JAK2 inhibition has different therapeutic effects according to myeloproliferative neoplasm development in mice

JAK2 inhibition therapy is used to treat patients suffering from myeloproliferative neoplasms (MPN). Conflicting data on this therapy are reported possibly linked to the types of inhibitors or disease type. Therefore, we decided to compare in mice the effect of a JAK2 inhibitor, Fedratinib, in MPN models of increasing severity: polycythemia vera (PV), post‐PV myelofibrosis (PPMF) and rapid post‐essential thrombocythemia MF (PTMF). The models were generated through JAK2 activation by the JAK2^V617F mutation or MPL constant stimulation. JAK2 inhibition induced a correction of splenomegaly, leucocytosis and microcytosis in all three MPN models. However, the effects on fibrosis, osteosclerosis, granulocytosis, erythropoiesis or platelet counts varied according to the disease severity stage. Strikingly, complete blockade of fibrosis and osteosclerosis was observed in the PPMF model, linked to correction of MK hyper/dysplasia, but not in the PTMF model, suggesting that MF development may also become JAK2‐independent. Interestingly, we originally found a decreased in the JAK2^V617F allele burden in progenitor cells from the spleen but not in other cell types. Overall, this study shows that JAK2 inhibition has different effects according to disease phenotypes and can (i) normalize platelet counts, (ii) prevent the development of marrow fibrosis/osteosclerosis at an early stage and (iii) reduce splenomegaly through blockage of stem cell mobilization in the spleen.     

Improved Diagnosis of the Transition to JAK2 V617F Homozygosity: The Key Feature for Predicting the Evolution of Myeloproliferative Neoplasms

Most cases of BCR-ABL1-negative myeloproliferative neoplasms (MPNs), essential thrombocythemia, polycythemia vera and primary myelofibrosis are associated with JAK2 ^V617F mutations. The outcomes of these cases are critically influenced by the transition from JAK2 ^V617F heterozygosity to homozygosity. Therefore, a technique providing an unbiased assessment of the critical allele burden, 50% JAK2 ^V617F, is highly desirable. In this study, we present an approach to assess the JAK2 ^V617F burden from genomic DNA (gDNA) and complementary DNA (cDNA) using one-plus-one template references for allele-specific quantitative-real-time-PCR (qPCR). Plasmidic gDNA and cDNA constructs encompassing one PCR template for JAK2 ^V617F spaced from one template for JAK2^{Wild Type} were constructed by multiple fusion PCR amplifications. Repeated assessments of the 50% JAK2^V617F burden within the dynamic range of serial dilutions of gDNA and cDNA constructs resulted in 52.53±4.2% and 51.46±4.21%, respectively. The mutation-positive cutoff was estimated to be 3.65% (mean +2 standard deviation) using 20 samples from a healthy population. This qPCR approach was compared with the qualitative ARMS-PCR technique and with two standard methods based on qPCR, and highly significant correlations were obtained in all cases. qPCR assays were performed on paired gDNA/cDNA samples from 20 MPN patients, and the JAK2 ^V617F expression showed a significant correlation with the allele burden. Our data demonstrate that the qPCR method using one-plus-one template references provides an improved assessment of the clinically relevant transition of JAK2 ^V617F from heterozygosity to homozygosity.     

Splenectomy Normalizes Hematocrit in Murine Polycythemia Vera

Splenic enlargement (splenomegaly) develops in numerous disease states, although a specific pathogenic role for the spleen has rarely been described. In polycythemia vera (PV), an activating mutation in Janus kinase 2 (JAK2^V617) induces splenomegaly and an increase in hematocrit. Splenectomy is sparingly performed in patients with PV, however, due to surgical complications. Thus, the role of the spleen in the pathogenesis of human PV remains unknown. We specifically tested the role of the spleen in the pathogenesis of PV by performing either sham (SH) or splenectomy (SPL) surgeries in a murine model of JAK2^V617F-driven PV. Compared to SH-operated mice, which rapidly develop high hematocrits after JAK2^V617F transplantation, SPL mice completely fail to develop this phenotype. Disease burden (JAK2^V617) is equivalent in the bone marrow of SH and SPL mice, however, and both groups develop fibrosis and osteosclerosis. If SPL is performed after PV is established, hematocrit rapidly declines to normal even though myelofibrosis and osteosclerosis again develop independently in the bone marrow. In contrast, SPL only blunts hematocrit elevation in secondary, erythropoietin-induced polycythemia. We conclude that the spleen is required for an elevated hematocrit in murine, JAK2^V617F-driven PV, and propose that this phenotype of PV may require a specific interaction between mutant cells and the spleen.     

Identification of oncostatin M as a JAK2 V617F-dependent amplifier of cytokine production and bone marrow remodeling in myeloproliferative neoplasms

The JAK2 mutation V617F is detectable in a majority of patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). Enforced expression of JAK2 V617F in mice induces myeloproliferation and bone marrow (BM) fibrosis, suggesting a causal role for the JAK2 mutant in the pathogenesis of MPNs. However, little is known about mechanisms and effector molecules contributing to JAK2 V617F-induced myeloproliferation and fibrosis. We show that JAK2 V617F promotes expression of oncostatin M (OSM) in neoplastic myeloid cells. Correspondingly, OSM mRNA levels were increased in the BM of patients with MPNs (median 287% of ABL, range 22-1450%) compared to control patients (median 59% of ABL, range 12-264%; P < 0.0001). OSM secreted by JAK2 V617F+ cells stimulated growth of fibroblasts and microvascular endothelial cells and induced the production of angiogenic and profibrogenic cytokines (HGF, VEGF, and SDF-1) in BM fibroblasts. All effects of MPN cell-derived OSM were blocked by a neutralizing anti-OSM antibody, whereas the production of OSM in MPN cells was suppressed by a pharmacologic JAK2 inhibitor or RNAi-mediated knockdown of JAK2. In summary, JAK2 V617F-mediated up-regulation of OSM may contribute to fibrosis, neoangiogenesis, and the cytokine storm observed in MPNs, suggesting that OSM might serve as a novel therapeutic target molecule in these neoplasms.     

JAK2 V617F Genotype Is a Strong Determinant of Blast Transformation in Primary Myelofibrosis

Purpose

The influence of JAK2 V617F mutation on blast transformation (BT) and overall survival (OS) in primary myelofibrosis (PMF) is controversial. In a large cohort of patients we applied competing risks analysis for studying the influence of JAK2V617F mutation on BT in PMF.

Patients and Methods

In 462 PMF–fibrotic type patients (bone marrow [BM] fibrosis grade >0) we computed the incidence of BT and death in the framework of Cox regression analysis and of Fine and Gray competing risks analysis for BT.

Results

At the Cox regression analysis, having either a wild-type (wt) or a homozygous JAK2V617F genotype were factors for BT (HR, 1.98 and 2.04, respectively, with respect to the heterozygous genotype), but not for OS. At the competing risks regression analysis, the risk for BT in wt and homozygous V617F patients increased with respect to Cox analysis, giving a sHR of 2.17 and 2.12, respectively. Correcting the results for the variables that could have influence on BT, JAK2V617F wt and homozygous genotypes remained independently associated with BT. In a validation cohort of 133 independent cases with PMF-prefibrotic type (BM fibrosis grade  = 0), the BT predictive model including JAK2V617F genotype and older age retained high discriminant capacity (C statistics, 0.70; 95% CI, 0.47 to 0.92).

Conclusion

The accumulation of mutated alleles in the JAK2V617F clone or the selective acquisition of a proliferative advantage in the wt clone are two relevant routes to BT in PMF. The influence of these results on treatment decisions with anti-JAK2 agents should be tested.     

JAK2 in Myeloproliferative Disorders Is Not Just Another Kinase

Myeloproliferative disorders (MPD) represent a subcategory of hematological malignancies and are characterized by a stem cell-derived clonal proliferation of myeloid cells including erythrocytes, platelets, and leucocytes. Traditionally, the term ‘MPD’ included chronic myeloid leukemia (CML), polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis with myeloid metaplasia (MMM). At present, these four disorders are referred to as ‘classic’ MPD and are distinguished from a spectrum of other MPD-like clinico-pathologic entities that are operationally classified as ‘atypical’ MPD. The oncogenic mutations(s) in classic MPD are unknown except for CML, which is associated with an activating mutation (Bcr/Abl) of the gene encoding for the Abl cytoplasmic protein kinase (PTK). In the last 3 months, a somatic point mutation of JAK2 (JAK2^V617F), the gene encoding for another cytoplasmic PTK was reported in the majority of patients with PV and approximately half of those with either ET or MMM. The same mutation was also found in a small number of patients with either atypical MPD or the myelodysplastic syndrome but not in normal controls, germline tissue including T lymphocytes, and patients with secondary erythrocytosis. In vitro, JAK2^V617F was associated with constitutive phosphorylation of JAK2 and its downstream effectors as well as induction of erythropoietin hypersensitivity in cell lines. In vivo, murine bone marrow transduced with a retrovirus containing JAK2^V617F induced erythrocytosis in the transplanted mice. Taken together, these observations suggest that JAK2^V617F is an acquired myeloid lineage-specific mutation that engenders a pathogenetic relevance for the PV phenotype in MPD.     

Increased Dkk‐1 plasma levels may discriminate disease subtypes in myeloproliferative neoplasms

Alterations in the bone marrow niche induced by abnormal production of cytokines and other soluble factors have been associated with disease progression in classical BCR‐ABL1 negative myeloproliferative neoplasms (MPN). Variations in circulating proteins might reflect local disease processes and plasma proteome profiling could serve to identify possible diagnostic and prognostic biomarkers. We employed a human cytokine array to screen for 105 distinct analytes in pooled plasma samples obtained from untreated young MPN patients (<35 years) with different clinical phenotypes and driver mutations, as well as from healthy individuals. Among molecules that exhibited significantly increased levels in MPN patients versus controls, the top of the list was represented by Dickkopf‐related protein 1 (Dkk‐1), which also showed the highest potential for discrimination between MPN subtypes. In the next step, a quantitative ELISA was used to measure plasma Dkk‐1 levels in 30 young‐onset MPN—10 essential thrombocythemia (ET), 10 polycythemia vera (PV), 10 pre‐fibrotic primary myelofibrosis (pre‐PMF)—and 10 controls. The results suggested that plasma Dkk‐1 levels could differentiate ET from pre‐PMF, in JAK2 V617F‐positive as well as in CALR‐positive patients, and also ET from PV in JAK2 V617F‐positive patients.     

The Polymorphisms in LNK Gene Correlated to the Clinical Type of Myeloproliferative Neoplasms

ObjectiveLNK is an adapter protein negatively regulating the JAK/STAT cell signaling pathway. In this study, we observed the correlation between variation in LNK gene and the clinical type of myeloproliferative neoplasms (MPN).MethodsA total of 285 MPN cases were recruited, including essential thrombocythemia (ET) 154 cases, polycythemia vera (PV) 76 cases, primary myelofibrosis (PMF) 19 cases, and chronic myeloid leukemia (CML) 36 cases. Ninety-three healthy individuals were used as normal controls. V617F mutation in JAK2 was identified by allele-specific PCR method, RT-PCR was used for the detection of BCR/ABL1 fusion gene, and mutations and variations in coding exons and their flanking sequences of LNK gene were examined by PCR-sequencing.ResultsMissense mutations of A300V, V402M, and R415H in LNK were found in 8 patients including ET (4 cases, all combined with JAK2-V617F mutation), PV (2 cases, one combined with JAK2-V617F mutation), PMF (one case, combined with JAK2-V617F mutation) and CML (one case, combined with BCR/ABL1 fusion gene). The genotype and allele frequencies of the three SNPs (rs3184504, rs111340708 and rs78894077) in LNK were significantly different between MPN patients and controls. For rs3184504 (T/C, in exon2), the T allele (p.262W) and TT genotype were frequently seen in ET, PV and PMF (P<0.01), and C allele (p.262R) and CC genotype were frequently seen in CML (P<0.01). For rs78894077 (T/C, in exon1), the T allele (p.242S) was frequently found in ET (P<0.05). For rs111340708 (TGGGGx5/TGGGGx4, in intron 5), the TGGGG x4 allele was infrequently found in ET, PMF and CML(P<0.01).ConclusionMutations in LNK could be found in some of MPN patients in the presence or absence of JAK2-V617F mutation. Several polymorphisms in LNK gene may affect the clinical type or the genetic predisposition of MPN.     

Recognizing and managing the expanded risk of tumor lysis syndrome in hematologic and solid malignancies

Background

Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) are Chronic Myeloproliferative Neoplasms (MPN) characterized by clonal myeloproliferation/myeloaccumulation without cell maturation impairment. The JAK2 V617F mutation and PRV1 gene overexpression may contribute to MPN physiopathology. We hypothesized that deregulation of the apoptotic machinery may also play a role in the pathogenesis of ET and PMF. In this study we evaluated the apoptosis-related gene and protein expression of BCL2 family members in bone marrow CD34⁺ hematopoietic stem cells (HSC) and peripheral blood leukocytes from ET and PMF patients. We also tested whether the gene expression results were correlated with JAK2 V617F allele burden percentage, PRV1 overexpression, and clinical and laboratory parameters.

Results

By real time PCR assay, we observed that A1, MCL1, BIK and BID, as well as A1, BCLW and BAK gene expression were increased in ET and PMF CD34⁺ cells respectively, while pro-apoptotic BAX and anti-apoptotic BCL2 mRNA levels were found to be lower in ET and PMF CD34⁺ cells respectively, in relation to controls. In patients' leukocytes, we detected an upregulation of anti-apoptotic genes A1, BCL2, BCL-X _L and BCLW. In contrast, pro-apoptotic BID and BIM _EL expression were downregulated in ET leukocytes. Increased BCL-X_L protein expression in PMF leukocytes and decreased BID protein expression in ET leukocytes were observed by Western Blot. In ET leukocytes, we found a correlation between JAK2 V617F allele burden and BAX, BIK and BAD gene expression and between A1, BAX and BIK and PRV1 gene expression. A negative correlation between PRV1 gene expression and platelet count was observed, as well as a positive correlation between PRV1 gene expression and splenomegaly.

Conclusions

Our results suggest the participation of intrinsic apoptosis pathway in the MPN physiopathology. In addition, PRV1 and JAK2 V617F allele burden were linked to deregulation of the apoptotic machinery.     

Molecular drug targets in myeloproliferative neoplasms: mutant ABL1, JAK2, MPL, KIT, PDGFRA, PDGFRB and FGFR1

Therapeutically validated oncoproteins in myeloproliferative neoplasms (MPN) include BCR-ABL1 and rearranged PDGFR proteins. The latter are products of intra- ( e.g. FIP1L1-PDGFRA) or inter-chromosomal ( e.g. ETV6-PDGFRB ) gene fusions. BCR-ABL1 is associated with chronic myelogenous leukaemia (CML) and mutant PDGFR with an MPN phenotype characterized by eosinophilia and in addition, in case of FIP1L1-PDGFRA, bone marrow mastocytosis. These genotype-phenotype associations have been effectively exploited in the development of highly accurate diagnostic assays and molecular targeted therapy. It is hoped that the same will happen in other MPN with specific genetic alterations: polycythemia vera ( JAK2 V617F and other JAK2 mutations), essential thrombocythemia ( JAK2 V617F and MPL5 15 mutations), primary myelofibrosis ( JAK2 V617F and MPL515 mutations), systemic mastocytosis ( KIT D816V and other KIT mutations) and stem cell leukaemia/lymphoma ( ZNF198-FGFR1 and other FGFR1 fusion genes). The current review discusses the above-listed mutant molecules in the context of their value as drug targets.     

Down-Regulation of the Epithelial Na+ Channel ENaC by Janus kinase 2

Janus kinase-2 (JAK2), a signaling molecule mediating effects of various hormones including leptin and growth hormone, has previously been shown to modify the activity of several channels and carriers. Leptin is known to inhibit and growth hormone to stimulate epithelial Na⁺ transport, effects at least partially involving regulation of the epithelial Na⁺ channel ENaC. However, no published evidence is available regarding an influence of JAK2 on the activity of the epithelial Na⁺ channel ENaC. In order to test whether JAK2 participates in the regulation of ENaC, cRNA encoding ENaC was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild type JAK2, gain-of-function ^V617FJAK2 or inactive ^K882EJAK2. Moreover, ENaC was expressed with or without the ENaC regulating ubiquitin ligase Nedd4-2 with or without JAK2, ^V617FJAK2 or ^K882EJAK2. ENaC was determined from amiloride (50 μM)-sensitive current (I _amil) in dual electrode voltage clamp. Moreover, I _amil was determined in colonic tissue utilizing Ussing chambers. As a result, the I _amil in ENaC-expressing oocytes was significantly decreased following coexpression of JAK2 or ^V617FJAK2, but not by coexpression of ^K882EJAK2. Coexpression of JAK2 and Nedd4-2 decreased I _amil in ENaC-expressing oocytes to a larger extent than coexpression of Nedd4-2 alone. Exposure of ENaC- and JAK2-expressing oocytes to JAK2 inhibitor AG490 (40 μM) significantly increased I _amil. In colonic epithelium, I _amil was significantly enhanced by AG490 pretreatment (40 μM, 1 h). In conclusion, JAK2 is a powerful inhibitor of ENaC.     

Abdominal venous thrombosis presenting in myeloproliferative neoplasm with JAK2 V617F mutation: a case report

Introduction

An unprovoked thombotic event in a patient is cause for further evaluation of an underlying hypercoaguable state. The investigation should include a thorough search, including checking for a variety of known inherited and acquired hypercoaguble states (protein C or S deficiency, anti-phospholipid antibodies, and anti-thrombin III deficiency) and gene mutations that predispose patients to an increased risk of clotting (for example, prothrombin gene 20210 mutation, factor V Leiden, and the JAK2 V617F mutation).

Case presentation

We report the case of a 38-year-old Caucasian woman with spontaneous, unprovoked abdominal venous thrombosis and demonstrate how testing for the JAK2 V617F mutation was useful in unmasking an underlying hypercoaguable state.

Conclusions

JAK2 V617F-positive myeloproliferative neoplasm was diagnosed. This case illustrates the importance of testing for JAK2 V617F in patients presenting with Budd-Chiari syndrome, even in the absence of overt hematologic abnormalities, in order to establish a diagnosis of underlying myeloproliferative neoplasm.     

Co‐targeting the PI3K/mTOR and JAK2 signalling pathways produces synergistic activity against myeloproliferative neoplasms

Aberrant JAK2 signalling plays a central role in myeloproliferative neoplasms (MPN). JAK2 inhibitors have proven to be clinically efficacious, however, they are not mutation‐specific and competent enough to suppress neoplastic clonal haematopoiesis. We hypothesized that, by simultaneously targeting multiple activated signalling pathways, MPN could be more effectively treated. To this end we investigated the efficacy of BEZ235, a dual PI3K/mTOR inhibitor, alone and in combination with the JAK1/JAK2 inhibitor ruxolitinib, in different preclinical models of MPN. Single‐agent BEZ235 inhibited the proliferation and induced cell cycle arrest and apoptosis of mouse and human JAK2V617F mutated cell lines at concentrations significantly lower than those required to inhibit the wild‐type counterpart, and preferentially prevented colony formation from JAK2V617F knock‐in mice and patients' progenitor cells compared with normal ones. Co‐treatment of BEZ235 and ruxolitinib produced significant synergism in all these in‐vitro models. Co‐treatment was also more effective than single drugs in reducing the extent of disease and prolonging survival of immunodeficient mice injected with JAK2V617F‐mutated Ba/F3‐EPOR cells and in reducing spleen size, decreasing reticulocyte count and improving spleen histopathology in conditional JAK2V617F knock‐in mice. In conclusion, combined inhibition of PI3K/mTOR and JAK2 signalling may represent a novel therapeutic strategy in MPN.     

Characterization of Glycoproteoforms of Integrins α2 and β1 in Megakaryocytes in the Occurrence of JAK2V617F Mutation-Induced Primary Myelofibrosis

Primary myelofibrosis (PMF) is a neoplasm prone to leukemic transformation, for which limited treatment is available. Among individuals diagnosed with PMF, the most prevalent mutation is the JAK2V617F somatic point mutation that activates the Janus kinase 2 (JAK2) enzyme. Our earlier reports on hyperactivity of β1 integrin and enhanced adhesion activity of the α2β1 complex in JAK2V617F megakaryocytes (MKs) led us to examine the new hypothesis that this mutation leads to posttranslational modification via changes in glycosylation. Samples were derived from immunoprecipitation of MKs obtained from Vav1-hJAK2^V617F and WT mice. Immunoprecipitated fractions were separated by SDS-PAGE and analyzed using LC-MS/MS techniques in a bottom-up glycoproteomics workflow. In the immunoprecipitate, glycopeptiforms corresponding to 11 out of the 12 potential N-glycosylation sites of integrin β1 and to all nine potential glycosylation sites of integrin α2 were observed. Glycopeptiforms were compared across WT and JAK2V617F phenotypes for both integrins. The overall trend observed is that JAK2V617F mutation in PMF MKs leads to changes in β1 glycosylation; in most cases, it results in an increase in the integrated area of glycopeptiforms. We also observed that in mutated MKs, changes in integrin α2 glycosylation were more substantial than those observed for integrin β1 glycosylation, a finding that suggests that altered integrin α2 glycosylation may also affect activation. Additionally, the identification of proteins associated to the cytoskeleton that were co-immunoprecipitated with integrins α2 and β1 demonstrated the potential of the methodology employed in this study to provide some insight, at the peptide level, into the consequences of integrin activation in MKs. The extensive and detailed glycosylation patterns we uncovered provide a basis for future functional studies of each site in control cells as compared to JAK2V617F-mutated cells. Data are available via ProteomeXchange with identifier PXD030550.     

Frequencies,Laboratory Features,and Granulocyte Activation in Chinese Patients with CALR-Mutated Myeloproliferative Neoplasms

Somatic mutations in the CALR gene have been recently identified as acquired alterations in myeloproliferative neoplasms (MPNs). In this study, we evaluated mutation frequencies, laboratory features, and granulocyte activation in Chinese patients with MPNs. A combination of qualitative allele-specific polymerase chain reaction and Sanger sequencing was used to detect three driver mutations (i.e., CALR, JAK2V617F, and MPL). CALR mutations were identified in 8.4% of cases with essential thrombocythemia (ET) and 5.3% of cases with primary myelofibrosis (PMF). Moreover, 25% of polycythemia vera, 29.5% of ET, and 48.1% of PMF were negative for all three mutations (JAK2V617F, MPL, and CALR). Compared with those patients with JAK2V617F mutation, CALR-mutated ET patients displayed unique hematological phenotypes, including higher platelet counts, and lower leukocyte counts and hemoglobin levels. Significant differences were not found between Chinese PMF patients with mutants CALR and JAK2V617F in terms of laboratory features. Interestingly, patients with CALR mutations showed markedly decreased levels of leukocyte alkaline phosphatase (LAP) expression, whereas those with JAK2V617F mutation presented with elevated levels. Overall, a lower mutant rate of CALR gene and a higher triple-negative rate were identified in the cohort of Chinese patients with MPNs. This result indicates that an undiscovered mutant gene may have a significant role in these patients. Moreover, these pathological features further imply that the disease biology varies considerably between mutants CALR and JAK2V617F.     

The platelet P2Y12 receptor under normal and pathological conditions. Assessment with the radiolabeled selective antagonist [3H]PSB-0413

Various radioligands have been used to characterize and quantify the platelet P2Y₁₂ receptor, which share several weaknesses: (a) they are metabolically unstable and substrates for ectoenzymes, (b) they are agonists, and (c) they do not discriminate between P2Y₁ and P2Y₁₂. We used the [³H]PSB-0413 selective P2Y₁₂ receptor antagonist radioligand to reevaluate the number of P2Y₁₂ receptors in intact platelets and in membrane preparations. Studies in humans showed that: (1) [³H]PSB-0413 bound to 425 ± 50 sites/platelet (K_D = 3.3 ± 0.6 nM), (2) 0.5 ± 0.2 pmol [³H]PSB-0413 bound to 1 mg protein of platelet membranes (K_D = 6.5 ± 3.6 nM), and (3) competition studies confirmed the known features of P2Y₁₂, with the expected rank order of potency: AR-C69931MX > 2MeSADP ≫ ADPβS > ADP, while the P2Y₁ ligand MRS2179 and the P2X₁ ligand α,β-Met-ATP did not displace [³H]PSB-0413 binding. Patients with severe P2Y₁₂ deficiency displayed virtually no binding of [³H]PSB-0413 to intact platelets, while a patient with a dysfunctional P2Y₁₂ receptor had normal binding. Studies in mice showed that: (1) [³H]PSB-0413 bound to 634 ± 87 sites/platelet (K_D = 14 ± 4.5 nM) and (2) 0.7 pmol ± 0.3 [³H]PSB-0413 bound to 1 mg protein of platelet membranes (K_D = 9.1 ± 5.3 nM). Clopidogrel and other thiol reagents like pCMBS or DTT abolished the binding both to intact platelets and membrane preparations. Therefore, [³H]PSB-0413 is an accurate and selective tool for radioligand binding studies aimed at quantifying P2Y₁₂ receptors, to identify patients with P2Y₁₂ deficiencies or quantify the effect of P2Y₁₂ targeting drugs.