Key Points Proinflammatory MKs from mice with GPS drive the extension of myelofibrosis, splenomegaly, and emperipolesis. The lack of preformed α-granules in Nbeal2−/− platelets leads to protection against cancer metastasis.

Somatic mutations in the endoplasmic reticulum chaperone calreticulin (CALR) are detected in approximately 40% of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF). Multiple different mutations have been reported, but all result in a +1-bp frameshift and generate a novel protein C terminus. In this study, we generated a conditional mouse knockin model of the most common CALR mutation, a 52-bp deletion. The mutant novel human C-terminal sequence is integrated into the otherwise intact mouse CALR gene and results in mutant CALR expression under the control of the endogenous mouse locus. CALR mice develop a transplantable ET-like disease with marked thrombocytosis, which is associated with increased and morphologically abnormal megakaryocytes and increased numbers of phenotypically defined hematopoietic stem cells (HSCs). Homozygous CALR mice developed extreme thrombocytosis accompanied by features of MF, including leukocytosis, reduced hematocrit, splenomegaly, and increased bone marrow reticulin. CALR HSCs were more proliferative in vitro, but neither CALR nor CALR displayed a competitive transplantation advantage in primary or secondary recipient mice. These results demonstrate the consequences of heterozygous and homozygous CALR mutations and provide a powerful model for dissecting the pathogenesis of CALR-mutant ET and PMF.

In Experiments 1 and 2, rats were exposed to two compound flavors, AXand BX, containing one flavor in common (X). Following this exposure phase, an aversion was conditioned to A in the experimental group by pairing its consumption with an injection of lithium, while a control group drank A without being poisoned. The effect of this treatment was to establish B as a conditioned inhibitor. In Experiment 1, experimental animals were slower than controls to condition an aversion to B when its consumption was paired with lithium (a retardation test of conditioned inhibition). In Experiment 2, B alleviated the suppression of intake of another flavor previously paired with lithium (a summation test). Experiments 3 and 4 established that these effects depended upon prolonged prior exposure to AX and BX.In their theory of stimulus representation, McLaren, Kaye, and Mackintosh (1989) suggested that exposure to two compound stimuli (AX and BX), containing a common element X, would permit the establishment of two types of associations between the elements of each compound. The first would be excitatory associations between the common element X and each unique element, A and B. The existence of such associations is evident from studies such as that of Rescorla and Cunningham (1978), employing compound flavors. Their effect will presumably be to enhance generalization between the two compounds: if AX is paired with an unconditioned stimulus (US), the ability of X to retrieve a representation of B could result in excitatory conditioning to B as well as to A on this trial. Acquired equivalence effects of this sort have been demonstrated by Hall (1989, 1991).It is well established, however, that prolonged exposure to AX and BX will often facilitate subsequent discrimination, rather than increase generalization between them (Hall, 1991). McLaren et al. (1989) attribute this perceptual learning etTect, in part, to differences in the associability of the common (X) and unique (A and B) elements, which occur as a consequence of greater latent inhibition of the former than of the latter. Several studies have provided good evidence for such a suggestion (e.g., Mackintosh, Kaye, & Bennett, 1991; Rodrigo, Chamizo, McLaren, & Mackintosh, 1994) see Experiments 3 and 4) concluded that such differential latent inhibition of common and unique elements could not be the only explanation of perceptual learning. They suggested that one additional mechanism was provided by a second set of associations that are formed between the elements of AX and BX-~specifically, inhibitory associations between A and 8. The establishment of excitatory associations between common and unique elements may cause X to retrieve a representation of B on AX trials (and of A on BX trials), but the presence of A signals the absence of the otherwise expected B (just as the presence of B signals the absence of the otherwise expected A), and according to standard associative theory, this should lead to the eventual establishment of inhibitory associations between A and B...

The principal morbidity and mortality in patients with essential thrombocythemia (ET) and polycythemia rubra vera (PV) stems from thrombotic events. Most patients with ET/PV harbor a JAK2V617F mutation, but its role in the thrombotic diathesis remains obscure. Platelet function studies in patients are difficult to interpret because of interindividual heterogeneity, reflecting variations in the proportion of platelets derived from the malignant clone, differences in the presence of additional mutations, and the effects of medical treatments. To circumvent these issues, we have studied a JAK2V617F knock-in mouse model of ET in which all megakaryocytes and platelets express JAK2V617F at a physiological level, equivalent to that present in human ET patients. We show that, in addition to increased differentiation, JAK2V617F-positive megakaryocytes display greater migratory ability and proplatelet formation. We demonstrate in a range of assays that platelet reactivity to agonists is enhanced, with a concomitant increase in platelet aggregation in vitro and a reduced duration of bleeding in vivo. These data suggest that JAK2V617F leads to intrinsic changes in both megakaryocyte and platelet biology beyond an increase in cell number. In support of this hypothesis, we identify multiple differentially expressed genes in JAK2V617F megakaryocytes that may underlie the observed biological differences.

Upon activation, platelets release a powerful cocktail of soluble and vesicular signals, collectively termed the "platelet releasate" (PR). Although several studies have used qualitative/quantitative proteomic approaches to characterize PR; with debated content and significant inter-individual variability reported, confident, and reliable insights have been hindered. Using label-free quantitative (LFQ)-proteomics analysis, a reproducible, quantifiable investigation of the 1U mL thrombin-induced PR from 32 healthy adults was conducted. MS proteomics data are available via ProteomeXchange, identifier PXD009310. Of the 894 proteins identified, 277 proteins were quantified across all donors and form a "core" PR. Bioinformatics and further LFQ-proteomic analysis revealed that the majority (84%) of "core" PR proteins overlapped with the protein composition of human platelet-derived exosomes. Vesicles in the exosomal-size range were confirmed in healthy-human PR and reduced numbers of similar-sized vesicles were observed in the PR of a mouse model of gray platelet syndrome, known to be deficient in platelet alpha-granules. Lastly, the variability of proteins in the PR was assessed, and reproducible secretion levels were found across all 32 healthy donors. Taken together, the PR contains valuable soluble and vesicular cargo and has low-population variance among healthy adults, rendering it a potentially useful platform for diagnostic fingerprinting of platelet-related disease.

Purpose:Primary ovarian insufficiency before the age of 40 years affects 1% of the female population and is characterized by permanent cessation of menstruation. Genetic causes include FMR1 expansion mutations. Previous studies have estimated mutation prevalence in clinical referrals for primary ovarian insufficiency, but these are likely to be biased as compared with cases in the general population. The prevalence of FMR1 expansion mutations in early menopause (between the ages of 40 and 45 years) has not been published.Methods:We studied FMR1 CGG repeat number in more than 2,000 women from the Breakthrough Generations Study who underwent menopause before the age of 46 years. We determined the prevalence of premutation (55–200 CGG repeats) and intermediate (45–54 CGG repeats) alleles in women with primary ovarian insufficiency (n = 254) and early menopause (n = 1,881).Results:The prevalence of the premutation was 2.0% in primary ovarian insufficiency, 0.7% in early menopause, and 0.4% in controls, corresponding to odds ratios of 5.4 (95% confidence interval = 1.7–17.4; P = 0.004) for primary ovarian insufficiency and 2.0 (95% confidence interval = 0.8–5.1; P = 0.12) for early menopause. Combining primary ovarian insufficiency and early menopause gave an odds ratio of 2.4 (95% confidence interval = 1.02–5.8; P = 0.04). Intermediate alleles were not significant risk factors for either early menopause or primary ovarian insufficiency.Conclusion:FMR1 premutations are not as prevalent in women with ovarian insufficiency as previous estimates have suggested, but they still represent a substantial cause of primary ovarian insufficiency and early menopause.

We therefore conclude that intermediate sized FMR1 CGG repeat alleles should not be considered a high-risk factor for POF based on current evidence.

Preexposure to two compound flavors (AX and BX) typically enhances their discriminability: An aversion conditioned to AXwill generalize less to BX,especially if the preexposure regime has involved altemated presentations ofAX and BX rather than presenting all AX trials before BX trials (or vice versa). One possible explanation ofthis finding is that altemating preexposure establishes inhibitory associations between the two unique features A and B, thus counteracting the generalization produced by excitatory associations between X and A and between X and B, which might result in either the retrieval of B on a conditioning trial to AX, or the retrieval of A on a test trial to BX.Three experiments on flavor aversion conditioning in rats tested these predictions. Experiment 1 suggested that the more important of these excitatory associations was that which allowed X to retrieve A on the test trial to BX. Experiment 2 suggested that the more important inhibitory association was that which allowed B to inhibit the representation of A on this test trial. Experiment 3 provided direct evidence of the role of this inhibitory B-IA association.Discrimination between two or more complex stimuli is often enhanced by prior exposure to one or more ofthem (see Hall, 1991, for a review). Such perceptualleaming effects have been weil established in flavor aversion conditioning, where discrimination between compound flavors such as mixtures of sucrose-Iemon and saline-lemon is enhanced by exposure to one or both ofthese flavors prior to conditioning an aversion to one and testing generalization to the other (see, e.g., Bennett, Wills, Wells, & Mackintosh, 1994;Mackintosh, Kaye, & Bennett, 1991;Symonds & Hall, 1995). Mackintosh et al. (1991) showed that this perceptua1 learning effect was dependent on the use of compound flavors sharing an element or feature in common. If an aversion was conditioned to saline alone, it did not generalize strongly to sucrose, and prior exposure to saline and sucrose did nothing to reduce the generalization that did occur. This is hardly surprising. According to one popular account, generalization between two stimuli occurs to the extent that they share elements in common (Estes, 1950). Ifwe represent two compound stimuli sharing elements in common as AX and BX, where A and Bare the elements unique to each stimulus, and X are those common to both, then conditioning to AX will generalize to BX because some of that conditioning accrues to the X elements shared by BX. There is, moreover, a very simple reason why preexposure to two compound stimuli, AX and BX, should reduce generalization between them (McLaren, Kaye, & Mackintosh, 1989). One of the best established consequences of preexposure to a stimulus that sub sequently serves as the conditional stimulus (CS) in a conditioning experiment is a retardation of subsequent conditioning to that stimulus-the phenomenon of latent inhibition (Lubow, 1989). But preexposure to both AX and BX will ensure twice as much preexposure to X as to A and B. It seems pr...