Organization of the cytoskeleton in resting, discoid platelets: preservation of actin filaments by a modified fixation that prevents osmium damage

This study evaluates the structural organization of the cytoskeleton within unactivated, discoid platelets. Previously, such studies have been difficult to interpret because of the ease with which platelets are stimulated, the sensitivity of actin filaments to cell extraction buffers, and the general problem of preserving actin filaments with conventional fixatives, compounded by the density of the cytoplasm in the platelet. In this study we have employed a new fixative containing lysine, which protects actin filaments against damage during fixation and thin-section processing. We used thick (0.25-micron) sections and conventional thin sections of extracted cells (fixed and lysed simultaneously by the addition of 1% Triton X-100 to the initial fixative) as well as thin sections of whole cells to examine three preparations of human platelets: discoid platelets washed by sedimentation; discoid platelets isolated by gel filtration; and circulating platelets collected by dripping blood directly from a vein into fixative. In all of these preparations, long, interwoven actin filaments were observed within the platelet and were particularly concentrated beneath the plasma membrane. These filaments appeared to be linked at irregular intervals to the membrane and to each other via short, approximately 20- to 50-nm-long cross-links of variable width. Although most filaments were outside the circumferential band of microtubules and the cisternae of the open canalicular system, individual filaments dipped down into the cytoplasm and were found between the microtubules and in association with other membranes. The ease with which single actin filaments can be seen in the dense cytoplasm of the human platelet after lysine/aldehyde fixation suggests the great potential of this new fixative for other cells.     

Actin cytoskeleton of resting bovine platelets

Actin filaments in resting discoid bovine platelets were examined by fluorescence and electron microscopy. Rhodamine-phalloidin staining patterns showed a characteristic wheel-like structure which consisted of a central small circle connected by several radial spokes to a large peripheral circle. This wheel-like structure was composed of actin filaments forming a characteristic arrowhead structure with heavy meromyosin from muscle. Actin filaments were densely arrayed in parallel with a marginal microtubule band and radiated out from the center to the periphery. Platelets treated with colchicine lost their marginal microtubule band but retained their wheel-like structure and normal discoid form. Cytochalasin B disrupted the wheel-like structure but not the marginal microtubule band or the normal discoid form. After simultaneous treatment with both cytochalasin B and colchicine, platelets lost their discoid shape. These results suggest that actin filaments and microtubules both play important roles in the maintenance of the discoid shape of resting bovine platelets.     

Biosynthesis of major platelet proteins in human blood platelets

We studied de novo protein biosynthesis in platelets of normal adult donors and in newly formed platelets isolated from splenectomized patients with idiopathic thrombocytopenic purpura (ITP). After metabolic labelling of platelet proteins, performed with different radiolabelled amino acids or carbohydrates, a tenfold increase in incorporation of radioactivity into trichloroacetic-acid-precipitable material was obtained with ITP platelets compared to control platelets. Electron microscopic studies of ITP platelets revealed the presence of rough endoplasmic reticulum and polyribosomes, providing morphological evidence for protein synthesis. SDS-PAGE of radiolabelled ITP platelet proteins followed by autoradiography showed that [35S]methionine and [3H]leucine were incorporated into almost all Coomassie-blue-stained proteins whereas [3H]carbohydrates only labelled a few bands. Using crossed-immunoelectrophoresis we identified some of the labelled platelet compounds and demonstrated that major membrane glycoproteins (GPIb, IIb, IIIa) and alpha-granule proteins, such as fibrinogen, thrombospondin, albumin and von Willebrand factor, were synthesized in newly formed circulating platelets.     

Stimulation of lymphocytes by platelet-antibody-complexes and their role in the pathogenesis of idiopathic thrombocytopenia

The effect of washed human platelets, platelet lysates, and platelet antibody complexes on 14C-thymidine incorporation by human lymphocytes was studied. For sensitization of platelets, HLA-specific alloantibodies as well as platelet autoantibodies were used. Lymphocytes for in vitro cultures were collected from unsensitized individuals, healthy women with proven fetomaternal immunization against HLA antigens and patients with idiopathic thrombocytopenic purpura (ITP). Unsensitized platelets have a dose-dependent inhibitory effect on the in vitro proliferation of normal lymphocytes induced by mitogens (PHA, ConA, PWM). Platelet antibody complexes (allo- and autoantibodies; allogenic and autologous lymphocyte-platelet combinations) did not stimulate 14C-thymidine incorporation. Lymphocytes from ITP patients showed a significantly reduced stimulatory response toward PHA compared to normal persons. These findings are discussed in the light of our present knowledge regarding the role of cellular immune reactions in the pathogenesis of ITP.     

Ionophore-induced disassembly of blood platelet microtubules: effect of cyclic AMP and indomethacin

Cytoplasmic calcium levels are believed to be important in blood platelet activation. Upon activation, the discrete marginal microtubule band, which maintains the discoid shape of non-activated platelets, becomes disrupted. Present studies demonstrate that the extent of assembly of the marginal microtubule band is related to cytoplasmic calcium levels. The divalent cationophore, A23187, causes platelet aggregation, secretion, and contraction by promoting calcium transport from intraplatelet storage sites into the cytoplasm. A23187 caused disassembly of platelet microtubules. Quantitation of electron micrographs revealed that numbers of microtubules were reduced by approximately 80% after A23187 treatment. Secondly, assembled microtubules in homogenates of platelets, in which microtubules were stabilized prior to homogenization, were decreased in favor of free tubulin in A23187-treated platelets. Thirdly, A23187 increased 14C-colchicine binding by intact platelets; this also indicated a shift in the microtubule subunit equilibrium to favor free, colchicine-binding tubulin subunits. In control experiments, A23187 did not affect the stability of platelet tubulin, the colchicine binding reaction, or the total tubulin content of platelets. Stimulation of colchicine binding depended on A23187 concentration (0.05-0.5 microM) and did not require extracellular calcium. A23187-stimulation of colchicine binding was blocked by dibutyryl cyclic AMP (0.80 mM) and/or 3-isobutyl-1-methylxanthine (50 microM) and by indomethacin (10 microM). Cyclic AMP or indomethacin also interferes with A23187-induced platelet activation, but indomethacin is not likely to completely inhibit the perturbation of intraplatelet calcium gradients by A23187. It is suggested that A23187-induced microtubule disassembly may be an indirect effect of calcium on microtubules.     

Actin filament content and organization in unstimulated platelets

The extent of actin polymerization in unstimulated, discoid platelets was measured by DNase I inhibition assay in Triton X-100 lysates of platelets washed at 37 degrees C by gel filtration, or in Triton X-100 lysates of platelets washed at ambient temperatures by centrifugation in the presence of prostacyclin. About 40% of the actin in the discoid platelets obtained by either method existed as filaments. These filaments could be visualized by electron microscopy of thin sections. Similar results were obtained when the actin filament content of discoid platelets was measured by sedimentation of filaments from Triton X-100 lysates at high g forces (145,000 g for 45 min). However, few of these filaments sedimented at the lower g forces often used to isolate networks of actin filaments from cell extracts. These results indicate that actin filaments in discoid cells are not highly crosslinked. Platelets isolated by centrifugation in the absence of prostacyclin were not discoid, but were instead irregular with one or more pseudopodia. These platelets also contained approximately 40-50% of their actin in a filamentous form; many of these filaments sedimented at low g forces, however, indicating that they were organized into networks. The discoid shape of these centrifuged platelets could be restored by incubating them for 1-3 h at 37 degrees C, which resulted in the reversal of filament organization. High g forces were then required for the sedimentation of the actin. Approximately 80-90% of the actin in platelets washed at 4 degrees C was filamentous; this high actin filament content could be attributed to actin polymerization during the preparation of the platelets at low temperatures. These studies show that platelet activation involves mechanisms for the structural reorganization of existing filaments, in addition to those previously described for mediating actin polymerization.     

Complement-dependent cytotoxic factor to megakaryocyte progenitors in sera from patients with idiopathic thrombocytopenic purpura

The influence of sera from patients with idiopathic thrombocytopenic purpura (ITP) was examined on colony formation from megakaryocyte (M) progenitors. Though incubation of marrow cells in Iscove's modified Dulbecco's medium (IMDM) containing 50% sera from several ITP patients stimulated M-colony formation in 8 of 13 cases, incubation in the sera from the patients and in baby rabbit serum as a source of complement significantly suppressed the colony formation. Experiments showed that sera of immunoglobulin G from ITP patients had significant complement-dependent cytotoxicity to M-progenitors in normal marrow cells or in the marrow cells from corresponding patients, but not to CFU-e, BFU-e or CFU-gm. Cytospin preparations of individually collected M-colonies from marrow cells treated with ITP patients' sera and complement revealed a reduction of megakaryocyte colonies containing cells of multilineages. These results indicate that autoantibodies detected in ITP patients can bind not only to platelets and megakaryocytes, but may also bind to M-progenitors.     

Motor-driven marginal band coiling promotes cell shape change during platelet activation

Platelets float in the blood as discoid particles. Their shape is maintained by microtubules organized in a ring structure, the so-called marginal band (MB), in the periphery of resting platelets. Platelets are activated after vessel injury and undergo a major shape change known as disc to sphere transition. It has been suggested that actomyosin tension induces the contraction of the MB to a smaller ring. In this paper, we show that antagonistic microtubule motors keep the MB in its resting state. During platelet activation, dynein slides microtubules apart, leading to MB extension rather than contraction. The MB then starts to coil, thereby inducing the spherical shape of activating platelets. Newly polymerizing microtubules within the coiled MB will then take a new path to form the smaller microtubule ring, in concerted action with actomyosin tension. These results present a new view of the platelet activation mechanism and reveal principal mechanistic features underlying cellular shape changes.     

Effect of cyclic AMP on human blood platelets: induction of pseudopod formation and protein phosphorylation

A variety of techniques, including immunofluorescence, electron microscopy and biochemical analysis, were used to examine shape changes and cytoskeletal reorganization of human blood platelets during treatment with N6,O2-dibutyryl adenosine 3',5'-cyclic monophosphoric acid (dbcAMP), and agent known to elevate the intracellular level of cyclic AMP (cAMP). Cytochemical analysis shows that the unstimulated platelets have a discoid shape with no obvious membrane projections. Platelets treated with dbcAMP produce pseudopod-like structures containing cytoskeletal proteins such as actin and microtubules. Biochemical analysis reveals that a 125,000 dalton phosphoprotein (P-125) is preferentially recruited into cytoskeletal fractions of platelets treated with dbcAMP. This protein, which is one of the substrates for cAMP-dependent kinase(s) and/or is closely associated with the cytoskeleton, may play an important role in regulating the shape changes and cytoskeletal reorganization that occur during the early stages of platelet activation.     

Platelets and Erythrocyte-Bound Platelets Bind Infectious HIV-1 in Plasma of Chronically Infected Patients

Chronic HIV-1 infection is associated with persistent viremia in most patients, but it remains unclear how free virus may survive the potential hostile effects of plasma. We investigated whether sites might exist on the surfaces of circulating blood cells for protection of infectious HIV-1 particles. Red blood cells (RBC) either from blood of uninfected normal individuals, or from blood obtained without EDTA from chronically infected HIV-1 patients, invariably contained a small number of RBC having attached platelets as determined by flow cytometry, light microscopy, and immunofluorescence microscopy. After mixing normal RBC with platelet-rich plasma, discrete populations of RBC, platelets, and complexes of platelets attached to RBC were purified by fluorescence-activated cell sorting. Upon incubation of purified cells or platelets with HIV-1 followed by washing and co-incubation with CD4-positive peripheral blood mononuclear cells (PBMC), platelets, and platelet-RBC complexes, but not platelet-free RBC, caused infection of PBMC. Infection was prevented by pre-treating the platelet-RBC complexes with EDTA. Plasma and RBC (comprising a RBC/platelet-RBC mixture) from chronically infected patients with low viral loads were also co-incubated with PBMC ex vivo to determine the presence of infectious HIV-1. All freshly isolated plasmas from the HIV-1-infected donors, obtained in the absence of anticoagulant, were noninfectious. Interestingly, the RBC from most of the patients caused cell-cell infection of PBMC that was prevented by stripping the RBC with EDTA. A monoclonal antibody to DC-SIGN partially inhibited cell-cell HIV-1 infection of PBMC by normal RBC pre-incubated with platelets and HIV-1. We conclude: (a) platelet-free EDTA-free plasma from chronically infected HIV-1 patients, although containing viral RNA, is an environment that lacks detectable infectious HIV-1; (b) platelets and platelet-RBC complexes, but not purified RBC, bind infectious HIV-1; (c) DC-SIGN, and possibly other C-type lectins, may represent binding sites for infectious HIV-1 on platelets and platelet-RBC complexes.     

Immunodiagnosis of platelet activation in immune thrombocytopenia through scFv antibodies cognate to activated IIb3 integrins

Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder characterized by low platelet count and presence of IgG autoantibodies to platelet surface glycoproteins, such as αIIbβ3 and GPIb/IX. Our previous work has shown that platelets in ITP patients exist in an activated state. Two different marker-based approaches are used to study the course of platelet activation: (1) binding of PAC-1 antibody, signifying a change in αIIbβ3 conformation, and (2) expression of P-selectin, signifying alpha granule content release from platelets. Here, we describe the development of a new scFv antibody (R38) that, compared with PAC-1, appears to better distinguish between platelets of ITP patients and healthy controls. Notably, R38 was generated using commercially sourced resting-state integrin that was coated on a microtiter plate. Its ability to distinguish between ITP patients and healthy controls thus suggests that inadvertent integrin activation caused by coating involves a conformational change and exposure of a cryptic epitope. This report also describes for the first time the potential use of an scFv antibody in the immunodiagnosis of platelet activation in ITP patients.     

Frozen/thawed platelets: importance of osmotic tolerance and platelet subpopulations.

Me2SO cryopreserved platelets circulate in vivo, reduce bleeding time, and have hemostatic properties but their functional recovery is only half that of the fresh material. Poor osmotic response is often reported as the cause of the freezing injury. Osmotic excursions on 1- and 5-day-old platelets have been studied. Platelets stored for 5 days have a lesser capability to regulate their volume particularly after an initial swelling. This is attributed to the reduction of discoid cell number, 80% vs 62% for 1-day-old and 5-day-old platelets, respectively. After freezing, hypotonic stress response is reduced from 86 to 39% for 1-day-old and 73 to 31% for 5-day-old platelets. This reduction in function is supported by a similar reduction of discoid cells from 80 to 40% for 1-day-old and 62 to 32% for 5-day-old platelets. The integrity of the cytoskeleton is critical for the osmotic response. Freezing recovery is significantly lowered in the presence of propylene glycol, which alters actin. This contrasts with the recovery of platelets treated with anti-aggregating agents. Platelets show a greater viability after freezing and thawing when PGI2 is added. It is postulated that freshly collected platelets, which are heterogeneous, contain populations of cells that are more sensitive to freezing than others. More tolerant cells remain discoid after freezing and are also less susceptible to storage lesions. Therefore, the maintenance of the integrity of the membrane and the cytoskeleton should be considered for the development of preservation methodologies.     

T-cell-mediated cytotoxicity toward platelets in chronic idiopathic thrombocytopenic purpura

Chronic idiopathic thrombocytopenic purpura (ITP) is a bleeding disorder that is characterized by increased platelet destruction and is believed to be autoantibody mediated. In this study, CD3+ T cells from ITP patients had increased expression of genes involved in cell-mediated cytotoxicity. In addition, cytotoxic cell-mediated lysis of autologous platelets was shown in active ITP. Our data suggest that T-cell-mediated cytotoxicity is an alternative mechanism for platelet destruction in ITP.     

Avian erythrocyte development: microtubules and the formation of the disk shape

The development of avian erythrocytes involves a spheroid to discoid transformation in shape. The disk shape of the young erythroid cells is dependent on the presence of microtubules in a marginal bundle in the early stages of postmitotic maturation. Disassembly of microtubules with colchicine, vincristine, sulfate or cold temperature produces the spheroidal shape. Erythrocytes which have acquired the flattened ellipsoidal shape do not alter their shape with disassembly of the microtubules. The number of microtubules decreases as cell maturation occurs. The correlation coefficient for the number of microtubular profiles in one end of erythrocytes and the concentration of ribosomes (cell age) is 0.88. Microtubules of immature erythrocytes disappear more rapidly at 0°C than do microtubules of mature cells.It is concluded that microtubules play little or no role in the maintenance of mature red cell shape; however, they play an important role in the development of the flat discoid shape of avian erythrocytes during maturation.     

Immunodiagnosis of platelet activation in immune thrombocytopenia through scFv antibodies cognate to activated IIb3 integrins

Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder characterized by low platelet count and presence of IgG autoantibodies to platelet surface glycoproteins, such as $$\alpha$$IIbβ3 and GPIb/IX. Our previous work has shown that platelets in ITP patients exist in an activated state. Two different marker-based approaches are used to study the course of platelet activation: (1) binding of PAC-1 antibody, signifying a change in αIIbβ3 conformation, and (2) expression of P-selectin, signifying alpha granule content release from platelets. Here, we describe the development of a new scFv antibody (R38) that, compared with PAC-1, appears to better distinguish between platelets of ITP patients and healthy controls. Notably, R38 was generated using commercially sourced resting-state integrin that was coated on a microtiter plate. Its ability to distinguish between ITP patients and healthy controls thus suggests that inadvertent integrin activation caused by coating involves a conformational change and exposure of a cryptic epitope. This report also describes for the first time the potential use of an scFv antibody in the immunodiagnosis of platelet activation in ITP patients.     

A lineage-restricted and divergent beta-tubulin isoform is essential for the biogenesis, structure and function of blood platelets

BACKGROUND: Mammalian megakaryocytes release blood platelets through a remarkable process of cytoplasmic fragmentation and de novo assembly of a marginal microtubule band. Cell-specific components of this process include the divergent beta-tubulin isoform beta1 that is expressed exclusively, and is the predominant isoform, in platelets and megakaryocytes. The functional significance of this restricted expression, and indeed of the surprisingly large repertoire of metazoan tubulin genes, is unclear. Fungal tubulin isoforms appear to be functionally redundant, and all mammalian beta-tubulins can assemble in a variety of microtubules, whereas selected fly and worm beta-tubulins are essential in spermatogenesis and neurogenesis. To address the essential role of beta1-tubulin in its natural context, we generated mice with targeted gene disruption. RESULTS: beta1-tubulin(-/-) mice have thrombocytopenia resulting from a defect in generating proplatelets, the immediate precursors of blood platelets. Circulating platelets lack the characteristic discoid shape and have defective marginal bands with reduced microtubule coilings. beta1-tubulin(-/-) mice also have a prolonged bleeding time, and their platelets show an attenuated response to thrombin. Two alternative tubulin isoforms, beta2 and beta5, are overexpressed, and the total beta-tubulin content of beta1-tubulin(-/-) megakaryocytes is normal. However, these isoforms assemble much less efficiently into platelet microtubules and are thus unable to compensate completely for the absence of beta1-tubulin. CONCLUSIONS: This is the first genetic study to address the essential functions of a mammalian tubulin isoform in vivo. The results establish a specialized role for beta1-tubulin in platelet synthesis, structure, and function.     

Detection of platelet-associated IgG in chronic immune thrombocytopenic purpura using antibody-coated polyacrylamide beads

Platelet-associated IgG (PAIgG) was detected by means of anti-human IgG coated polyacrylamide beads ("Immunobeads") technique in 32 patients with chronic ITP. Both a direct test (with in vivo sensitized platelets) and an indirect test (with in vitro loaded platelets) were carried out. The percent of rosette forming beads was both in the direct test (41.2%) and in the indirect test (32.6%) significantly higher in the cases of chronic ITP patients than in the controls (2.5% and 3.2%, respectively). These results confirm the diagnostic value of this new, relatively simple and rapid method in routine clinical practice.     

Proof of an association between Helicobacter pylori and idiopathic thrombocytopenic purpura in Latin America

BACKGROUND: Association between Helicobacter pylori and idiopathic thrombocytopenic purpura (ITP) has been found in Japan and in some European countries. It has also been shown that eradication of H. pylori can increase platelet counts in patients with ITP. The aims of this study were to determine the prevalence of H. pylori infection in patients with ITP in Colombia, and the effect of bacterial eradication on their platelet counts. MATERIALS AND METHODS: Between December 1998 and April 2006, a total of 32 patients diagnosed with ITP were included in the study. Controls were age and sex matched. RESULTS: H. pylori infection in patients with ITP was significantly higher (p = .00006) than in control individuals (90.6% and 43.8%, respectively), as determined by (13)C-urea breath test. A significant association between H. pylori infection and ITP was found (p < .0003), with an odds ratio (OR) of 13.15 (95%CI: 3.24-53.29). Multivariate analysis for the association between H. pylori and ITP showed an OR of 20.44 (95%CI: 3.88-107.49) for women and 19.28 (95%CI: 2.03-183.42) for individuals over 50 years. All 29 H. pylori-positive patients with ITP received eradication treatment. After a median follow up of 12.2 months, 80.8% had a recovery in platelet counts. CONCLUSIONS: According to these results and others from different countries where H. pylori infection rates are high, patients with ITP should be initially tested for H. pylori status, and if present, infection should be eradicated before initiating a drastic conventional ITP treatment. An algorithm for the study and management of patients with ITP in the post-Helicobacter era is presented.     

Circulating platelet activation in healthy subjects and in some pathological conditions. Method of study and results

Current clinical methods for evaluating platelet function are artful tests which study the effects of various stimuli on platelets, whereas the clinician is much more interested in methods evaluating the activation of circulating platelets. The hallmark of activation of platelets is their shape change, i.e. the transformation of the platelets from smooth disks into spiny spheres; the aggregation begins when 30% of platelets are activated. In 1138 subjects (384 healthy individuals and 854 patients with various pathological conditions with high thrombotic risk) we have investigated circulating platelet activation and circulating platelet aggregates by fixation of blood cells in a glutaraldehyde mixture and by evaluation of platelet shape change and aggregates on a phase-contrast microscope. The method is precise, accurate and suitable for clinical purposes.     

Megakaryocyte fragments and the microtubule coil

We have examined megakaryocyte process fragments that migrate out of bone marrow explants after a short period of incubation and assume a beaded form, consisting of 2 or more putative platelets. The fragmentation appears to occur in vivo and supports the proposal that platelet liberation does not always occur in a sequential manner from the distal ends of megakaryocyte processes. Transmission electron microscopy revealed that microtubules were generally oriented longitudinally in the process fragments. Rarely, a microtubule coil was found in a terminally located putative platelet. The observations favour the view that the marginal coil of microtubules, which is a characteristic of circulating platelets, does not usually form until after platelets have been liberated.