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Low TLT-1 and collagen receptor α2 in FPD/AML
Table 1. Features of familial platelet disorder with predisposition to acute myelogenous leukemia patients.
Pedigree/ Sex/Age Platelet count MPV patient (years) (x109/L) (fL)
ISTH-BAT
(score) ADP
Platelet aggregation (%)
EPI COL AA
60±7 40±4 60±5
65±9 60±20 70±10 8±4 33±3 53±10 12±5 28±4 28±4
RUNX1 mutation
p.R174Q
p.R174Q p.R139X p.R139X
RUNX1 RNA levels
1.13*
0.92*
0.46*
2.66*
3.2**
2.9**
2.4**
AII-1
AII-2 BII-2 BIII-1 DII-1 DIII-1
DIII-3
M/38 90
F/39 116 F/37 130 M/2 106 F/51 145 M/30 115
F/27 94
9.6 1 55±7
10.6 2 37±2
9 1 24±4
10.2 2 40±10
11.3 5 31±2
10.5 6 30±3
10.4 4 34±9
14±6 20±0 29±40 p.T219Rfs*8 15±0 8±7 4±6 p.T219Rfs*8 19±2 21±6 34±41 p.T219Rfs*8
MPV: mean platelet volume; ISTH-BAT: International Society on Thrombosis and Hemostasis-bleeding assessment tool; ADP: adenosine diphosphate;;EPI: epinephrine; COL: col- lagen; AA: arachidonic acid; nd: not done. Pedigrees A,2 B2 and D8,11 were previously described. Reference values for MPV, 8.9-12.5 fL. Platelet aggregation in response to 2 μM ADP, 1 μM epinephrine, 4 μg/mL collagen, and 1mM arachidonic acid was performed as described,8,9 and expressed as maximal light transmission percentage; Mean ± Standard Deviation values are shown;reference values,75±9,78±9.5,77±11 and 79±9%,respectively.*Microarray data in patient megakaryocytes; **quantitative polymerase chain reaction data in patient platelets, relative to healthy subjects (n=4), set as 1.
Results
Gene expression profiling of shRUNX1-transduced and familial platelet disorder with predisposition to acute myelogenous leukemia megakaryocytes reveals downregulation of TREML1 and ITGA2
To identify RUNX1-targets that could be involved in FPD/AML platelet dysfunction, we first performed tran- scriptome analysis of mature (CD41+CD42+) MK cultured from four patients, two carrying the R174Q mutation, and two with the R139X mutation. We then analyzed the tran- scriptome of MK cultured from normal leukapheresis- derived CD34+ cells transduced with shRUNX1 at days 6 and 7 of culture in order to detect RUNX1 targets involved in late stages of MK differentiation and, more particularly, in proplatelet formation and platelet function. A signifi- cant increase in 43 genes and a decrease in 61 genes was shown in both FPD/AML and shRUNX1-transduced MK (Figure 1A and B and Online Supplementary Table S3A-F). Analysis of up-regulated genes did not show any potential candidates for FPD/AML platelet dysfunction (Online Supplementary Table S4A and Online Supplementary Figure S1). In contrast, analysis of GO pathways revealed two down-regulated genes, TREML1 and ITGA2, that were of interest regarding their role in platelet biology (Figure 1C and D and Online Supplementary Table S4B).
TREML1 codes for TLT-1, which represents an immunoreceptor tyrosine-based inhibition motif (ITIM)- containing receptor exclusively expressed in MK and platelets, where it is stored in α-granules.14 It undergoes surface translocation upon platelet activation14 and has been recently proposed to represent a more rapid and sensitive marker of platelet activation compared to P-selectin.15 TLT-1 ectodomain is released following platelet activation, leading to a naturally occurring soluble fragment (sTLT-1), which represents an abundant con- stituent of the platelet sheddome.16 Unlike other platelet ITIM receptors, the non-canonical TLT-1 has activating effects.17 Early work showed that, in a transiently trans- fected RBL-2H3 cell line, TLT-1 acts as a co-stimulatory receptor enhancing FceRI-mediated calcium signaling through recruitment of Src homology 2 domain-contain- ing tyrosine phosphatase (SHP)-2 to its cytoplasmic ITIM domain.18 In human platelets, incubation with a blocking anti-TLT-1 antibody was shown to inhibit platelet aggre- gation triggered by thrombin,19 whereas, conversely,
sTLT-1 enhances platelet aggregation triggered by a vari- ety of classic platelet agonists.17 Fibrinogen represents the only known ligand for TLT-1 and has been shown to bind both the full-length protein as well as the soluble form,17 and to favor fibrinogen deposition in vivo in a murine model of acute lung injury.20 Although the precise mecha- nism of TLT-1 action still has to be clearly defined, it has been proposed that, during platelet aggregation, fibrino- gen is cross-linked by TLT-1, facilitating platelet-fibrino- gen interactions and higher-order platelet aggregates, in concert with GPIIbIIIa.17 In addition, TLT-1 has been shown to interact through its cytoplasmic domain with ERM (ezrin/radixin/moesin) proteins, potentially linking fibrinogen to the platelet cytoskeleton.17 The essential role of TLT-1 in platelet function is revealed in Treml1-/- mice, which display mild thrombocytopenia, decreased platelet aggregation, and prolonged bleeding time.17 In addition to its role in hemostasis, sTLT-1 released by platelets reduces inflammation and organ damage during sepsis by counteracting leukocyte activation and platelet-neu- trophil crosstalk.21
ITGA2 encodes the α2 subunit of collagen receptor α2b1, which, in concert with GPVI, mediates the platelet- collagen interaction at sites of vascular injury required for stable platelet adhesion.22 Integrin α2b1 is essential when platelets are exposed to monomeric collagen, whereas it plays a supportive role for fibrillar collagen, where GPVI is the central receptor. The complementary interplay between both collagen receptors is required for an optimal platelet response to collagen. Upregulation of ITGA2 was demonstrated in K562 cells transduced with a RUNX1 expression vector, suggesting RUNX1 may regulate ITGA2 expression.23
Thus, we next assessed TREML1 and ITGA2 expression profile during in vitro megakaryopoiesis and showed that TREML1 and ITGA2 mRNA levels increase during normal MK differentiation (Figure 1E). Using real-time PCR, we confirmed that TREML1 and ITGA2 mRNAs are decreased in mature MK after shRNA-mediated RUNX1 inhibition (Figure 1F). Moreover, transduction of a WT RUNX1 cDNA carrying a mutation that does not change the amino acid sequence but prevents its recognition by shRUNX1 (RUNX1mut) was able to rescue the inhibition in TREML1 and ITGA2 induced by shRUNX1 (Figure 1G), further demonstrating the relationship between RUNX1 and both TREML1 and ITGA2.
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