Familial platelet disorder with predisposition to acute myelogenous leukemia (FPD/ AML) is an autosomal dominant familial platelet disorder characterized by thrombocytopenia and a propensity to develop AML. Mutation analyses of RUNX1 in 3 families with FPD/AML showing linkage to chromosome 21q22.1 revealed 3 novel heterozygous point mutations (K83E, R135fsX177 (IVS4 ؉ 3delA), and Y260X). Functional investigations of the 7 FPD/ AML RUNX1 Runt domain point mutations described to date (2 frameshift, 2 nonsense, and 3 missense mutations) were performed. Consistent with the position of the mutations in the Runt domain at the RUNX1-DNA interface, DNA binding of all mutant RUNX1 proteins was absent or significantly decreased. In general, missense and nonsense RUNX1 proteins retained the ability to heterodimerize with PEBP2␤/CBF␤ and inhibited transactivation of a reporter gene by wild-type RUNX1. Colocalization of mutant RUNX1 and PEBP2␤/CBF␤ in the cytoplasm was observed. These results suggest that the sequestration of PEBP2␤/CBF␤ by mutant RUNX1 may cause the inhibitory effects. While haploinsufficiency of RUNX1 causes FPD/AML in some families (deletions and frameshifts), mutant RUNX1 proteins (missense and nonsense) may also inhibit wild-type RUNX1, possibly creating a higher propensity to develop leukemia. This is consistent with the hypothesis that a second mutation has to occur, either in RUNX1 or another gene, to cause leukemia among individuals harboring RUNX1 FPD/AML mutations and that the propensity to acquire these additional mutations is determined, at least partially, by the initial RUNX1 mutation. IntroductionThe most frequent mutations associated with leukemia are recurrent somatic chromosomal translocations or inversions, many of which involve the polyomavirus enhancer-binding protein or core-binding factor transcriptional regulation complex (PEBP2/CBF). Several translocations involve the ␣ subunit of this complex, the RUNX1 gene (also called AML1, CBF␣2, or PEBP2␣B) on chromosome 21q22.1 (t(8;21), t(3;21), and t(12;21)). Additionally, the ␤ subunit of the complex, PEBP2␤ also called CBF␤, is disrupted in inv(16)(p13;q22). 1 An abundance of evidence points to the existence of genes that predispose to hematologic malignancies. However, large multiple-generation families with hematologic malignancies alone are rare. 2 Only 2 loci for familial hematologic malignancies have been identified to date, 1 on chromosome 21q22.1 3 and the other on 16q22. 4,5 These loci contain RUNX1 and PEBP2␤/CBF␤, respectively.Studies of families that demonstrate single-gene inheritance for leukemia predisposition should help to identify the genes and mechanisms involved in the first steps of leukemia development. The autosomal dominant familial platelet disorder (FPD)/ AML (acute myelogenous leukemia; Online Mendelian Inheritance in Man no. 601399) is a good model to validate this hypothesis because, in addition to developing thrombocytopenia, patients show a propensity for progression to myelodysplasia and acute myeloid leuke...

Platelets are non-nucleated cells that play central roles in the processes of hemostasis, innate immunity, and inflammation; however, several reports show that these distinct functions are more closely linked than initially thought. Platelets express numerous receptors and contain hundreds of secretory products. These receptors and secretory products are instrumental to the platelet functional responses. The capacity of platelets to secrete copious amounts of cytokines, chemokines, and related molecules appears intimately related to the role of the platelet in inflammation. Platelets exhibit non-self-infectious danger detection molecules on their surfaces, including those belonging to the “toll-like receptor” family, as well as pathogen sensors of other natures (Ig- or complement receptors, etc.). These receptors permit platelets to both bind infectious agents and deliver differential signals leading to the secretion of cytokines/chemokines, under the control of specific intracellular regulatory pathways. In contrast, dysfunctional receptors or dysregulation of the intracellular pathway may increase the susceptibility to pathological inflammation. Physiological vs. pathological inflammation is tightly controlled by the sensors of danger expressed in resting, as well as in activated, platelets. These sensors, referred to as pathogen recognition receptors, primarily sense danger signals termed pathogen associated molecular patterns. As platelets are found in inflamed tissues and are involved in auto-immune disorders, it is possible that they can also be stimulated by internal pathogens. In such cases, platelets can also sense danger signals using damage associated molecular patterns (DAMPs). Some of the most significant DAMP family members are the alarmins, to which the Siglec family of molecules belongs. This review examines the role of platelets in anti-infection immunity via their TLRs and Siglec receptors.

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The mechanism of collagen-induced human platelet activation was examined using Ca 2؉ , Na ؉ , and the pHsensitive fluorescent dyes calcium green/fura red, sodium-binding benzofuran isophthalate, and 2 ,7 -bis(2-car- Collagen is the most thrombogenic component of the subendothelium (1). Following vascular damage, collagen is exposed to circulating platelets and both acts as a substrate for the adhesion of platelets (2-4) and induces platelet activation (4). The prevailing evidence proposes that two receptors are involved in the platelet response to collagen; integrin ␣ 2 ␤ 1 acts to adhere platelets to collagen, allowing platelets to interact with the lower affinity receptor glycoprotein VI, which is mainly responsible for platelet activation (3,5).Many of the platelet responses to collagen progress simultaneously when platelets adhere to collagen. At high concentrations, collagen activation of platelets has been shown to proceed through activation of phospholipase C␥2 and subsequent cleavage of phosphatidylinositol 4,5-bisphosphate into inositol 1,4,5-trisphosphate and 1,2-diacylglycerol (6, 7). Inositol 1,4,5-trisphosphate induces the release of calcium from the dense tubular system (8, 9), whereas 1,2-diacylglycerol activates protein kinase C (10). The collagen-induced inositol 1,4,5-trisphosphate-mediated increase in [Ca 2ϩ ] i is accompanied by an influx of calcium from the extracellular milieu (11, 12). 1,2-Diacylglycerol and calcium mediate the characteristic platelet activation responses such as shape change, granule secretion, and aggregation.At lower concentrations, many of the effects of collagen are enhanced by its production of thromboxane A 2 (TXA) 1 (6, 13-15). The collagen-induced increase in [Ca 2ϩ ] i can be decreased by inhibiting the production of TXA via the pretreatment of platelets with cyclooxygenase inhibitors such as aspirin (11,16,17).Calcium is an important second messenger in the platelet activation cascade. At rest, a [Ca 2ϩ ] i of ϳ100 nM is maintained by a balance between the leak of Ca 2ϩ into the platelet and the concurrent efflux of free Ca 2ϩ across the plasma membrane of the platelet and accumulation in intracellular stores (18,19). Ca 2ϩ is moved out across the plasma membrane through the actions of the plasma membrane Ca 2ϩ -ATPase and the Na ϩ / Ca 2ϩ exchanger (NCX). Plasma membrane Ca 2ϩ -ATPases are membrane-inserted enzymes that use the energy of ATP hydrolysis to move Ca 2ϩ against its gradient and across the membrane. The NCX is capable of moving Ca 2ϩ into or out of the platelet cytosol in exchange for Na ϩ (20, 21). In the resting state, the NCX removes Ca 2ϩ from the platelet cytosol. Internally, Ca 2ϩ is transported into the dense tubular system by the sarco/endoplasmic reticulum Ca 2ϩ -ATPases 2b and 3 (22,23).In response to a moderate dose of collagen (10 g/ml), ϳ70% of the increase in [Ca 2ϩ ] i is due to the influx of Ca 2ϩ from the extracellular milieu, with the remainder as a function of Ca 2ϩ release from the dense tubular system (12). Because voltagega...

The levels and expression of the proteins CD63 and granulophysin in platelets from control and from a Hermansky-Pudlak syndrome subject (a condition characterized by dense granule and lysosomal deficiencies and the accumulation of ceroid-like material in reticuloendothelial cells) were examined. Immunofluorescence studies indicated that anti-CD63 and anti-granulophysin antibodies recognized similar numbers of granules; coapplication of antibodies did not identify more granules than the individual antibodies. Significantly fewer granules were recognized in Hermansky-Pudlak syndrome platelets than in control using either antibody. Immunoblotting studies demonstrated that anti-CD63 and anti-granulophysin antibodies apparently recognize the same protein, which was deficient in Hermansky-Pudlak platelets. Analysis by fluorescence-activated cell sorter (FACS) showed biphasic expression of CD63 and granulophysin after thrombin stimulation of control but not Hermansky-Pudlak platelets. Anti-CD63 effectively blocked detection of the protein by anti-granulophysin using immunofluorescence, ELISA, immunoblotting, and FACS analysis. Amino-terminal sequencing over the first 37 amino acids revealed that granulophysin was homologous to CD63, melanoma antigen ME491, and pltgp4O. These results suggest that granulophysin and CD63 are possibly identical proteins. This is the first report of a protein present in platelet dense granules, lysosomes, and melanocytes, but deficient in a patient with Hermansky-Pudlak syndrome. (J. Clin. Invest. 1993.