Microtubule coils in spread blood platelets

The fate of the circumferential bundle of microtubules in activated platelets has been a subject of disagreement. Thin sections of stimulated platelets fixed at multiple intervals following exposure to aggregating agents have revealed that the circumferential band is constricted into a tight ring around centrally concentrated organelles. However, studies of detergent-resistant platelet cytoskeletons fixed and either negatively stained or critical point dried after activation on polylysine-coated grids have revealed that microtubule rings disappear, leaving only fragments in the peripheral cytoplasm of spread cells. The present study has employed immunofluorescence on glass slides and the whole mount technique with detergent extraction and either negative staining or critical point drying to evaluate the fate of microtubules in surface-activated platelets treated with or without the microtubule stabilizing agent, taxol. Significant numbers of microtubule coils were visible in control and taxol-treated platelets stained indirectly with a fluorescein-coupled antibody to tubulin 30 to 60 minutes after surface activation on glass. Coils of microtubules were also visible in dendritic forms and in significant numbers of spread platelets on negatively stained or critical point dried whole mounts in the electron microscope. The findings support the concept that microtubule disassembly is not an integral step in early phases of platelet activation.     

Isolation of microtubule coils from normal human platelets

The blood platelet is the only human cell known to have a circumferential band of microtubules. However, the mechanisms involved in assembly of the multi-looped coil, its interaction with the cell membrane to support discoid shape, and constriction into tight rings around centrally concentrated organelles in activated platelets are unknown. Separation of the microtubule rings from intact platelets would permit new approaches to solution of these questions. The present study has used simultaneous detergent extraction and fixation to isolate intact microtubule coils in significant numbers from suspended platelets for the first time. Isolated coils closely resembled the circumferential band observed in thin sections of plastic embedded platelets and in platelets prepared by the negative-stain whole-mount method. Enough microtubule coils could be recovered from suspensions of concentrated platelets to permit counting and quantitation on microscope grids. Results of this study will permit new approaches to clarification of the structural physiology of platelet microtubule coils.     

Isolation of circumferential microtubules from platelets without simultaneous fixation

Circumferential bands of microtubules (MT) support the discoid shape of resting platelets and participate with the contractile apparatus in shape change and internal contraction following activation. Elucidation of interactions between the circumferential coils and proteins of the stable and contractile cytoskeleton is essential for understanding MT function in platelet physiology. A previous investigation demonstrated that the circumferential rings can be isolated intact from resting platelets following simultaneous exposure to glutaraldehyde and Triton X-100. However, the use of fixation prevented the characterization of protein interactions. The present study has circumvented this problem by developing a procedure for isolating intact microtubule coils from detergent-treated platelets without the use of fixative agents. Incubation of the platelets for intervals of 30 to 60 minutes with the microtubule-stabilizing agent taxol preserved the circumferential bundle after extraction with Triton X-100 even after washing five times. The procedure has made it possible to carry out protein studies on isolated microtubule rings and associated proteins.     

Visualization of microtubule growth in living platelets reveals a dynamic marginal band with multiple microtubules

The marginal band of microtubules maintains the discoid shape of resting blood platelets. Although studies of platelet microtubule coil structure conclude that it is composed of a single microtubule, no investigations of its dynamics exist. In contrast to previous studies, permeabilized platelets incubated with GTP-rhodamine-tubulin revealed tubulin incorporation at 7.9 (± 1.9) points throughout the coil, and anti-EB1 antibodies stained 8.7 (± 2.0) sites, indicative of multiple free microtubules. To pursue this result, we expressed the microtubule plus-end marker EB3-GFP in megakaryocytes and examined its behavior in living platelets released from these cells. Time-lapse microscopy of EB3-GFP in resting platelets revealed multiple assembly sites within the coil and a bidirectional pattern of assembly. Consistent with these findings, tyrosinated tubulin, a marker of newly assembled microtubules, localized to resting platelet microtubule coils. These results suggest that the resting platelet marginal band contains multiple highly dynamic microtubules of mixed polarity. Analysis of microtubule coil diameters in newly formed resting platelets indicates that microtubule coil shrinkage occurs with aging. In addition, activated EB3-GFP–expressing platelets exhibited a dramatic increase in polymerizing microtubules, which travel outward and into filopodia. Thus, the dynamic microtubules associated with the marginal band likely function during both resting and activated platelet states.     

Effects of a microtubule stabilizing agent on the response of platelets to vincristine

The discoid shape of blood platelets is supported by a circumferential bundle of microtubules. Removal of the microtubules by an antimitotic drug, vincristine, is associated with loss of lentiform appearance, formation of tubulin paracrystals, a depressed response to aggregating agents, and impaired secretory activity. Recent studies have suggested that the action of vincristine on platelet secretion and aggregation is directly related to its action on microtubules, while other work had indicated that the antimitotic drug prevents the release reaction by inhibiting prostaglandin synthesis. The present study has examined the influence of taxol, a microtubule stabilizing agent, on the response of platelets to vincristine. Taxol completely prevented vincristine- induced shape change, microtubule disassembly, and tubulin paracrystal formation, even at concentrations one-tenth that of the antimitotic drug. Pretreatment with vincristine to dissociate microtubules and convert tubulin to crystals before exposure to taxol did not affect altered shape or tubulin paracrystals, but did cause assembly of free pools of tubulin into tubular polymers. Studies of physiology confirmed that vincristine, in amounts that remove microtubules, depresses platelet aggregation and secretion, effects that could be overcome by increasing agonist concentration. Although completely preventing microtubule dissociation, taxol had no corrective influence on vincristine-induced inhibition of platelet function. Biochemical studies revealed that vincristine concentrations that disassembled microtubules and blocked secretion did not inhibit conversion of 14C- arachidonic acid to thromboxane B2. The findings suggest that vincristine inhibits platelet function through some mechanism other than disassembling microtubules, but the other mechanism does not involve inhibition of prostaglandin synthesis.     

Immunogold staining of microtubules in resting and activated platelets

A circumferential microtubule is known to support the discoid form of resting platelets, but its fate following exposure of the cells to aggregating agents is uncertain. The present study has employed an immunocytochemical approach to follow the fate of the circumferential microtubule in activated platelets. Monoclonal antibodies to tubulin and to vinculin and a polyclonal antibody to actin were incubated with isolated microtubule coils and stained with staphylococcal protein A coupled to immunogold in order to test their specificity. Thin sections of glycolmethacrylate embedded platelets before and after exposure to thrombin for 15, 30 and 60 s were stained with antibodies to tubulin and actin. Immunogold particles showed a high specificity for isolated MT coils stained for tubulin, modest intensity for actin, and none for vinculin. Gold particles were randomly distributed in thin sections of resting and activated platelets stained for actin. Immunogold was limited to the circumferential microtubule in resting platelets and constricted coils in thrombin-activated cells. The number of gold particles in areas of cytoplasm away from microtubules in platelets stained with antitubulin antibody increased slightly following thrombin activation, but the change was not significant. Results support the concept that microtubule coils supporting the discoid form of resting platelets do not dissolve following exposure of the cells to potent agonists.     

beta-Dystroglycan modulates the interplay between actin and microtubules in human-adhered platelets

To maintain the continuity of an injured blood vessel, platelets change shape, secrete granule contents, adhere, aggregate, and retract in a haemostatic plug. Ordered arrays of microtubules, microfilaments, and associated proteins are responsible for these platelet responses. In full-spread platelets, microfilament bundles in association with other cytoskeleton proteins are anchored in focal contacts. Recent studies in migrating cells suggest that co-ordination and direct physical interaction of microtubules and actin network modulate adhesion development. In platelets, we have proposed a feasible association between these two cytoskeletal systems, as well as the participation of the dystrophin-associated protein complex, as part of the focal adhesion complex. The present study analysed the participation of microtubules and actin during the platelet adhesion process. Confocal microscopy, fluorescence resonance transfer energy and immunoprecipitation assays were used to provide evidence of a cross-talk between these two cytoskeletal systems. Interestingly, beta-dystroglycan was found to act as an interplay protein between actin and microtubules and an additional communication between these two cytoskeleton networks was maintained through proteins of focal adhesion complex. Altogether our data are indicative of a dynamic co-participation of actin filaments and microtubules in modulating focal contacts to achieve platelet function.     

Platelet spherocytosis: a new bleeding disorder

The present study evaluated a child with thrombocytopenia and a rare congenital bleeding disorder associated with platelet spherocytosis. Differential interference phase contrast microscopy (DIC) revealed that his platelets were spherical in form. Examination of thin sections in the electron microscope showed that his platelets were nearly devoid of microtubules (MT) and microtubule coils (MTC) that support the discoid shape in 100% of normal platelets. Immunofluorescence with a monoclonal antibody to tubulin, the precursor protein of MT, revealed bright rings in normal cells and diffuse fluorescence of patient platelets. The brightness of the fluorescence emitted by patient platelets was comparable to the diffuse fluorescence of normal platelets after chilling to dissolve intact MT, suggesting normal and patient cells contained comparable amounts of tubulin. Exposure of patient platelets to Taxol, an agent that stabilizes and induces MT, caused MT formation in 82% of patient platelets and MTC development in 11%, resulting in their conversion to discs. Glycoproteins GPIIb/IIIa and GPIb were present on patient platelets, and the cells contained normal numbers of dense bodies, ruling out storage pool disease. The patient's platelets adhered to and spread normally on glass but failed to undergo rapid, irreversible aggregation when stirred with agents that produced a complete response in normal discoid platelets, even when the patient's platelets were concentrated. The poor response of spherical platelets was associated with failure to become irregular and extend long filopodia. Thus, the spherical shape of patient platelets may contribute to the thrombocytopenia and to the clinical bleeding symptoms.     

Fine Structural Alterations Induced in Platelets by Adenosine Diphosphate

Blood platelets exposed to the nucleotide adenosine diphosphate (ADP)lose their discoid appearance and adhere to one another. The basis for theADP induced shape change has remained obscure. In the present study thestructural transformations of platelets under the influence of ADP have beenexamined in the electron microscope. Loss of lentiform appearance is closelyrelated to a marked reorganization of the platelet hyaloplasm. The marginalbundle of microtubules decreases in circumference and is moved into theinterior of the platelet. Granules and other organelles are transported to thecell center where they are enveloped within a web of microtubules and microfilaments. These changes are completely reversed after the influence of ADPdisappears. The movement of the circumferential bundle of microtubulesfrom its position under the cell wall appears to be the principle cause forthe loss of discoid shape. Constriction of hyaloplasmic elements followed bycomplete recovery of unaltered appearance are indicative of a reversible waveof contraction occurring in the platelet hyaloplasm. Thus the change in plateletsurface contour after exposure to ADP is not due to a direct modification ofthe cell wall by ADP, but to a contractile wave which ADP indirectly triggers in the substance of the cells.

Submitted on March 28, 1967 Accepted on November 1, 1967     

An Ultrastructural Basis for the Shape Changes Induced in Platelets by Chilling

Blood platelets normally circulate in the form of flat discs. Under conditionswhich promote aggregation, platelets transform from discs to irregular spheresand become sticky. Exposure of platelets to low temperature for brief periodscauses similar shape changes. The present study demonstrates that the alterations induced in platelets by chilling are related to disappearance of a marginal bundle of microtubules, which has recently been described in platelets.Rewarming platelets which have previously been chilled results in recoveryof both the marginal band of tubules and platelet disc shape. Thus the bundleof microtubules in platelet hyaloplasm appears intimately associated with discshape of unaltered cells. Factors which influence platelet shape changes maydo so by acting on the supporting marginal band of microtubules.

Submitted on February 27, 1967 Accepted on May 18, 1967     

Influence of intracellular chelating agents on formation of spike-like pseudopods by human platelets

Recent reports have suggested that spike-like pseudopods develop on platelets loaded with intracellular chelating agents during glass activation, and proposed that the extensions are caused by an unusual organization of newly assembled actin filaments. The present study has reexamined this hypothesis. Platelets loaded with one of the chelating agents, Quin II or BAPTA, seldom formed spike-like pseudopods when exposed to glass at 37 C for 30-60 min. However, when chilled and rewarmed the Quin II-or BAPTA-loaded platelets readily developed one or several spike-like extensions after a 30-60-min exposure to glass or on shaking in suspension. Thin section and negative-stain electron microscopy demonstrated that the major constituents of spike-like pseudopods were microtubules. Unusual coils of actin filaments were not observed. The observation was confirmed by immunofluorescence microscopy employing an anti-tubulin antibody and fluorescein-conjugated antimouse IgG. Cytochalasin B, an agent that inhibits new actin filament formation had virtually no effect on spike formation by chilled-rewarmed, Quin II- or BAPTA-loaded cells, whereas prior exposure to vincristine, an agent that dis-assembles microtubules and prevents their reformation, blocked spike development. Taxol, a drug that stabilizes microtubules and prevents their disassembly by cold or vincristine, prevented spike formation. Results indicate that microtubule assembly is the major cause of spike-like pseudo-pod formation, and the increased assembly may be due to binding of free cytoplasmic calcium by intracellular chelating agents.     

Comparison of platelet function and morphology in patients undergoing percutaneous coronary intervention receiving bivalirudin versus unfractionated heparin versus clopidogrel pretreatment and bivalirudin

We hypothesized that direct thrombin inhibition could attenuate platelet activation and release of soluble CD40 ligand (sCD40L), a marker of inflammation, during percutaneous coronary intervention (PCI). To assess platelet function under flow conditions with bivalirudin versus unfractionated heparin (UFH), we employed the cone and plate(let) analyzer (CPA) assay in drug-spiked blood samples from volunteers (n = 3) in vitro, and then in PCI patients who received bivalirudin alone (n = 20), UFH alone (n = 15), and clopidogrel pretreatment plus bivalirudin (n = 15). Scanning electron microscopy was employed to image bivalirudin or UFH-treated platelets to determine whether platelet function observations had a morphologic explanation. Enzyme immunoassay was used to measure sCD40L levels in PCI patients. In vitro, bivalirudin decreased platelet surface coverage; UFH increased platelet surface coverage. In PCI patients, bivalirudin alone decreased platelet surface coverage, UFH alone increased platelet surface coverage, and clopidogrel pretreatment plus bivalirudin additively reduced platelet surface coverage. Unlike UFH, bivalirudin did not activate platelets in SEM studies. Bivalirudin alone or coupled with clopidogrel significantly reduced plasma sCD40L in PCI patients. In conclusion, our findings suggest that under flow conditions, bivalirudin alone or coupled with clopidogrel may have an antiplatelet effect versus UFH alone during PCI. These data suggest that bivalirudin and UFH may confer an anti-inflammatory effect by reducing sCD40L during PCI.     

Actin filaments and microtubule dual‐granule transport in human adhered platelets: the role of α‐dystrobrevins

Upon activation with physiological stimuli, human platelets undergo morphological changes, centralizing their organelles and secreting effector molecules at the site of vascular injury. Previous studies have indicated that the actin filaments and microtubules of suspension‐activated platelets play a critical role in granule movement and exocytosis; however, the participation of these cytoskeleton elements in adhered platelets remains unexplored. α‐ and β‐dystrobrevin members of the dystrophin‐associated protein complex in muscle and non‐muscle cells have been described as motor protein receptors that might participate in the transport of cellular components in neurons. Recently, we characterized the expression of dystrobrevins in platelets; however, their functional diversity within this cellular model had not been elucidated. The present study examined the contribution of actin filaments and microtubules in granule trafficking during the platelet adhesion process using cytoskeleton‐disrupting drugs, quantification of soluble P‐selectin, fluorescence resonance transfer energy analysis and immunoprecipitation assays. Likewise, we assessed the interaction of α‐dystrobrevins with the ubiquitous kinesin heavy chain. Our results strongly suggest that microtubules and actin filaments participate in the transport of alpha and dense granules in the platelet adhesion process, during which α‐dystrobrevins play the role of regulatory and adaptor proteins that govern trafficking events.     

Influence of intracellular chelating agents on formation of spike-like pseudopods by human platelets

Recent reports have suggested that spike-like pseudopods develop on platelets loaded with intracellular chelating agents during glass activation, and proposed that the extensions are caused by an unusual organization of newly assembled actin filaments. The present study has reexamined this hypothesis. Platelets loaded with one of the chelating agents, Quin II or BAPTA, seldom formed spike-like pseudopods when exposed to glass at 37°C for 30–60 min. However, when chilled and rewarmed the Quin II-or BAPTA-loaded platelets readily developed one or several spike-like extensions after a 30–60-min exposure to glass or on shaking in suspension. Thin section and negative-stain electron microscopy demonstrated that the major constituents of spike-like pseudopods were microtubules. Unusual coils of actin filaments were not observed. The observation was confirmed by immunofluoresence microscopy employing an anti-tubulin antibody and fluorescein-conjugated antimouse IgG. Cytochalasin B, an agent that inhibits new actin filament formation had virtually no effect on spike formation by chilled-rewarmed, Quin II- or BAPTA-loaded cells, whereas prior exposure to vincristine, an agent that disassembles microtubules and prevents their reformation, blocked spike development. Taxol, a drug that stabilizes microtubules and prevents their disassembly by cold or vincristine, prevented spike formation. Results indicate that microtubule assembly is the major cause of spike-like pseduopod formation, and the increased assembly may be due to binding of free cytoplasmic calcium by intracellular chelating agents.     

Cytoskeletal F-actin,not the circumferential coil of microtubules,regulates platelet dense-body granule secretion

Cytoskeletal F-actin assembly and microtubule reorganization are principal cellular events responsible for activation-induced platelet shape change; however, their roles in regulating platelet secretion have remained controversial. Herein, label-free microelectrochemistry techniques and pharmacological approaches are used to probe the role of F-actin and the microtubule in platelet dense-body secretion. Altered microtubule integrity via exposure to paclitaxel or vincristine had no effect on serotonin release in platelet suspensions. Disruption of F-actin by cytochalasin D (CytoD) or latrunculin A (LatA) substantially enhanced the rate of serotonin release, while inhibition of the F-actin-dependent platelet motor protein myosin IIA by blebbistatin had no effect. CytoD-treated platelets also showed enhanced serotonin quantal secretion rate. These results clearly indicate that F-actin, but not the microtubule, regulates platelet dense-body secretion and does so by serving as a physical barrier. This study also demonstrates the promise of microelectrochemistry for giving important insight into platelet quantal secretion mechanisms in future studies.     

Cytoskeletal F-actin, not the circumferential coil of microtubules, regulates platelet dense-body granule secretion

Cytoskeletal F-actin assembly and microtubule reorganization are principal cellular events responsible for activation-induced platelet shape change; however, their roles in regulating platelet secretion have remained controversial. Herein, label-free microelectrochemistry techniques and pharmacological approaches are used to probe the role of F-actin and the microtubule in platelet dense-body secretion. Altered microtubule integrity via exposure to paclitaxel or vincristine had no effect on serotonin release in platelet suspensions. Disruption of F-actin by cytochalasin D (CytoD) or latrunculin A (LatA) substantially enhanced the rate of serotonin release, while inhibition of the F-actin-dependent platelet motor protein myosin IIA by blebbistatin had no effect. CytoD-treated platelets also showed enhanced serotonin quantal secretion rate. These results clearly indicate that F-actin, but not the microtubule, regulates platelet dense-body secretion and does so by serving as a physical barrier. This study also demonstrates the promise of microelectrochemistry for giving important insight into platelet quantal secretion mechanisms in future studies.     

Formin proteins in megakaryocytes and platelets: regulation of actin and microtubule dynamics

The platelet and megakaryocyte cytoskeletons are essential for formation and function of these cells. A dynamic, properly organised tubulin and actin cytoskeleton is critical for the development of the megakaryocyte and the extension of proplatelets. Tubulin in particular plays a pivotal role in the extension of these proplatelets and the release of platelets from them. Tubulin is further required for the maintenance of platelet size, and actin is the driving force for shape change, spreading and platelet contraction during platelet activation. Whilst several key proteins which regulate these cytoskeletons have been described in detail, the formin family of proteins has received less attention. Formins are intriguing as, although they were initially believed to simply be a nucleator of actin polymerisation, increasing evidence shows they are important regulators of the crosstalk between the actin and microtubule cytoskeletons. In this review, we will introduce the formin proteins and consider the recent evidence that they play an important role in platelets and megakaryocytes in mediating both the actin and tubulin cytoskeletons.     

Automated Plateletpheresis Does Not Cause an Increase in Platelet Activation in Volunteer Donors

Abstract: Activation of platelets during collection and storage has been implicated as a major cause of the platelet storage lesion. In this study, we investigated the effect of an automated plateletpheresis procedure on the in vivo platelet activation in 20 volunteer donors. Peripheral blood samples were collected immediately before and after plateletpheresis on the Haemonetics V50 Blood Cell Separator. Activation of platelets was determined by quantitating the amount of platelet P-selectin (CD62) expression using a whole blood method on flow cytometry. Adenosine diphosphate (ADP), collagen, and ristocetin induced platelet aggregations were also measured on a whole blood impedance aggregometer. Plateletpheresis caused a significant decrease in the CD62-positive platelet percentage and aggregation responses to 3 agonists. We concluded that the plateletpheresis procedure did not cause an increase in platelet activation in donors. Further studies are required to elucidate whether activated platelets are collected during the procedure or removed from the circulation of the donor and replaced by resting platelets, activated platelets bind to leukocytes or endothelial cells, and the plateletpheresis procedure is a powerful stimulus for platelet activation.—     

Investigation of the mechanisms of monoclonal antibody-induced platelet activation.

Antiplatelet antibodies can activate platelets causing platelet aggregation and the release reaction. However, the pathway of activation by these antibodies is unknown and several potential mechanisms are possible. In this report, we describe studies investigating potential pathways of platelet activation by IgG antibodies. We tested 16 different IgG monoclonal antibodies (MoAbs) against a variety of platelet surface components and found that six antibodies were capable of causing platelet aggregation and release. These included MoAbs against glycoprotein (GP) IIb/IIIa, CD9, GPIV, and two other not well-characterized platelet components. There was no relationship between the number of platelet binding sites and the ability of an MoAb to activate the platelets. By adding intact and F(ab')2 preparations of the MoAb to control or Fc receptor-blocked platelets, we found that in all instances the MoAbs initiated platelet activation via interacting with the platelet Fc receptors. Clustering of the platelet protein components using a secondary antibody did not cause activation. Studies into the pathway of Fc-dependent activation demonstrated that the MoAbs were capable of activating platelets by occupying Fc receptors on adjacent platelets (interplatelet activation), as well as on the same platelet (intraplatelet activation).