Autologous bone marrow transplantation in autoimmune arthritis restores immune homeostasis through CD4+CD25+Foxp3+ regulatory T cells

Despite the earlier use of potent immunosuppressive or cytostatic drugs and the recent emergence of biologicals as treatment for human autoimmune diseases (AIDs), some patients still remain unresponsive to treatment. To those severely ill patients, autologous bone marrow transplantation (aBMT) is applied as a last resource, leading to disease remission in a majority of patients. The underlying mechanism of action of aBMT is still largely unknown. Here, we showed that regulatory T cells (Tregs) play a role in the natural disease course of proteoglycan-induced arthritis (PGIA) and in disease remission by aBMT. aBMT led to an initial phase of rapid disease improvement corresponding with a relative increase in CD4⁺CD25⁺ T cells. At this time, the CD4⁺CD25⁺ cells did not yet show an increase in Foxp3 expression and showed less potent suppression. After this initial improvement, disease relapsed but stabilized at a level below the severity before aBMT. This second phase was actively regulated by potently suppressive CD4⁺CD25⁺Foxp3⁺ Tregs. This work provided further insight into the role of Tregs in restoration of the immune balance by aBMT and can open the way to explore therapeutic interventions to further improve treatment of AID and disease relapses.      

Splenic CD19-CD35+B220+ cells function as an inducer of follicular dendritic cell network formation

Follicular dendritic cells (FDCs) form a reticular FDC network in the lymphoid follicle that is essential for the retention and presentation of native antigens in the form of antigen-antibody immune complexes (ICs) to B cells during secondary immune response. Although the presence of migrating precursors of FDCs has been hypothesized, their entity has not been elucidated. Here we report the identification of murine splenic CD19^–CD11c^–CD35⁺B220⁺ cells as an inducer of FDC network formation. We demonstrated that CD19^–CD11c^–CD35⁺B220⁺ cells, together with stromal cells, had the remarkable ability to form lymphoid-follicle–like structures that contained B220⁺FDC-M1⁺ reticular cells originally derived from CD19^–CD11c^–CD35⁺B220⁺ cells in the CD35⁺ reticulum. Our results indicate that CD19^–CD11c^–CD35⁺B220⁺ cells function as an inducer of FDC network formation and that the interaction between CD19^–CD11c^–CD35⁺B220⁺ cells and stromal cells is required to initiate lymphoid follicle formation.      

Characterization of clonogenic multiple myeloma cells

The identity of the cells responsible for the initiation and maintenance of multiple myeloma (MM) remains unclear largely because of the difficulty growing MM cells in vitro and in vivo. MM cell lines and clinical specimens are characterized by malignant plasma cells that express the cell surface antigen syndecan-1 (CD138); however, CD138 expression is limited to terminally differentiated plasma cells during B-cell development. Moreover, circulating B cells that are clonally related to MM plasma cells have been reported in some patients with MM. We found that human MM cell lines contained small (< 5%) subpopulations that lacked CD138 expression and had greater clonogenic potential in vitro than corresponding CD138⁺ plasma cells. CD138^- cells from clinical MM samples were similarly clonogenic both in vitro and in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice, whereas CD138⁺ cells were not. Furthermore, CD138^- cells from both cell lines and clinical samples phenotypically resembled postgerminal center B cells, and their clonogenic growth was inhibited by the anti-CD20 monoclonal antibody rituximab. These data suggest that MM "stem cells" are CD138^- B cells with the ability to replicate and subsequently differentiate into malignant CD138⁺ plasma cells.      

Antigen targeting to endosomal pathway in dendritic cell vaccination activates regulatory T cells and attenuates tumor immunity

Lymphoma cells are malignant cells of the T- or B-cell lineage that often express many surface markers inappropriately, yet are not recognized as abnormal by the immune system. We modeled this situation by inoculating ovalbumin-expressing E.G7-OVA lymphoma cells into mice that expressed ovalbumin as a self antigen in pancreatic islets, and investigated the efficacy of dendritic cell (DC) vaccination in these mice. Although vaccination with DC-expressing ovalbumin induced strong cytotoxic T-cell immunity, which led to clearance of E.G7-OVA lymphoma cells in naive C57BL/6 mice, DC vaccination was ineffective in mice expressing ovalbumin as a self antigen. Antigen modification to increase its processing via the endosomal processing pathway dramatically increased CD4 T-cell activation but paradoxically, impaired the protective effect of DC vaccination even in naive mice. Depletion of CD25⁺ T cells (regulatory T cells [Tregs]) prior to vaccination restored the efficacy of DC vaccination and allowed eradication of lymphoma also in mice expressing ovalbumin as a self antigen. We conclude that lymphoma cells may be eradicated using DC vaccination if activation of CD25⁺ Tregs is simultaneously inhibited, and that intentionally enhanced endosomal antigen processing in DC vaccines may shift the balance from CD4 T-cell help toward stimulation of Tregs.      

Dexamethasone-based regimens versus melphalan-prednisone for elderly multiple myeloma patients ineligible for high-dose therapy

Dexamethasone alone increases life expectancy in patients with relapsed multiple myeloma (MM); however, no large randomized study has compared dexamethasone and dexamethasone-based regimens with standard melphalan-prednisone in newly diagnosed MM patients ineligible for high-dose therapy. In the Intergroupe Francophone du Myélome (IFM) 95-01 trial, 488 patients aged 65 to 75 years were randomized between 4 regimens of treatment: melphalan-prednisone, dexamethasone alone, melphalan-dexamethasone, and dexamethasone-interferon alpha. Response rates at 6 months (except for complete response) were significantly higher among patients receiving melphalan-dexamethasone, and progression-free survival was significantly better among patients receiving melphalan (P < .001, for both comparisons), but there was no difference in overall survival between the 4 treatment groups. Moreover, the morbidity associated with dexamethasone-based regimens was significantly higher than with melphalan-prednisone, especially for severe pyogenic infections in the melphalan-dexamethasone arm and hemorrhage, severe diabetes, and gastrointestinal and psychiatric complications in the dexamethasone arms. Overall, these results indicated that dexamethasone should not be routinely recommended as first-line treatment in elderly patients with MM. In the context of the IFM 95-01 trial, the standard melphalan-prednisone remained the best treatment choice when efficacy and patient comfort were both considered. These results might be useful in the context of future combinations with innovative drugs.      

Distinct functional significance of Akt and mTOR constitutive activation in mantle cell lymphoma

Functional characterization of signaling pathways that critically control mantle cell lymphoma (MCL) cell growth and survival is relevant to designing new therapies for this lymphoma. We herein demonstrate that the constitutive activation of Akt correlates with the expression of the phosphorylated, inactive form of PTEN. Phosphatidyl-inositol-3 kinase (PI3-K)/Akt or mammalian target of rapamycin (mTOR) inhibition decreased the growth of both primary MCL cultures and established cell lines and antagonizes the growth-promoting activity of CD40 triggering and IL-4. These effects are mediated by nuclear accumulation of the p27^Kip1 inhibitor induced by down-regulation of the p45^Skp2 and Cks1 proteins, which target p27^Kip1 for degradation. Moreover, Akt inhibition down-regulated cyclin D1 by promoting its proteasome-dependent degradation driven by GSK-3. Intriguingly, mTOR inhibition affected cyclin D1 proteolysis only in MCL cells in which GSK-3 is under the direct control of mTOR, suggesting that different MCL subsets could be differently responsive to mTOR inhibition. Finally, PI3-K/Akt inhibitors, but not rapamycin, induced variable levels of caspase-dependent apoptosis and reduced telomerase activity. These results indicate that Akt and mTOR activation have distinct functional relevance in MCL and suggest that targeting Akt may result in more effective therapeutic effects compared with mTOR inhibition.      

Knock-in of an internal tandem duplication mutation into murine FLT3 confers myeloproliferative disease in a mouse model

Constitutive activation of FMS-like tyrosine kinase 3 (FLT3) by internal tandem duplication (ITD) mutations is one of the most common molecular alterations known in acute myeloid leukemia (AML). To investigate the role FLT3/ITD mutations play in the development of leukemia, we generated a FLT3/ITD knock-in mouse model by inserting an ITD mutation into the juxtamembrane domain of murine Flt3. FLT3^wt/ITD mice developed myeloproliferative disease, characterized by splenomegaly, leukocytosis, and myeloid hypercellularity, which progressed to mortality by 6 to 20 months. Bone marrow (BM) and spleen from FLT3^wt/ITD mice had an increased fraction of granulocytes/monocytes and dendritic cells, and a decreased fraction of B-lymphocytes. No sign of acute leukemia was observed over the lifetime of these mice. BM from FLT3^wt/ITD mice showed enhanced potential to generate myeloid colonies in vitro. BM from FLT3^wt/ITD mice also produced more spleen colonies in the in vivo colony-forming unit (CFU)–spleen assay. In the long-term competitive repopulation assay, BM cells from FLT3^wt/ITD mice outgrew the wild-type competitor cells and showed increased myeloid and reduced lymphoid expansion activity. In summary, our data indicate that expression of FLT3/ITD mutations alone is capable of conferring normal hematopoietic stem/progenitor cells (HSPCs) with enhanced myeloid expansion. It also appears to suppress B lymphoid maturation. Additional cooperative events appear to be required to progress to acute leukemia.      

Transgenic expression of JAK2V617F causes myeloproliferative disorders in mice

The JAK2^V617F mutation was found in most patients with myeloproliferative disorders (MPDs), including polycythemia vera, essential thrombocythemia, and primary myelofibrosis. We have generated transgenic mice expressing the mutated enzyme in the hematopoietic system driven by a vav gene promoter. The mice are viable and fertile. One line of the transgenic mice, which expressed a lower level of JAK2^V617F, showed moderate elevations of blood cell counts, whereas another line with a higher level of JAK2^V617F expression displayed marked increases in blood counts and developed phenotypes that closely resembled human essential thrombocythemia and polycythemia vera. The latter line of mice also developed primary myelofibrosis-like symptoms as they aged. The transgenic mice showed erythroid, megakaryocytic, and granulocytic hyperplasia in the bone marrow and spleen, displayed splenomegaly, and had reduced levels of plasma erythropoietin and thrombopoietin. They possessed an increased number of hematopoietic progenitor cells in peripheral blood, spleen, and bone marrow, and these cells formed autonomous colonies in the absence of growth factors and cytokines. The data show that JAK2^V617F can cause MPDs in mice. Our study thus provides a mouse model to study the pathologic role of JAK2^V617F and to develop treatment for MPDs.      

The role of angiopoietins in the development of endothelial cells from cord blood CD34+ progenitors

Circulating endothelial progenitors contribute to neovascularization at sites of injury and tumorigenesis in postnatal life. Yet, the molecular mechanisms initiating the endothelial developmental program of these precursors remain elusive. Here we provide evidence that endothelial development from progenitors circulating in human cord blood requires angiopoietins, a set of growth factors also involved in vascular branching during embryogenesis. We show that cord blood cells with the potential for endothelial development reside in a CD34⁺CD11b⁺ subset capable of autonomously producing and binding angiopoietins. Functionally, endogenous angiopoietin-1 regulates initial endothelial cell commitment, whereas angiopoietin-2 enhances expansion of the endothelial cell progeny. These findings suggest a role for angiopoietins as regulators of endothelial development from circulating progenitors and imply a function of angiopoietins at distinct developmental steps in postnatal angiogenesis.      

Radiologic and nuclear events: contingency planning for hematologists/oncologists

Untoward events involving radioactive material, either accidental or intentional, are potentially devastating. Hematologists and oncologists are uniquely suited to help manage radiation victims, as myelosuppression is a frequent complication of radiation exposure. In the aftermath of a large event, such as a nuclear detonation, there may be a national call for surge capacity that involves hematologists/oncologists across the country in the disaster response. In preparation, the National Marrow Donor Program and American Society for Blood and Marrow Transplantation have established the Radiation Injury Treatment Network (RITN), a voluntary consortium of transplant centers, donor centers, and umbilical cord blood banks. RITN is partnered with the Office of the Assistant Secretary for Preparedness and Response in the United States Department of Health and Human Services to develop treatment guidelines, educate healthcare professionals, coordinate situation response, and provide comprehensive evaluation and care for radiation injury victims. We outline the current plans for event response and describe scenarios, including catastrophic events that would require extensive support from hematologists/oncologists across the country. In addition, we highlight important reference resources and discuss current efforts to develop medical countermeasures against radiation toxicity. Practitioners and institutions across the country are encouraged to become involved and participate in the planning.      

Hematopoietic stem cell transplantation (HSCT) in children with juvenile myelomonocytic leukemia (JMML): results of the EWOG-MDS/EBMT trial

Allogeneic hematopoietic stem cell transplantation (HSCT) is the only proven curative therapy for juvenile myelomonocytic leukemia (JMML). We, the European Working Group on Childhood MDS (EWOG-MDS) and the European Blood and Marrow Transplantation (EBMT) Group, report the outcome of 100 children (67 boys and 33 girls) with JMML given unmanipulated HSCT after a preparative regimen including busulfan, cyclophosphamide, and melphalan. Forty-eight and 52 children received transplants from an HLA-identical relative or an unrelated donor (UD), respectively. The source of hematopoietic stem cells was bone marrow, peripheral blood, and cord blood in 79, 14, and 7 children, respectively. Splenectomy had been performed before HSCT in 24 children. The 5-year cumulative incidence of transplantation-related mortality and leukemia recurrence was 13% and 35%, respectively. Age older than 4 years predicted an increased risk of disease recurrence. The 5-year probability of event-free survival for children given HSCT from either a relative or a UD was 55% and 49%, respectively (P = NS), with median observation time of patients alive being 40 months (range, 6 to 144). In multivariate analysis, age older than 4 years and female sex predicted poorer outcome. Results of this study compare favorably with previously published reports. Disease recurrence remains the major cause of treatment failure. Outcome of UD-HSCT recipients is comparable to that of children receiving transplants from an HLA-identical sibling. (Blood. 2005;105:410-419)      

The centrosome index is a powerful prognostic marker in myeloma and identifies a cohort of patients that might benefit from aurora kinase inhibition

Centrosome amplification is common in myeloma and may be involved in disease pathogenesis. We have previously derived a gene expression–based centrosome index (CI) that correlated with centrosome amplification and was an independent prognostic factor in a small cohort of heterogeneously treated patients. In this study, we validated the prognostic significance of the CI in 2 large cohorts of patients entered into clinical trials and showed that a high CI is a powerful independent prognostic factor in both newly diagnosed and relapsed patients, whether treated by intensive therapy (total therapy II) or novel agents (bortezomib). Tumors with high CI overexpressed genes coding for proteins involved in cell cycle, proliferation, DNA damage, and G₂-M checkpoints, and associated with the centrosome and kinetochore/ microtubules. In particular, aurora kinases are significantly overexpressed in patients with high CI, with concordant increase in protein expression. Human myeloma cell lines with higher CI are more responsive to treatment with a novel aurora kinase inhibitor. Aurora kinase may represent novel therapeutic targets in these patients with very poor prognosis.      

Thalidomide for treatment of multiple myeloma: 10 years later

Thalidomide, bortezomib, and lenalidomide have recently changed the treatment paradigm of myeloma. In young, newly diagnosed patients, the combination of thalidomide and dexamethasone has been widely used as induction treatment before autologous stem cell transplantation (ASCT). In 2 randomized studies, consolidation or maintenance with low-dose thalidomide has extended both progression-free and overall survival in patients who underwent ASCT at diagnosis. In elderly, newly diagnosed patients, 3 independent randomized studies have reported that the oral combination of melphalan and prednisone plus thalidomide (MPT) is better than the standard melphalan and prednisone (MP). These studies have shown better progression-free survival, and 2 have shown improved overall survival for patients assigned to MPT. In refractory-relapsed disease, combinations including thalidomide with dexamethasone, melphalan, doxorubicin, or cyclophosphamide have been extensively investigated. The risks of side effects are greater when thalidomide is used in combination with other drugs. Thromboembolism and peripheral neuropathy are the major concern. The introduction of anticoagulant prophylaxis has reduced the rate of thromboembolism to less than 10%. Immediate thalidomide dose reduction or discontinuation when paresthesia is complicated by pain or motor deficit has decreased the severity of neuropathy. Future studies will define the most effective or the best sequence of combinations which could improve life expectancy.      

Activated human NK and CD8+ T cells express both TNF-related apoptosis-inducing ligand (TRAIL) and TRAIL receptors but are resistant to TRAIL-mediated cytotoxicity

The expression of tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL) and TRAIL receptors was investigated in resting and cytokine-activated purified primary human natural killer (NK) and CD8⁺ T cells. Resting NK and CD8⁺ T cells expressed the mRNA for all TRAIL receptors, but TRAIL-R4 was the only receptor clearly detectable on the surface of both cell types. NK cells were activated by interleukin 2 (IL-2) or IL-15, whereas CD8⁺ T cells were activated by phytohemagglutinin (PHA) + IL-2 followed by IL-2 alone for up to 10 days. On activation, both cell types rapidly expressed TRAIL-R2 and TRAIL-R3, whose expression peaked at day 10 of culture. TRAIL-R1, however, was never expressed at any time point examined, whereas the expression of TRAIL-R4, which showed a progressive increase in CD8⁺ T cells, remained constant in NK cells. Notwithstanding the expression of TRAIL-R2, recombinant TRAIL did not show any cytotoxic activity on either NK or CD8⁺ T cells. Both resting and activated NK and CD8⁺ T cells were found to express high levels of the 2 isoforms of c-FLIP (cellular Fas-associated death domain protein [FADD]–like IL-1–converting enzyme [FLICE]–inhibitory protein). Small interference RNA-mediated inhibition of c-FLIP expression in NK cells abrogated their resistance to the apoptotic effect of soluble TRAIL. Thus, once activated the major cytotoxic effector cells are potentially sensitive to TRAIL but are physiologically protected from its apoptotic action by intracellular level of c-FLIP.      

Highly efficient lentiviral-mediated human cytokine transgenesis on the NOD/scid background

Human neo-organ formation from stem cells can only be assayed by in vivo xenotransplantation. The human nonobese diabetic–severe combined immunodeficient (HuNOD/scid) CD34⁺ cell transplantation is a model that allows examination of hematopoietic tissue formation, although human hematopoietic cell maturation is abortive. Conventional humanization of the cytokine microenvironment has depended on generation of human cytokine-transgenic mice in strains appropriate for conventional plasmid microinjection, followed by backcrossing, a costly and time-consuming approach. Lentiviral vector infection of single-cell embryos was recently reported to produce transgenic animals. Using this approach, we have generated direct human granulocyte-macrophage colony-stimulating factor (hGM-CSF) transgenic mice from lentivirus-microinjected NOD/scid embryos, with 68% efficiency and 100% penetrance; this allowed us to obtain NOD/scid transgenic mice with considerable savings of resources. This powerful technique should assist in producing novel mouse models for the study of human blood cell lineage development and other human neo-organs from stem cell xenotransplantation for which a similar "humanization" rationale may be required.      

Efficient marking of human cells with rapid but transient repopulating activity in autografted recipients

Short-term hematopoietic reconstituting cells have been identified in mice, nonhuman primates, and among human cells that engraft xenogeneic hosts. We now present clonal marking data demonstrating a rapid but unsustained contribution of cultured human autografts to the initial phase of hematologic recovery in myeloablated patients. Three patients received transplants of granulocyte colony-stimulating factor–mobilized autologous peripheral blood (PB) cells, of which a portion (8%-25% of the CD34⁺ cells) had been incubated in vitro with growth factors (5 days) and clinical grade LN retrovirus (3-5 days). More than 9% of the clonogenic and long-term culture-initiating cells harvested were transduced. Semiquantitative and linear amplification-mediated polymerase chain reaction analyses of serial PB samples showed that marked white blood cells appeared in all 3 patients within 11 days and transiently constituted up to 0.1% to 1% of those produced in the first month. However, within another 2 to 9 months, marked cells had permanently decreased to very low levels. Analysis of more than 50 vector insertion sites showed none of the clones detected in the first month were active later. Eighty percent of inserts were located within or near genes, 2 near CXCR4. These findings provide direct evidence of cells with rapid but transient repopulating activity in patients and demonstrate their efficient transduction in vitro.      

IL-2 and IL-21 confer opposing differentiation programs to CD8+ T cells for adoptive immunotherapy

IL-2 and IL-21 are closely related cytokines that might have arisen by gene duplication. Both cytokines promote the function of effector CD8⁺ T cells, but their distinct effects on antigen-driven differentiation of naive CD8⁺ T cells into effector CD8⁺ T cells are not clearly understood. We found that antigen-induced expression of Eomesodermin (Eomes) and maturation of naive CD8⁺ T cells into granzyme B- and CD44-expressing effector CD8⁺ T cells was enhanced by IL-2, but, unexpectedly, suppressed by IL-21. Furthermore, IL-21 repressed expression of IL-2Ra and inhibited IL-2–mediated acquisition of a cytolytic CD8⁺ T-cell phenotype. Despite its inhibitory effects, IL-21 did not induce anergy, but instead potently enhanced the capacity of cells to mediate tumor regression upon adoptive transfer. In contrast, IL-2 impaired the subsequent antitumor function of transferred cells. Gene expression studies revealed a distinct IL-21 program that was characterized phenotypically by increased expression of L-selectin and functionally by enhanced antitumor immunity that was not reversed by secondary in vitro stimulation with antigen and IL-2. Thus, the efficacy of CD8⁺ T cells for adoptive immunotherapy can be influenced by opposing differentiation programs conferred by IL-2 and IL-21, a finding with important implications for the development of cellular cancer therapies.      

Band 3 Courcouronnes (Ser667Phe): a trafficking mutant differentially rescued by wild-type band 3 and glycophorin A

We describe a mutation in human erythrocyte band 3 (anion exchanger 1; SLC4A1) causing both hereditary spherocytosis and distal renal tubular acidosis. The proband developed a transfusion-dependent, hemolytic anemia following birth. Immunoblotting showed band 3 was reduced to approximately 35% of wildtype; other proteins of the band 3/Rh macrocomplex were also reduced. DNA sequence analysis revealed a novel homozygous mutation, c.2000C>T, leading to the amino acid substitution Ser667Phe. The parents were heterozygous for the same mutation. Sulfate influx in the patient's erythrocytes was approximately 40% wild type. The mutant band 3 produced very little chloride influx when expressed in Xenopus oocytes. Influx was partially rescued by coexpression of glycophorin A and also rescued by coexpression of wild-type band 3. At 2 years of age, an ammonium chloride challenge showed the child has incomplete distal renal tubular acidosis (dRTA). Stable expression of mutant kidney band 3 in both nonpolarized and polarized Madin-Darby canine kidney cells showed that most of the mutant protein was retained in the endoplasmic reticulum. Overall our results suggest that the Ser667Phe does not affect the anion transport function of band 3, but causes a trafficking defect in both erythrocytes and kidney cells.      

The JAK2 617V>F mutation triggers erythropoietin hypersensitivity and terminal erythroid amplification in primary cells from patients with polycythemia vera

The JAK2 617V>F mutation is frequent in polycythemia vera (PV) and essential thrombocythemia (ET). Using quantitative polymerase chain reaction (PCR), we found that high levels of JAK2 617V>F in PV correlate with increased granulocytes and high levels of hemoglobin and endogenous erythroid colony formation. We detected normal progenitors and those that were heterozygous or homozygous for the mutation by genotyping ET and PV clonal immature and committed progenitors. In PV patients, we distinguished homozygous profiles with normal, heterozygous, and homozygous progenitors from heterozygous profiles with only heterozygous and normal progenitors. PV patients with a heterozygous profile had more mutated, committed progenitors than did other PV and ET patients, suggesting a selective amplification of mutated cells in the early phases of hematopoiesis. We demonstrated that mutated erythroid progenitors were more sensitive to erythropoietin than normal progenitors, and that most homozygous erythroid progenitors were erythropoietin independent. Moreover, we observed a greater in vitro erythroid amplification and a selective advantage in vivo for mutated cells in late stages of hematopoiesis. These results suggest that, for PV, erythrocytosis can occur through two mechanisms: terminal erythroid amplification triggered by JAK2 617V>F homozygosity, and a 2-step process including the upstream amplification of heterozygous cells that may involve additional molecular events.      

Peripheral tachykinins and the neurokinin receptor NK1 are required for platelet thrombus formation

Platelets play an important role in hemostasis, with inappropriate platelet activation being a major contributor to debilitating and often fatal thrombosis by causing myocardial infarction and stroke. Although current antithrombotic treatment is generally well tolerated and effective, many patients still experience cardiovascular problems, which may reflect the existence of alternative underlying regulatory mechanisms in platelets to those targeted by existing drugs. In this study, we define a role for peripherally distributed members of the tachykinin family of peptides, namely substance P and the newly discovered endokinins A and B that are present in platelets, in the activation of platelet function and thrombus formation. We have reported previously that the preferred pharmacologically characterized receptor for these peptides, the NK1 receptor, is present on platelets. Inhibition or deficiency of the NK1 receptor, or SP agonist activity, resulted in substantially reduced thrombus formation in vitro under arterial flow conditions, increased bleeding time in mice, and a decrease in experimentally induced thromboembolism. Inhibition of the NK1 receptor may therefore provide benefit in patients vulnerable to thrombosis and may offer an alternative therapeutic target.