Biomedical subjects

Hervé Falet

Publications and source records attributed to Hervé Falet.

8 recordsLinked to original sources

PubMed2003

Role for phosphoinositide 3-kinase in Fc gamma RIIA-induced platelet shape change.

Platelets transform from disks to irregular spheres, grow filopodia, form ruffles, and spread on surfaces coated with anti-Fc gamma RIIA antibody. Fc gamma RIIA cross-linking leads to a tenfold increase in actin filament barbed end exposure and robust actin assembly. Activation of the small GTPases Rac and Cdc42 follows Fc gamma RIIA cross-linking. Shape change, actin filament barbed end exposure, and quantifiable actin assembly require phosphoinositide 3-kinase (PI3-kinase) activity and a rise in intracellular calcium. PI3-kinase inhibition blocks activation of Rac, but not of Cdc42, and diminishes the association of Arp2/3 complex and CapZ with polymerized actin. Furthermore, addition of constitutively active D-3 phosphorylated polyphosphoinositides or recombinant PI3-kinase subunits to octylglucoside-permeabilized platelets elicits actin filament barbed end exposure by releasing gelsolin and CapZ from the cytoskeleton. Our findings place PI3-kinase activity upstream of Rac, gelsolin, and Arp2/3 complex activation induced by Fc gamma RIIA and clearly distinguish the Fc gamma RIIA signaling pathway to actin filament assembly from the thrombin receptor protease-activated receptor (PAR)-1 pathway.

Actin-Related Protein 3↗

PubMed2003

Mechanisms and implications of platelet discoid shape.

The platelet marginal band consists of a single peripheral microtubule (MT) that is wound in 8 to 12 coils and maintains discoid cell shape. About 90% of beta-tubulin in the marginal band is of the divergent, megakaryocyte (MK)/platelet-restricted beta1 isoform. beta1-tubulin-null mice show reduced proplatelet formation, thrombocytopenia, and platelet spherocytosis. Here, we show that structural abnormalities in resting beta1-tubulin-/- platelets include frequent kinks and breaks in the marginal band. Platelets derived from mice lacking the transcription factor GATA1 show similar defects, probably as a direct consequence of absent beta1-tubulin. beta1-tubulin+/- platelets have normal ratios of beta-tubulin isotypes but the marginal band is half the normal thickness, which is sufficient to maintain elliptical cell shape. Thus, a threshold 50% or less of the normal amount of beta1-tubulin is required to preserve marginal band integrity and cell shape. beta1-tubulin-/- platelets have normal size and contents and show no defects in serotonin release or aggregation. Accordingly, the apparently isolated spherocytosis allows investigation of the role of discoid platelet shape in hemostasis. On agonist stimulation, the disorganized MTs in beta1-tubulin-/- platelets fail to condense into central rings and instead are dispersed in short bundles and linear arrays. Nevertheless, intravital microscopy and flow chamber studies demonstrate full functionality of these spherocytic platelets under physiologic shear conditions. Together, these findings highlight the essential requirements of the MK/platelet-restricted beta1-tubulin isoform in platelet structure and suggest that spherocytosis does not impair many aspects of platelet function.

Animals↗

PubMed2003

The clearance mechanism of chilled blood platelets.

Platelet transfusion is a very common lifesaving medical procedure. Not widely known is the fact that platelets, unlike other blood cells, rapidly leave the circulation if refrigerated prior to transfusion. This peculiarity requires blood services to store platelets at room temperature, limiting platelet supplies for clinical needs. Here, we describe the mechanism of this clearance system, a longstanding mystery. Chilling platelets clusters their von Willebrand (vWf) receptors, eliciting recognition of mouse and human platelets by hepatic macrophage complement type 3 (CR3) receptors. CR3-expressing but not CR3-deficient mice exposed to cold rapidly decrease platelet counts. Cooling primes platelets for activation. We propose that platelets are thermosensors, primed at peripheral sites where most injuries occurred throughout evolution. Clearance prevents pathologic thrombosis by primed platelets. Chilled platelets bind vWf and function normally in vitro and ex vivo after transfusion into CR3-deficient mice. Therefore, GPIb modification might permit cold platelet storage.

Animals↗

PubMed2003

Platelet moesin interacts with PECAM-1 (CD31).

Platelet activation results information of filopodia and cell spreading by extension of lamellae. Moesin is a member of the ezrin/radixin/moesin (ERM) family of proteins, which localize in cell extensions like filopodia and function as cross-linkers between the actin cytoskeleton and the plasma membrane. Here we investigated whether the adhesion molecule PECAM-1 (CD31) is a membrane-binding partner for moesin in platelets. Our data show that moesin co-immunoprecipitated with PECAM-1 in lysates from thrombin-stimulated, but not resting platelets. Furthermore, PECAM-1 co-localized with moesin at the cell periphery and in filopodia of glass-activated platelets. Our observations suggest that moesin may play a role in platelet adhesion, linking PECAM-1 with the actin cytoskeleton.

Actins↗

PubMed2003

Structural requirements of SLP-76 in signaling via the high-affinity immunoglobulin E receptor (Fc epsilon RI) in mast cells.

The adapter SLP-76 plays an essential role in Fc epsilon RI signaling, since SLP-76(-/-) bone marrow-derived mast cells (BMMC) fail to degranulate and release interleukin-6 (IL-6) following Fc epsilon RI ligation. To define the role of SLP-76 domains and motifs in Fc epsilon RI signaling, SLP-76(-/-) BMMC were retrovirally transduced with SLP-76 and SLP-76 mutants. The SLP-76 N-terminal and Gads binding domains, but not the SH2 domain, were critical for Fc epsilon RI-mediated degranulation and IL-6 secretion, whereas all three domains are essential for T-cell proliferation following T-cell receptor (TCR) ligation. Unexpectedly, the three tyrosine residues in SLP-76 critical for TCR signaling, Y112, Y128, and Y145, were not essential for IL-6 secretion, but were required for degranulation and mitogen-activated protein kinase activation. Furthermore, a Y112/128F SLP-76 mutant, but not a Y145F mutant, strongly reconstituted mast cell degranulation, suggesting a critical role for Y145 in Fc epsilon RI-mediated exocytosis. These results point to important differences in the function of SLP-76 between T cells and mast cells.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Importance of free actin filament barbed ends for Arp2/3 complex function in platelets and fibroblasts.

We investigated the effect of actin filament barbed end uncapping on Arp23 complex function both in vivo and in vitro. Arp23 complex redistributes rapidly and uniformly to the lamellar edge of activated wild-type platelets and fibroblasts but clusters in marginal actin filament clumps in gelsolin-null cells. Treatment of gelsolin-null platelets with the negative dominant N-WASp C-terminal CA domain has no effect on their residual actin nucleation activity, placing gelsolin actin filament severing, capping, and uncapping function upstream of Arp23 complex nucleation. Actin filaments capped by gelsolin or the gelsolin homolog CapG fail to enhance Arp23 complex nucleation in vitro, but uncapping of the barbed ends of these actin filaments restores their ability to potentiate Arp23 complex nucleation. We conclude that Arp23 complex contribution to actin filament nucleation in platelets and fibroblasts importantly requires free barbed ends generated by severing and uncapping.

Actin-Related Protein 2↗

PubMed2002

Normal Arp2/3 complex activation in platelets lacking WASp.

Arp2/3 complex is believed to induce de novo nucleation of actin filaments at the edge of motile cells downstream of WASp family proteins. In this study, the signaling pathways leading to Arp2/3 complex activation, actin assembly, and shape change were investigated in platelets isolated from patients with Wiskott-Aldrich Syndrome (WAS), that is, who lack WASp, and in WASp-deficient mouse platelets. WASp-deficient human and mouse platelets elaborate filopodia, spread lamellae, and assemble actin, identical to control WASp-expressing platelets. Human platelets contain 2 microM Arp2/3 complex, or 8600 molecules/cell. Arp2/3 complex redistributes to the edge of the lamellae and to the Triton X-100-insoluble actin cytoskeleton of activated WASp-deficient platelets. Furthermore, the C-terminal CA domain of N-WASp, which sequesters Arp2/3 complex, inhibits by half the actin nucleation capacity of octylglucoside-permeabilized and activated WAS platelets, similar to its effect in WASp-expressing cells. Along with WASp, platelets express WAVE-2 as a physiologic activator of Arp2/3 complex and a small amount of N-WASp. Taken together, our findings show that platelets activate Arp2/3 complex, assemble actin, and change shape in the absence of WASp, indicating a more specialized role for WASp in these cells.

Actin-Related Protein 2↗

PubMed2002

Purification of salmon thrombin and its potential as an alternative to mammalian thrombins in fibrin sealants.

A method to produce highly purified thrombin from salmon blood is described, and a series of biochemical, cell biologic, and biophysical assays demonstrate the functional similarities and some differences between salmon and human thrombins. Salmon thrombin with specific activity greater than 1000 units/mg total protein can be prepared by modifications of the methods used for purification of human thrombin. Using a synthetic substrate based on the human fibrinogen A-alpha polypeptide sequence as an indicator of enzymatic activity, salmon and human thrombin preparations contain similar specific activities per mass of purified protein. Salmon thrombin activates human fibrinogen and initiates the formation of fibrin clots whose structure and rheologic properties are indistinguishable from those of human fibrin clotted by human thrombin. Salmon thrombin also activates human platelets. Approximately 10 times higher activities are needed for the same rate of platelet aggregation compared to human thrombin, and some aspects of platelet activation, most notably phosphatidylserine exposure, are diminished relative to the effects of human thrombin. This latter finding suggests that salmon thrombin may not activate all of the receptors that are targets of human thrombin, although it does appear to activate signals that are sufficient to produce normal rates of activation and aggregation as measured by conventional aggregometry. Together with the recent purification of salmon fibrinogen and its application in mammalian wound healing, the availability of salmon thrombin allows the formulation of biological sealants devoid of any exogenous mammalian proteins and so may aid the design of materials with increased safety from infectious disease transmission.

Animals↗