Large Number Of Platelets Research Articles

Platelet-rich plasma enhances human osteoblast-like cell proliferation and differentiation

Platelet-rich plasma (PRP) is widely used to promote tissue healing. However, there is no concrete evidence for the biological effects of PRP. This study evaluated the biological effects of PRP on the proliferation and differentiation of 2 human osteoblast-like cell lines. Human osteosarcoma cell lines HOS and SaOS-2 were used in this study. PRP was prepared from freshly drawn human venous blood containing a large number of platelets. The MTT assay was used to examine the effects of PRP on osteoblast viability. To evaluate the growth and differentiation, alkaline phosphatase activity was assessed and the expression of procollagen type I, osteopontin, and osteoprotegerin mRNA was measured using semiquantitative reverse transcriptase-polymerase chain reaction. Further, core binding factor alpha 1 (cbfa1/Runx2/AML3/PebpalphaA), a critical regulator of osteoblast differentiation, was also determined. The administration of PRP enhanced the viability of HOS and SaOS-2 cells in a dose-dependent manner. Alkaline phosphatase activity was suppressed during the cell growth phase, but was strongly enhanced when the cells reached confluence. Semiquantitative reverse-transcription polymerase chain reaction analysis showed that PRP enhanced the levels of procollagen type I, osteopontin, osteoprotegerin, and core binding factor alpha 1 (cbfa1) mRNA. These results suggest that PRP has a favorable effect on human osteoblast-like cells, and acts both to enhance bone regeneration and as an activator in wound healing.

Autologous fibrin glue in peripheral nerve regeneration in vivo

The activity of several growth factors on peripheral nerve regeneration is reported. Autologous fibrin glue contains a large number of platelets, which release significant quantities of growth factors. In order to understand the role of autologous fibrin glue in peripheral nerve regeneration, a 15-mm rabbit peroneal nerve defect was repaired using a vein graft filled with autologous fibrin glue. Axonal regeneration was examined using histological and electrophysiological methods. The extent of axonal regeneration was superior when treated with autologous fibrin glue. Our data suggest that fibrin nets formed by fibrinogen, in combination with growth factors present in autologous fibrin glue, might effectively promote peripheral nerve regeneration in nerve defects.

Inherited Thrombocytopenias: Molecular Mechanisms

Each megakaryocyte forms 10(3) platelets and 10(11) platelets are replenished daily. The unique and amazing mechanisms that allow megakaryocytes to become giant and polyploid and to release such a large number of platelets are still poorly understood. The study of inherited thrombocytopenias offers the possibility to gain new information on these processes because several different forms, deriving from defective megakaryocytic commitment, differentiation, maturation, or platelet formation, have been identified. Moreover, in the presence of some genetic defects, megakaryocytes produce platelets with a shortened life span. In this review, we summarize what we have learned about inherited thrombocytopenias in the last few years.

Production of functional platelets by differentiated embryonic stem (ES) cells in vitro

AbstractMegakaryocytes and functional platelets were generated in vitro from murine embryonic stem (ES) cells with the use of a coculture system with stromal cells. Two morphologically distinctive megakaryocytes were observed sequentially. Small megakaryocytes rapidly produced proplatelets on day 8 of the differentiation, and large hyperploid megakaryocytes developed after day 12, suggesting primitive and definitive megakaryopoiesis. Two waves of platelet production were consistently observed in the culture medium. A larger number of platelets was produced in the second wave; 104 ES cells produced up to 108 platelets. By transmission electron microscopy, platelets from the first wave were relatively rounder with a limited number of granules, but platelets from the second wave were discoid shaped with well-developed granules that were indistinguishable from peripheral blood platelets. ES-derived platelets were functional since they bound fibrinogen, formed aggregates, expressed P-selectin upon stimulation, and fully spread on immobilized fibrinogen. These results show the potential utility of ES-derived platelets for clinical applications. Furthermore, production of gene-transferred platelets was achieved by differentiating ES cells that were transfected with genes of interest. Overexpression of the cytoplasmic domain of integrin β3 in the ES-derived platelets prevented the activation of αIIbβ3, demonstrating that this system will facilitate functional platelet studies. (Blood. 2003;102:4044-4051)

Mean platelet volume and the number of large platelets in atherosclerotic patients

Mean platelet volume and the number of large platelets in atherosclerotic patients

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.

Megakaryocytes and platelets in the spleen of the dromedary camel (Camelus dromedarius).

This study was carried out on spleens of clinically healthy camels (n = 35) of both sexes (0.5-15 years) by routine histology, electron microscopy and immunohistochemistry using 12 anti-bovine platelet antibodies. Megakaryocytes were observed in the red pulp. Their number decreased with age: they were found in the spleens of all camels under 8 years old but only in 57% of camels over 8 years old. Only two antibodies (IVA37 and IVA38) cross-reacted with camel platelets. A large number of platelets were found in the splenic cords and the marginal zone. Ultrastructurally, the platelets were oval in shape surrounded by a plasma membrane, and their cytoplasm was rich in glycogen and contained less dense granules. Microtubules and microfilaments were found at their periphery. Several platelets were observed in the red pulp. There are similarities in some surface antigens of bovine and camel platelets. The presence of megakaryocytes in the camel spleen indicates a thrombopoietic function of the spleen until adulthood but that this decreases with age thereafter.

Transfusions of granulocyte-colony-stimulating factor-mobilized granulocyte components to allogeneic transplant recipients: analysis of kinetics and factors determining posttransfusion neutrophil and platelet counts.

Granulocyte-colony-stimulating factor (G-CSF) is a safe and effective agent for mobilization of neutrophils in normal donors, consistently resulting in cell yields per leukapheresis (LA) procedure that are superior to those with other agents. LA components also contain platelets, whose clinical relevance is unknown. This study describes the kinetics of and analyzes the factors determining the ANC and platelet count increments seen with each of three transfusions of granulocytes collected from HLA-matched sibling donors receiving G-CSF (n = 10; maximum of 3 LA procedures/donor). The transfusions were given to recipients (n = 10) on alternate days beginning. Day 1 after allogeneic bone marrow transplant (BMT). Significant, sustained increments in the recipient ANCs were observed after the transfusion of G-CSF-mobilized LA components. The mean peak posttransfusion increments in the ANCs were 1195, 729, and 631 per microL with transfusion of donor LA components on Days 1, 3, and 5, respectively. The length of time that the mean posttransfusion ANC was at or above the baseline (pretransfusion) value was 25 to 37 hours, depending on the post-BMT day when the component was administered. No consistent relationship was observed between LA component granulocyte dose, baseline recipient ANC, or temperature elevation and post-transfusion ANC increments. Large numbers of platelets (mean, 2.55 x 10(11)) were present in LA components, and this resulted in significant increments from baseline in the mean platelet count 1 hour after LA component transfusions. Between Days 1 and 7, the duration of severe neutropenia was shorter and the percentage of patients requiring nondonor platelet transfusions was less in study patients who received LA component transfusions than in a similar historical control group who did not. The transfusion of G-CSF-mobilized, HLA-matched LA components to allogeneic BMT recipients resulted in significant and sustained increments in the ANC and the platelet count. Within the range examined, a relationship between neutrophil dose and an increment in the ANC was not demonstrated.

The effect of temperature and shear rate on platelet aggregation.

Samples of whole blood were obtained from male volunteers and exposed to combinations of shear rates and temperatures representative of cardiopulmonary bypass (CPB) in a modified computer-controlled concentric cylinder rotational viscometer for a period of 100 s. Blood sampled from the chamber was fixed in paraformaldehyde, stained with CD41 and analysed by flow cytometry. Only platelet-positive particles were acquired, each individual cell, or aggregate of cells, identified by analysis of its fluorescence and forward light scatter characteristics. Little platelet aggregation was observed at shear rates of less than 4000 s(-1) for temperatures of greater than 24 degrees C, but large numbers of aggregates were formed at all temperatures at 4000 s(-1) (p<0.05), with more aggregates forming at 24 and 30 degrees C than at 37 and 42 degrees C (p<0.05). We conclude that the process of aggregation is dependent on both temperature and shear rate. We note that a large number of platelets become involved in aggregates under conditions of temperature and shear-rate typical of CPB.

Biphasic, organ-specific, and strain-specific accumulation of platelets induced in mice by a lipopolysaccharide from Escherichia coli and its possible involvement in shock.

Platelets contain a large amount of 5-hydroxytryptamine (5HT, serotonin). Intravenous injection into BALB/c mice of a Boivin's preparation of lipopolysaccharide (LPS) from Escherichia coli induced rapid 5HT accumulation in the lung (within 5 min) and slow 5HT accumulation in the liver (2 to 5 h later). The rapid response required high doses of LPS (more than 0.1 mg/kg). On the basis of 5HT measurements, 70% or more of the platelets which disappeared from the blood appeared to have accumulated rapidly in the lung, and a large number of platelets were found there by electron microscopy. A shock, which was manifested by crawling, convulsion, or prostration, followed shortly after the rapid accumulation of 5HT in the lung. On the other hand, the slow accumulation of 5HT in the liver could be induced by much lower doses of LPS (1 microg/kg or less), even when given by intraperitoneal injection. This 5HT accumulation appears to be a reflection of platelet accumulation in the liver (Y. Endo and M. Nakamura, Br. J. Pharmacol. 105:613-619, 1992). The combination of a low dose of LPS with D-galactosamine amplified the hepatic accumulation of 5HT, and the mice developed a severe hepatic congestion resulting in death. The rapid response was not induced at all in C3H/HeN mice. These results and comparison with other LPS preparations indicate that some component(s) of LPS from E. coli induces a biphasic, organ-specific and strain-specific accumulation of platelets, and it is proposed that this effect is involved in the development of shock.

Mean platelet volume improves upon the megathrombocyte index but cannot replace the blood film examination in the evaluation of thrombocytopenia.

The measurement of the number of platelets larger than 3 microns (megathrombocyte index) is the first element in the evaluation of thrombocytopenia. This is currently performed by counting the number of large platelets on the peripheral blood film. The MPV (mean platelet volume) is an automated measurement of the platelet volume. This study examines the mean values, correlations, sensitivity, specificity and the receiver operating characteristic curve (comparison of two tests) to determine which of these tests better separates the production state. For increased vs. decreased production, the MPV was 10.0 + 1.9 fL and 8.0 + 1.5 fL (P < .0001) respectively and the megathrombocyte index (MEGA) was 19.0 + 17.6% and 11.5 + 14.9% (P < .007) respectively. The correlation with the state of production was better for MPV (R = .47) than for MEGA (R = .20). For the MPV a sensitivity of 80% occurred with the MPV > or = 8.4 fL with a specificity of 71%. For a MEGA > or = 6%, the sensitivity was 80% but the specificity was 43%. For any MPV the sensitivity and specificity were better than for any MEGA. The Receiver Operating Characteristic Curve demonstrated that the MPV is a better test than the MEGA for separating the production into increased and decreased states. The MPV is a better test than the MEGA and will add to, but not replace, examination of the peripheral blood film in the diagnosis of thrombocytopenia.

Initial validation and quality control of platelet concentrates prepared by apheresis

Platelet concentrate (PC) prepared by apheresis is a widely used product in the practice of transfusion medicine. New machines, new protocols and/or disposables for existing machines, as well as new storage containers for the PC are being introduced with great frequency. Furthermore, additional steps such as filtration are being introduced between preparation and storage. The goal of this paper is to discuss the wide range of laboratory measurements which might be used by manufacturers of apheresis centers to validate a new procedure prior to its introduction into clinical practice. In addition, the discussion should shed light on what procedures might be used as quality control assessments by an apheresis center during the ongoing utilization of a procedure which has previously been validated. Once a PC has been prepared, it must be stored for finite period of time. A new procedure may provide a PC with a large number of platelets and few contaminating leukocytes but poor potential for storage. Therefore, any new preparative procedure must specify how long the PC may be stored and what the conditions of storage are to be. The laboratory assays to be described should be applied at the end of that storage interval.

Serotonin and beta-thromboglobulin release reaction from platelet as triggered by interaction with polypeptide derivatives.

The effect of wettability of 14 polypeptide derivatives upon adhesion and activation of platelets was investigated with reference to release reactions from adhered platelets, using radioisotope labeling and radioimmunoassay method. The serotonin release was more significant from platelets adhered to polymer materials to which a large number of platelets are adhered. However, no clear relationship was found between adhesion of platelets and beta-thromboglobulin release from adhered platelets. Therefore, stimuli inducing serotonin release and beta-thromboglobulin release were considered to be from different origins. The trend in beta-thromboglobulin release was well correlated with the extent of morphological change of adhered platelets as observed by scanning electron microscope. It was suggested that the determination of released beta-thromboglobulin in association with the measurement of platelet adhesion could be useful for evaluation of blood compatibility of materials.

Kinetics and mobilization from the spleen of indium-111-labeled platelets during platelet apheresis.

The rate and extent of platelet mobilization from the spleen were measured, and their relationship to the removal of platelets from the peripheral blood during discontinuous flow platelet apheresis was determined in four normal volunteers. Autologous platelets were labeled with Indium-111-oxine and in vivo whole body and organ In-111 radioactivity quantitated with a scintillation camera and a computer-assisted imaging system. Dynamic changes in splenic radioactivity were monitored during 12 cycles of platelet apheresis. The number of platelets harvested and changes in whole body and blood In-111 activity were determined during the procedure. The platelet life-span was estimated, and the sites of sequestration of labeled platelets was measured. Platelet apheresis removed a mean of 64 percent of platelets in the circulation; i.e., 48 percent of all platelets in the body. During the procedures, 28.0 +/- 9.4 percent In-111-labeled platelets in the body were removed, splenic radioactivity decreased by 36.5 +/- 13.2 percent, and whole body activity decreased by 34.5 +/- 9.7 percent. In-111 activity in the spleen and whole body decreased in parallel, indicating a dynamic equilibrium between these pools. The life-span of the labeled platelets was 226 +/- 25 hours, similar to that of normal subjects. The major sites of sequestration of senescent platelets were the spleen (37.9 +/- 20%) and liver (30.3 +/- 5.6%); this is similar to that found in normal subjects. We conclude that as platelets are removed from the peripheral blood, the blood pool is rapidly and effectively replenished from the splenic platelet pool. These two pools are in dynamic equilibrium and permit removal of large numbers of platelets without resultant thrombocytopenia. Platelet apheresis does not adversely effect platelet life-span, and the sequestration pattern in the reticuloendothelial system is normal.

Factors influencing yield of plateletpheresis by discontinuous centrifugation.

Plateletphereses performed by the Haemonetics Model 30 Blood Processor under a fixed condition were retrospectively analyzed. The mean platelet yield was 5.97 X l0 (ll) (+/- l.46 X l0 (ll). Prepheresis platelet count was significantly correlated to platelet yield (p less than 0.001), but hematocrit was not (p greater than 0.1). Blood-drawing time showed a significant correlation (p less than 0.01) and may suggest than an infusion rate of 60 ml/minute is a technique of choice. A large number of platelets (% platelet recovery = 73.0 +/- l4.l%) was discarded in order to remove erythrocytes and leukocytes from the initial product. The recovery was negatively correlated to the percentage removal of leukocytes (p less than 0.01), which means that a platelet product of good quality not containing leukocytes can be prepared at the sacrifice of a large quantity of platelets. It is desirable to devise a simple and practical technique to obtain pure platelets without loss.

Function and morphology of platelets produced for transfusion by intermittent‐flow centrifugation plateletpheresis or combined platelet‐ leukapheresis

The effects on platelet function of intermittent-flow centrifugation pheresis were measured employing platelets collected from ten donors by combined platelet-leukapheresis with hydroxyethyl starch (LP) and from ten by plateletpheresis (PP) by similar techniques except without starch. Greater numbers of platelets were produced by LP than by PP. Aggregation of platelets collected by both LP and PP was normal (did not differ from prepheresis baseline) to collagen and to 10(-5)M adenosine diphosphate (ADP). Slight impairment to aggregation with 2 X 10(-6)M ADP and 5.5 X 10(-5)M epinephrine occurred with both techniques. These abnormalities, however, were significantly less severe in platelets collected by LP. Platelet morphology by electron microscopy was nearly normal, although glycogen granules were absent in LP platelets. Thus, hydroxyethyl starch, at doses currently used during a single LP, does not enhance abnormalities of platelet aggregation over those expected to result from PP alone. Actually, LP platelets function better in vitro than those collected by PP. Large numbers of platelets can be harvested by LP, and their use as a component that is comparable to PP platelets may mean improved efficiency and reduced costs for pheresis centers.

Myeloproliferative disease in the cat: A granulocytic and megakaryocytic disorder

Abstract The history, clinical, laboratory and post-mortem findings of a myeloproliferative disorder in a cat are described. The main features in peripheral blood were the presence of a high proportion of cells with abnormal morphology, and a large number of platelets, many of which were large and had immature staining characteristics. The abnormal cells had some characteristics of the granulocytic cell line. There was a proliferation of megakaryocytes within the spleen. The findings were characteristic of a granulocytic leukaemia with some evidence of a megakaryocytic myelosis.

Heterogeneity of Human Platelets. VI. Correlation of Platelet Function With Platelet Volume

Platelet aggregation velocity was measured with an aggregometer in platelet-rich plasma from 34 normal subjects following addition of ADP, collagen, or epinephrine. Platelet volume parameters [mean, mode, megathrombocyte index (large platelets)] were determined with a model B Coulter Counter attached to a P64 Channel Analyzer with electronic recorder and correlated with platelet count. A negative linear relationship was found between platelet count and platelet volume (r = –0.53, p 0.1, respectively). Platelet volume distribution curves of residual nonaggregated platelets revealed a relative absence of larger platelets. Thus, platelet aggregation velocity was proportional to platelet volume, but correlated best with the number of large platelets or megathrombocyte index, particularly with ADP-induced platelet aggregation. It is suggested that the clinical strategy for treatment of platelet disorders would be better directed toward evaluation of platelet size and function rather than number, and that experimental as well as therapeutic procedures for the harvesting of platelets be directed toward a method of collection in which a portion of the megathrombocyte population is not routinely discarded.

Structure of the spleen in idiopathic thrombocytopenic purpura.

The structure of the spleens of 12 patients with idiopathic thrombocytopenic purpura (ITP) who underwent splenectomy was studied. The white pulp was characterized by the presence of large numbers of lymphatic nodules containing highly reactive germinal centers. The marginal zone contained large numbers of vessels surrounded by plasma cells. These findings imply active antibody production and are consistent with the concept that the spleen is a major source of antiplatelet antibody production in ITP. Large numbers of platelets in various stages of degradation were seen in cords, particularly in the marginal zone. They appeared both extracellularly and with the cytoplasm of macrophages. It appeared that platelet destruction was initiated by focal cytoplasmic degradation and resulted in formation of cellular debris. These findings indicate that the spleen plays a fundamental role in the pathogenesis of ITP, involving synthesis of antiplatelet antibody, localization of platelets in a milieu rich in this antibody, and subsequently, phagocytosing of immune-damaged platelets.

In Vitro Platelet Reaction with Isolated Glomerular Basement Membrane

SummaryPurified canine glomerular basement membrane (BM) induced platelet aggregation when added to heparinized platelet-rich plasma (PRP) of the rat. Platelet aggregation induced by BM suspension was compared with that induced by collagen submicroscopically. When aliquots of BM or collagen suspension of the same concentration (2 mg/ml saline) was incubated with PRP, platelet-BM reaction was far less pronounced than platelet-collagen reaction. Platelet-BM reaction was slow and aggregates were small; swelling and pseudopod formation were obvious but platelet degranulation was limited. With high concentration (5 mg/ml saline) of BM suspension, the aggregation time was shortened and aggregates were generally composed of larger number of platelets. Treatment of BM with collagenase abolished the platelet-BM reaction. It is postulated that similar “platelet aggregating” elements in collagen are present in BM; but the quantity of these elements is less in a BM than in a collagen molecule.