Francisca Ferrer-Marin

Antiplatelet therapy versus observation in low-risk essential thrombocythemia with CALR mutation

The role of antiplatelet therapy as primary prophylaxis of thrombosis in low-risk essential thrombocythemia has not been studied in randomized clinical trials. We assessed the benefit/risk of low-dose aspirin in 433 patients with low-risk essential thrombocythemia (271 with a CALR mutation, 162 with a JAK2V617F mutation) who were on antiplatelet therapy or observation only. After a follow up of 2215 person-years free from cytoreduction, 25 thrombotic and 17 bleeding episodes were recorded. In CALR-mutated patients, antiplatelet therapy did not affect the risk of thrombosis but was associated with a higher incidence of bleeding (12.9 versus 1.8 episodes per 1000 patient-years, P=0.03). In JAK2V617F-mutated patients, low-dose aspirin was associated with a reduced incidence of venous thrombosis with no effect on the risk of bleeding. Coexistence of JAK2V617F-mutation and cardiovascular risk factors increased the risk of thrombosis, even after adjusting for treatment with low-dose aspirin (incidence rate ratio: 9.8; 95% confidence interval: 2.3–42.3; P=0.02). Time free from cytoreduction was significantly shorter in CALR-mutated patients with essential thrombocythemia than in JAK2V617F-mutated ones (median time 5 years and 9.8 years, respectively; P=0.0002) and cytoreduction was usually necessary to control extreme thrombocytosis. In conclusion, in patients with low-risk, CALR-mutated essential thrombocythemia, low-dose aspirin does not reduce the risk of thrombosis and may increase the risk of bleeding.

Impaired leucocyte activation is underlining the lower thrombotic risk of essential thrombocythaemia patients with CALR mutations as compared with those with the JAK2 mutation.

concentrate in the management of severe protein C deficiency – experience from 12 centres. British Journal of Haematology, 164, 414–421. Pai, N., Shetty, S. & Ghosh, K. (2010) Protein C (PROC) gene mutations in two Indian families with purpura fulminans. Annals of Hematology, 89, 835–836. Stenson, P.D., Mort, M., Ball, E.V., Shaw, K., Phillips, A.D. & Cooper, D.N. (2014) The Human Gene Mutation Database: building a comprehensive mutation repository for clinical and molecular genetics, diagnostic testing and personalized genomic medicine. Human Genetics, 133, 1–9. Tait, R.C., Walker, I.D., Reitsma, P.H., Islam, S.I., McCall, F., Poort, S.R., Conkie, J.A. & Bertina, R.M. (1995) Prevalence of protein C deficiency in the healthy population. Thrombosis and Haemostasis, 73, 87–93. Takahashi, T., Shinohara, K., Nawata, R., Wakiyama, M. & Hamasaki, N. (1999) A novel mutation of the protein C gene with a frameshift deletion of 3 base pair (3380AGG) in exon 6 in type 1 deficiency associated with arterial and venous thrombosis. American Journal of Hematology, 62, 260–261.

Correction of anaemia on dialysis: did we forget physiology?

A frequent complication in patients with kidney diseases,chronic anaemia is commonly treated with erythropoietin-stimulating agents (ESA). At present, the optimal target forhaemoglobin (Hb) concentration is, however, unknown.The purpose of this communication is to plead for a lessaggressive correction of nephrogenic anaemia, fully justi-fied by the adaptive mechanisms that patients on dialysisare prompt to set off in order to increase their tolerance toanaemia. First, their delivery of oxygen from Hb is oppor-tunely facilitated. Second, beyond a certain threshold, ris-ing Hb concentration with ESA does not mean increasingtissue oxygenation. Although the physiological adaptationin patients with kidney failure has been known for decades,it has been constantly and startlingly ignored in the designof studies aiming at a full correction of anaemia and an‘irrational’ normalizationofHbwiththeuseofESA.Flawed in essence, these studies also demonstrate the dan-gerousness of a generic approach.Historically, the food and drug administration (FDA) ap-proved the prescription of recombinant erythropoietin(rhuEPO) in 1989 on the basis of a signal study published2 years earlier showing that rHuEPO could eliminate theneedfortransfusionsinpatientswithend-stagerenaldisease(ESRD), and thereby protect them from immunologicsensitization, infection and iron overload [1]. Twenty yearslater, on 16 February 2010, the same FDA announced thatESA should actually be prescribed under a‘risk manage-ment programme’, known as the Risk Evaluation and Miti-gation Strategy: data collected in longitudinal clinicalstudies had indeed repeatedly shown that in patients withrenal disease or with solid tumours, complete correction ofanaemiawith ESA had increased the risk of death, nonfatalmyocardial infarction [2–4], fatal or nonfatal stroke andblood clots [4]. In retrospect, thus, excessive normalizationofHblevelswithESAmayhaveimprovedthequalityoflifeofpatientswithESRD[5,6]butattheexpenseofadecreasein their ‘quantity’ of life [3]. The reasons to avoid bloodtransfusion still prevail, though, and a low Hb will increasethe risk for cardiovascular events in these patients [7], aswell. Since experimental findings have strongly suggestedthat the side effects of ESA are essentially related to a risein haematocrit and not to some off-target properties of thedrugs [8], it is now time to revise our target downwards [9].Themajorfunctionofthecentralcirculationistotransportoxygen from the lungs to the peripheral tissues at a rate thatsatisfies overall oxygen consumption. There is no storagesystem for oxygen (O

Identification of two novel mutations in RASGRP2 affecting platelet CalDAG-GEFI expression and function in patients with bleeding diathesis

Abstract The RASGRP2 gene encodes the Ca2+ and DAG-regulated guanine nucleotide exchange factor I (CalDAG-GEFI), which plays a key role in integrin activation in platelets and neutrophils. We here report two new RASGRP2 variants associated with platelet dysfunction and bleeding in patients. The homozygous patients had normal platelet and neutrophil counts and morphology. Platelet phenotyping showed: prolonged PFA-100 closure times; normal expression of major glycoprotein receptors; severely reduced platelet aggregation response to ADP and collagen (both patients); aggregation response to PAR1 and arachidonic acid markedly impaired in one patient; PMA-induced aggregation unaffected; platelet secretion, clot retraction, and spreading minimally affected. Genetic analysis identified two new homozygous variants in RASGRP2: c.706C>T (p.Q236X) and c.887G>A (p.C296Y). In both patients, CalDAG-GEFI protein was not detectable in platelet lysates, and platelet αIIbβ3 activation, as assessed by fibrinogen binding, was greatly impaired in response to all agonists except PMA. Patient neutrophils showed normal integrin expression, but impaired Mn2+-induced fibrinogen binding. In summary, we have identified two new RASGRP2 mutations that can be added to this rapidly growing form of inherited platelet function disorder.

An atypical IgM class platelet cold agglutinin induces GPVI-dependent aggregation of human platelets

Platelet cold agglutinins (PCA) cause pseudothrombocytopenia, spurious thrombocytopenia due to ex vivo platelet clumping, complicating clinical diagnosis, but mechanisms and consequences of PCA are not well defined. Here, we characterised an atypical immunoglobulin (Ig)M PCA in a 37-year-old woman with lifelong bleeding and chronic moderate thrombocytopenia, that induces activation and aggregation of autologous or allogeneic platelets via interaction with platelet glycoprotein (GP)VI. Patient temperature-dependent pseudothrombocytopenia was EDTA-independent, but was prevented by integrin αIIbβ3 blockade. Unstimulated patient platelets revealed elevated levels of bound IgM, increased expression of activation markers (P-selectin and CD63), low GPVI levels and abnormally high thromboxane (TX)A2 production. Patient serum induced temperature- and αIIbβ3-dependent decrease of platelet count in allogeneic donor citrated platelet-rich plasma (PRP), but not in PRP from Glanzmanns thrombasthenia or afibrinogenaemia patients. In allogeneic platelets, patient plasma induced shape change, P-selectin and CD63 expression, (14)C-serotonin release, and TXA2 production. Activation was not inhibited by aspirin, cangrelor or blocking anti-Fc receptor (FcγRIIA) antibody, but was abrogated by inhibitors of Src and Syk, and by a soluble GPVI-Fc fusion protein. GPVI-deficient platelets were not activated by patient plasma. These data provide the first evidence for an IgM PCA causing platelet activation/aggregation via GPVI. The PCA activity persisted over a five-year follow-up period, supporting a causative role in patient chronic thrombocytopenia and bleeding.

Evaluation of Novel Platelet Polymorphisms in Stroke. Dichotomic Effect of rs5443 in GNB3

Dear Editor, Our group recently characterized two single-nucleotide polymorphisms (SNPs), rs4366150 and rs1787566, on the genes encoding lysophosphatidic acid receptor-1 (LPAR1) and myosin VB (MYO5B), respectively, which are associated with platelet reactivity, in a cohort of 286 healthy children.1 Furthermore, the recently identified SNPs rs5443 (on the gene encoding guanine nucleotide binding protein 3, GNB3) and rs3737224 (on the gene encoding platelet endothelial aggregation receptor 1, PEAR1)2,3 may also be considered as potential new genetic factors implicated in platelet function. However, the role of these SNPs in thrombosis or hemorrhage disorders has either not been addressed, or is still controversial. Given the functional effect of the four aforementioned SNPs, we aimed to determine their potential role in 1) the development of thrombosis in patients with ischemic stroke (IS) or 2) bleeding in patients with intracranial hemorrhage [subarachnoid hemorrhage (SAH) and intracerebral hemorrhage (ICH)]. For this purpose, the presence of the four SNPs was determined in consecutive patients who survived an IS and in patients who suffered from an intracranial hemorrhage. The cohort with IS according to the Trial of ORG 10172 in Acute Stroke Treatment criteria were patients enrolled in the Unit of Neurology in Reina Sofia Hospital (Murcia, Spain). We also recruited 611 healthy controls from the general population from blood donors (n=377) and traumatology and ophthalmology patients undergoing minor outpatient surgery (n=234). Cohorts with SAH and ICH were enrolled over a 3-year period and are described in a previous report from our group.4 All subjects were Caucasians and gave their informed consent to enter the study, which was approved by the local Ethics Committee and was performed according to the Declaration of Helsinki. Genomic DNA was isolated from whole blood samples according to standard procedures, and DNA was amplified using Taqman probes from Life Technologies (Madrid, Spain). Genotyping of the different cohorts for the different SNPs showed that they were all in Hardy-Weinberg equilibrium (not shown). The general characteristics of patients and controls are given in Table 1. The univariate analyses revealed no association (p>0.05) between the presence of rs4366150 (LPAR1), rs1787566 (MYO5B), and rs3737224 (PEAR1), and the development of IS, SAH, or ICH (Table 1). Nevertheless, there was a trend toward significance for the rs5443 (T allele) SNP in GNB3 as a risk factor in the development of IS (p=0.087) and as a protective factor in SAH (p=0.071). No association between rs5443 (T allele) and development of ICH was found (p=0.781). Multivariate analysis taking into account risk factors (age, sex, and hypertension) confirmed that the T allele of rs5443 was an independent protective factor against the development of SAH [odds ratio (OR)=0.608, 95% confidence interval (CI)=0.383-0.964, p=0.034], but excluded an effect on the risk of developing IS (p=0.152) (Table 1). We further refined our study by segregating by gender. The results showed that while in women the T allele of rs5443 was not associated with IS (p=0.713), there was a statistically significant, almost three-fold increase in the risk of developing IS among men without diabetes mellitus (OR=2.72, 95% CI=1.116-6.612, p=0.028). Indeed, the issue of sexual dimorphism in the occurrence of cardiovascular disease is well known, and the role of hormones in IS has also been addressed in several studies.5 Table 1 Clinical features and prevalence of selected risk factors in case-control study Interestingly, our finding indicating that the T allele rs5443 SNP has an impact on both IS and SAH sheds light on the potential duality of SNPs as simultaneously being both risk and protective factors for different diseases. This concept of dichotomy has already been proposed by our group for other SNPs, such as factor V Leiden thrombophilia or FVII-323 Del/Ins, in the presence of which a phenotype of thrombosis or of bleeding may appear according to the presence of specific conditions and other risk factors.4 Future studies in larger cohorts are necessary to clarify the exact role of the rs5443 SNP in GNB3 as a factor capable of modulating the individual thrombotic risk.

Evaluation of two‐step haemoglobin screening with HemoCue for blood donor qualification in mobile collection sites

Inaccuracy of fingerstick haemoglobin compromises donors health and losses blood donations. We evaluated the benefit of double haemoglobin screening with HemoCue.

Significant Hypo-Responsiveness to GPVI and CLEC-2 Agonists in Pre-Term and Full-Term Neonatal Platelets and following Immune Thrombocytopenia.

Neonatal platelets are hypo-reactive to the tyrosine kinase-linked receptor agonist collagen. Here, we have investigated whether the hypo-responsiveness is related to altered levels of glycoprotein VI (GPVI) and integrin α2β1, or to defects in downstream signalling events by comparison to platelet activation by C-type lectin-like receptor 2 (CLEC-2). GPVI and CLEC-2 activate a Src- and Syk-dependent signalling pathway upstream of phospholipase C (PLC) γ2. Phosphorylation of a conserved YxxL sequence known as a (hemi) immunotyrosine-based-activation-motif (ITAM) in both receptors is critical for Syk activation. Platelets from human pre-term and full-term neonates display mildly reduced expression of GPVI and CLEC-2, as well as integrin αIIbβ3, accounted for at the transcriptional level. They are also hypo-responsive to the two ITAM receptors, as shown by measurement of integrin αIIbβ3 activation, P-selectin expression and Syk and PLCγ2 phosphorylation. Mouse platelets are also hypo-responsive to GPVI and CLEC-2 from late gestation to 2 weeks of age, as determined by measurement of integrin αIIbβ3 activation. In contrast, the response to G protein-coupled receptor agonists was only mildly reduced and in some cases not altered in neonatal platelets of both species. A reduction in response to GPVI and CLEC-2, but not protease-activated receptor 4 (PAR-4) peptide, was also observed in adult mouse platelets following immune thrombocytopenia, whereas receptor expression was not impaired. Our results demonstrate developmental differences in platelet responsiveness to GPVI and CLEC-2, and also following immune platelet depletion leading to reduced Syk activation. The rapid generation of platelets during development or following platelet depletion is achieved at the expense of signalling by ITAM-coupled receptors.