52 does not inhibit platelet aggregation induced by tethered ligand peptides. The TR34-52 peptide is a thrombin substrate, with cleavage occurring at the R41-S42 bond as judged by high performance liquid chromatography (HPLC) and platelet aggregation analysis. Anti-TR34-52 prevented cleavage of the TR34-52 peptide, suggesting that the antibody prevents platelet activation, at least in part, by preventing cleavage of the thrombin receptor. These data, although indirect, provide additional support for a thrombin activation mechanism involving thrombin cleavage of the receptor; in addition, they provide new evidence indicating that receptor cleavage is followed by loss of the N-terminal peptide, and insertion of the tethered ligand into a protected domain.

Substituting isoserine for serine in the thrombin receptor activation peptide SFLLRN confers resistance to aminopeptidase M-induced cleavage and inactivation.

Peptides containing sequences derived from the new NH2 terminus of the seven-transmembrane domain thrombin receptor after thrombin cleavage can activate platelets directly. We recently demonstrated that such peptides are readily cleaved and inactivated by plasma, serum, and endothelial cell-associated aminopeptidase M. The rapid degradation and inactivation of the peptides makes it difficult to assess dose-response relationships precisely or to conduct long term incubations with cells in the presence of plasma or serum. To overcome these problems, we first substituted D-serine for the NH2-terminal L-serine in an active peptide ligand (SFLLRNPNDKY). The D-serine derivative resisted degradation in plasma, but it is less than 1% as active as the L-serine peptide. Substituting a racemic mixture of the beta-amino acid isoserine for serine in a related peptide ligand (SFLLRN) produced an active peptide ((iso-S)FLLRN) that is approximately 15-20% as potent as SFLLRN as judged by platelet aggregation. To assess the stability of the peptides in plasma, SFLLRN (1 mM) was incubated with 50% plasma for various periods of time; after 15 min, 65% of the peptide remained intact, and after 2 h only 4% remained intact. Loss of aggregating activity paralleled the loss of intact peptide. In contrast, even after 2 h of incubation with plasma, 83% of the (iso-S)FLLRN remained intact and the aggregating activity was essentially unchanged. Qualitative differences in the patterns of platelet aggregation produced by the peptides were also observed. Thus, the distinct pattern of aggregation followed by rapid disaggregation observed with submaximal aggregating doses of SFLLRN was less evident with (iso-S)FLLRN, and inhibition of aminopeptidase M by amastatin enhanced aggregation in platelet-rich plasma induced by SFLLRN but not (iso-S)FLLRN. The (iso-S)FLLRN peptide should permit improved analysis of the effects of constant levels of peptide-induced thrombin receptor stimulation in the presence of plasma or serum, or when testing the effects of the peptide on cells that contain surface-associated aminopeptidase M.

The thrombin receptor extracellular domain contains sites crucial for peptide ligand-induced activation.

A thrombin receptor (TR) demonstrating a unique activation mechanism has recently been isolated from a megakaryocytic (Dami) cell line. To further study determinants of peptide ligand-mediated activation phenomenon, we have isolated, cloned, and stably expressed the identical receptor from a human umbilical vein endothelial cell (HUVEC) library. Chinese hamster ovary (CHO) cells expressing a functional TR (CHO-TR), platelets, and HUVECs were then used to specifically characterize alpha-thrombin- and peptide ligand-induced activation responses using two different antibodies: anti-TR34-52 directed against a 20-amino acid peptide spanning the thrombin cleavage site, and anti-TR1-160 generated against the NH2-terminal 160 amino acids of the TR expressed as a chimeric protein in Escherichia coli. Activation-dependent responses to both alpha-thrombin (10 nM) and peptide ligand (20 microM) were studied using fura 2-loaded cells and microspectrofluorimetry. Whereas preincubation of CHO-TR with anti-TR34-52 abolished only alpha-thrombin-induced [Ca2+]i transients, preincubation with anti-TR1-160 abrogated both alpha-thrombin- and peptide ligand-induced responses. This latter inhibitory effect was dose dependent and similar for both agonists, with an EC50 of approximately 90 micrograms/ml. Anti-TR1-160 similarly abolished peptide ligand-induced [Ca2+]i transients in platelets and HUVECs, whereas qualitatively different responses characterized by delayed but sustained elevations in [Ca2+]i transients were evident using alpha-thrombin. Platelet aggregation to low concentrations of both ligands was nearly abolished by anti-TR1-160, although some shape change remained; anti-TR34-52 only inhibited alpha-thrombin-induced aggregation. These data establish that a critical recognition sequence for peptide ligand-mediated receptor activation is contained on the NH2-terminal portion of the receptor, upstream from the first transmembrane domain. Furthermore, alpha-thrombin-induced activation of HUVECs and platelets may be partially mediated by an alternative mechanism(s) or receptor(s).