Thrombocytopenia-associated mutations in Ser/Thr kinase MASTL deregulate actin cytoskeleton dynamics in platelets

MASTL, a Ser/Thr kinase that inhibits PP2A-B55 complexes during mitosis, is mutated in autosomal dominant thrombocytopenia. However, the connections between the cell cycle machinery and this human disease remain unexplored. We report here that, whereas Mastl ablation in megakaryocytes prevented proper maturation of these cells, mice carrying the thrombocytopenia-associated mutation developed thrombocytopenia as a consequence of aberrant activation and survival of platelets. Activation of mutant platelets was characterized by hyper-stabilized pseudopods mimicking the effect of PP2A inhibition and actin polymerization defects. These aberrations were accompanied by abnormal hyper-phosphorylation of multiple components of the actin cytoskeleton and were rescued both in vitro and in vivo by inhibiting upstream kinases such as PKA, PKC, or AMPK. These data reveal an unexpected role of Mastl in actin cytoskeleton dynamics in postmitotic cells, and suggest that the thrombocytopenia-associated mutation in MASTL is a pathogenic dominant mutation that mimics decreased PP2A activity resulting in altered phosphorylation of cytoskeletal regulatory pathways.We thank Peter Storz (Mayo Clinic; Jacksonville, FL) for sharin g reagents and Sheila Rueda for her support with the management of the mouse colony. B.H. and R.S.-M. were supported by the Juan de la Cierva Programme from the Spanish M inistry of Economy and Competitiveness (MINECO). M.T. was supported by Foundation La Caixa. A.E.B. was supported by the Programa de Empleo Juvenil, Comunidad de M adrid. M.A.-F. received a young investigator g rant from MINECO (SAF2014-60442- JIN; co-financed by FEDER funds). P.G.dF. was supported by Fundació la Marató de TV3 (project 080121 and project 20153031). J.M. was supported by the Ramon y Cajal programme (MINECO; RYC-2012-10651). M.M. lab. is supported by grants from the MINE CO (SAF2015- 69920-R), Programa iLUNG from the Comunidad de Madrid (B2017/BM D-3884), and Worldwide Cancer Research (15-0278). CNIO is a Severo Ochoa Cen ter of Excellence (MINECO awards SEV-2015-0510)S

Variabilidad y expresión génica de PROS1, GAS6 y los receptores TAM en patología vascular

"Enfermedad vascular" es un término general para describir al conjunto de patologías que afectan a los sistemas vascular y circulatorio, entre las que se encuentran las enfermedades analizadas en esta tesis: la enfermedad tromboembólica, la aterosclerosis y la enfermedad cerebrovascular. Todas ellas son enfermedades complejas en las que intervienen múltiples factores genéticos y ambiéntales y se ha apuntado al posibilidad de que compartan mecanismos patogénicos comunes (cambien y Tiret,2005). En este contexto las principales proteínas estudiadas en esta tesis, la proteína S (PS) y el factor 6 especifico de parada celular o Growth Arrested Specfic gene 6 (GAS6), no solo están relacionadas con estas enfermedades vasculares, sino que comparten características comunes a nivel de estructura y función proteica y de estructura génica, ya que son parálogas, es decir, los genes que codifican para ambas proteínas provienen de un gen ancestral común, que se ha diversificado de forma independiente durante el proceso evolutivo. El trabajo que se presenta a continuación se fundamenta en la evidencia de una imbricación, a diferentes niveles, entre los sistemas inflamatorio y hemostático con consecuencias en las enfermedades estudiadas, esto es la trombosis, la enfermedad aterosclerótica y la enfermedad cerebrovascular asociada a aterosclerosis carotidea. La proteína S es bien conocida por su actividad anticoagulante y protectora del desarrollo de trombosis venosas (Castoldi y Hackeng, 2008). GAS6, mediante su interacción con receptores tirosina quinasa TAM (TYRO3,AXL y MERTK), se ha asociado a distintas enfermedades complejas, como enfermedad cardio y cerebrovascular, cáncer y enfermedades inflamatorias, entre otras (Bellido- Martin y Garcia de Frutos, 2008). La descripción de la anticoagulante, en la homeostasis vascular. Este trabajo profundiza en la patología molecular asociada a la deficiencia de proteína S y la trombosis venosa y explora el papel de esta proteína, así como la de su paráloga GAS6 y los receptores celulares de ambas, os receptores TAM, en la aterosclerosis carotidea y la enfermedad cerebrovascular.Peer Reviewe

GAS6 in systemic inflammatory diseases: with and without infection

Vitamin K-dependent proteins are not only essential regulators of blood coagulation. A recent paper in Critical Care describes the levels of the vitamin K-dependent GAS6 and the soluble form of its receptor Axl in plasma from patients with sepsis of systemic inflammation. The results confirm that GAS6 is elevated during septicemia, but the fact that inflammatory conditions without infection produce a similar effect suggests it is inflammation that induces the synthesis of GAS6, rather than the interactions with bacteria or other infectious agents. The soluble form of the GAS6 receptor Axl was induced less compared with the effect observed in GAS6. This is important as the two proteins form an inactive complex in plasma, suggesting that a functional GAS6 form could be synthesized under these conditions. GAS6 has been proposed as a broad regulator of the innate immune response. GAS6 synthesis is therefore likely to be a regulatory mechanism during systemic inflammation. Recent advances provide the necessary tools for further research, including genetic screenings of the components of this system. © 2010 BioMed Central Ltd.Peer Reviewe

Effect of the Gas6 c.834+7G>A polymorphism and the interaction of known risk factors on AMD pathogenesis in hungarian patients

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al.Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly in the developed world. Numerous genetic factors contribute to the development of the multifactorial disease. We performed a case-control study to assess the risk conferred by known and candidate genetic polymorphisms on the development of AMD. We searched for genetic interactions and for differences in dry and wet AMD etiology. We enrolled 213 patients with exudative, 67 patients with dry AMD and 106 age and ethnically matched controls. Altogether 12 polymorphisms in Apolipoprotein E, complement factor H, complement factor I, complement component 3, blood coagulation factor XIII, HTRA1, LOC387715, Gas6 and MerTK genes were tested. No association was found between either the exudative or the dry form and the polymorphisms in the Apolipoprotein E, complement factor I, FXIII and MerTK genes. Gas6 c.834+7G>A polymorphism was found to be significantly protective irrespective of other genotypes, reducing the odds of wet type AMD by a half (OR = 0.50, 95%CI: 0.26-0.97, p = 0.04). Multiple regression models revealed an interesting genetic interaction in the dry AMD subgroup. In the absence of C3 risk allele, mutant genotypes of both CFH and HTRA1 behaved as strongly significant risk factors (OR = 7.96, 95%CI: 2.39 = 26.50, p = 0.0007, and OR = 36.02, 95%CI: 3.30-393.02, p = 0.0033, respectively), but reduced to neutrality otherwise. The risk allele of C3 was observed to carry a significant risk in the simultaneous absence of homozygous CFH and HTRA1 polymorphisms only, in which case it was associated with a near-five-fold relative increase in the odds of dry type AMD (OR = 4.93, 95%CI: 1.98-12.25, p = 0.0006). Our results suggest a protective role of Gas6 c.834+7G>A polymorphism in exudative AMD development. In addition, novel genetic interactions were revealed between CFH, HTRA1 and C3 polymorphisms that might contribute to the pathogenesis of dry AMD. © 2012 Losonczy et al.This study was supported by by the Hungarian Research Fund OTKA K68616 (to A.B.) and by the Social Renewal Operational Programme TAMOP 4.2.1./ B-091/1/KONV-2010-0007 project (to A.B. and I.B.).Peer Reviewe

Association study between polymorphims in GAS6-TAM genes and carotid atherosclerosis.

et al.Carotid atherosclerosis (CA) is one of the most common causes of stroke, and recent studies suggest that pathways initiated by the interaction of the plasma vitamin K-dependent protein GAS6 with the tyrosine kinase receptors TYRO3, AXL and MERTK (TAM) may have a relevant role in atherogenesis. Furthermore, our previous studies indicated an association between GAS6 and stroke. The aim of this study was to analyse the genetic association between SNPs and haplotypes in GAS6-TAM genes and CA. We performed a case-control study with 233 CA patients confirmed by nuclear magnetic resonance angiography and 202 patients who suffered from cardioembolic (non atherogenic) stroke. For all included subjects information on established risk factors was available. Genotyping of 16 selected tagSNPs was performed by real-time PCR, using either FRET or TaqMan probes. Adjusted logistic regression (LR) analyses indicated that rs2289743 in TYRO3 and rs869016 in MERTK were associated to CA, decreasing its risk (OR [95%CI]=0.39 [0.16-0.94] and OR [95%CI]=0.31 [0.14-0.69], respectively). Linkage disequilibrium results were consistent with the haplotype blocks described in HapMap and adjusted LR analyses revealed that the haplotype ACAA in MERTK, containing the minor allele of the associated SNP, was also associated to CA. No association was observed with GAS6 and AXL variants, which suggests that CA is not the mechanism underlying the reported association between GAS6 and stroke. The association between TYRO3 and MERTK variants and carotid atherosclerosis found in this study reinforces a physiological role of the GAS6-TAM pathway in atherogenesis. © Schattauer 2010.Peer Reviewe

Molecular basis of protein S deficiency

Protein S deficiency (PSD) has been the most difficult to study among the classical inherited thrombophilic factors. This is in part due to the peculiar biology of protein S (PS), which has an anticoagulant role but no enzymatic activity, and because it interacts with plasma components that function in both haemostasis and inflammation. Clinically, it also has been difficult to define and standardise valuable assays to determine PS status and implication in thrombosis. Despite these drawbacks, at present heterozygous PS deficiency is well established as an autosomal dominant trait associated with an increased risk of thrombosis from data on familial and population studies. Almost two-hundred mutations have been characterised in PROSI, and approximately 30% of them have been characterised in vitro, clarifying the mechanisms leading to PSD. Furthermore, recent studies on the presence of large deletions in PROSI have increased the number of PSD associated to PROSI mutations. Finally, the discovery of new functions for PS, both in the anticoagulant system as well as in the interaction with cellular components through receptor tyrosine kinases, is broadenin the importance of this molecule in the context of biomedicine. © 2007 Schattauer GmbH, Stuttgart.This study has been partly supported by grants SAF2004–07539 and SAF2004–00543, from the Spanish Ministerio de Educación y Ciencia, ISCIII 01/1468, ISCIII network C03/07 and ISCIII PI051149 from the Instituto de Salud Carlos III. B.H. is recipient of a grant from the Institut d’Investigació Biomèdica de Bellvitge (IDIBELL 06/IDB-001).Peer Reviewe

Role of cyclin-dependent kinase 6 (CDK6) in platelet function and hemostasis in mice

Trabajo presentado en el 23rd International Congress on Thrombosis, celebrado en Valencia, España, del 14 al 17 de mayo de 2014[Background] The cyclin-dependent kinase Cdk6 has been implicated in integrin signaling and cytoskeletal reorganization. In the present study we characterized the possible function of Cdk6 in platelets.[Methods] Mice deficient in Cdk6 (cdk6¿/¿) were compared to wild type strains in several hemostatic and platelet functional test in vivo and in vitro.[Results] Cdk6 and cyclin D1 are present in human and murine platelets, suggesting a specific role in hemostasis in addition to their function in cell cycle regulation. Isolated murine platelets derived from cdk6¿/¿ mice showed decreased aggregation after stimulation with the platelet agonists ADP, collagen and thrombin. This was reflected in a delayed and/or weaker activation of the Akt and Erk kinase pathways compared to wild type platelets. Cdk6-deficient platelets showed a moderate impairment in their ability to spread on fibrinogen-coated surfaces, showing alterations in the dynamics of their activation process. In whole blood, cdk6 ¿/¿ platelets formed less aggregates under flow conditions. Also, the clots formed by Cdk6¿/¿ platelets in response to thrombin showed delayed retraction. The platelet deficiency caused a prolongation in tail-bleeding times in Cdk6 ¿/¿ animals and protected against arterial and venous thrombosis. In turn, lack of Cdk6 resulted in reduced mortality in pulmonary embolism models initiated by collagen and epinephrine.[Conclusions] Platelet Cdk6 seems to have a role in platelet activation that results in an impaired platelet response, which is reflected in mice hemostasis. Therapies using Cdk6 as target should consider the influence in hemostasis that could be derived from these effects.Peer Reviewe

Functional characterization of twelve natural PROS1 mutations associated with anticoagulant protein S deficiency

[Background]: The molecular mechanisms by which PROS1 mutations result in protein S deficiency are still unknown for many of the mutations, particularly for those that result in a premature termination codon. The aim of this study was to analyze the functional relevance on mRNA and protein expression of 12 natural PROS1 mutations associated with protein S deficiency.[Design and methods]: Five mutations were nonsense, three were small frameshift deletions, one was c.258,259AG>GT at the 3' end of exon 3, one was p.M640T and the last two were c.-7C>G and p.L15H, found in double heterozygosis as [c.-7C>G;44T>A]. The apparently neutral variant p.R233K was also analyzed. PROS1 cDNA was assessed by reverse transcriptase polymerase chain reaction of platelet mRNA. Expression of mutant proteins was determined by site-directed mutagenesis and analyses of transiently transfected PROS1 mutants in COS-7 cells.[Results]: Only cDNA from the normal allele was observed from the five nonsense mutations, the frameshift deletion c.1731delT and from c.258,259AG>GT. Both the normal and the mutated alleles were observed from [c.-7C>G;44T>A], c.187,188delTG and p.M640T. Transient expression analyses of PROS1 mutants whose mRNA was normally expressed revealed greatly reduced secretion of p.L15H and c.1272delA, mild secretion values of p.M640T and normal secretion levels of c.-7C>G and, as expected, p.R233K.[Conclusions]: Whereas the main cause of quantitative protein S deficiency associated with missense mutations is defective synthesis, stability or secretion of the mutated protein, the main mechanism for the deficiency associated with mutations that generate a premature termination codon is not the synthesis of a truncated protein, but the exclusion of the mutated allele, probably by nonsense-mediated mRNA decay.Funding: we acknowledge grants ISCIII 01/1468, ISCIII network C03/07 and ISCIII PI051149, from the Instituto de Salud Carlos III; SAF 2001-1059 and SAF2004-07539, from the Spanish Ministry of Education and Science; and grant 2002-PIR-00333 from the AGAUR, Generalitat de Catalunya. BH is the recipient of a grant from the Institut d’Investigació Biomèdica de Bellvitge (IDIBELL06/IDB-001).Peer reviewe

Association of specific haplotypes of GAS6 gene with stroke

The product of the growth arrest-specific gene 6 (GAS6), a ligand or tyrosine kinase receptors, is a vitamin K-dependent protein, structurally related to anticoagulant protein S. Gas6-deficient mice are protected against thrombosis, demonstrating the importance of this protein in the cardiovascular system. In a preliminary study on GAS6 polymorphisms and atherothrombotic disease we found an association between the AA genotype of the c.834+7G>A GAS6 polymorphism and stroke. In order to further explore this association by considering GAS6 haplotypes and the main stroke subtypes,457 patients with ischemic stroke, 199 with hemorrhagic stroke and 150 asymptomatic controls were genotyped for eight GAS6 polymorphisms and other genetic markers in the same genome region. Association was measured by logistic regression analysis. The THESIAS program was used to measure linkage disequilibrium and haplotype frequencies. In univariate analysis, the GAS6 c.834+7AA genotype was found associated with decreased risk for stroke (OR: 0.59; 95%Cl:0.37-0.93). After adjustment for vascular risk factors, association was maintained when stroke subtypes affecting the microvasculature such as lacunar stroke and deep haemorrhage, were grouped together (OR: 0.44; 95%Cl: 0.21-0.90). Furthermore, haplotype analysis revealed that association was even stronger when the c.834+7A allele was present in a specific haplotype (CACA) of four GAS6 polymorphisms. From these results we conclude that the A allele of the GAS6 c.834+7G>A polymorphism and more specifically, the CACA haplotype, is less prevalent in patients with stroke, suggesting a protective role for stroke of this haplotype. © 2007 Schattauer GmbH, Stuttgart.Peer Reviewe

Role of sodium tungstate as a potential antiplatelet agent.

Purpose: Platelet inhibition is a key strategy in the management of atherothrombosis. However, the large variability in response to current strategies leads to the search for alternative inhibitors. The antiplatelet effect of the inorganic salt sodium tungstate (Na2O4W), a protein tyrosine phosphatase 1B (PTP1B) inhibitor, has been investigated in this study.Methods: Wild-type (WT) and PTP1B knockout (PTP1B-/-) mice were treated for 1 week with Na2O4W to study platelet function with the platelet function analyzer PFA-100, a cone-and-plate analyzer, a flat perfusion chamber, and thrombus formation in vivo. Human blood aliquots were incubated with Na2O4W for 1 hour to measure platelet function using the PFA-100 and the annular perfusion chamber. Aggregometry and thromboelastometry were also performed.Results: In WT mice, Na2O4W treatment prolonged closure times in the PFA-100 and decreased the surface covered (%SC) by platelets on collagen. Thrombi formed in a thrombosis mice model were smaller in animals treated with Na2O4W (4.6±0.7 mg vs 8.9±0.7 mg; P<0.001). Results with Na2O4W were similar to those in untreated PTP1B -/- mice (5.0±0.3 mg). Treatment of the PTP1B-/- mice with Na2O4W modified only slightly this response. In human blood, a dose-dependent effect was observed. At 200 mM, closure times in the PFA-100 were prolonged. On denuded vessels, %SC and thrombi formation (%T) decreased with Na2O4W. Neither the aggregating response nor the viscoelastic clot properties were affected.Conclusion: Na2O4W decreases consistently the hemostatic capacity of platelets, inhibiting their adhesive and cohesive properties under flow conditions in mice and in human blood, resulting in smaller thrombi. Although Na2O4W may be acting on platelet PTP1B, other potential targets should not be disregarded © 2015 Fernández-Ruiz et al.This work was supported by the Secretaría de Estado de Investigación, Desarrollo e Innovación, and the Ministerio de Economía y Competitividad of Spain (SAF2011-28214 and SAF2010-19527); the Red de Investigación Cardiovascular, Instituto de Salud Carlos III, of Spain (RD12/0042/0016); and the Government of Catalonia (2009 SGR 1426). BH is a recipient of a Juan de la Ciervas’ grant from the Instituto de Salud Carlos III (JCI-2011-10417)Peer Reviewe