Labelling autologous platelets with 111In tropolonate for platelet kinetic studies: limitations imposed by thrombocytopenia

An in vitro method of radiolabelling platelets with 111In tropolonate in plasma has been devised enabling imaging and cell kinetic studies to be performed in patients with thrombocytopenia (TP) using autologous, rather than donor, platelets. Platelets from 10 TP patients, with platelet counts ranging from 4-91 x 10(9)/l, were labelled in 50% plasma with 111In tropolonate, containing the optimum tropolone concentration of 2 x 10(-4) mol/l, at platelet concentrations ranging from 0.08-4.5 x 10(9)/ml resulting in labelling efficiencies (LE) between 51 and 86%. In 4 patients, red cells contaminated the platelets but this was corrected for by measuring the proportion of 111In on the platelets prior to injection and in the post-injection blood samples. Platelet recovery (PR), mean platelet life-span (MPLS) and platelet production rate (PPR) were calculated and splenic and hepatic images were taken. The results clearly show that useful clinical data can be obtained by this method even in patients with severe TP.     

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.     

Substantial expression of glycoproteins IX and V on the platelet surface from a patient with Bernard-Soulier syndrome

Summary. Flow cytometric studies demonstrated that the platelets from a patient with Bernard-Soulier syndrome expressed substantial amounts of both GPIX and GPV, although in reduced amounts.     

A case of Bernard-Soulier syndrome: study of platelet glycoprotein Ib in a kindred

The Bernard-Soulier syndrome is characterized by low platelet counts, abnormally large (giant) platelets, and impaired or absent platelet aggregation by the inducer antibiotic ristocetin. The recent discovery of the inherited biochemical defect and the deficient synthesis of platelet glycoprotein Ib (GP-Ib), has contributed greatly to the understanding of the disease. We report a case of the Bernard-Soulier syndrome presenting with bleeding from the pharynx after adenotomy. The patient and nearest family members were studied by a novel immunoperoxidase method for quantification of platelet glycoprotein Ib using a specific monoclonal antibody (AN51).     

Intrinsic impaired proplatelet formation and microtubule coil assembly of megakaryocytes in a mouse model of Bernard-Soulier syndrome

Background

Giant platelets and thrombocytopenia are invariable defects in the Bernard-Soulier syndrome caused by deficiency of the GPIb-V-IX complex, a receptor for von Willebrand factor supporting platelet adhesion to the damaged arterial wall. Various properties of this receptor may be considered potential determinants of the macrothrombocytopenia.

Design and Methods

To explore the underlying mechanisms of the disease, megakaryopoiesis was studied in a mouse model deficient in GPIbβ. Megakaryocytes were initially characterized in situ in the bone marrow of adult mice, after which their capacity to differentiate into proplatelet-bearing cells was evaluated in cultured fetal liver cells.

Results

The number of megakaryocyte progenitors, their differentiation and progressive maturation into distinct classes and their level of endoreplication were normal in GPIbβ^−/− bone marrow. However, the more mature cells exhibited ultrastructural anomalies with a thicker peripheral zone and a less well developed demarcation membrane system. GPIbβ^−/− megakaryocytes could be differentiated in culture from Lin⁻ fetal liver cells in normal amounts but the proportion of cells able to extend proplatelets was decreased by 41%. Moreover, the GPIbβ^−/− cells extending proplatelets displayed an abnormal morphology characterized by fewer pseudopodial extensions with thicker shaft sections and an increased diameter of the terminal coiled elements. GPIbβ^−/− released platelets were larger but retained a typical discoid shape. Proplatelet formation was similarly affected in bone marrow explants from adult mice examined by videomicroscopy. The marginal microtubular ring contained twice as many tubulin fibers in GPIbβ^−/− proplatelet buds in cultured and circulating platelets.

Conclusions

Altogether, these findings point to a role of the GPIb-V-IX complex intrinsic to megakaryocytes at the stage of proplatelet formation and suggest a functional link with the underlying microtubular cytoskeleton in platelet biogenesis.     

A flow cytometric assay using mepacrine for study of uptake and release of platelet dense granule contents

Summary. Diagnosis of platelet dense granule storage pool disease and release defects at present requires a combination of studies including lumiaggregometry, conventional platelet aggregation, radioactive serotonin uptake and release, and electron microscopy. Flow cytometric methods have been developed to study platelet activation, aggregation, and α-granule protein release. Here, we have investigated the use of flow cytometry for analysis of platelet dense granule content uptake and release using mepacrine as a fluorescent marker. Mepacrine (quinacrine) is rapidly taken up and localized in dense granules of platelets. For the assay, as little as 20 μl of blood from a fingerstick collected without anticoagulant or venous blood collected in 3.8% sodium citrate were diluted 1:40 with 2 ml Hanks balanced salt solution (BSS). 300 μl of this cell suspension were incubated with mepacrinc alone, or simultaneously with a mouse monoclonal antibody to human platelet glycoprotein IIb (Tab), used as a platelet-specific marker. The bound monoclonal antibody was then indirectly labelled with the fluorochrome, RED670. 100 μl of the sample were further diluted with Hanks BSS for one- or two-colour flow cytometric analysis. To verify that mepacrine uptake was related to platelet dense granule content, platelets of beige mice, a strain with dense granule deficiency, were examined. Their mepacrine uptake was substantially decreased compared to that of normal mice. Decreased mepacrine uptake also was demonstrated in platelets of a patient with Hermansky-Pudlak syndrome in which a deficiency of platelet dense granules is characteristic. In both human and mouse platelets, mepacrine uptake was proportional to platelet size. Thrombin induced mepacrine release in a dose-dependent manner from 0.003 to 0.4 U/ml. Therefore both platelet uptake and release of mepacrine can be readily detected by flow cytometry. Flow cytometry provides an attractive alternative to aggregation and radioactive serotonin as methods to study defects in platelet dense granule function.     

Flow cytometric analysis of platelets from childrenwith the Wiskott-Aldrich syndrome reveals defects in platelet development, activation and structure

The pathophysiology of platelet dysfunction in the Wiskott-Aldrich immune deficiency syndrome (WAS) remains unclear. Using flow cytometry, we have characterized the functional properties of platelets from 10 children with WAS. Patients with WAS had thrombocytopenia, small platelets, increased platelet-associated IgG and reduced platelet-dense granule content. Levels of reticulated 'young' platelets were normal in the WAS patients. Although the mean numbers of platelet glycoprotein (GP) Ib, GPIIbIIIa and GPIV molecules per platelet appeared lower in WAS patients than in healthy controls, analysis of similar-sized platelets revealed the mean number of GPIb molecules per platelet to be comparable in patients and normal controls. Surface GPIIbIIIa and GPIV expression was, however, significantly lower on the WAS platelets than on normal platelets. Compared with normal platelets, WAS platelets showed a reduced ability to modulate GPIIbIIIa expression following thrombin stimulation. In addition, thrombin- and ADP-induced expression of CD62P and CD63 was defective in WAS platelets. Phallacidin staining of the WAS platelets revealed less F-actin content than in normal platelets. Together, these data suggest that the reduced platelet number and function in WAS reflects, at least in part, a defect in bone marrow production as well as an intrinsic platelet abnormality.     

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.     

Synthesis of osteoprotegerin and RANKL by megakaryocytes is modulated by oestrogen

To investigate the mechanisms by which megakaryocytes (MKs) may influence bone remodelling, CD34(+) cells were cultured for 6, 9 and 12 d with or without 17beta-oestradiol (E) and immunolocalized for osteoprotegerin (OPG), receptor activator of nuclear factor (NF)-kappaB ligand (RANKL) and CD61. Specific protein expression was measured quantitatively by image analysis. Fluorescence-based immunocytochemistry was used to co-localize OPG and RANKL with CD61. OPG and RANKL mRNA was assessed in CD61(+) cells with or without E at 24 and 48 h. At 6 d, OPG and RANKL expression was unchanged by E treatment. At 9 d, the E-treated cultures with maturing MKs showed a 1.72-fold (P < 0.01) increase in OPG expression and a 1.8-fold (P < 0.01) reduction in RANKL. Maximal OPG expression was seen at 12 d with a threefold induction of expression (P < 0.001), whilst RANKL levels were further suppressed by 2.3-fold compared with controls (P < 0.001). CD61 co-localized with OPG and RANKL. mRNA data were consistent with that of protein, with a 90-fold induction in OPG expression and a 34-fold suppression of RANKL expression by E (P < 0.001). Thus, E stimulates megakaryocytopoiesis and modulates OPG and RANKL expression, providing evidence that MKs may play a role in bone remodelling and, in particular, in E-induced changes in osteoclastogenesis and bone resorption.     

Giant electron-dense chains,clusters and granules in megakaryocytes and platelets with normal dense bodies: an inherited thrombocytopenic disorder I. Megakaryocytes

A woman and her male child were referred because of life-long thrombocytopenia, moderately increased platelet size, and absence of laboratory findings suggesting immune thrombocytopenia or defective platelet function. Evaluation of their platelets in the electron microscope revealed the presence of organelles never seen before in human cells. The present study has focused on megakaryocyte pathology to be sure the aberrant platelet organelles originated in the parent cell. There were two different types of organelles developing in megakaryocytes from our patients unrelated to the formed organelles in the large cells. No relationship could be identified between the aberrant structures and alpha granules, lysosomes or dense bodies. One of the large organelles was intensely electron opaque and appeared to arise from small dense fragments forming in segments of endoplasmic reticulum. The second, target-like organelle also appeared to develop in the endoplasmic reticulum. Its smallest precursors were hexagonal fragments with a granular layer inside a more electron-dense outer layer. Fusion of these elements resulted in formation of typical target-like organelles. Ultimately the target-like organelles became electron opaque and difficult to distinguish from the large dense bodies. Other features of these unusual structures were identified in platelets from the two patients and will be discussed in a subsequent report.     

Organ distribution and fate of human platelets: studies of asplenic and splenomegalic patients

Little is known about the organ distribution and fate of human platelets. We investigated the kinetics, organ distribution, and fate of autologous 111In-oxine-labeled platelets in 12 normal volunteers, four asplenic subjects, and four patients with splenomegaly. The initial recovery of infused 111In-platelets from the circulation was 97.8 +/- 9.8% (means +/- SD) for asplenic subjects and 26.3 +/- 5.9% for splenomegalic patients as compared to 59.2 +/- 9.3% for normal controls. The mean platelet survival times as derived from the multiple-hit model were 9.2 +/- 1.0 days for asplenics and 6.2 +/- 0.6 days for splenomegalic subjects (8.4 +/- 0.8 days for normals). At 30 min postinfusion, 79.4 +/- 19.2% of the infused 111In-platelets pooled in the spleen of splenomegalic subjects and 42.7 +/- 12.2% in normal controls. There was 7.1 +/- 2.0, 12.6 +/- 3.7, and 29.3 +/- 8.4% pooling in the liver of splenomegalic, normal, and asplenic subjects, respectively. At 10 days postinfusion, 37 and 24% of the 111In-platelets were sequestered in the spleen and liver of normal control subjects, respectively. Similar figures for splenomegalic subjects were 71 and 14%, respectively. In asplenic subjects, 89% was sequestered in the liver. We conclude that spleen and liver are the primary sites of platelet destruction, accounting for 61% of infused 111In-platelets in normal volunteers and 85% in splenomegalics, while the liver is the primary site of platelet destruction, accounting for 89% in asplenic subjects.     

Molecular characterization of two mutations in platelet glycoprotein (GP) Ib alpha in two Finnish Bernard-Soulier syndrome families

Bernard-Soulier syndrome (BSS) is a rare hereditary bleeding disorder and macrothrombocytopenia which is caused by a defect in the platelet glycoprotein Ib/IX/V (GP Ib/IX/V) complex, the receptor for von Willebrand factor and thrombin. Here we report the molecular basis of the classical form of BSS in two unrelated Finnish patients, both with a life-long history of severe bleeding. Flow cytometry and immunoblotting showed no expression of GP Ib/IX, GP Ib alpha, GP Ib beta or GP IX (less than 10%) in the patients' platelets. No expression of GP V (< 10%) was observed in propositus 1, but a residual amount was found in propositus 2 (24%). DNA sequencing analysis revealed that propositus 1 was compound heterozygous for a two-base-pair deletion at Tyr505(TAT) and a point mutation Leu129(CTC)Pro(CCC) in the GP Ib alpha gene. Propositus 2 was homozygous for the Tyr505(TAT) deletion. The nine relatives who were heterozygous for either of the mutations also had low levels of GP Ib alpha (74-90%). Hence, Bernard-Soulier patients homozygous or compound heterozygous for Tyr505(TAT) are severely affected. Interestingly, both mutations have independently been found in three other families in previous reports, suggesting their ancient age or mutational 'hot spot'.     

Activation during preparation of therapeutic platelets affects deterioration during storage: a comparative flow cytometric study of different production methods

Three different separation methods, all using centrifugation, are routinely used to prepare therapeutic platelet concentrates from human donor blood. Platelet concentrates derived from platelet-rich plasma (PRP-PC), buffy coat (BC-PC) and apheresis (AP-PC) were investigated at the end of production, and over an 8 d storage period. Change in platelet surface markers were measured by flow cytometry, using fluorescein-conjugated antibodies to fibrinogen, P-selectin (CD62P), GPIIb–IIIa (CD41), GPIbα (CD42b) and GPV (CD42d), and fluorescein-conjugated Annexin V was used to measure expression of anionic phospholipid.
All concentrates showed some changes during preparation but PRP-PC underwent the greatest changes with significantly higher levels of P-selectin ( P  < 0.001) and bound Annexin V ( P  = 0.001) than AP-PC or BC-PC, and lower levels of GPIbα ( P  = 0.002) and GPV ( P  < 0.001). These changes were attributable to component separation rather than venesection. These markers all continued to change on storage with a strong positive correlation between the changes seen during production and those after 5 d storage. PRP-PC continued to show the greatest changes whereas BC-PC showed the least. Fibrinogen was bound to 40–50% of platelets in all preparations and this did not alter significantly on storage whereas total expression of GPIIb–IIIa remained unchanged throughout.
There was no evidence that the platelet surface changes were thrombin-mediated and leucocyte depletion of BP-PC by filtration had no effect on the changes. It is proposed that the deterioration of platelet concentrates during storage may be related to activation occurring during preparation. 'Whole blood' flow cytometry using a panel of fluorescein-labelled reagents provides an informative method for evaluating platelet concentrates.     

Diagnosis of platelet function disorders: A standardized,rational, and modular flow cytometric approach

A high proportion of patients with mucocutaneous bleeding diathesis and suspected inherited or acquired platelet disorder remain without diagnosis even after comprehensive laboratory testing. Since flow cytometry allows investigation of resting and activated platelets on the single cell level by requiring only minimal amounts of blood, this method has become an important assay within the diagnostic algorithm, especially in pediatrics. We therefore developed a standardized and modular flow cytometric approach that contributes to clarify impaired platelet function in a rational step-by-step manner.

Due to simultaneous analysis of four fluorophores in a basic panel design, we are able to readily detect the most common and clinically significant platelet disorders: Glanzmann thrombasthenia or Glanzmann-like diseases (fibrinogen receptor GPIIb-IIIa), Bernard–Soulier syndrome (von Willebrand-factor receptor complex GPIb-IX-V) and less well characterized β1-integrins that serve as the collagen, laminin or fibronectin receptor (CD29-CD49b, e and f, respectively). Platelet reactivity was investigated in response to the agonists adenosine diphosphate (ADP) and thrombin receptor activator peptide 6 (TRAP6) in suboptimal and optimal concentrations by quantifying surface expression of activation markers CD62P and CD63 as well as binding of PAC-1 antibody to the high affinity conformation of the fibrinogen receptor. For advanced diagnostic questions, several further modules were implemented: (i) calcium mobilization for evaluation of early signal transduction, (ii) a kinetically resolved mepacrine assay for estimation of delta-granule content and release, and (iii) a module to determine platelet reactivity upon additional agonists like the thromboxane A₂-analogue U46619 or collagen. Blood withdrawn from a healthy control cohort allowed generating preliminary standard values for all parameters. The modules were validated by analysis of patients with known or suspected platelet defects (leukocyte-adhesion deficiency type III, Wiskott–Aldrich syndrome, acute myeloid leukemia, sickle cell disease and chronic immune thrombocytopenia).     

Optimally functional fluorescein isothiocyanate-labelled fibrinogen for quantitative studies of binding to activated platelets and platelet aggregation

Dynamic and quantitative studies of the binding of fibrinogen (Fg) to its receptor, GPIIb–IIIa, on activated platelets, leading to platelet aggregation, are best studied with fluorescently-labelled Fg by flow cytometry. Due to conflicting reports on the functionality of FITC-labelled Fg, we have developed a reproducible and 'mild' labelling of fibrinogen with FITC-celite at pH 7.4–8.5 for direct and dynamic studies of specific Fg binding to activated platelets evaluated for native platelet-rich plasma, for washed platelets, and for activated, fixed platelets. We have demonstrated the equivalence of FITC-labelled and unlabelled Fg for binding to activated GPIIb–IIIa receptors, and in the rate and extent of mediating platelet aggregation. We found that FITC-Fg labelled at pH ≥9 had reduced to absent specific binding to activated platelets, whether using soluble FITC or FITC-celite. The FITC-labelled Fg must be diluted 3-fold with unlabelled Fg when evaluating maximal Fg binding to activated platelets in order to prevent autoquenching of the FITC-Fg which leads to underestimation of Fg levels. The dissociation constant ( K _D) of Fg on stable preparations of activated, fixed platelets, determined with FITC-Fg binding to platelets by flow cytometry, was in the range reported for ¹²⁵I-labelled Fg, 70–255 n m with B_max = 10 000–25 000 Fg per platelet ( n   = 20). The FITC-Fg was used to monitor Fg binding to activated platelets directly in plasma, as well as to evaluate platelet subpopulations which maximally bind Fg according to the concentration of ADP used as activator. It is expected that this 'mildly' labelled FITC-Fg will stimulate further studies of platelet activation directly in native anticoagulated blood/plasma, for both basic and clinical research.     

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.     

Platelet aggregates as markers of platelet activation: Characterization of flow cytometric method suitable for clinical applications

The present paper describes a flow cytometric method for assay of platelet aggregates (PAg) in blood. This method combines and simplifies previously reported techniques, simultaneously enumerating PAg formed upon platelet activation, their expression of activation marker CD62P (P-selectin), and their content of bound leukocytes (LPAg). The sensitivity of this method to low levels of agonists (ADP, collagen) is compared to conventional aggregometry and some features of platelet-leukocyte interaction are explored. The results were: (1) ADP or collagen induced a dose-dependent increase in PAg number and corresponding decline in free platelets. The ED₅₀ for ADP (0.15 μM) and for collagen (0.2 μg/mL) was about 1/20 the ED₅₀ found by aggregometry, indicating 20-fold greater sensitivity. (2) At higher concentrations, the fraction of PAg with bound leukocytes (LPAg) increased to 60–70%. This rise correlated with PAg size and CD62P expression, but not with the number of PAg formed. (3) The response of whole blood (WBD) to agonists was qualitatively different from that of platelet-rich plasma (PRP): in WBD the population of CD62P+ PAg was much higher than in PRP and the population of CD62P+ free platelets was much lower. This implies that leukocytes rapidly recruit activated platelets. (4) Desmopressin (DDAVP) at 5 nmol/L induced a significant rise in activated (CD62P+) PAg and platelets, even though no effect of DDAVP could be detected by conventional aggregometry; this further confirms that DDAVP acts directly on platelets. (5) Plasma samples from TTP patients induced a rise in PAg when added to normal PRP, though little or no effect could be detected by aggregometry. In summary, the flow cytometric method described here appears useful for detecting low levels of platelet activation and provides information on platelet leukocyte interaction, potentially important in identifying and differentiating thrombogenic states. Since it is rapid and economical, it is well suited for clinical implementation. Am. J. Hematol. 57:33–42, 1998. © 1998 Wiley-Liss, Inc.     

Platelet-activating factor is a weak platelet agonist: Evidence from normal human platelets and platelets with congenital secretion defects

We have examined the effects of a novel platelet agonist, platelet activating factor (PAF), on human platelets. Irreversible aggregation and ¹⁴C-serotonin secretion in response to PAF (10^?5 M) was found to be dependent on both thromboxane production and secreted adenosine diphosphate (ADP). Liberation of arachidonic acid (AA) from membrane-bound phospholipids is a prerequisite step in platelet thromboxane production. Studies with ³H-AA-labeled platelets revealed that PAF (10^?5 M) was a weak stimulus for the mobilization of AA. In addition, PAF (10^?5M) was found to be a weak inducer of thromboxane synthesis (mean = 6 pmol/10⁸ platelets) as compared to thrombin 5 U/ml (mean = 177 pmol/10⁸ platelets), measured using a radioimmunoassay for thromboxane B₂. Formation of phosphatidic acid is an early step in stimulus-response coupling in platelets. Our studies indicate that PAF is a weak stimulus for phosphatidic acid formation as well. To obtain further insights into its action, we examined the effect of PAF on platelets from three groups of patients with congenital secretion defects: patients with the storage pool deficiency, those with impaired thromboxane synthesis due to impaired liberation of AA from phospholipids, and those with impaired secretion despite normal granule stores and thromboxane production. The response to PAF was impaired in all patients, providing further evidence that PAF-induced platelet activation is dependent on secreted ADP and thromboxane A₂ synthesis, and occurs by mechanisms common to a number of agonists. Overall, these studies indicate that PAF is a weak platelet agonist.