Sustained thrombocytopenia in mice: serial studies of megakaryocytes and platelets

We studied thrombopoiesis in mice after the experimental induction of sustained, immune thrombocytopenia with platelet antiserum (PAS). Utilizing light and electron microscopy and a digital image analyzer to determine platelet sectional areas, we examined platelets and megakaryocytes (MK) after 120 h of sustained, severe thrombocytopenia (120CT) and during recovery from thrombocytopenia at 48 h (48R), 72 h (72R), and 120 h (120R) after cessation of administration of PAS. Mean platelet volume (MPV), determined by electrical impedance, also was measured at each time point. Platelets at 120CT (platelet count less than 50,000/microliter), 48R (platelet count 100-200,000/microliter), and 72R (platelet count approximately 1 x 10(6)/microliter) were significantly larger in sectional area than control platelets and contained increased profiles of endoplasmic reticulum and Golgi cisternae, a lower concentration of surface-connected canalicular system, and occasional membrane complexes. The largest median platelet sectional area was detected at 48R and was the largest median value observed in response to either chronic or acute thrombocytopenia. At 120R, most platelets were normal in size and cytoplasmic appearance, although some large cells remained present in the circulation. MPV paralleled the morphometric changes in platelet sectional area. MK were increased in number at 120CT, 48R, 72R, and 120R. In addition, at least half of the MK examined at 48R contained small areas of cytoplasm, devoid of organelles, that were interspersed between larger areas of organelle-filled, undemarcated cytoplasm. The modal bone marrow megakaryocyte ploidy class, determined using two-color fluorescence-activated flow cytometry, shifted from 16N to 32N in response to sustained thrombocytopenia. In contrast, during recovery and development of rebound thrombocytosis, the relative frequency of 8N megakaryocytes was significantly increased. Because there was no consistent correlation between megakaryocyte cytoplasmic characteristics and platelet morphology, these data support the hypothesis that platelet formation is not determined by compartmentalization of MK cytoplasm into platelet areas as MK mature in the bone marrow, but involves a rearrangement of MK cytoplasm immediately prior to platelet release.     

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.     

Parasinusoidal location of megakaryocytes in marrow: A determinant of platelet release

Megakaryocytopoiesis occurs in the hematopoietic (extravascular) compartment of marrow. Thus, platelets must traverse the wall of the vascular sinuses of marrow to enter the circulation. We have examined mouse and rat marrow, fixed by rapid immersion so as to maintain anatomical relationships as close to the natural state as possible. Quantitative transmission electron microscopy (TEM) of random transections of femurs established that megakaryocytes reside less than 1 μ from a marrow sinus wall with a probability unlikely to be the result of chance (P < 0.001). An intimate relationship exists between the megakaryocyte periphery and the abluminal surface of the endothelial lining cell. At the time of platelet release megakaryocyte cytoplasm invaginates and penetrates the endothelial lining cell. The penetrating cytoplasm is detached and enters the marrow circulation. From their dimensions in comparison to circulating platelets, the released cytoplasm represents a packet of platelets that undergoes further fragmentation in the circulation. The parasinusoidal location of megakaryocytes and the process of sinus-wall penetration and platelet delivery was observed by TEM and scanning electron microscopy. These studies provided quantitative support for a specific anatomical arrangement of megakaryocytes in marrow. Moreover, the process of platelet release appears to be a physiological form of metastasis with invasion of vascular walls and vascular spread of cells, that are in this case amitotic.     

Gray platelet syndrome: immunoelectron microscopic localization of fibrinogen and von Willebrand factor in platelets and megakaryocytes

An immunogold method was used for investigating the subcellular localization of von Willebrand factor (vWF) and fibrinogen (Fg) in platelets and cultured megakaryocytes from normal subjects and from three patients with the gray platelet syndrome (GPS), a rare congenital disorder characterized by the absence of alpha-granules. In normal platelets at rest, vWF was detected exclusively in alpha-granules, with a characteristic distribution: gold particles were localized at one pole of each labeled granule, outlining the inner face of its membrane. vWF was distributed similarly in the alpha-granules of megakaryocytes at day 12 of culture, where it was also found in small vesicles near the Golgi complex. In contrast, Fg was observed in the whole matrix of all platelet alpha-granules but not in the nucleoids. In platelets from three patients with GPS, vWF and Fg were distributed homogeneously in the rare normal alpha-granules, which could be recognized by their size, and also in small granules identified as abnormal alpha-granules, which were similar in size to the small, possibly immature granules present in normal megakaryocytes. In addition, in some unstimulated platelets, Fg labeling was associated with dense material in the lumen of the surface-connected canalicular system (SCCS). At day 12 of culture, megakaryocytes from the patients with GPS contained some small alpha-granules labeled for Fg and vWF identical to those found in mature platelets. The majority of alpha-granules of normal size appeared partially or completely empty. Thus, we conclude that vWF is distributed differently from Fg in normal alpha-granules, and that unstimulated platelets from patients with GPS contain Fg and vWF in a population of small granules identifiable as abnormal alpha-granules only by immunoelectron microscopy. In addition, the presence of Fg in the SCCS of gray platelets suggests a spontaneous release of the alpha- granule content.     

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.     

Localization of platelet osteonectin at the internal face of the alpha-granule membranes in platelets and megakaryocytes.

Osteonectin is a 32-Kd phosphoglycoprotein originally described in bone but also found in platelets. Platelet and bone osteonectin are different both structurally and immunologically. We have previously shown that platelet osteonectin, by binding to thrombospondin, is involved in the secretion-dependent phase of the platelet aggregation process. In this study, we used antiosteonectin antibodies in combination with immunogold labeling to investigate by electron microscopy the fine localization of osteonectin within normal and gray platelets. Using both a polyclonal and monoclonal antibody ON3, osteonectin was specifically located at the internal face of alpha-granule membranes within normal platelets. Osteonectin was not distributed within all alpha-granules, probably because of its low platelet content. In addition, using immunofluorescence, osteonectin could also be detected in immature and mature megakaryocytes with a granular pattern of staining, suggesting that osteonectin is synthesized by megakaryocytes. Using platelets from two patients with gray platelet syndrome, osteonectin was absent within all abnormal small alpha-granules, but was detected in some rare normal-sized alpha-granules. In separate double-label studies, thrombospondin and von Willebrand factor did not colocalize with osteonectin in resting platelets. However, osteonectin was located at the inner face of the alpha-granules, as it is for alpha-granule membrane protein GMP-140 and glycoprotein IIb-IIIa. These results, taken together with the fact that monoclonal antibodies to osteonectin bind only to the surface of activated platelets, suggest that platelet osteonectin is redistributed to the cell surface during fusion of alpha-granule membranes with the plasma membrane.     

Ultrastructural localization of platelet factor 4 in rat megakaryocytes and platelets by gold-labeled antibody detection

The pattern of distribution of platelet factor 4 (PF-4) was studied in rat platelets and megakaryocytes following immunohistochemical labeling with a monoclonal antibody (2E7) specific for PF-4 and visualization by a protein A-gold complex. We observed a heterogeneity in PF-4 expression among alpha granules with a minority of them being unlabeled by immunoelectron microscopy, a pattern similar in both mature, circulating platelets and developing megakaryocytes. Furthermore, the majority of the labeled alpha granules in both cell types displayed a unique, eccentric localization of the PF-4 that was mainly over the nucleoid region within each granule. This localization is similar to the microtubular localization in alpha granules reported previously for von Willebrand factor and yet distinct from the reported random distribution of fibrinogen. We also observed significant labeling of small vesicular structures in developing megakaryocytes that may be involved in the transport of PF-4 and its packaging in platelet alpha granules. This new information is important in relating patterns of PF-4 biogenesis in megakaryocytes to conditions of alpha granular dysfunction in platelets.     

Original article: evaluation of platelet function by flow cytometric measurement of ligand binding

Rapid and relevant evaluation of platelet function is often clinically important. By means of fluorescent labelled chicken antibodies (which do not bind to Fc-receptors) against fibrinogen and von Willebrand factor and flow cytometry, we have determined the time course of ligand association to platelets after stimulation with adenosine 5'-diphosphate and ristocetin respectively. The expression of guanosine 5'-phosphate (GMP)-140 was also measured. We have applied this technique to evaluate platelet function during platelet storage and cardiopulmonary bypass. There was a significant reduction of the binding of fibrinogen and von Willebrand factor and significantly increased expression of GMP-140 after 9 days of storage. Changes in metabolic variables such as lactate accumulation, glucose consumption and decrease in pH confirm that the functional impairment is due to a large extent to a deteriorated platelet metabolism. No significant differences were found between samples taken before and during cardiopulmonary bypass, but there was a tendency towards increased ligand binding as well as increased expression of GMP-140 at the end of cardiopulmonary bypass. The flow cytometric technique that is described may be useful for evaluation of platelet function and platelet activation in vivo.     

Cocaine activates platelets and increases the formation of circulating platelet containing microaggregates in humans

OBJECTIVE—To determine whether there is evidence of platelet activation following in vivo cocaine administration in humans, as cocaine abuse is associated with myocardial infarction and stroke, and platelet activation leading to thrombosis is a possible mechanism.
SETTING—University hospital.
DESIGN AND SUBJECTS—Following a randomised, double blind crossover design, 14 healthy volunteers were studied twice, receiving cocaine (2 mg/kg intranasally) once and placebo once. Flow cytometric analysis of P-selectin expression (an α granule membrane protein found on the surface of activated platelets), quantification of the platelet specific proteins platelet factor 4 and β thromboglobulin, and measurement of platelet containing microaggregate and platelet microparticle (fragment) formation were used to assess platelet activation. Circulating von Willebrand factor antigen (vWF) was measured to evaluate a possible role of endothelial stimulation concurrent with platelet activation.
RESULTS—There was an increase in both platelet factor 4 (mean (SD), 16 (7) to 39 (22) IU/ml, p = 0.04) and β thromboglobulin (70 (20) to 98 (26) IU/ml, p < 0.01) at 120 minutes following cocaine administration. Platelet containing microaggregate formation was increased at 40 minutes (from 47 (3.2)% to 54 (2.0)%, p < 0.001) and 80 minutes (55 (2.5)%, p = 0.04). Bleeding time decreased following cocaine from 10 (1) to 9 (1) minutes (p = 0.07). No changes in any of the measured variables were noted following placebo administration.
CONCLUSIONS—Cocaine exposure causes platelet activation, α granule release, and platelet containing microaggregate formation. These data support the view that cocaine, even at the relatively low doses commonly self administered by occasional abusers, may promote thrombosis and predispose healthy individuals to ischaemic events. Platelet inhibitors should be considered early in any patient with suspected cocaine related ischaemia.


Keywords: platelets; cocaine; flow cytometry; myocardial infarction     

Diagnostic significance of the number of bone marrow megakaryocytes in cases with decreased platelet production evidenced by platelet kinetic study

In 18 patients with decreased platelet production proved by reduced platelet turnover, the numbers of megakaryocytes in clot section of marrow aspirate were counted. Platelet counts ranged from 19,000 to 128,000/microliters. The sternal marrow showed decrease of megakaryocytes only in 3 out of 18 cases while megakaryocytes in the iliac marrow were reduced in 6 out of 11 cases. Only one of 11 cases showed decrease of megakaryocyte in both marrows examined. On the contrary, four cases showed normal number of megakaryocytes at both sites. Of 9 cases with normal or increased number of megakaryocytes in sternal and/or iliac marrow, no case showed morphologically abnormal megakaryocytes and only one case had the reduced number of platelet-forming megakaryocytes. Results suggested that the number of megakaryocytes in bone marrow especially in the sternum was not reliable for establishing the diagnosis of decreased platelet production, and that about 50% of patients with thrombocytopenia due to hypoproduction of platelets could be diagnosed pertinently only after the study of platelet kinetics.     

Occurrence of platelet basic protein, a precursor of low affinity platelet factor 4 and beta-thromboglobulin, in human platelets and megakaryocytes

beta-thromboglobulin antigen, a platelet-specific secreted protein, occurs in three forms: platelet basic protein, low affinity platelet factor 4, and beta-thromboglobulin. The combined level of beta-thromboglobulin antigen in megakaryocytes, measured by radioimmunoassay, was 13 +/- 7 micrograms/10(6) cells (SD, n = 6). The relative proportions of the three forms of beta-thromboglobulin antigen present within platelets and megakaryocytes were determined in cells lysed with trichloroacetic acid to minimize artifactual proteolysis. Samples were analyzed by isoelectric focusing in polyacrylamide gel with quantitative immunological detection on a nitrocellulose transfer of the gel. In platelets, the major species found was low affinity platelet factor 4 with precursor platelet basic protein as only 25% +/- 11% (SD, n = 16) of total beta-thromboglobulin antigen. In megakaryocytes partially purified both from normal bone marrow aspirates and from whole marrow specimens obtained after surgery, platelet basic protein was a higher proportion of beta-thromboglobulin antigen (49% +/- 13% SD, n = 11) than was the case in platelets. beta-thromboglobulin itself was never detected under the conditions of cell lysis used. Our results suggest that platelet basic protein is synthesized in megakaryocytes and that its cleavage is associated with an earlier stage of cell development than simply maturation to platelets. Further support for the precursor status of platelet basic protein was found in the expression of predominantly this antigenic form in a human erythroleukemia cell line.     

Expression of stanniocalcin-1 in megakaryocytes and platelets

Stanniocalcin-1 (STC) is a 56-kDa homodimeric glycoprotein hormone originally found in fish, in which it regulates calcium/phosphate homeostasis and protects against toxic hypercalcaemia. The recently characterized human STC is 80% similar to fish STC. We have earlier reported a high expression of STC in terminally differentiated human and rodent brain neurones, and found that STC contributes to the maintenance of their integrity. Here, we report that mature megakaryocytes and platelets display high STC content. K562 cells, induced to megakaryocytoid differentiation in vitro, acquired expression of STC, which was not seen in untreated K562 cells or cells induced to erythroid differentiation.     

Human platelet antigen-1a antibodies induce the release of the chemokine RANTES from human platelets

BACKGROUND AND OBJECTIVE: Binding of human platelet antigen-1a (HPA-1a)-specific antibodies to target platelets can trigger platelet activation and mediator release. Here we tested the effect of HPA-1a antibody-containing sera on platelet release of the chemokine RANTES (regulated on activation, normal, T-cell expressed, and presumably secreted) in vitro. PATIENTS AND METHODS: HPA-1a-containing sera obtained from 11 mothers delivered of an infant with neonatal alloimmune thrombocytopenia (NAIT) and from six patients with post-transfusion purpura (PTP) were incubated with HPA-1a/a target platelets. Antibody-induced release of soluble RANTES was determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: A significant release of soluble RANTES was induced by four out of the 17 sera. Two out of the four reactive sera were obtained from mothers who were delivered of a baby with NAIT and the remaining two sera were from patients with PTP. Chemokine release was specific for binding of anti-HPA-1a to the platelet membrane, as none of the reactive sera induced the release of soluble RANTES when incubated with HPA-1b/b platelets. The blockade of platelet-expressed Fc gamma receptor type II (FcgammaRII) inhibited anti-HPA-1a-mediated RANTES release when incubated with the reactive sera of patients with NAIT, but not when platelets were incubated with sera of patients with PTP. CONCLUSION: Our findings suggest that anti-HPA-1a antibody-induced release of platelet-derived RANTES can play a role in adverse reactions in alloimmunized patients.     

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.     

Mechanisms of platelet release: in vivo studies and in vitro modeling

Abstract

Mechanisms related to platelet release in the context of the bone marrow niche are not completely known. In this review we discuss what has been discovered about four critical aspects of this process: 1) the bone marrow niche organization, 2) the role of the extracellular matrix components, 3) the mechanisms by which megakaryocytes release platelets and 4) the novel approaches to mimic the bone marrow environment and produce platelets ex vivo.     

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