Comparison of sea turtle thrombocyte aggregation to human platelet aggregation in whole blood

The endangered sea turtles are living "fossils" that afford us an opportunity to study the hemostatic process as it likely existed millions of years ago. There are essentially no data about turtle thrombocyte aggregation prior to our studies. Thrombocytes are nucleated cells that serve the same hemostatic functions as the anucleated mammalian platelet. Sea turtle thrombocytes aggregate in response to collagen and beta-thrombin. Ristocetin induces an agglutination/aggregation response indicating the presence of a von Willebrand-like receptor, GPIb, found in all mammalian platelets. Samples treated with alpha-thrombin plus gamma-thrombin followed by ristocetin results in a rapid, stronger response than ristocetin alone. These responses are inhibited by the RGDS peptide that blocks fibrinogen cross-linking of mammalian platelets via the fibrinogen receptor, GPIIb/IIIa. Three platelet-like proteins, GPIb, GPIIb/IIIa and P-selection are detected in sea turtle thrombocytes by fluorescence activated cell sorting. Turtle thrombocytes do not respond to ADP, epinephrine, serotonin, thromboxane A2 mimetic, U46619, trypsin, or alpha-thrombin and gamma-thrombin added alone. Comparison of hemostasis in sea turtles to other vertebrates could provide a framework for understanding the structure/function and evolution of these pathways and their individual components.     

Chicken thrombocytes in culture: lymphocyte-conditioned medium delays apoptosis.

Chicken thrombocytes are nucleated cells, analogs to mammalian platelets. These cells are involved in hemostasis, phagocytosis and secretion of specific products. Most of the properties of avian thrombocytes have been established in experiments that employed recently isolated blood cells. Attempts to cultivate these cells for a long period of time under optimal culture conditions for peripheral blood cells were unsuccessful; thrombocytes died after 24 h of cultivation unlike macrophages cocultured with them. Here we investigate the reasons and type of thrombocyte death in culture. Thrombocytes were separated from peripheral blood of roosters and cultured for 48 h. The influence of different culture conditions on thrombocyte viability was studied. Cells were cultured as adherent cell monolayers or under agitation (preventing adherence), in the presence or lack of lymphocytes or their soluble factors, and various concentrations of fetal bovine serum. After 24 h in standard culture thrombocytes displayed cytoplasm and chromatin condensation, DNA cleaved into oligonucleosomal fragments and unaltered mitochondria. These results strongly suggest that thrombocytes suffer an apoptotic cell death in culture. Apoptosis could be delayed by culturing thrombocytes in the presence of lymphocytes or their soluble factors.     

Shape transformation and cytoskeletal reorganization in activated non-mammalian thrombocytes

The nucleated thrombocytes of non-mammalian vertebrates are partially flattened, ovoid cells morphologically distinct from mammalian platelets, and the extent of their functional equivalence is unknown. To test whether they resemble platelets in having similar F-actin-based post-activation stages, rapid fixation/extraction/labeling methods were developed to reveal cytoskeletal organization in dogfish thrombocytes by confocal microscopy. Unactivated cells contained cortical F-actin plus denser F-actin co-localizing with outer marginal band (MB) microtubules. In the post-activation sequence, determined for the first time by continuous observation of individual thrombocytes following thrombin perfusion, cells rounded and blebbed, spread, and eventually flattened extensively. The MB twisted and then became disorganized, with microtubule bundles remaining centrally located and associated with nuclear clefts. In contrast, F-actin occupied blebs and outward-spreading cytoplasm, initially in spiky projections, then predominantly in stress fibers, and inhibitors of F-actin assembly or myosin ATPase blocked shape changes. Thus, the post-activation stages and cytoskeletal events observed in nucleated thrombocytes were found to parallel those of platelets.     

Identification of fibronectin in chicken thrombocytes.

Cell attachment and spreading of chicken embryo fibroblasts and three mammalian cell lines were promoted by using thrombocyte secretion products (TSP) prepared from chicken thrombocytes which are analogous to mammalian platelets. The cell attachment activity following TSP treatment was interfered with by the addition of a synthetic peptide containing the RGD sequence from the cell attachment region of fibronectin (FN). Cell attachment on TSP-coated plates was inhibited by the addition of anti-chicken FN rabbit antiserum, but not by anti-chicken vitronectin (VN) rabbit antiserum. By immunoblotting, immunofluorescent test and sensitive enzyme linked immunosorbent assay using an anti-chicken FN antiserum, the presence of FN in thrombocytes and the release of FN from the cells were demonstrated. The release of FN from thrombocytes was partially induced by the in vitro cultivation of the cells and was further promoted by treatment of the cells with thrombin.     

Soluble factors differ in platelets derived from separate niches: a pilot study comparing the secretome of peripheral blood and bone marrow platelets

Background aimsPlatelet-rich plasma (PRP) and bone marrow aspirate are commonly used in orthobiologics for their anti-inflammatory, anabolic/regenerative and immunomodulatory characteristics via platelet degranulation and cell secretions. Although platelets are derived from megakaryocytes in the bone marrow, no attention has been paid to the potential benefits of bone marrow platelets and whether their contents differ from aging platelets in peripheral blood.MethodsIn the present study, leukocyte-poor peripheral blood-derived platelets in plasma (LPP) and leukocyte-poor bone marrow platelets in plasma (BMP) were prepared from six donors, activated with calcium chloride, incubated and sampled at day 0, day 3 and day 6. LPP and BMP are platelet preparations intended to evaluate the respective platelet secretomes but are not classified as conventional PRPs, as they are not concentrated to the extent necessary to meet the qualifying criteria. At each time point, 15 growth and immunomodulatory factors were quantitated in LPP and BMP: platelet-derived growth factor AA, basic fibroblast growth factor/fibroblast growth factor 2, granulocyte-macrophage colony-stimulating factor, hepatocyte growth factor, macrophage colony-stimulating factor, stem cell factor, vascular endothelial growth factor, tumor necrosis factor alpha, IL-1β, interferon gamma, IL-4, IL-10, IL-1 receptor antagonist protein, IL-12p40 and arginase-1.ResultsThe results illustrate that platelets derived from bone marrow have a unique secretome profile compared with those derived from peripheral blood, with significant differences in anti-inflammatory cytokines, which are associated with monocyte polarization.ConclusionsUltimately, bone marrow-derived platelets may be useful as a stand-alone orthobiologic or as an effective adjuvant to autologous cell therapies where anti-inflammatory and anabolic processes are desired, especially with respect to monocyte function.     

Clinical aspects of control in therapy with platelet inhibitors]

46 patients with ischaemic heart disease were treated with Micristin (20-40 mg/kg) or a combination of Micristin and Propranolol (80-120 mg/die). The values of bleeding time, the platelet factor 4 in lysate of thrombocytes or in plasma of patients as well as the soluble fibrin monomer complexes were investigated. They showed no obvious correlation to the clinical findings.     

Trout thrombocytes contain 12- but not 5-lipoxygenase activity

Fish thrombocytes are thought to be the evolutionary forerunners of mammalian platelets. Thrombocyte preparations made by conventional methods, such as density gradient centrifugation, contain other cell types such as neutrophilic granulocytes and lymphocytes that could interfere with subsequent experiments. In this study, rainbow trout thrombocytes were separated by density gradient centrifugation and further purified by magnetic cell sorting (MACS) using the thrombocyte specific monoclonal antibody, 30D8. Thrombocyte purity was assessed by reactivity to 30D8 using flow cytometry and immunocytochemistry. Following purification by density gradient centrifugation, thrombocytes were 66.9±9.2% (mean value±S.E.M., n=3) pure. Further purification by MACS significantly increased thrombocyte purity to 97.3±0.6%, whereas only 1.4% of the MACS ?ve fraction were identified as these cells. Incubation of thrombocytes isolated by density gradient alone with calcium ionophore, A23187, generated a range of eicosanoids derived from arachidonic or eicosapentaenoic acids, namely, leukotriene (LT)B₄, LTB₅, lipoxin (LX)A₄, LXA₅, 12-hydroxyeicosatetraenoic acid (12-HETE) and 12-hydroxyeicosapentaenoic acid (12-HEPE). A similar eicosanoid generation profile was observed for cells in the MACS ?ve fraction; however, MACS +ve cells (thrombocytes) generated no 4 or 5 series LT or LX but did generate significant amounts of the 12-lipoxygenase (LO) products, 12-HETE and 12-HEPE. These results indicate that trout thrombocytes contain no demonstrable 5-LO activity and like their mammalian counterparts possess 12-LO activity.     

Platelet responses in health and disease

Summary This article summarizes recent ultrastructure findings from our laboratory and documents some of the information accumulated primarily since 1975 from many laboratories. Special attention is given to documentation by scanning electron microscopy which affords insight into platelet activation (adhesion, aggregation, release/secretion) and especially platelet-vessel wall interactions. Structural physiology of platelets is considered in some detail as a basis for understanding platelet disorders contributing to clinical problems of thrombosis and hemorrhage. The impaired ability of vonWillebrand platelets to adhere to injured vessel wall is reported using the human umbilical vein perfusion model. Relationships between platelets and blood coagulation factors focus on the exquisite sensitivity of platelets to minute amounts of thrombin. Unmasking of platelet factor 3 sites is identified on activated platelets, after glutaraldehyde fixation, by their reaction to latex bearing anti-platelet factor 3 markers. The basis for platelet-collagen interactions is reviewed. Conditions for and possible mechanisms behind platelet interaction with vessel wall are discussed. Ex vivo flowing blood-vessel wall models offer opportunities for improved understanding of the platelets role(s) in vascular diseases.     

Nucleated thrombocytoid cells

Summary The so-called nucleated thrombocytes of the domestic fowl (and mallard) were analyzed intravitally by use of phase- and interference-contrast microscopy with regard to their morphology and functional state, and in comparison to other avian blood cells and mammalian blood platelets. Since nucleated thrombocytoid cells of birds do not differ in size from white blood cells, they are automatically included in differential cell counts. They can easily be confused with other white blood cells and even with erythrocyte ghosts, which also appear to be hemostatically active elements. However, the nucleated thrombocytoid cells display a characteristic, definite morphology. The platelet-like spreading process and elongation of aging cells in vitro to spindle forms deserve special attention. A technique of counting live avian blood cells was used to determine their values in chickens of different sexes and ages.Supported by the Deutsche Forschungsgemeinschaft     

Avian thrombocyte thrombospondin

A high molecular weight glycoprotein (450,000) was obtained from thrombin-treated duck thrombocytes by barium citrate adsorption technique followed by heparin-agarose affinity chromatography. Amino acid composition (high number of acidic amino acids and cystine) as well as carbohydrate contents (1.3 per cent hexosamine, 0.9 per cent sialic acid and 1.5 per cent hexose) showed similarity to mammalian platelet thrombospondin.     

Role of Platelets in Neuroinflammatory Disorders. A Review

Platelets, or thrombocytes, are important players in inflammation, wound healing, initiation of immune response and regeneration in the organism. Disruption of the blood-brain barrier occurs during certain neurological disorders, such as brain trauma, Alzheimer’s disease or stroke, when blood cells including platelets can invade nervous tissue. However, the role of platelets in the context of neuroinflammation remains understudied. Recent studies have shown that activated platelets release a wide set of coagulative and vascular factors during neurovascular pathologies in the central nervous system. Moreover, platelets stimulate immunity and regulate inflammation in the central nervous system. Trophic and growth factors stored in platelets may play a role in neuronal regeneration. Activated platelets release neurotransmitters, serotonin, dopamine, histamine, and glutamate, and can modify neuronal cell activity in neuropathologies. This review focuses on the major aspects and mechanisms of platelet functions in neuroinflammation, and therapeutic potential of platelets for treatment of neurodegenerative disorders.     

Binding of fibronectin to alpha-granule-deficient platelets

Most of the proposed functions for fibronectin involve its interaction with cells, yet the molecular nature of cellular fibronectin binding site(s) has remained obscure. Thrombin induces saturable platelet binding sites for plasma fibronectin and concurrently stimulates surface expression of a number of platelet alpha-granule constituents including thrombospondin and fibrin which are known to interact with fibronectin. To test the hypothesis that these (or other alpha-granule proteins) mediate plasma fibronectin binding, we used platelets of patients with the Gray Platelet Syndrome. These cells were deficient in thrombospondin, beta-thromboglobulin, platelet factor 4, fibronectin, and fibrinogen as measured in radioimmunoassay. They also had reduced von Willebrand factor content as judged by immunofluorescence. At plasma fibronectin inputs from 0.03 to 3 times the apparent kilodalton, these Gray platelets bound virtually identical quantities of fibronectin as normal cells. Thus, platelets containing 1,500 molecules of thrombospondin per platelet could bind more than 100,000 molecules of plasma fibronectin per cell following thrombin stimulation. These data preclude any simple model in which newly surface expressed thrombospondin (or other alpha-granule protein) functions as the major thrombin-stimulated plasma fibronectin receptor in this cell type.     

Effect of serine proteinases on the intracellular dynamics of membrane lipids and aggregation of human thrombocytes

Room temperature tryptophan phosphorescence (RTTP) of human platelets in suspension was studied. The spectral and kinetic parameters of RTTP of thrombocytes were determined. By using the RTTP, the effect of serine proteases: thrombin, trypsin, and alpha-chymotrypsin at low concentrations (0-50 micrograms/ml) on the slow internal dynamics of membrane proteins was studied. It was shown that short-term incubation of platelets with proteases induces changes in the internal dynamics of membrane proteins in situ, and the magnitude of this effect correlates well with the extent of platelet aggregation. The functional role of the changes in the internal dynamics of membrane proteins and their relationship with intracellular signal transduction were discussed.     

Platelets induce human umbilical vein endothelial cell proliferation through P-selectin

We studied whether platelets could participate in the endothelial cell monolayer regeneration in the case of a vessel damage. Incorporation of [3H]-thymidine into the DNA of human umbilical vein endothelial cells (HUVECs) was measured after 48 h of co-incubation with platelets. The effect of platelets was compared to that of platelet-free supernatants from thrombin-activated platelets that had secreted their active granule constituents. Platelets dose-dependently induced HUVEC proliferation. Platelets preactivated by thrombin induced similar proliferation as did unactivated platelets (proliferation factor = 7 - 8), indicating that preactivation of platelets was not required. Platelets fixed with paraformaldehyde had no effect, suggesting that the platelet mitogenic effect required a mobile, alive membrane. Ketanserine and suramin reduced by at most 30 % the platelet-induced proliferation; supernatants of thrombin-activated platelets caused only minor proliferation (proliferation factor = 2), suggesting that secreted 5-hydroxytryptamine and growth factors poorly contributed to the proliferative effect. When the co-incubation was performed in the presence of an anti P-selectin antibody, the platelet-induced HUVEC proliferation was inhibited. The results suggest that platelet adhesion participate in the control of the endothelial regeneration and that platelet P-selectin is a molecular determinant of the proliferative signal.     

The Blood Platelet as a Model for Regulating Blood Coagulation on Cell Surfaces and Its Consequences

Platelets actively participate in regulating thrombin production following physical or chemical injury to blood vessels. Injury to blood vessels initiates activation of the large numbers of platelets that appear in the subendothelium where they become exposed to tissue factor and to molecules adhesive for platelets and normally found in the extracellular matrix. The complex of plasma factor VIIa with extravascular tissue factor both initiates and localizes thrombin production on platelets and on extravascular cells. Thrombin production at these sites in turn enhances platelet activation and the subsequent hemostatic plug formation to minimize bleeding. Thrombin production and platelet activation also initiate the process of wound healing requiring thrombin-dependent cell activation and platelet-dependent formation of new blood vessels (angiogenesis). Activated platelets release from their storage granules several proteins and other factors that regulate local thrombin formation and the responses of blood vessel cells to injury to assure hemostasis and effective wound healing. Failure to localize and adequately regulate thrombin production and/or platelet activation can have pathological consequences, including the development and propagation of atherosclerosis and enhancement of tumor development. The primary basis for the pathological consequences of the failure to adequately regulate thrombin production is that the multi-functional thrombin activates several types of cells to initiate their mitogenesis. Mitogenesis precedes many of the undesirable consequences of poorly regulated thrombin production and platelet activation. In addition, activated platelets release a variety of products which influence the functions of several cell types to the extent that inadequate regulation of platelet activation (by excessive thrombin production) could contribute to the pathogenesis of acute and chronic arterial thrombosis and to tumor development. Activated platelets participate in tumor development by releasing several factors that positively (and negatively) regulate blood vessel formation.     

Biological properties of a hepatocyte growth factor from rat platelets

In an accompanying communication we demonstrated that about half of the potency of rat serum to stimulate DNA synthesis in cultured adult rat hepatocytes resides in a polypeptidelike substance from the platelets. A lysate of rat platelets was able to restore the potency of platelet-poor rat serum, whereas a lysate of human platelets inhibited thymidine incorporation by the hepatocytes. Moreover, addition to these cultures of either highly purified human platelet-derived growth factor (PDGF) or human platelet factor 4 (PF-4) failed to influence DNA synthesis either alone or in the presence of rat or human platelet-poor serum, which is required for expression of PDGF activity. Unlike the human platelet factors, rat platelet lysate (RPL) was moderately active by itself and was augmented equally well by platelet-poor serum from either source. At concentrations below 5%, platelet-poor serum from hypophysectomized rats was as potent as that from normal rats in augmenting RPL activity. This suggests that, unlike PDGF, which is not activated by hypophysectomized rat serum, the hepatotrophic component of RPL does not require the presence of exogenous somatomedins for activity, but interacts instead with other plasma constituents or with somatomedins produced by the hepatocytes in vitro. Rat platelets do, however, appear to contain PDGF or its rat equivalent in addition to the hepatocyte growth factor, since if they are heated to 100 degrees C for 10 min, their ability to stimulate nuclear labeling in confluent BALB/c 3T3 cells is not impaired, while their ability to stimulate DNA synthesis in rat hepatocytes is destroyed. These studies indicate that the hepatocyte growth factor from rat platelets differs from PDGF in its biological as well as physical characteristics, but that rat platelets also contain PDGF or an equivalent substance.     

Determination of HLA antigens in serum and thrombocytes using microneutralization and microabsorption tests

The microneutralization and microabsorption test is described for identifying HLA-antigens in the serum and on thrombocytes. The incubation time for neutralization and absorption amounts to 3 hours at 20-25 degrees C. Absorption is made by platelet suspension at a concentration of 300 000 to 500,000 thrombocytes per mm3. In the majority of cases satisfactory results can be achieved by a neutralizing ratio (absorption ratio) of 1: 1. Some HLA sera show a deviating ability of neutralization with those HLA-antigens contained in sera and absorption with appropriate platelets; this behaviour is not connected with the titre of HLA-cytotoxins. Depending on the case there will be a varying content of HLA-antigens in sera and on thrombocytes of individuals. Moreover there is no regular conformity concerning the content of HLA-antigens in the serum and on the thrombocytes of a single person.     

1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholines: influence on aggregation and [3H]serotonin release of human thrombocytes

1-O-Alkyl-2-O-acetyl-sn-glycero-3-phosphocholines (platelet activating factor, PAF) aggregate human thrombocytes in a concentration dependent fashion. After a short lag-phase, maximum aggregation is reached within 2 min. PAF releases serotonin from human thrombocytes within 1 min. Indomethacin and creatine phosphate (CP)/creatine phosphokinase (CPK) are able to inhibit the second phase of the aggregation by PAF, while xylocain reduces both the first and second phase of aggregation of human thrombocytes. Hirudine neither influences the first nor the second phase of aggregation by PAF.