The fate of mitochondria during platelet activation.

Blood platelets undergo several successive motor-driven reorganizations of the cytoskeleton when they are recruited to an injured part of a vessel. These reorganizations take place during the platelet activation phase, the spreading process on the injured vessel or between fibrin fibers of the forming clot, and during clot retraction. All these steps require a lot of energy, especially the retraction of the clot when platelets develop strong forces similar to those of muscle cells.

The Role of LIM Kinases during Development: A Lens to Get a Glimpse of Their Implication in Pathologies.

The organization of cell populations within animal tissues is essential for the morphogenesis of organs during development. Cells recognize three-dimensional positions with respect to the whole organism and regulate their cell shape, motility, migration, polarization, growth, differentiation, gene expression and cell death according to extracellular signals. Remodeling of the actin filaments is essential to achieve these cell morphological changes.

Asymmetrical Forces Dictate the Distribution and Morphology of Platelets in Blood Clots.

Primary hemostasis consists in the activation of platelets, which spread on the exposed extracellular matrix at the injured vessel surface. Secondary hemostasis, the coagulation cascade, generates a fibrin clot in which activated platelets and other blood cells get trapped. Active platelet-dependent clot retraction reduces the clot volume by extruding the serum.

Two Antagonistic Microtubule Targeting Drugs Act Synergistically to Kill Cancer Cells.

Paclitaxel is a microtubule stabilizing agent and a successful drug for cancer chemotherapy inducing, however, adverse effects. To reduce the effective dose of paclitaxel, we searched for pharmaceutics which could potentiate its therapeutic effect. We screened a chemical library and selected Carba1, a carbazole, which exerts synergistic cytotoxic effects on tumor cells grown in vitro, when co-administrated with a low dose of paclitaxel.

Marginal band microtubules are acetylated by αTAT1.

The discoid shape of resting platelets is maintained by a peripheral, circular bundle of microtubules called marginal band. Marginal band microtubules are acetylated on lysine 40 of the alpha-tubulin subunits. We have previously shown that the deacetylase HDAC6 is responsible for tubulin deacetylation in platelets and that the hyperacetylated state of the microtubules in platelets correlates with faster activation/spreading kinetics, pointing to a regulatory role of this modification.

β1 integrins mediate the BMP2 dependent transcriptional control of osteoblast differentiation and osteogenesis.

Osteoblast differentiation is a highly regulated process that requires coordinated information from both soluble factors and the extracellular matrix. Among these extracellular stimuli, chemical and physical properties of the matrix are sensed through cell surface receptors such as integrins and transmitted into the nucleus to drive specific gene expression. Here, we showed that the conditional deletion of β1 integrins in the osteo-precursor population severely impacts bone formation and homeostasis both in vivo and in vitro.

ICAP-1 monoubiquitylation coordinates matrix density and rigidity sensing for cell migration through ROCK2-MRCKα balance.

Cell migration is a complex process requiring density and rigidity sensing of the microenvironment to adapt cell migratory speed through focal adhesion and actin cytoskeleton regulation. ICAP-1 (also known as ITGB1BP1), a β1 integrin partner, is essential for ensuring integrin activation cycle and focal adhesion formation. We show that ICAP-1 is monoubiquitylated by Smurf1, preventing ICAP-1 binding to β1 integrin.

Calcium and calmodulin-dependent serine/threonine protein kinase type II (CaMKII)-mediated intramolecular opening of integrin cytoplasmic domain-associated protein-1 (ICAP-1α) negatively regulates β1 integrins.

Focal adhesion turnover during cell migration is an integrated cyclic process requiring tight regulation of integrin function. Interaction of integrin with its ligand depends on its activation state, which is regulated by the direct recruitment of proteins onto the β integrin chain cytoplasmic domain. We previously reported that ICAP-1α, a specific cytoplasmic partner of β1A integrins, limits both talin and kindlin interaction with β1 integrin, thereby restraining focal adhesion assembly.

Expression of 14 von Willebrand factor mutations identified in patients with type 1 von Willebrand disease from the MCMDM-1VWD study.

Background: Candidate von Willebrand factor (VWF) mutations were identified in 70% of index cases in the European study 'Molecular and Clinical Markers for the Diagnosis and Management of type 1 von Willebrand Disease'. The majority of these were missense mutations.

Objectives: To assess whether 14 representative missense mutations are the cause of the phenotype observed in the patients and to examine their mode of pathogenicity.

Molecular characterization of four ADAMTS13 mutations responsible for congenital thrombotic thrombocytopenic purpura (Upshaw-Schulman syndrome).

ADAMTS13 mutations S203P, R268P, R507Q and A596V were previously identified in French patients with hereditary thrombotic thrombocytopenic purpura (TTP) (Upshaw-Schulman syndrome). Mutated recombinant (r) ADAMTS13 were transiently expressed in COS-7 cells and characterized in comparison with wild-type (WT) rADAMTS13. ADAMTS13 antigen was qualitatively and quantitatively estimated by electrophoretic analysis and ELISA.

Effect of von Willebrand disease type 2B and type 2M mutations on the susceptibility of von Willebrand factor to ADAMTS-13.

Background: von Willebrand disease (VWD) type 2 is associated with mutations in von Willebrand factor (VWF) that affect its secretion, multimeric pattern, affinity for platelet receptors and clearance of the protein. While increased proteolysis by a disintegrin-like and metalloprotease with thrombospondin type 1 motifs-13 (ADAMTS-13) has been clearly established for VWF type 2A, only little is known about VWF types 2B and 2M in this regard.

Objectives: Sensitivity of wild-type (WT) and mutated recombinant (r) VWF to proteolysis by ADAMTS-13 was investigated to better understand the role of this process in the pathophysiology of VWD.

Impaired dimerization of von Willebrand factor subunit due to mutation A2801D in the CK domain results in a recessive type 2A subtype IID von Willebrand disease.

The CK domain of von Willebrand factor (VWF) is involved in the dimerization of the protein. We identified the homozygous substitution A2801D of the CK domain in two siblings. Patients had low levels of VWF in plasma, abnormal ristocetin-induced binding to platelets and abnormal multimeric pattern with a lack of high molecular weight (HMW) forms and the presence of intervening bands between normal multimers.

Thrombotic thrombocytopenic purpura associated with von Willebrand factor-cleaving protease (ADAMTS13) deficiency in children.

The physiopathology of thrombotic thrombocytopenic purpura (TTP) has been clarified since 1998, when it was shown that TTP in adults was most often associated with an acquired deficiency of von Willebrand factor-cleaving protease (ADAMTS13) due to autoantibodies, whereas TTP in children was most often associated with a hereditary autosomal recessive severe deficiency of ADAMTS13. The hereditary form of TPP (Upshaw-Schulman syndrome) is a very rare but life-threatening disease if adequate treatment (plasma therapy) is not administered. First manifestations occur before age 10 in two thirds of cases and as soon as birth in most cases.

Mutations C1157F and C1234W of von Willebrand factor cause intracellular retention with defective multimerization and secretion.

The D3 domain of von Willebrand factor (VWF) is involved in the multimerization process of the protein through the formation of disulfide bridges. We identified heterozygous substitutions, C1157F and C1234W, in the VWF D3 domain in two unrelated families with unclassified and type 2A von Willebrand disease, respectively. VWF was characterized by a low plasmatic level, an abnormal binding to platelet GPIb and a high capacity of secretion from endothelial cells following DDAVP infusion.

Ten candidate ADAMTS13 mutations in six French families with congenital thrombotic thrombocytopenic purpura (Upshaw-Schulman syndrome).

ADAMTS13, the specific von Willebrand factor (VWF)-cleaving metalloprotease, prevents the spontaneous formation of platelet thrombi in the microcirculation by degrading the highly adhesive ultralarge VWF multimers into smaller forms. ADAMTS13 severe enzymatic deficiency and mutations have been described in the congenital thrombotic thrombocytopenic purpura (TTP or Upshaw-Schulman syndrome), a rare and severe disease related to multivisceral microvascular thrombosis. We investigated six French families with congenital TTP for ADAMTS13 enzymatic activity and gene mutations.

An experimental model to study the in vivo survival of von Willebrand factor. Basic aspects and application to the R1205H mutation.

To explore the molecular basis of von Willebrand factor (VWF) clearance, an experimental model employing VWF-deficient mice was developed. Biodistribution was examined by the injection of radiolabeled VWF, which was primarily directed to the liver with minor amounts in other organs. Disappearance of VWF from plasma was characterized by a rapid initial phase (t((1/2))alpha = 13 min) and a slow secondary phase (t((1/2))beta = 3 h), with a mean residence time (MRT) of 2.

A new mutation, S1285F, within the A1 loop of von Willebrand factor induces a conformational change in A1 loop with abnormal binding to platelet GPIb and botrocetin causing type 2M von Willebrand disease.

We report the identification of a new mutation in exon 28 of the von Willebrand factor (VWF) gene in two related patients with type 2M von Willebrand disease (VWD). The molecular abnormality changes the Ser 1285 to Phe within the A1 loop of VWF. The S1285F mutation was reproduced by site-directed mutagenesis on the full-length VWF cDNA.

Two clusters of charged residues located in the electropositive face of the von Willebrand factor A1 domain are essential for heparin binding.

The VWF A1 domain seems to possess two heparin binding regions (residues 565-587 and 633-648) displaying positively charged amino acids, but the overall polyanion-A1 domain interaction scheme remains essentially elusive. To probe this molecular reaction as well as the role of electrostatic forces in VWF-heparin interaction, we performed mutagenesis and molecular modeling experiments. Fifteen mutated rVWFs were expressed [R571A, K572A, R573A, K585A, R571A/K572A/R573A, R578A/R579A, R578A/R579A/K585A, R571A/K572A/R573A/R578A/R579A/K585A (6A), K642G, K643G, K644G, K645G, K642G/K645G, K643G/K644G, and K642G/K643G/K644G/K645G (4G)].

Identification of a new type 2M von Willebrand disease mutation also at position 1324 of von Willebrand factor.

Type 2M von Willebrand disease (VWD) refers to variants with decreased platelet-dependent function that is not associated with the loss of high molecular weight (HMW) von Willebrand factor (VWF) multimers. This category includes the so-called "phenotype B" responsible for inexistent ristocetin-induced but normal botrocetin-induced binding of VWF to platelet glycoprotein lb. The missense mutation G1324S was identified in the first patient reported to display "phenotype B".

Selective inactivation of Von Willebrand factor binding to glycoprotein IIb/IIIa and to inhibitor monoclonal antibody 9 by site-directed mutagenesis.

Introduction: The purpose of this study was to assess the requirement for the RGD sequence of von Willebrand factor (VWF) for its binding to the beta3 chain of integrins and the structural basis for the specificity of monoclonal antibody (MoAb) 9 which specifically binds to VWF and inhibits this interaction.

Material And Methods: : Seven point mutations were introduced into VWF by site-directed mutagenesis. Mutated recombinant VWF were tested for their multimeric pattern and their ability to bind to purified GPIIb/IIIa, thrombin-activated platelets and MoAb 9.

Defect of heparin binding in plasma and recombinant von Willebrand factor with type 2 von Willebrand disease mutations.

The aim of our study was to characterise heparin-binding properties of mutated von Willebrand factor (VWF) in 24 patients plasmas with type 2 von Willebrand disease (VWD). and in 15 recombinant VWF (rVWF) with the corresponding mutations. Binding of mutated rVWF or plasma VWF was compared to that of WT-rVWF or normal pool plasma VWF.

Type 2 von Willebrand disease causing defective von Willebrand factor-dependent platelet function.

Type 2 von Willebrand disease causing defective von Willebrand factor-dependent platelet function comprises mainly subtypes 2A, 2B and 2M. The diagnosis of type 2 von Willebrand disease may be guided by the observation of a disproportionately low level of ristocetin cofactor activity or collagen-binding activity relative to the von Willebrand factor antigen level. The decreased platelet-dependent function is often associated with an absence of high molecular weight multimers (types 2A and 2B), but the high molecular weight multimers may also be present (type 2M and some type 2B), and supranormal multimers may exist (as in the Vicenza variant).