Wistar Furth Rat Megakaryocytes Lack Dense Compartments and Intercellular Plaques,Membranous Structures Rich in Cytoskeletal Proteins

Wistar Furth (WF) rats have an abnormal thrombopoietic phenotype with morphologically aberrant megakaryocytes, larger than normal mean platelet volume, and platelet alpha-granule protein deficiency. Here, ultrastructural comparisons of WF rat megakaryocytes to those of rats (Wistar) with normal platelet formation during stimulated megakaryocytopoiesis following 5-fluorouracil administration, have revealed a previously unrecognized membrane structure in normal rat megakaryocytes, and two additional abnormalities in WF megakaryocytes. The novel structures were zones of electron density on the cytoplasmic face of apposed plasma membranes of adjoining normal megakaryocytes. These modified focal adhesion-type contacts were distributed at intervals between adjacent megakaryocytes, and were spaced by deposits of extracellular material. These structures also were present between apposed plasma membranes of Wistar rat megakaryocytes in unperturbed marrows, but were absent between megakaryocytes of WF rats. The second WF rat megakaryocyte abnormality is the absence of cytoplasmic dense compartments, another specialized membranous structure that is continuous with the megakaryocyte demarcation membrane system. Both the intercellular plaques and dense compartments of Wistar rat megakaryocytes were found to be rich in cytoskeletal proteins including actin, α-actinin, talin, and vinculin as indicated by ultrastructural immunogold labeling. We hypothesize that an abnormality in cytoskeletal protein function may be responsible for the lack of these structures in the WF rat.     

Lipid and membrane fluidity abnormalities in platelets and megakaryocytes of the hereditary macrothrombocytopenic Wistar Furth rat.

Biochemical and functional abnormalities of megakaryocytes and platelets were studied in Wistar Furth (WF) rats which have genetically determined macrothrombocytopenia and megakaryocytopenia, and were compared with their counterparts in Sprague-Dawley (SD) rats. Both megakaryocytes and platelets synthesized phospholipids from [14C]acetate. WF and SD megakaryocytes incorporated 0.27 and 0.29 nmol acetate per 10(6) cells, respectively. Phosphatidylcholine (PC) accounted for 64% and 58% of the PL radioactive label in megakaryocytes of SD and WF rats, respectively, (P less than 0.05), while 69% of labeled activity was associated with PC of SD platelets compared to 60% found in PC of WF platelets (P less than 0.01). In WF platelets a significant increase in the levels of lysophosphatidylcholine (6.1% vs. 3.0%) was observed. WF platelets had substantially higher levels of esterified cholesterol, triglycerides, ceramides and a 3-fold increase in the total protein per platelet compared to SD platelets. The fatty acid composition of WF platelet PC showed quantitative abnormalities. Plasma lecithin-cholesterol acyl transferase activity and platelet function monitored by the uptake and release of [14C] serotonin showed nonsignificant variations between SD and WF rats. Compared with the control, platelet membrane fluidity, measured by fluorescence polarization using platelets labeled with 1,6-diphenyl-1,3,5-hexatriene, was significantly decreased in the WF rats.     

Disruption of microtubules in vivo by vincristine induces large membrane complexes and other cytoplasmic abnormalities in megakaryocytes and platelets of normal rats like those in human and Wistar Furth rat hereditary macrothrombocytopenias

Abnormal organization of platelet microtubules is associated with abnormal platelet formation in hereditary macrothrombocytopenias such as the gray platelet syndrome, May-Hegglin anomaly, and Epstein's syndrome, and that of the Wistar Furth rat, suggesting that aberrant microtubule organization may contribute to defective platelet formation in these clinical entities. Here, we examined the consequence of microtubule disruption on the organization of megakaryocyte cytoplasmic organelles using the microtubule depolymerizing agent, vincristine (VCR). Wistar rat bone marrow was fixed and processed for transmission electron microscopy after VCR administration alone, after 5-fluorouracil (5-FU) administration alone, or after 5-FU followed by intravenous injection of 0.1–1.0 mg/kg VCR for intervals of 30 min to 8 hr. 5-FU was given to increase megakaryocyte frequency to facilitate ultrastructural evaluations. VCR alone or in combination with 5-FU caused formation of large membrane complexes in the cytoplasm of Wistar rat megakaryocytes at all dosages studied, identical to those found in megakaryocytes of human hereditary macrothrombocytopenias and the Wistar Furth rat. The proportion of megakaryocytes with these large membrane complexes increased with time after 5-FU and VCR, and was maximal (～two-third of megakaryocytes) at VCR dosages of 0.75–1.0 mg/kg. The majorityof megakaryocytes displayed other abnormalities, including blebbing of plasma membranes, an increased number of dense compartments, dilated demarcation membrane (DMS) channels, which contained dense material immunocytochemically identified as secreted α-granule proteins, and an increased incidence of emperipolesis. Rats administered 5-FU alone did not demonstrate these abnormalities, with the exception of an increase in dense compartments. Platelets from rats treated with VCR aloene or 5-FU and VCR also showed abnormalities including membrane complexes, rounded shape, formation of tubulin paracrystals, development of membrane blebs, and the presence of proteinaceous material within the cisternae of the surface-connected canalicular system (SCCS). The membrane complexes in platelets of 5-FU-, VCR-treated Wistar rats as well as untreated Wistar Furth rats were composed of elements of both the SCCS and dense tubular system; membrane complexes in megakaryocytes of 5-FU-, VCR-treated rats were composed of both DMS and smooth endoplasmic reticulum. We conclude that intact microtubules play a major role in the organization of the megakaryocyte DMS and may contribute to the stability of megakaryocyte α-granules. © 1995 Wiley-Liss, Inc.     

In vitro stimulation of megakaryocyte maturation by megakaryocyte stimulatory factor

Counterflow centrifugal elutriation and Percoll density gradient centrifugation were employed to prepare cell populations from rat bone marrow that were selectively enriched in the cytoplasmically immature megakaryocytes and depleted of the most mature megakaryocytes. The incorporation of [14C]leucine into the platelet-specific alpha-granule protein, platelet factor 4, as well as the incorporation of [35S]sulfate into platelet proteoglycans synthesized by the maturing megakaryocytes were monitored as markers of cytoplasmic maturation. Rat platelet factor 4 was specifically isolated and characterized by its high affinity for heparin-Sepharose and its amino-terminal sequence homology to human and rabbit platelet factor 4. The [35S]sulfate-labeled proteoglycans were primarily composed of chondroitin 4-sulfate glycosaminoglycans and were identified as platelet granule components by their ability to be secreted by megakaryocytes in response to thrombin or A23187. The production of both components was increased as much as 3-fold in a dose-dependent manner by the addition of picomolar concentrations of purified megakaryocyte stimulatory factor, without a concomitant increase in general protein synthesis. The above results suggest that the megakaryocyte stimulatory factor may regulate the synthesis of platelet granule components by megakaryocytes and hence control the rate and/or extent of cytoplasmic maturation during megakaryocyte development.     

Incorporation of a circulating protein into alpha granules of megakaryocytes

In order to determine whether or not proteins circulating in plasma can be incorporated into megakaryocytes and platelets, horseradish peroxidase (HRP) was injected intravenously into guinea pigs and these cells were examined for uptake by cytochemistry and electron microscopy. Enriched samples of megakaryocytes enabled ultrastructural analysis of large numbers of these rare bone marrow cells. In megakaryocytes, more than 50% of alpha granules contained HRP between 75 minutes and 7 hours after injection. At 24 hours, 25% of the megakaryocyte granules were peroxidase positive; less were so by 48 hours and none at 4 days. Thus, the findings demonstrate that a circulating protein can be endocytosed by megakaryocytes and rapidly packaged into alpha granules. A precipitous drop in circulating platelet numbers was observed 45 minutes after injection. At this time, circulating platelets showed the tracer only on the platelet plasma membrane, and none in platelet granules. Platelet numbers increased to 35% by 7 hours and only the platelet granules contained HRP. These platelets secreted the HRP stored in granules in response to thrombin. Unfortunately, our present studies do not allow us to distinguish between direct endocytosis by the platelet and/or shedding of new platelets from recently labeled megakaryocytes. Our studies are the first to demonstrate an endocytic pathway by which megakaryocytes can incorporate a circulating protein into alpha granules. An important physiologic implication of this endocytic pathway is the possible origin of certain alpha granule proteins from plasma.     

Sulfated proteoglycans and sulfated proteins in guinea pig megakaryocytes and platelets in vivo. Relevance to megakaryocyte maturation and platelet activation

This study has examined changes in proteoglycan synthesis during megakaryocyte maturation in vivo. Guinea pigs were injected with Na235SO4, and megakaryocytes and platelets were isolated from 3 h to 5 days later. The proteoglycans and other sulfated molecules in both cells were characterized at each time point by gel filtration, ion-exchange chromatography, gel electrophoresis, and chemical and enzymatic digestions. Two populations of chondroitin 6-sulfate proteoglycans were found by DEAE-Sephacel chromatography. The major fraction was eluted with 4 M guanidine hydrochloride and the minor fraction with 4 M guanidine HCl, 2% Triton X-100. The Kav of the major proteoglycan peak in the platelets at 1 day after injection was 0.18-0.20 on Sepharose CL-6B and decreased gradually to 0.12 by 3 days, when proteoglycan radioactivity per cell was maximal. The peak for megakaryocyte proteoglycans at 3 h was broad, with Kav = 0.1-0.2. The appearance of different portions of the proteoglycan peak in platelets coincided with their disappearance from megakaryocytes. Proteoglycan size was a function of glycosaminoglycan chain length. The proteoglycans eluted with Triton X-100 from DEAE-Sephacel (Kav = 0.04-0.07 on Sepharose CL-6B) were not labeled in platelets until 2 days after injection. Our data suggest that megakaryocytes synthesize different-sized chondroitin sulfate proteoglycans at different stages of development. The proteoglycans of the major fraction were released from platelets in response to thrombin, and a small amount was released by ADP. The proteoglycans of the Triton X-100 eluate were not released by thrombin or ADP. About 20% of the sulfate radioactivity was incorporated into molecules that appear to be sulfated proteins and were not released by thrombin or ADP.     

Phospholipid composition of rat megakaryocytes and its rearrangement in platelets

Rat platelets and their megakaryocyte precursors were examined for phospholipid composition. (1) The phospholipid composition of rat megakaryocytes, which were enriched and prepared from bone marrow cells, was almost identical to that of platelets. (2) The subclass composition of choline-containing glycerophospholipids (CGP) of rat megakaryocytes differed significantly from that of platelets: 1-alkenyl-2-acyl glycerophosphocholine (GPC) in megakaryocytes accounted for 29% of the total, whereas that in platelets was only 7%. (3) Rat platelets contained a larger amount of arachidonic acid than megakaryocytes, especially in ethanolamine-containing glycerophospholipids (EGP). (4) [32P]Phosphoric acid was significantly incorporated into megakaryocytes, whereas platelets showed little incorporation. On the other hand, the uptake of [3H]arachidonic acid into platelet phospholipids was about 15-times higher than that observed with megakaryocytes. (5) As reported previously for other blood cells, such as neutrophils and macrophages, the radioactivity of labeled arachidonic acid incorporated into CGP of platelets decreased, whereas that incorporated into EGP increased during a subsequent chase period. Hardly any such change was observed with megakaryocytes. These results suggest that the phospholipid composition of rat platelets is mainly determined at the time of thrombopoiesis, whereas the composition of molecular species is remodeled during circulation after thrombopoiesis.     

A platelet disorder due to a structural abnormality of membrane glycoprotein Ib

In a patient with mild bleeding symptoms, decreased platelet spreading and defective aggregation, an abnormal membrane glycoprotein (GP) was detected. Staining and surface labelling characteristics, cleavage by calcium-activated protease and decreased content of normal GP Ib in the platelets of the patient and 3 related carriers of the abnormal glycopeptide suggest that the additional component is a genetic GP Ib variant structurally characterized by an increase of about 20,000 in apparent Mr of the large subunit GP Ib alpha. The observed hereditary membrane GP abnormality although present in heterozygous state may be causally related to the defect of platelet function.     

Mast cell exocytosis: evidence that granule proteoglycan processing is not coupled to degranulation

It has been hypothesized that the dissolution of mast cell granules at the time of degranulation results from proteoglycan cleavage coupled to exocytosis. To address this hypothesis, we studied granule proteoglycan before and after exocytosis in dog mastocytoma cells, which solubilize granule contents during exocytosis. 35S-labeled proteoglycans were extracted from unstimulated whole cells and cell degranulation supernatant. Sequential anion-exchange and gel filtration chromatography, followed by specific glycosaminoglycan digestion, identified chondroitin sulfate and heparin glycosaminoglycan and proteoglycan in unstimulated cells and degranulated material alike. Glycosaminoglycan type and charge density in degranulation supernatant were unchanged compared with unstimulated cells. There was no decrease in proteoglycan size with cell activation and exocytosis. Thus, granule release and solubilization does not appear to require exocytosis-coupled degradation of granule proteoglycans. Release in association with high-m.w. proteoglycans may serve to limit rates of diffusion and activity of proteases and other mast cell mediators.     

Animal models with inherited hematopoietic abnormalities as tools to study thrombopoiesis

Animals with hereditary abnormalities of hematopoiesis are quite useful in the study of regulatory pathways of megakaryocytopoiesis and platelet formation. Seven such animal models are analyzed here. The Wistar Furth rat has been recently discovered to have reduced platelet number, but large mean platelet volume, and is, therefore, a model of hereditary macrothrombocytopenia. Study of the Wistar Furth rat may help to elucidate the process of platelet formation. Two mouse mutants the S1/S1d and W/Wv, have macrocytic anemia with reduced megakaryocyte number, but normal platelet count. In these mice, the platelet count is maintained by increased platelet production per megakaryocyte. These models demonstrate that factors other than platelet level are monitored in the feedback regulation of megakaryocytopoiesis and platelet production, and further study should lead to a better understanding of the regulation of megakaryocyte size. The Belgrade rat has severe microcytic anemia with decreased megakaryocyte number. Megakaryocyte size is increased, but platelet count is moderately reduced and thus the megakaryocyte-platelet picture resembles that of severe iron deficiency anemia. A more in depth examination of this model should delineate the effects of iron deficiency and hypoxia on megakaryocytopoiesis. The grey collie dog has cyclic hematopoiesis with large asynchronous fluctuations in all blood cell counts at approximately 2-week intervals. Megakaryocytes have not been studied. This model should be a tool to define the relationships between hematopoietic growth factors and differentiation of the various hematopoietic cell lineages. The br/br rabbit has a transient disturbance in fetal megakaryocytopoiesis and brachydactyly due to spontaneous amputation. Further study of this model may provide a better understanding of fetal megakaryocyte development and establish whether an association exists between the abnormal megakaryocytes and the limb amputations. The nude mouse with its severe T-lymphocyte deficiency has been studied to ascertain whether T cells play a regulatory role in normal and acute thrombocytopenia-stimulated megakaryocytopoiesis. The question of whether T cells or their products are responsible for reactive thrombocytosis in chronic inflammation could be examined with this model. These animal mutants have provided and should continue to provide important models for understanding the regulation of megakaryocytopoiesis and platelet production.     

Regulation of megakaryocytes in W/Wv mice

W/Wv mice were injected with antiplatelet serum to produce thrombocytopenia or with platelet transfusions to induce thrombocytosis. The responses of their platelets and megakaryocytes were followed to determine if proliferative abnormalities of the megakaryocytic system would be detected. W/Wv mice responded normally to the stimulation from thrombocytopenia with rebound thrombocytosis, macromegakaryocytosis, and macrothrombocytosis. The megakaryocytes of these mice became smaller than normal in response to post-thrombocytopenic rebound thrombocytosis but not to transfusion-induced thrombocytosis. Thus, endogenous thrombocytosis appeared to be a more potent suppressor of megakaryocyte growth than exogenous. These results failed to reveal an effective abnormality of the thrombocytopoietic regulatory system of W/Wv mice in spite of their intrinsically reduced numbers of megakaryocytes and the well known defect of stem cell proliferation. Thrombocytopoietic regulation appeared, therefore, to occur mainly at the committed, rather then pluripotential, stem cell level, and normal responses of the platelet system were observed in spite of severe abnormalities at the pluripotential stem cell level.     

Characterization of the sulfated glycosaminoglycan on the surface and in the storage granules of rabbit platelets.

Rabbit platelets were labeled in vivo with 35S for characterization of platelet sulfated glycosaminoglycan. When rabbit platelets were aggregated by ADP, sulfated proteoglycan was lost from the platelet surface although no release of granule contents occurred. The sulfated proteoglycan contained in the granules of platelets pretreated with ADP was subsequently released by treatment with thrombin. The 35S-labeled proteoglycan from both sources was isolated by gel filtration and the glycosaminoglycan portion of the proteoglycan was characterized as chondroitin 4-sulfate by examining the products of digestion with hyaluronidase, chondroitinase AC and ABC, and chondro-4- and 6-sulfatases; by identification of the hexosamine as N-acetylgalactosamine; by determination of a 1 : 1 : 1 molar ratio of N-acetylgalactosamine, uronic acid and inorganic sulfate; and by cetylpyridinium chloride cellulose chromatography. In these studies, the use of 35S-labeled proteoglycan made possible detection and quantification of much smaller amounts of material than would be possible with unlabeled material. Chondroitin 4-sulfate was the only sulfated glycosaminoglycan identified in the proteoglycan lost from the platelet surface during ADP-induced aggregation and in the proteoglycan released from the granules when the platelets were exposed to thrombin.     

Autophagic regulation of platelet biology

Platelets, developed from megakaryocytes, are characterized by anucleate and short-life span hemocyte in mammal vessel. Platelets are very important in the cardiovascular system. Studies indicate the occurrence of autophagy platelets and megakaryocytes. Moreover, abnormal autophagy decreases the number of platelets and suppresses platelet aggregation. In addition, mitophagy, as a kind of selective autophagy, could inhibit platelet aggregation under oxidative stress or hypoxic, whereas promote platelet aggregation after reperfusion. Finally, autophagy regulates hemorrhagic and thrombosis diseases by influencing the number and function of platelets. In this paper, the role of autophagy in platelets and megakaryocytes, as well as coupled with the promotive or inhibitory role of hemorrhagic and thrombosis diseases are elucidated. Therefore, autophagy may be a potentially therapeutic target in modulating the platelet-related diseases.     

Aspirin inhibits rat megakaryocyte thromboxane synthesis

This study examines the question of whether the aspirin-induced delay in the recovery of platelet cyclooxygenase pathway activity, as measured by RIA of thromboxane B₂, results from a direct effect on megakaryocyte cyclooxygenase. From our measurement of recovery of TXB₂ and information on megakaryocyte transit time in rats, we propose that thromboxane synthesis may represent a relatively late step in the differentiation of megakaryocytes. Megakaryocyte thromboxane production was depressed by 70% and that of platelets by 85% at two hr after 20 mg/kg oral aspirin dissolved in DMSO. Full megakaryocyte thromboxane recovery occurred by 72 hr and preceded complete platelet thromboxane recovery by 24 hr. Whereas megakaryocyte thromboxane synthesis showed substantial recovery by 36 hr after aspirin, platelet recovery did not begin for 24 hr and achieved a maximal recovery rate over the following 12 hr. This finding is consistent with predictions based upon human data for both megakaryocyte labeling studies and post-aspirin platelet recovery. We conclude from our data and from estimates of megakaryocyte maturation times in marrow, that thromboxane synthesis develops in rat megakaryocytes after approximately 48 hr of cytoplasmic differentiation toward platelet shedding. This metabolic capacity therefore serves as a marker of megakaryocyte differentiation.     

Characterization of platelet abnormalities of Tester Moriyama (TM) rats with storage pool deficiency

Platelet abnormalities of Tester Moriyama (TM) rats, which have prolonged bleeding time with normal platelet count, were characterized by comparison with those of fawn-hooded (FH) rats with platelet storage pool deficiency (SPD). Morphologically, the dense granules were virtually lacking in platelets from TM and FH rats. Platelets from TM and FH rats aggregated in response to adenosine diphosphate (ADP), but failed to have secondary aggregation. In contrast, platelet aggregation was completely absent in response to 1 to 20 micrograms of collagen/ml, although partial aggregation was observed at the higher dosage of 50 micrograms/ml. Normal amounts of platelet membrane glycoproteins IIb/IIIa were expressed in TM and FH rats, but platelet adenosine triphosphate (ATP) and ADP contents were lower than those in platelets from control Wistar rats. Platelet ATP-to-ADP ratio of TM and FH rats was significantly higher than that of Wistar rats. Serotonin content in platelets from TM and FH rats was 20 to 25% that of Wistar rat platelets. These results suggested that platelet abnormalities of TM rats are a typical characteristic of platelet SPD and are similar to those of FH rats, which are genetically different from TM rats. Therefore, TM rats may serve as a useful animal model for the study of platelet SPD.     

Cellular events in tolerance. II. Thymus-bone marrow cell cooperation in the immune response to BSA in Wistar Furth rats

Normal bone marrow cells from Wistar Furth rats were competent to transfer immune responsiveness to bovine serum albumin to thymectomised, irradiated, syngeneic recipients. When the bone marrow cells were taken from donors thymectomised early in life they were incompetent, but competence was restored by addition of normal thymus cells. It was concluded that normal Wistar Furth bone marrow cells contain some thymus-derived cells. Thymus cells from tolerant donors were less effective in cooperation with bone marrow cells, however the thymus cells appeared less tolerant than their donors.     

Increased immunoreactive neuropeptide Y in platelets of spontaneously hypertensive rats (SHR)

A high concentration of immunoreactive neuropeptide Y was observed in rat platelets using a specific and sensitive radioimmunoassay for neuropeptide Y. Three kinds of high performance liquid chromatography combined with radioimmunoassay for neuropeptide Y showed that immunoreactive neuropeptide Y in rat platelets is identical to rat authentic neuropeptide Y. To investigate the pathological role of platelet neuropeptide Y in genetic hypertensive rats, the platelet content and plasma concentration of neuropeptide Y were measured by a sensitive radioimmunoassay for rat neuropeptide Y in 5-, 10- and 15-wk old spontaneously hypertensive rat and age-matched Wistar Kyoto rat. Platelet content of immunoreactive neuropeptide Y in 5-, 10- and 15-wk old spontaneously hypertensive rat was higher than that in Wistar Kyoto rat at each age. No difference was observed in plasma concentration of immunoreactive neuropeptide Y between spontaneously hypertensive rat and Wistar Kyoto rat at each age.     

Serglycin is essential for maturation of mast cell secretory granule

To address the biological function of the scarcely studied intracellular proteoglycans, we targeted the gene for serglycin (SG), the only known committed intracellular proteoglycan. SG-/- mice developed normally and were fertile, but their mast cells (MCs) were severely affected. In peritoneum there was a complete absence of normal granulated MCs. Furthermore, peritoneal cells and ear tissue from SG-/- animals were devoid of the various MC-specific proteases. However, mRNA for the proteases was present in SG+/+, SG+/-, and SG-/- tissues, indicating that SG is essential for the storage, but not expression, of the MC proteases. Experiments, in which the differentiation of bone marrow stem cells into mature MCs was followed, showed that secretory granule maturation was compromised in SG-/- cells. Moreover, SG+/+ and SG+/- cells, but not SG-/- cells, synthesized proteoglycans of high anionic charge density. Taken together, we demonstrate a key role for SG proteoglycan in MC function.     

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.     

Detection of neuropeptide Y-like immunoreactivity and messenger RNA in rat platelets: the effects of vinblastine, reserpine, and dexamethasone on NPY expression in blood cells.

Rat plasma contains high basal levels (220 pmol/liter) of neuropeptide Y (NPY)-like immunoreactivity (LI) compared to pig (30 pmol/liter) and man (25 pmol/liter). The platelet-enriched fraction (PEF), obtained from rat blood contained 10,061 pmol/g NPY-LI. However, in human and pig blood, the PEF contained very low levels of NPY-LI. Gradient centrifugation of rat blood showed the highest concentration of NPY-LI (10.8 +/- 0.4 pmol/g) in the platelet fraction. The mononuclear cell fraction contained 1.64 +/- 0.16 pmol/g, whereas only 0.56 +/- 0.06 pmol/g of NPY-LI was found in the red blood cell/polymorphonuclear cell fraction. Characterization of NPY-LI in rat plasma and platelets by high-pressure liquid chromatography showed one predominating peak which coeluted with synthetic NPY (1-36) as well as three minor peaks, one of which coeluted with oxidized NPY. Analysis of NPY messenger RNA (mRNA) in bone marrow of the rat revealed a 0.79-kb-long NPY mRNA. This size is intermediate to the 0.82-kb NPY mRNA in brain and the 0.76-kb NPY mRNA in spleen. The highest level of NPY mRNA in rat blood was found in the mononuclear cell fraction but NPY mRNA was also detected in the platelet fraction. No NPY mRNA was detected in bone marrow or blood from pig and rabbit or from human blood or bone marrow. Forty-eight hours after treatment of rats with vinblastine the content of NPY mRNA and NPY-LI in rat blood was decreased, while the level of NPY-LI in bone marrow was markedly enhanced. Reserpine treatment caused an increase in NPY mRNA content in bone marrow and spleen. After administration of dexamethasone the level of NPY mRNA increased in both spleen and peripheral blood cells with increased NPY-LI content in the spleen. It is concluded that in addition to megakaryocytes in spleen and bone marrow, platelets and possibly also lymphocytes/monocytes in peripheral blood of the rat contain NPY mRNA and peptide. The expression of NPY mRNA in bone marrow, spleen, and blood is influenced by vinblastine, reserpine, and dexamethasone.