Karin Sadoul

This study reveals a previously unrecognized mechanism by which platelets retract and compact fibrin fibers. Using a newly developed 2D fiber-retraction assay, we observed an initial “gearwheel” pattern of actin–myosin organization in spread platelets with associated, extracellular fibrin patches that appear to form an initiation complex for fibrin fiber attachment and rearrangement. The final outcome of this process results in two morphologically different platelet populations. Spread platelets with coiled fibers around and above their pseudo-nucleus. Other platelets are only partially spread on the glass surface and are surrounded by tightly packed fibrin fibers around bulbous protrusions (“bulbs”), mirroring the architecture of platelets and adjacent fibers within a retracted clot. Thus, the observed compaction process might also take place during clot retraction in order to reduce clot volume, stiffen the clot and enhance wound repair. Apart from pulling on fibers like on a rope, platelets actively wrap fibrin fibers into compact structures, similar to balls of wool. Besides DNA packaging, this represents a new example of a natural fiber compaction mechanism. Using a combination of 3D clot-retraction and 2D fiber-retraction assays, expansion and electron microscopy, live imaging and mathematical modeling, we show that platelets use an actomyosin-driven motion to gather and loop fibrin fibers around the base of bulbous protrusions. These bulbs form when a platelet becomes trapped between fibrin fibers during 3D clot retraction or 2D fiber-retraction assays. These findings complement and extend earlier models of platelet-mediated fibrin fiber retractions, offering new insight into how platelets mechanically organize fibrin fibers.

This study reveals a previously unrecognized mechanism by which platelets retract and compact fibrin fibers. Using a newly developed 2D fiber-retraction assay, we observed an initial “gearwheel” pattern of actin–myosin organization in spread platelets with associated, extracellular fibrin patches that appear to form an initiation complex for fibrin fiber attachment and rearrangement. The final outcome of this process results in platelets surrounded by tightly packed fibrin fibers, mirroring the architecture of platelets and adjacent fibers within a retracted clot. Thus, the observed compaction process might also take place during clot retraction in order to reduce clot volume, stiffen the clot and enhance wound repair. Apart from pulling on fibers like on a rope, platelets actively wind-up fibrin fibers into compact structures, similar to balls of wool. Besides DNA packaging, this represents a new example of a natural fiber compaction mechanism. Using a combination of 3D clot-retraction and 2D fiber-retraction assays, expansion and electron microscopy, live imaging and mathematical modeling, we show that platelets use an actomyosin-driven swirling motion to gather and loop fibrin fibers around the base of bulbous protrusions (“bulbs”). These bulbs form when a platelet becomes trapped between fibrin fibers during clot retraction or 2D fiber-retraction assays. These findings complement and extend earlier models of platelet-mediated fibrin fiber retractions, offering new insight into how platelets mechanically organize fibrin fibers.

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. Platelets can produce energy through glycolysis and mitochondrial respiration. However, although resting platelets have only 5 to 8 individual mitochondria, they produce adenosine triphosphate predominantly via oxidative phosphorylation. Activated, spread platelets show an increase in size compared with resting platelets, and the question arises as to where the few mitochondria are located in these larger platelets. Using expansion microscopy, we show that the number of mitochondria per platelet is increased in spread platelets. Live imaging and focused ion beam–scanning electron microscopy suggest that a mitochondrial fission event takes place during platelet activation. Fission is Drp1 dependent because Drp1-deficient platelets have fused mitochondria. In nucleated cells, mitochondrial fission is associated with a shift to a glycolytic phenotype, and using clot retraction assays, we show that platelets have a more glycolytic energy production during clot retraction and that Drp1-deficient platelets show a defect in clot retraction.

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. Cofilin is an important binding protein for these filaments; it increases their elasticity in terms of flexion and torsion and also severs them. The activity of cofilin is spatiotemporally inhibited via phosphorylation by the LIM domain kinases 1 and 2 (LIMK1 and LIMK2). Phylogenetic analysis indicates that the phospho-regulation of cofilin has evolved as a mechanism controlling the reorganization of the actin cytoskeleton during complex multicellular processes, such as those that occur during embryogenesis. In this context, the main objective of this review is to provide an update of the respective role of each of the LIM kinases during embryonic development.

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. Thus, the clot architecture changes with time of contraction, which may have an important impact on the healing process and the dissolution of the clot, but the precise physiological role of clot retraction is still not completely understood. Since platelets are the only actors to develop force for the retraction of the clot, their distribution within the clot should influence the final clot architecture. We analyzed platelet distributions in intracoronary thrombi and observed that platelets and fibrin co-accumulate in the periphery of retracting clots in vivo. A computational mechanical model suggests that asymmetric forces are responsible for a different contractile behavior of platelets in the periphery versus the clot center, which in turn leads to an uneven distribution of platelets and fibrin fibers within the clot. We developed an in vitro clot retraction assay that reproduces the in vivo observations and follows the prediction of the computational model. Our findings suggest a new active role of platelet contraction in forming a tight fibrin- and platelet-rich boundary layer on the free surface of fibrin clots.

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. Carba1 targets the colchicine binding-site of tubulin and is a microtubule-destabilizing agent. Catastrophe induction by Carba1 promotes paclitaxel binding to microtubule ends, providing a mechanistic explanation of the observed synergy. The synergistic effect of Carba1 with paclitaxel on tumor cell viability was also observed in vivo in xenografted mice. Thus, a new mechanism favoring paclitaxel binding to dynamic microtubules can be transposed to in vivo mouse cancer treatments, paving the way for new therapeutic strategies combining low doses of microtubule targeting agents with opposite mechanisms of action.

(1) Background: Platelets were postulated to constitute the trigger of liver regeneration. The aim of this study was to dissect the cellular interactions between the various liver cells involved in liver regeneration and to clarify the role of platelets. (2) Methods: Primary mouse liver sinusoidal endothelial cells (LSECs) were co-incubated with increasing numbers of resting platelets, activated platelets, or platelet releasates. Alterations in the secretion of growth factors were measured. The active fractions of platelet releasates were characterized and their effects on hepatocyte proliferation assessed. Finally, conditioned media of LSECs exposed to platelets were added to primary hepatic stellate cells (HSCs). Secretion of hepatocyte growth factor (HGF) and hepatocyte proliferation were measured. After partial hepatectomy in mice, platelet and liver sinusoidal endothelial cell (LSEC) interactions were analyzed in vivo by confocal microscopy, and interleukin-6 (IL-6) and HGF levels were determined. (3) Results: Co-incubation of increasing numbers of platelets with LSECs resulted in enhanced IL-6 secretion by LSECs. The effect was mediated by the platelet releasate, notably a thermolabile soluble factor with a molecular weight over 100 kDa. The conditioned medium of LSECs exposed to platelets did not increase proliferation of primary hepatocytes when compared to LSECs alone but stimulated hepatocyte growth factor (HGF) secretion by HSCs, which led to hepatocyte proliferation. Following partial hepatectomy, in vivo adhesion of platelets to LSECs was significantly increased when compared to sham-operated mice. Clopidogrel inhibited HGF secretion after partial hepatectomy. (4) Conclusion: Our findings indicate that platelets interact with LSECs after partial hepatectomy and activate them to release a large molecule of protein nature, which constitutes the initial trigger for liver regeneration.

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 HDAC6KO platelets correlates with faster activation/spreading kinetics, pointing to a regulatory role of this modification. So far, the question about the reverse enzyme, responsible for tubulin acetylation in platelets, has remained unanswered. Several enzymes have been described as having tubulin acetylation activity. Here we identify αTAT1 as the enzyme responsible for the acetylation of marginal band microtubules. We show that αTAT1 deficiency has only minor consequences for platelet production and function. A residual tubulin acetylation level in αTAT1 deficient platelet lysates suggests the presence of an additional tubulin-acetylating enzyme that is unable to acetylate marginal band microtubules.

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 drugs which could potentiate its therapeutic effect. We have screened a chemical library and selected Carba1, a carbazolone, which exerts synergistic cytotoxic effects on tumor cells grownin vitro, when co-administrated with a low dose of paclitaxel. Carba1 targets the colchicine binding-site of tubulin and is a microtubule-destabilizing agent. The Carba1-induced modulation of microtubule dynamics increases the accumulation of fluorescent paclitaxel inside microtubules, providing a mechanistic explanation of the observed synergy between Carba1 and paclitaxel. The synergistic effect of Carba1 with paclitaxel on tumor cell viability was also observedin vivoin xenografted mice. Thus, a new mechanism favoring paclitaxel accumulation in microtubules can be transposed toin vivomouse cancer treatments, paving the way for new therapeutic strategies combining low doses of microtubule targeting agents with opposite mechanisms of action.

Although live imaging of dynamic processes in platelets is a challenging task, several important observations have been published during the last 20 years. We will discuss the amazing insights that have been achieved, the difficulties that can be encountered as well as some questions still open and the future technical perspectives.