Circulating Megakaryocytes and Platelet Release in the Lung

1. Megakaryocytes were demonstrated in central venous blood of each of23 patients who underwent cardiac catheterization. Cell counts ranged from0.7 to 5.9 megakaryocytes per ml. of blood; the equivalent of one-third ofthese cells were considered to contain a full complement of cytoplasm. It hasbecome evident that megakaryocytes are normal constituents of blood.

2. In an attempt to quantify megakaryocyte migration from the bone marrow it was calculated that from 20-50 per cent of the mature megakaryocytepopulation enters the blood and ultimately reaches the lungs. The possibilitythat all megakaryocytes migrate from the marrow is not precluded with certainty by these studies.

3. It was estimated that from 7-17 per cent of the body’s platelets are released in the pulmonary capillaries. If all megakaryocytes migrate from thebone marrow, then as much as 33 per cent of the platelet population is delivered to the blood in the lungs.

Submitted on January 15, 1965 Accepted on March 13, 1965     

Bernard-Soulier syndrome: quantitative characterization of megakaryocytes and platelets by flow cytometric and platelet kinetic measurements

Abstract: Platelets and megakaryocytes have been characterized in a Bernard-Soulier syndrome (BSS) kindred with respect to glycoprotein (GP) membrane receptors and measurements of thrombocytopoiesis. The index patient exhibited lifelong bleeding tendency, moderate thrombocytopenia (35 × 10⁹/1), giant platelets (mean platelet volume 12.5 μm³ compared to 7.5 ± 1.5 μm³ in normals), absent ristocetin-induced platelet agglutination and absent binding of von Willebrand factor (vWF). Flow-cytometric analysis revealed absent platelet binding (0–2%) of monoclonal antibodies (mAb, LJ-P3, LJ-Ibl and LJ-Ibl0) directed against distinct epitopes on membrane GPIbα of the GPIb-IX complex, and normal binding of LJ-P4 mAb directed against GPIIb/IIIa complex (relative to increased platelet surface area). Marrow megakaryocytes also failed to express GPIb-IX complex, but demonstrated normal expression of GPIIb/IIIa. Among 6 asymptomatic family members, the patient's mother and 2 of his 4 children exhibited approximately 50% binding of anti-GPIbα mAb to their platelets by both flow cytometry and direct binding studies using ¹²⁵I-vWF, ¹²⁵I-LJ-Ibl and ¹²⁵I-LJ-Ibl0 mAb. Marrow megakaryocytes were increased in the average cell volume and cytoplasmic granularity with a corresponding increase in ploidy (46% > 16N compared to 22 ± 5% in normal individuals), a pattern typical of megakaryocytes stimulated by thrombocytopenia. Autologous ¹¹¹In-platelet life span was shortened to 4.1 days (compared with 9.5 ± 0.5 days in normal subjects), and the turnover of platelet mass in the circulation was near normal. The data directly demonstrate that the platelet membrane GPIb-IX defect in BSS originates in megakaryocytes at all levels of cell maturation, and exclude the possibility that the receptor abnormality is acquired during cell maturation or after platelets are released into the circulation. Since marrow megakaryocytes exhibited cellular changes consistent with stimulated megakaryocytopoiesis, these results also suggest that thrombocytopenia in this kindred of BSS is a consequence of both decreased platelet survival and ineffective platelet production.     

Expression of adhesion antigens of human bone marrow megakaryocytes, circulating megakaryocytes and blood platelets.

There is evidence that mature megakaryocytes migrate into sinusoids, enter the blood and fragment in the vascular bed. We wondered whether differences in expression of adhesion antigens could be associated with the egress of megakaryocytes from bone marrow into the peripheral blood or the fragmentation into platelets. Megakaryocytes from human marrow were purified by counterflow centrifugal elutriation followed by a glycoprotein Ib-dependent agglutination procedure. Megakaryocytes from central venous blood and pulmonary arteries were purified by counterflow centrifugal elutriation alone. Adhesion antigens were labelled in an immunohistochemical assay. Both bone marrow megakaryocytes and platelets from healthy volunteers stained > 75% positive for CD36, CD41, CD42, Cdw49b (alpha subunit VLA2), Cdw49e (alpha subunit VLA5), Cdw49f (alpha subunit VLA6) and CD62. Circulating megakaryocytes, although > 75% positive for CD41, had, unlike platelets and bone marrow megakaryocytes, a reduced and remarkable heterogeneous (5-100% positive) labelling with antibodies against Cdw49b, Cdw49e, Cdw49f. These results could be confirmed by comparing the bone marrow megakaryocytes, circulating megakaryocytes and platelets from 7 patients that were recovered and processed at the same time. Morphologically mature, circulating megakaryocytes have, unlike bone marrow megakaryocytes, a heterogeneous expression of adhesion antigens, especially of Cdw49b, Cdw49e, and Cdw49f.     

Isolation of megakaryocytes from human placentae

Shedding of cytoplasm from circulating megakaryocytes (MKs) within the pulmonary vasculature suggests the lungs are an important site for normal platelet production. Fetal lungs receive only a minor fraction of the circulating blood volume. The placenta may act as a site for intrauterine platelet formation. Isolation of MKs from fetal vessels within the placenta has not been previously reported. Immediately after delivery, 3 human placentae were subjected to forward and retrograde perfusion across the placental capillary bed on the fetal side. MKs in perfusates were harvested by 'whole blood filtration' and identified by morphological and immunochemical methods. All perfusates yielded MKs. Qualitatively MKs with copious cytoplasm were more commonly found in perfusates collected from fetal arteries compared with those from fetal veins. This is consistent with filtration of MKs and fragmentation of their cytoplasm within the placental microcirculation to produce platelets. Perfusion of human placentae followed by filtration of perfusates is a useful technique for harvesting fetal MKs and permitting further elucidation of their physiological role.     

BclxL overexpression in megakaryocytes leads to impaired platelet fragmentation

Fragmentation of polyploid megakaryocytes into platelets has great relevance for blood homeostasis. Apoptotic cell death is a highly regulated genetic program, which has been observed in mature megakaryocytes fragmenting into platelets. The antiapoptotic protein BclxL has been reported as up-regulated during megakaryocytic differentiation in vitro, but absent during late megakaryopoiesis. Our study focused on examining BclxL levels in megakaryocytes in vivo and in assessing the effect of its overexpression in transgenic mice (via the platelet factor 4 [PF4] promoter) on megakaryocyte development and platelet fragmentation. Interestingly, in the wild-type and less in PF4-driven transgenic mice, BclxL was not detected in a fraction of the large mature megakaryocytes, suggesting a regulation on the protein level. BclxL overexpression was associated with a moderate increase in megakaryocyte number, with no significant change in ploidy level or platelet counts. When the mice were challenged by induction of immune thrombocytopenia, the rate of platelet recovery was significantly slower in the transgenic mice as compared with controls. Moreover, proplatelet formation in vitro by transgenic megakaryocytes was limited. Transgenic megakaryocytes displayed poorly developed platelet demarcation membranes and cell margin extensions. Our study indicates that regulated expression of BclxL in megakaryocytes is important for the development of cells with a high potential to fragment into platelets.     

Immune thrombocytopenia: antiplatelet autoantibodies inhibit proplatelet formation by megakaryocytes and impair platelet production in vitro

Primary immune thrombocytopenia is an autoimmune disease mediated by antiplatelet autoantibodies that cause platelet destruction and suppression of platelet production. In vitro effects of autoantibodies on megakaryocyte production and maturation have been reported recently. However, the impact of these autoantibodies on crucial megakaryocyte functions, proplatelet formation and subsequent platelet release, has not been evaluated. We examined the effects of serum and IgG from 19 patients with immune thrombocytopenia using day 8 or 9 megakaryocytes (66.3 ± 10.6% CD41⁺), derived from cord blood hematopoietic stem cells (CD34⁺). The number of proplatelet-bearing megakaryocytes, the number of platelets released in the culture, total megakaryocyte numbers, ploidy pattern and caspase activation were measured at various times after treatment. After 5 days of treatment the number of proplatelet-bearing megakaryocytes was significantly decreased by 13 immune thrombocytopenia autoantibodies relative to the control group (P<0.0001) and this decrease was accompanied by a corresponding reduction of platelet release. Other features, including total megakaryocyte numbers, maturation and apoptosis, were not affected by immune thrombocytopenia antibodies. Treating the megakaryocytes with the thrombopoietin receptor agonists romiplostim and eltrombopag reversed the effect of the autoantibodies on megakaryocytes by restoring their capacity to form proplatelets. We conclude that antiplatelet antibodies in immune thrombocytopenia inhibit proplatelet formation by megakaryocytes and hence the ability of the megakaryocytes to release platelets. Treatment with either romiplostim or eltrombopag regenerates proplatelet formation from the megakaryocytes.     

Histochemical demonstration of 5-hydroxytryptamine in platelets and megakaryocytes

A histochemical fluorescence method for the demonstration of biogenicmonoamines was applied to the smear preparation of peripheral blood plateletsand bone marrow megakaryocytes of rabbits and humans. The fluorescenceobtained was identified as 5-HT by histochemical and pharmacologic criteria.With this technic, the following results were obtained: (1) A large amountof 5-HT was present in platelets and in mature platelet-forming megakaryocytes. (2) Only a small amount of 5-HT was demonstrable in the intermediate maturation forms of megakaryocytes with lack of platelet budding.

The possibility that the 5-HT detected was derived from (a) transport of5-HT formed elsewhere, and/or (b) 5-HT formation from 5-HTP in themegakaryocytes themselves during their maturation was discussed.

Submitted on October 24, 1966 Accepted on February 20, 1967     

Hemodynamic and functional consequences of intravascular platelet aggregation in skeletal muscle

The effect of ADP-induced platelet aggregation upon blood flow, O₂-consumption, and performance was investigated in isolated and autoperfused working canine gastrocnemius muscles. During steady state work a mean blood flow of 87.1 ± 33.7 ml × min^?1 × 100 g^?1 and a mean O₂-consumption of 7.89 ± 4.03 ml × min^?1 × 100 g^?1 was found. The average amplitude of muscle contraction was 10.4 ± 4.1 mm. Changes of these parameters due to the ADP-infusion were found to depend upon the number of platelets which remained in the muscles during infusion. On the other hand this so-called platelet loss was significantly correlated with the arterial platelet count. In animals with a low arterial platelet count (below 10⁴/mm³) platelet loss did not exceed 2 × 10⁹ cells × min^?1 × 100 g^?1. Blood flow as well as venous O₂-saturation increased during ADP-infusion. In an experiment with a high platelet loss (14.3 × 10⁹ cells × min^?1 × 100 g^?1) according to a platelet count of 390 × 10³/mm³ muscle contraction and O₂-consumption were reduced to approximately 30% of their preinfusion values. These changes, however, only were present during the time of ADP-infusion. After its termination the muscles started to recover immediately reaching the control values within 5 min.     

Microcytic thrombocytosis, small megakaryocytes, platelet lipids and hyperreactivity to collagen, lymphocytopenia, eosinophilia, and low blood volume in genetically hyperlipidemic rabbits.

Three- to 15-month-old rabbits with Watanabe heritable hyperlipidemia (WHHL) were tested to determine if hematological abnormalities would accompany the known hyperlipidemia and deficiency of receptors for low-density lipoprotein; the findings were compared to those of New Zealand white (NZW) rabbits of the same ages. WHHL plasma cholesterol and triglyceride levels were always greater than or equal to 6x those of NZW, and both were lower in older than in younger WHHL rabbits. From age 7 to 18 months, WHHL platelet counts were higher than those of age-matched NZW; the average for all WHHL was 1.45x that for NZW (p = 0.001). Average WHHL mean platelet volume (MPV) was 0.94x that for NZW (p = 0.025), with a tendency for greater microcytosis to occur at more advanced ages; electron microscopy supported the small size of WHHL platelets. WHHL platelet mass per microliter of blood (platelet count x MPV) was 1.39x that of NZW (p = 0.001), with differences occurring after the age of 6 months. The average WHHL blood volume was 18.3% less than in NZW (33.5 vs 41.0 ml/kg body weight) (p = 0.00005), so platelet count and mass per kilogram of body weight were similar in the two strains. The predominant ploidy of mature megakaryocytes from each strain was 32N; megakaryocytes were smaller in WHHL than NZW due to a smaller size of 32N cells (p = 0.002). Total leukocyte counts were the same in WHHL and NZW rabbits, but eosinophils were 32% higher (p = 0.037) and lymphocytes 34% lower (p = 0.008) in WHHL. Hematocrits and reticulocytes did not differ. Platelet-free cholesterol was 1.2x, esterified cholesterol 11.3x, phospholipids 0.9x, triglyceride 2.4x, and free cholesterol/phospholipid molar ratio 1.29x corresponding values in NZW platelets. WHHL platelets released more serotonin in response to a small dose (5 micrograms/ml) of collagen than did NZW platelets. These findings suggest that: 1) megakaryocytopoiesis and leukopoiesis are affected by lipid metabolism and/or LDL receptors, and 2) platelet production may be regulated more by the total mass of platelets than by their concentration in the blood.     

Membrane proteins on human megakaryocytes and platelets identified by monoclonal antibodies

We describe five monoclonal antibodies that react with four discrete antigens present on human platelets. Antibodies B2.12 and B59.2 precipitate the glycoprotein IIb-IIIa complex from radiolabeled platelet membrane extracts and inhibit platelet aggregation induced by adenosine diphosphate (ADP), collagen, or epinephrine. The antigen recognized by the two antibodies is present on megakaryocytes but either absent entirely or expressed in small amounts on platelets from Glanzmann's thrombasthenic patients. The antigen recognized by antibody B37.3 is absent from thrombasthenic platelets. Antibody B1.12 reacts with an antigen shared by platelets and 20% of peripheral blood lymphocytes and is a potent inducer of platelet aggregation. Antibody B2.10 reacts specifically with platelets and megakaryocytes but does not affect platelet functions. Thus, these reagents are useful tools in diagnostic and functional studies of both normal and abnormal platelets.     

Occurrence of Megakaryocytes in Various Vessels and Their Retention in the Pulmonary Capillaries in Man

A total of 17 patients with hypertension undergoing renal vein or adrenal vein catheterization were investigated in order to ascertain the number of megakaryocytes in blood from the inferior vena cava, the femoral artery and a cubital vein. On an average 11.9, 3.8, and 4.5 megakaryocytes per ml were found, respectively. In blood from the inferior vena cava, 30 % of the megakaryocytes had copious cytoplasm, while megakaryocytes in arterial and cubital venous blood had sparse or no visible cytoplasm. It was demonstrated that ? of the megakaryocytes were retained in the pulmonary circulation and that at least 70% of the platelets could derive from megakaryocytes in central venous blood or the pulmonary circulation. It was found that megakaryocytes pass through a life cycle in which the differentiation take place in the bone marrow, platelet release occurs mainly in central venous blood and in the pulmonary circulation and the destruction of the megakaryocyte nucleus take place outside the bone marrow, especially in the pulmonary circulation.     

Methods for genetic modification of megakaryocytes and platelets

During recent decades there have been major advances in the fields of thrombosis and haemostasis, in part through development of powerful molecular and genetic technologies. Nevertheless, genetic modification of megakaryocytes and generation of mutant platelets in vitro remains a highly specialized area of research. Developments are hampered by the low frequency of megakaryocytes and their progenitors, a poor efficiency of transfection and a lack of understanding with regard to the mechanism by which megakaryocytes release platelets. Current methods used in the generation of genetically modified megakaryocytes and platelets include mutant mouse models, cell line studies and use of viruses to transform primary megakaryocytes or haematopoietic precursor cells. This review summarizes the advantages, limitations and technical challenges of such methods, with a particular focus on recent successes and advances in this rapidly progressing field including the potential for use in gene therapy for treatment of patients with platelet disorders.     

Canine lung uptake of plasma and platelet serotonin

Platelet-endothelial interaction and serotonin release were studied in 20 in vitro pump perfused lung lobes. Indicator dilution studies were performed in sequence using an injectate composed of indocyanine green and platelets labelled with either ¹⁴C-serotonin (¹⁴C-5-HT) or ⁵¹Cr. In control experiments all tracer transit times were equivalent (¹⁴C-5-HT, ⁵¹Cr and indocyanine green) without entrapment of the tracer or platelets by the system. In six lungs with normal pulmonary function platelet transit times were not prolonged and no platelet entrapment was observed. These lungs took up to 72% of the total ¹⁴C-5-HT contained in the injected platelets and plasma. The remaining 14 lobes showed variable degrees of pulmonary insufficiency such as elevated intrapulmonary shunt, low compliance and high vascular resistance; platelet entrapment was related to these phenomena (p 0.01). Six lobes were studied with ¹⁴C-5-HT, and despite entrapment of 22% of the injected platelets and transit time prolongations no 5-HT uptake was observed. The platelet-plasma ¹⁴C-5-HT ratio was higher in the effluent blood than in the injectate. The data suggest that in the pump perfused lung lobe with normal pulmonary function: (1) Serotonin released from platelets is rapidly taken up by the pulmonary circulation during a single transit through the lung lobe; (2) the serotonin uptake occurs without any observable prolongation of platelet time. In the pump perfused lung lobe with abnormal pulmonary function: (1) Serotonin released from the platelets is not taken up by the pulmonary circulation despite significant platelet entrapment; (2) the released serotonin is removed by the circulating platelets.     

Origin of Pulmonary Megakaryocytes

1. Evidence is presented which indicates that pulmonary megakaryocytesdo not originate in the lungs but elsewhere in the body and are carried tothe lungs in the venous blood.

2. Some megakaryocytes in the lungs evidently deliver platelets to theblood.

3. Surgery is a potent stimulus to megakaryocyte production; the numbersof megakaryocytes found in the lung postoperatively is significantly increased.

Submitted on July 3, 1964 Accepted on September 19, 1964     

Identification of primary lysosomes in human megakaryocytes and platelets

The presence of lysosomal enzymes in human platelets is well documented; the identity of the "lysosome," however, has been the subject of some disagreement. In order to determine the time of appearance and subcellular localization of two lysosomal enzymes in megakaryocytes (MK) and platelets, we examined normal human bone marrow and blood by electron microscopy and cytochemistry. Acid phosphatase (AcPase) was present in the Golgi region in the youngest recognizable MK, as well as in those with a considerable degree of cytoplasmic maturation. Heavy reaction product was usually confined to one or two Golgi-associated cisternae and coated vesicles; other Golgi cisternae were sometimes lightly reactive. In mature MK, reaction product was limited to vesicles of variable size, but smaller than alpha-granules. Another lysosomal enzyme, arylsulfatase (AS), was localized in similar small vesicles in MK of all stages; it could not be demonstrated in the Golgi complex. Vesicles containing AS were also found in about 25% of platelet profiles, whereas vesicles containing AcPase were found in only about 15% of platelet profiles. The alpha-granules of all MK and platelets examined were negative for both enzymes. We conclude that the enzyme-containing vesicles in these cells constitute the lysosomes and that they are distinct from other platelet organelles. Since there was no evidence that they had participated in any digestive event, we believe that they are primary lysosomes, whose contents are secreted during platelet aggregation and the release reaction.     

Plasma thrombopoietin levels in thrombocytopenic states: implication for a regulatory role of bone marrow megakaryocytes

To evaluate the diagnostic value of thrombopoietin (TPO, c-mpl ligand) measurements, and clarify the regulatory mechanisms of TPO in normal and in thrombocytopenic conditions, the plasma TPO concentration was determined in normal individuals (n = 20), umbilical cord blood (n = 40), chronic idiopathic thrombocytopenic purpura (ITP; n = 16), in severe aplastic anaemia (SAA; n = 3), chemotherapy-induced bone marrow hypoplasia (n = 10), myelodysplastic syndrome (MDS; n = 11), and sequentially during peripheral blood progenitor cell transplantation (n = 7). A commercially available ELISA and EDTA-plasma samples were used for the analysis. The plasma TPO concentration in the normals and umbilical cord blood were 52 ± 12 pg/ml and 66 ± 12 pg/ml, respectively. The corresponding values in patients with SAA and chemotherapy-induced bone marrow hypoplasia were 1514 ± 336 pg/ml and 1950 ± 1684 pg/ml, respectively, and the TPO concentration, measured sequentially after myeloablative chemotherapy and peripheral blood progenitor cell transplantation, was inversely related to the platelet count. In contrast, the plasma TPO recorded in patients with ITP (64 ± 20 pg/ml) and MDS (68 ± 23 pg/ml) were only slightly higher than normal levels. In conclusion, TPO levels were significantly elevated in patients in which bone marrow megakaryocytes and platelets in circulation were markedly reduced, whereas TPO levels were normal in ITP patients, and only slightly increased in the MDS patients. These latter patients displayed a preserved number of megakaryocytes in bone marrow biopsies. Our data support the suggestion that megakaryocyte mass affects the plasma TPO concentration. In thrombocytopenic patients a substantially increased plasma TPO implies deficient megakaryocyte numbers. However, TPO measurements do not distinguish between ITP and thrombocytopenia due to dysmegakaryopoiesis, as seen in MDS patients.     

Human megakaryocytes. III. Characterization in myeloproliferative disorders

Abnormal proliferation of the megakaryocytic line was observed in the marrow tissue from patients with myeloproliferative disorders. Megakaryocytes were identified by immunofluorescence using distinct platelet protein markers. Plasma factor VIII antigen (factor VIII:AGN) and platelet glycoproteins IIb and IIIa were detected in normal mature and early megakaryocytes, as well as in a morphologically heterogeneous population of low density marrow cells regarded as atypical megakaryocytes. Atypical megakaryocytes were defined as oval/round 14- 35-micron diameter blast-like mononuclear/multinucleated cells bearing platelet protein markers with distinct morphological features, including cytoplasmic vacuolation, variable nuclear/cytoplasmic ratios, and variable cytoplasmic granulation. Atypical megakaryocytes were observed in most chronic myelogenous leukemia (CML) patients and in two patients with polycythemia vera, representing between 60 and 1,840 cells/10(4) cells (less than 1.050 g Percoll/cu cm). No atypical megakaryocytes were found in (a) 20 normal controls, (b) two patients with essential thrombocythemia, (c) a patient with thrombocytosis secondary to acute bleeding, and (d) in two patients with CML. Atypical megakaryocytes appear to represent a single-cell population, as demonstrated by a series of double immunofluorescence assays using combinations of five different antiplatelet protein sera. There was a statistically significant correlation between the frequency of atypical megakaryocytes and the presence of immature forms of myeloid cells in blood. Analyses of Fc IgG receptors conducted with two different immunofluorescence systems have demonstrated that phenotypic similarities existed between atypical megakaryocytes and myeloproliferative platelet proteins and differentiation markers on megakaryocytes are useful in elucidating the pathophysiologic alterations occurring in the megakaryocytic compartment in patients with myeloproliferative disorders.     

Characteristics of a novel rat anti-mouse platelet monoclonal antibody: application to studies of megakaryocytes.

Using murine platelets as an immunogen, a rat monoclonal antibody (designated 4A5) that recognizes only murine blood platelets and marrow megakaryocytes was developed. The extent of binding of 4A5 to platelets was dependent upon their state of activation. Following phorbol ester, ionophore, or thrombin stimulation of resting platelets, a decrease of > 50% in the binding of 4A5 was observed by flow cytometry. This decrease in antibody binding to the platelets was accompanied by an increase in antibody released into the platelet-free supernatant following platelet activation. When platelets were first radioiodinated, followed by activation and incubation of the platelet-free supernatant with 4A5-derivatized beads, no precipitable counts were observed compared with control resting platelets. This suggests that antibody release was related to an activation-dependent conformational change in the 4A5 epitope. Following solubilization of biotinylated platelets, 4A5 bound to an 80-kd membrane protein. Immunohistochemical studies with 4A5 showed that megakaryocytes could be identified both in vitro and ex vivo. When marrow was first stained histochemically with 4A5 followed by staining for acetylcholinesterase, the distribution of stained cells was similar. Flow cytometric analysis using 4A5 and propidium iodide showed that the antibody could be used to identify megakaryocytes for ploidy analysis in vivo or in vitro. 4A5 was capable of inducing a moderate thrombocytopenia in mice compared with polyclonal anti-platelet serum. When bound to plastic or to magnetic beads, 4A5 could be used to purify murine megakaryocytes to homogeneity. The data suggest that monoclonal antibody 4A5 will be useful in quantitative studies of murine platelets and megakaryocytes.     

Comparative analyses of megakaryocytes derived from cord blood and bone marrow

Thrombocytopenia is typically observed in patients undergoing cord blood transplantation. We hypothesized that delayed recovery of the platelet count might be caused by defects in the megakaryocytic differentiation pathway of cord blood progenitors. To test this hypothesis, we compared the features of in vitro megakaryocytopoiesis between cord blood progenitors and those in bone marrow cells after isolation of CD34+ cells as progenitors. The proliferative responses of the progenitors in cord blood are higher than those in bone marrow cells in the presence of interleukin (IL)-3, stem cell factor (SCF) and thrombopoietin (TPO). However, the ability to generate mature megakaryocytes was higher in bone marrow progenitors than in cord blood in the same in vitro culture system, when examined by the expression of CD41, polyploidy and proplatelet formation. Furthermore, an earlier induction of c-mpl protein, a receptor for TPO, was observed in the progenitors from bone marrow than in those from cord blood in the presence of SCF and IL-3. Therefore, the ability to generate mature megakaryocytes in bone marrow progenitors is superior to that in cord blood, and the delayed engraftment of platelets after cord blood transplantation might be attributed to the features of cord blood megakaryocyte progenitors.     

Original article: relaxin reduces the number of circulating platelets and depresses platelet release from megakaryocytes: studies in rats

In previous studies we have shown that relaxin (RLX), a peptide hormone produced predominantly during pregnancy, is a powerful inhibitor of platelet aggregation. The current study shows that RLX, given systemically to rats, also affects the number of circulating platelets and their release by spleen megakaryocytes. Male albino rats were treated acutely or chronically with RLX, and the effects of the peptide evaluated by platelet count and morphological analysis of spleen megakaryocytes. The results obtained show that RLX causes a decrease of circulating platelets, which is already appreciable 1 h after a single administration and becomes significant upon a May treatment with the peptide (790000 ± 18000/ml in the RLX-treated rats versus 865 000 ± 23 000/ml in the controls; P<0.01). Coincidentally, spleen megakaryocytes show clearcut changes consistent with a depression of platelet release, namely the appearance of a thick peripheral cytoplasmic halo, filled with actin microfilaments and without platelet territories, and a virtual absence of images of thrombocytopoiesis. Based on the properties of RLX in inhibiting hemostasis, recognized in previous investigations and further proved by the present study, a role of the peptide is proposed in counteracting the hypercoagulable state which currently takes place during normal pregnancy.