[Show abstract][Hide abstract] ABSTRACT: MYH9-Related Disorders are a group of rare autosomal dominant platelet disorders presenting as nonsyndromic forms characterized by macrothrombocytopenia with giant platelets and leukocyte inclusion bodies or as syndromic forms combining these hematological features with deafness and/or nephropathy and/or cataracts. They are caused by mutations in the MYH9 gene encoding the nonmuscle myosin heavy chain II-A (NMMHC-IIA). Until now, at least 49 MYH9 mutations have been reported in isolated cases or small series but only rarely in large series. We report the results of an 8-year study of a large cohort of 109 patients from 37 sporadic cases and 39 unrelated families. We have identified 43 genetic variants, 21 of which are novel to our patients. A majority, 33 (76.7%), were missense mutations and six exons were preferentially targeted, as previously published. The other alterations were three deletions of one nucleotide, one larger deletion of 21 nucleotides, and one duplication. For the first time, a substitution T>A was found in the donor splice site of intron 40 (c.5765+2T>A). Seven patients, four from the same family, had two genetic variants. The analysis of the genotype-phenotype relationships enabled us to improve the knowledge of this heterogeneous but important rare disease.
[Show abstract][Hide abstract] ABSTRACT: Pregnancy in women with inherited thrombocytopenias is a major matter of concern as both the mothers and the newborns are potentially at risk of bleeding. However, medical management of this condition cannot be based on evidence because of the lack of consistent information in the literature. To advance knowledge on this matter, we performed a multicentric, retrospective study evaluating 339 pregnancies in 181 women with 13 different forms of inherited thrombocytopenia. Neither the degree of thrombocytopenia nor the severity of bleeding tendency worsened during pregnancy, and the course of pregnancy was not different from that of healthy subjects in terms of miscarriages, fetal bleeding and preterm births. The degree of thrombocytopenia in the babies was similar to that in the mother. Only 7 of 156 affected newborns had delivery-related bleeding, but two of them died of cerebral hemorrhage. The frequency of delivery-related maternal bleeding ranged from 6.8 to 14.2% depending on the definition of abnormal blood loss, suggesting that the risk of risk of abnormal blood loss was increased with respect to the general population. However, no mother died or received hysterectomy to arrest bleeding. The search for parameters predicting delivery-related bleeding in the mother suggested that hemorrhages requiring blood transfusion were more frequent in women with history of severe bleedings before pregnancy and with platelet count at delivery lower than 50 x 10(9)/L.
[Show abstract][Hide abstract] ABSTRACT: Mutations in the MYH9 gene cause autosomal dominant MYH9-related diseases (MYH9-RD) that associate macrothrombocytopenia with various other clinical conditions. The mechanisms giving rise to giant platelets remain poorly understood.
To study the proplatelet formation (PPF) derived from megakaryocytes (MK) generated in vitro from eleven patients with MYH9-RD with different mutations, compared to controls.
PPF from cultured patients MKs was evaluated with or without blebbistatin or the ROCK inhibitor Y27632. Myosin IIA and actin distribution were studied in spreading MKs on different surfaces by immunoconfocal analysis. Kinetic studies of contractility were performed on spreading MKs and the impact of blebbistatin on the maturation of the patients' MKs was evaluated by electron microscopy.
We show that in vitro MKs of eleven patients formed significantly fewer proplatelets than controls. MKs from MYH9-RD displayed an abnormal spreading on polylysine, fibronectin and collagen, with a disorganized actin network and a marked increase in stress fiber formation. Traction force microscopy studies demonstrated an elevated level of contractile forces in adherent mutated MKs. The myosin II inhibitor blebbistatin and the ROCK inhibitor Y27632 both rescued the proplatelet formation defect and normalized the ultrastructural characteristics of MYH9-RD MKs. Altogether, our results show that in MYH9-RD, mutations modify the overall MYH9 function and provoke a proplatelet defect through an excess of actomyosin contractility in spreading MKs; results which may promote new therapeutic strategies aimed at reducing this actomyosin contractility. This article is protected by copyright. All rights reserved.
Full-text · Article · Oct 2013 · Journal of Thrombosis and Haemostasis
[Show abstract][Hide abstract] ABSTRACT: The endothelial protein C receptor (EPCR) plays a crucial role in the anticoagulant and anti-inflammatory effects of the protein C pathway, whereas its soluble form (sEPCR) exhibits opposite properties. High plasma levels of sEPCR have been observed in subjects carrying the A3 haplotype of PROCR, the EPCR gene. Elevated plasma levels of sEPCR were also recently reported in women with preeclampsia (PE), a multisystemic syndrome involving inflammation, endothelial dysfunction and thrombosis. To determine whether this increase is genetically mediated or acquired, we analyzed sEPCR levels and the A3 haplotype distribution in 145 preeclamptic women and 145 age- and term-matched women with normal pregnancies enrolled in a case-control study. Plasma sEPCR levels were higher in the women with PE than in the controls, and this difference was not due to A3 haplotype over-representation. We observed a positive correlation between sEPCR levels and two markers of endothelium activation/damage (von Willebrand factor and soluble thrombomodulin), and a trend towards a third (sVCAM1). We also found an association between sEPCR levels in the highest quartile and the PE risk. The modest increase of sEPCR levels, together with the correlation with other endothelium activation/damage markers, suggest that it is more an innocent bystander of the endothelium activation/damage than an actor in PE.
No preview · Article · Aug 2011 · Thrombosis Research
[Show abstract][Hide abstract] ABSTRACT: The endothelial cell protein C receptor also exists in soluble form in plasma (sEPCR), resulting from ADAM17 cleavage. Elevated sEPCR levels are observed in subjects carrying the A3 haplotype, which is characterized by a Ser219Gly substitution in the transmembrane domain, rendering the receptor more sensitive to cleavage. Because sEPCR production is not completely blocked by metalloprotease inhibition, we looked for another mechanism. Comparing mRNA expression patterns and levels in A3 and non-A3 cells from 32 human umbilical cord veins, we detected a truncated mRNA in addition to the full-length mRNA. This truncated mRNA was 16 times more abundant in A3 human umbilical vein endothelial cells than in non-A3 human umbilical vein endothelial cells and encoded a protein lacking the transmembrane domain. We stably expressed a recombinant form of this protein (rEPCRisoform) and a protein mimicking the plasma sEPCR (rEPCRsol). Functional studies of the purified recombinant proteins revealed that the rEPCRisoform bound to recombinant protein C with similar affinity than rEPCRsol and that it also inhibited the anticoagulant activity of APC. Trace amounts of the EPCR isoform were found in the plasma of A3 subjects. These results suggest that the sEPCRisoform could contribute to the regulatory effect of sEPCR in plasma.
[Show abstract][Hide abstract] ABSTRACT: The endothelial cell protein C (PC) receptor (EPCR) facilitates PC activation by the thrombin-thrombomodulin complex. A soluble form of this receptor (sEPCR) found in plasma inhibits both activated PC (aPC) activity and PC activation by competing for PC with membrane-associated EPCR. Elevated sEPCR levels are found in approximately 20% of healthy subjects, but the mechanisms underlying this interindividual variability are unknown. We measured sEPCR levels in 100 healthy male volunteers, and observed 2 phenotypic groups of subjects. The temporal stability of sEPCR levels suggested genetic control. Extensive analysis of the EPCR gene in these subjects revealed 13 polymorphisms in complete linkage disequilibrium; these defined 3 haplotypes, 1 of which (A3) was strongly associated with high sEPCR levels. The high constitutive sEPCR levels observed in A3 haplotype carriers might reduce the efficiency of the PC system and predispose these subjects to venous thrombosis. By studying 338 patients with venous thrombosis and 338 age- and sex-matched healthy subjects, we found that the A3 haplotype was overrepresented in the patients. In multivariate analysis, subjects carrying the A3 haplotype had an increased risk of thrombosis (odds ratio [OR] = 1.8; P =.004). Thus, the A3 haplotype, which is associated with elevated plasma sEPCR levels, is a candidate risk factor for venous thrombosis.
[Show abstract][Hide abstract] ABSTRACT: Protein S (PS) possesses a sex-hormone-binding globulin (SHBG)-like domain in place of the serine-protease domain found in other vitamin K-dependent plasma proteins. This SHBG-like domain is able to bind a complement fraction, C4b-binding protein (C4b-BP). To establish whether the PS SHBG-like domain can fold normally in the absence of other domains, and to obtain information on the specific functions of this region, we expressed the PS SHBG-like domain alone or together with its adjacent domain EGF4. The folding of the two recombinant modules was studied by analyzing their binding to C4b-BP. The apparent dissociation constants of this interaction indicated that both recombinant modules adopted the conformation of native PS, indicating that the PS SHBG-like region is an independent folding unit. We also obtained the first direct evidence that the SHBG-like domain alone is sufficient to support the interaction with C4b-BP. In addition, both recombinant modules were able to bind Ca2+ directly, as shown by the migration shift in agarose gel electrophoresis in the presence of Ca2+, together with the results of equilibrium dialysis and the functional effect of Ca2+ on the C4b-BP/PS interaction, confirming the presence of one Ca2+ binding site within the SHBG-like domain. Neither recombinant module exhibited activated protein C (aPC) cofactor activity in a clotting assay, suggesting that the PS SHBG-like region must be part of the intact molecule for it to contribute to aPC cofactor activity, possibly by constraining the different domains in a conformation that permits optimal interaction with aPC.
Preview · Article · Mar 2003 · European Journal of Biochemistry
[Show abstract][Hide abstract] ABSTRACT: The protein C (PC) pathway plays a critical role in the regulation of coagulation and modulation of inflammation. An endothelial cell receptor (named EPCR) was recently discovered, and was shown to bind the zymogen as well as the active form of PC (aPC) with identical affinities. The identification of this receptor allowed several elusive points to be clarified. Up to now, the anticoagulant PC pathway was thought to act essentially in the microcirculation while discovery of EPCR at the surface of large vessels highlights also the role of this pathway on the entire vascular system. Indeed, the activation rate of EPCR-bound PC by the thrombin-thrombomodulin (IIa-TM) complex is three to five-fold higher than that of circulating PC by the IIa-TM complex, counterbalancing the apparent low concentration of thrombomodulin in large vessels. In addition, aPC binding to EPCR decreases tissue-factor expression, thus limiting procoagulant activity of enclothelial cells. This receptor could be one of the missing links explaining the anti-inflammatory effect of aPC. A recent study, evidenced several intracellular mechanisms elucidating the anti-inflammatory effects of aPC infusion, and EPCR could be responsible for signal transduction. EPCR is related to MHC class I molecules, a family of transmembrane proteins able to bind peptides. The bindinq of PC to EPCR is mostly conditioned by the Gla domain of PC, and is independent of the presence of anionic phospholipids. Thus, the presence of procoagulant surface is not a prerequisite for PC activation. As for thrombomodulin, a soluble form of EPCR (sEPCR) is present under physiological conditions, and corresponds to a truncated form of the cellular protein released from the vascular wall by a constitutive proteolysis. The sEPCR is able to bind PC as well as aPC with identical Kd. It increases slightly, the catalytic activity of aPC toward chomogenic substrates. Soluble EPCR also modulates the inflammatory, response, by binding to CD11b-CD18 and PR-3 proteins expressed by activated neutrophils, thus precluding neutrophil adhesion. Additional investigations are required to further evaluate the importance of this receptor in human pathology. However, gene knock-out experiments in mice and in vivo studies in different animal models, indicate that EPCR should play a critical role in both coagulation and inflammation.