Phosphotyrosine proteins in platelets from patients with storage pool disease: direct relation between granule defects and defective signal transduction

BACKGROUND AND OBJECTIVES: Storage pool diseases (SPD) are heterogeneous disorders associated with an abnormal presence of intraplatelet granules, which cause mild to moderate bleeding diathesis. We investigated signaling through tyrosine phosphorylation of proteins occurring in platelets with total or partial absence of dense- and alpha-granules in response to activation. DESIGN AND METHODS: We included a patient with severe delta-SPD, a patient with severe alpha-SPD or gray platelet syndrome, and six patients with partial deficiency of dense or a-granules. SPD was confirmed by electron microscopy evaluation of platelet ultrastructure. Platelet function was evaluated by bleeding time determination and conventional aggregometry. Platelet suspensions were activated with collagen and thrombin to analyze changes in tyrosine phosphorylation of proteins by electrophoresis and Western-blotting. RESULTS: Bleeding times were prolonged in all the patients included. Aggregation responses were slightly decreased in delta-SPD and normal in the rest of patients. Tyrosine phosphorylation in platelets from patients with partial forms of SPD was equivalent to that observed in control platelets, absent in response to collagen and thrombin activation in delta-SPD, and deficient only to thrombin activation in alpha-SPD. INTERPRETATION AND CONCLUSIONS: Tyrosine phosphorylation of proteins in activated platelets is highly dependent on the substances contained in the dense-granules and moderately dependent on those contained in the alpha-granules. A minimum amount of intraplatelet granules ensures signaling through tyrosine phosphorylation of proteins.     

A Flow Cytometric Approach to Quantitative Estimation of Platelet Surface Immunoglobulin G

Flow cytometry (FC) was used to estimate platelet-surface IgG (PSIgG) by quantifying the fluorescence of platelets incubated with a fluorescein isothio-cyanate (FLTC)-labelled polyclonal goat anti-human IgG antibody or FITC-labelled non-immune goat IgG. Results were expressed as relative fluorescence intensity (RFI) defined as the ratio of specific fluorescence (mean fluorescence of platelets incubated with the FITC anti-IgG) over non-specific fluorescence (mean fluorescence of platelets incubated with FITC non-immune goat IgG). A normal range was formed by analysing platelets from 71 healthy subjects. Platelets from 16 patients with a firm clinical diagnosis of immune-mediated thrombocytopenia had a mean RFI significantly higher (p <0.001) than the controls, whereas platelets from 9 patients thought to have non-immune thrombocytopenia had an RFI not significantly different from the normal controls. From a prospectively studied group of 62 patients with no clinically obvious cause for their thrombocytopenia or impaired platelet function 35.5% had raised PSIgG. In order to express the results as number of IgG molecules per platelet, reference curves were created by using FC to nieasurc PSIgG of platelets coated with known amounts of a chimeric IgG (human IgG with murine hypervariable region) monoclonal antibody to the glycoprotein IIb-IIIa complex. Normal platelets had an average 1,463 (SD = 927) molecules of PSIgG. In patients with immune-mediated thrombocytopenia the levels ranged from 690 to 32,328 (mean 11,535) molecules per platelet.
Flow-cytometric PSIgG estimation was sensitive, fast and easy to perform and therefore suitable for both research and clinical service purposes.     

A Flow Cytometric Approach to Quantitative Estimation of Platelet Surface Immunoglobulin G

Flow cytometry (FC) was used to estimate platelet-surface IgG (PSIgG) by quantifying the fluorescence of platelets incubated with a fluorescein isothio-cyanate (FLTC)-labelled polyclonal goat anti-human IgG antibody or FITC-labelled non-immune goat IgG. Results were expressed as relative fluorescence intensity (RFI) defined as the ratio of specific fluorescence (mean fluorescence of platelets incubated with the FITC anti-IgG) over non-specific fluorescence (mean fluorescence of platelets incubated with FITC non-immune goat IgG). A normal range was formed by analysing platelets from 71 healthy subjects. Platelets from 16 patients with a firm clinical diagnosis of immune-mediated thrombocytopenia had a mean RFI significantly higher (p <0.001) than the controls, whereas platelets from 9 patients thought to have non-immune thrombocytopenia had an RFI not significantly different from the normal controls. From a prospectively studied group of 62 patients with no clinically obvious cause for their thrombocytopenia or impaired platelet function 35.5% had raised PSIgG. In order to express the results as number of IgG molecules per platelet, reference curves were created by using FC to nieasurc PSIgG of platelets coated with known amounts of a chimeric IgG (human IgG with murine hypervariable region) monoclonal antibody to the glycoprotein IIb-IIIa complex. Normal platelets had an average 1,463 (SD = 927) molecules of PSIgG. In patients with immune-mediated thrombocytopenia the levels ranged from 690 to 32,328 (mean 11,535) molecules per platelet.
Flow-cytometric PSIgG estimation was sensitive, fast and easy to perform and therefore suitable for both research and clinical service purposes.     

Inherited giant platelet disorders

Giant platelet disorders (GPD) refer to rare, usually inherited states characterized by abnormally large platelets, thrombocytopenia and bleeding tendency of variable severity. This review summarizes major clinical and laboratory features of three GPDs (Bernard-Soulier syndrome, May-Hegglin anomaly and gray platelet syndrome). Differential diagnosis between immunological thrombocytopenia and GPDs is important. Although rare, giant platelet disorders should be borne in mind, since bleeding tendency in some individuals may be severe and knowledge of bleeding diathesis is of importance before delivery or surgical procedures also in less symptomatic individuals.     

Chronic isolated macrothrombocytopenia with autosomal dominant transmission: a morphological and qualitative platelet disorder

Abstract: We studied 47 subjects belonging to 13 unrelated families with a history of mild haemorrhagic diathesis and chronic thrombocytopenia. 36 patients presented some degree of thrombocytopenia: 7/36 (19%) had slight thrombocytopenia (100–150×10⁹/L); 26/36 (72%) had mild thrombocytopenia (50–100×10⁹/L) and 3/36 (8%) had severe thrombocytopenia (<50×10⁹/L). No correlation was observed between platelet count and the degree of haemorrhagic diathesis, which was mild in the majority of patients. Transmission was autosomal dominant. Platelet anisocytosis, increased percentage of large platelets and absence of leukocyte inclusions were observed in 26/30 (87%) of the examined blood smears. The ultrastructural appearance of platelets was normal. Megakaryocytes appeared normal in number in 10/10 patients, but showed asynchronous nuclear-cytoplasm maturation and mainly nonlobulated nuclei. Platelet aggregation was studied in 26 patients and either increased or decreased curves were variably observed in response to different aggregating agents. Platelet-associated IgG (PAIgG) was increased in 18/31 (58%) patients, while serum autoantibodies against platelet glycoproteins Ib/IX or IIb/IIIa were demonstrable in only 1 case. An increased expression of platelet surface glycoproteins Ib and IIb/IIIa, as studied by murine monoclonal antibodies binding in 17 cases, was observed. Platelet survival performed by 111In-oxine-labelled autologous platelets was normal in the 3 studied patients. Congenital macrothrombocytopenia confirms to be a distinct clinical disorder for which the name of “chronic isolated hereditary macrothrombocytopenia” is proposed.     

Type IIB Tampa: a variant of von Willebrand disease with chronic thrombocytopenia, circulating platelet aggregates, and spontaneous platelet aggregation

Type IIB von Willebrand disease is characterized by enhanced ristocetin- induced platelet aggregation and absence of large von Willebrand factor multimers from plasma. An alteration of the von Willebrand factor molecule resulting in increased reactivity with platelets appears to be the basis for these abnormalities. We have now identified a new variant of type IIB von Willebrand disease in a family in which the four affected members also have chronic thrombocytopenia, in vivo platelet aggregate formation, and spontaneous platelet aggregation in vitro. In spite of repeatedly prolonged bleeding times and persistent thrombocytopenia, their bleeding diathesis is only moderate.     

Acute profound thrombocytopenia without bleeding complications after abciximab administration

Abciximab (c7E3) is the Fab fragment of the chimeric monoclonal antibody directed against glycoprotein IIb/IIIa found on the surface of platelets. It is the first FDA-approved platelet receptor glycoprotein inhibitor. Severe thrombocytopenia (defined as platelet count < 50 x 109/L) on first administration is a rare complication, occurring in only 1.6% of patients in the EPIC (Evaluation of 7E3 for the Prevention of Ischemic Complications) study. Bleeding complications were the rule in these thrombocytopenic patients as reported by EPIC investigators, Kereiakes et al. and Berkowitz et al. Platelet transfusions are required to reverse the bleeding diathesis. Intravenous IgG has not reportedly been helpful in reversing thrombocytopenia, and experience with corticosteroids is not reported in the literature. We report a case of acute profound thrombocytopenia following abciximab administration treated with platelet transfusion augmented by intravenous corticosteroids, in whom there was no bleeding complication.     

Bernard-Soulier syndrome: diagnosis by an ELISA method using monoclonal antibodies in 2 new unrelated patients

Two unrelated patients with Bernard-Soulier syndrome and their relatives were studied. The patients demonstrated severe bleeding diathesis, the relatives were asymptomatic. The propositi showed the characteristic abnormalities of the syndrome: thrombocytopenia, a percentage of giant platelets higher than 65%, prolonged bleeding time and defective platelet aggregation to ristocetin and bovine plasma. On the contrary, in the heterozygotes, the typical abnormalities were not fully evident. We introduce a simple ELISA method for the precise definition of both homozygous and heterozygous states for the syndrome by the quantitation of platelet glycoprotein (GP) Ib. Specific binding of monoclonal antibodies anti-platelet GPIb was performed both by direct binding of radioiodinated antibody and by ELISA. Comparable results were obtained. In fact, we demonstrated near absence of GPIb in the 2 propositi and about half the amount in the heterozygotes studied compared to normal platelets.     

Qualitative Platelet Defects with Reduced Life-Span in Acute Leukaemia*

Summary: Qualitative platelet abnormalities, as well as thrombocytopenia, may contribute to the haemorrhagic diathesis in acute leukaemia. The prevalence of impaired platelet function, as reflected by aggregation response to ADP, adrenaline and Ristocetin, and platelet factor III activity, was assessed in 21 patients with acute leukaemia. During the phases of active disease or partial remission, impaired aggregation and/or defective platelet factor lll release occurred in eleven of fifteen patients with active disease and in three of five patients in partial remission. In complete remission, platelet function was normal. In patients with active disease or in partial remission, defective platelet function was associated with reduced autologous platelet life-spans, whilst normal platelet function was coupled with normal platelet survival. Although the mechanisms for the production of thrombocytopenia in acute leukaemia are multifactorial, it is suggested that the acquisition of defective platelets with associated reduced survival may play a contributory role.     

Differentiation of patients with subtype IIb-like von Willebrand''s disease by means of perfusion experiments with reconstituted blood

Four unrelated patients with a bleeding diathesis (bleeding time longer than 30 min), some spontaneous platelet aggregation, thrombocytopenia and large platelets, had decreased levels of factor VIII-von Willebrand factor (FVIII-VWF) related properties and impaired platelet adherence to human artery subendothelium. The largest multimers of plasma FVIII-VWF were absent and enhanced ristocetin induced platelet aggregation in platelet-rich plasma was observed. 'Pseudo-von Willebrand's disease' was excluded, because FVIII-VWF from normal subjects did not initiate platelet aggregation. Perfusion studies with reconstituted blood, consisting of respectively patient platelets in normal plasma or normal platelets in patient plasma, yielded evidence for an intrinsic platelet abnormality in two out of the four patients and a plasma defect in the other two patients. Similar experiments with platelets and plasma from four patients with von Willebrand's disease (VWD) subtype I and four patients with VWD subtype IIa showed that the impaired platelet adherence in blood from these patients was caused by a plasma defect. These experiments indicate that patients with laboratory findings in many respects similar to those originally reported for patients with VWD subtype IIb may represent a heterogeneous group of individuals. The data derived from our study imply that perfusion experiments with reconstituted blood offer a new approach in characterization of these patients, which may be more relevant for the treatment of the patients than characterization by ristocetin induced adsorption of FVIII-VWF to platelets.     

Platelet proteome and function in X−linked thrombocytopenia with thalassemia and in silico comparisons with gray platelet syndrome

In X-linked thrombocytopenia with thalassemia (XLTT; OMIM 314050), caused by the mutation p.R216Q in exon 4 of the GATA1 gene, male hemizygous patients display macrothrombocytopenia, bleeding diathesis and a b-thalassemia trait. Herein, we describe findings in two unrelated Swedish XLTT families with a bleeding tendency exceeding what is expected from the thrombocytopenia. Blood tests revealed low P-PAI-1 and P-factor 5, and elevated S-thrombopoietin levels. Transmission electron microscopy showed diminished numbers of platelet a- and dense granules. The proteomes of isolated blood platelets from five male XLTT patients, compared to five sex- and agematched controls, were explored. Quantitative mass spectrometry showed alterations of 83 proteins (fold change ≥±1.2, q<0.05). Of 46 downregulated proteins, 39 were previously reported to be associated with platelet granules. Reduced protein levels of PTGS1 and SLC35D3 were validated in megakaryocytes of XLTT bone marrow biopsies by immunohistochemistry. Platelet function testing by flow cytometry revealed low dense- and a-granule release and fibrinogen binding in response to ligation of receptors for ADP, the thrombin receptor PAR4 and the collagen receptor GPVI. Significant reductions of a number of agranule proteins overlapped with a previous platelet proteomics investigation in the inherited macrothrombocytopenia gray platelet syndrome. In contrast, Ca²⁺ transporter proteins that facilitate dense granule release were downregulated in XLTT but upregulated in gray platelet syndrome. Ingenuity pathway analysis showed altered coagulation system and protein ubiquitination pathways in the XLTT platelets. Collectively, the results revealed protein and functional alterations affecting platelet a- and dense granules in XLTT, probably contributing to bleeding.     

Wiskott—Aldrich Syndrome: Qualitative Platelet Defects and Short Platelet Survival

S ummary . Investigations of the haemostatic functions in three patients with the Wiskott-Aldrich syndrome are presented. All patients had severe thrombocytopenia and prolonged bleeding times. The platelets had abnormal morphology with reduced size and variations of shape. Electron microscopy revealed ultrastructural abnormalities with a reduced number of organelles, and many of the platelets contained large numbers of tubules. Platelet electrophoretic mobility in citrated plasma was not reduced by collagen, and platelet aggregation by collagen and ADP was deficient. Biochemical studies revealed a lack of the storage pool of adenine nucleotides. Platelet adhesiveness in vitro in whole blood was reduced. Platelet factor-3 release by kaolin, ADP and freezing and thawing was normal in one and reduced in another of the patients.
Platelet survival studies showed a normal survival time of normal donor platelets in one patient, while autologous platelets had a markedly reduced survival time in two of the patients. The bone marrow contained normal numbers of megakaryocytes. By electron microscopy of the bone marrow, blood platelets were found to be phagocytosed by macrophages and reticulum cells. The main cause of the thrombocytopenia is most probably incrcased peripheral destruction of platelets. It is suggested that the qualitatively defective platelets are recognized in the reticulo-endothelial system as foreign particles and phagocytosed.     

Severe platelet dysfunction in hairy cell leukemia with improvement after splenectomy

A patient with hairy cell leukemia developed purpura not attributable to thrombocytopenia. We found markedly reduced platelet aggregation responses and malondialdehyde production, decreased serotonin uptake, and depleted dense granule contents. Ultrastructural studies showed that most platelets had few or nor granules. All of the clinical and laboratory studies of platelet function and morphology improved after splenectomy. These findings indicate that qualitative defects in platelet function occurring in hairy cell leukemia may cause clinically important bleeding and that the bleeding diathesis may be ameliorated by splenectomy.     

Bleeding due to thrombocytopenia in acute leukemias and reevaluation of the prophylactic platelet transfusion policy

Prophylactic platelet administration is indicated at counts below 20 X 10(9)/l. The bleeding tendency and severity were compared between thrombocytopenic patients with acute-lymphocytic leukemia (ALL) and acute non-lymphocytic leukemia (ANLL) in the ranges of 10-20 X 10(9)/l platelets, while prophylactic platelet administration was given only below 10 X 10(9)/l. The bleeding tendency for ALL was quite similar at platelet counts above or below 10 X 10(9)/l. The bleeding tendency was significantly lower (p less than 0.001) when the platelets were above this level in ANLL patients. When the thrombocytopenia was caused by chemotherapy, the bleeding was significantly lower in both types of leukemia above 10 X 10(9)/l (p less than 0.05 for ALL, p less than 0.001 for ANLL) as compared with lower counts. When the thrombocytopenia was caused by leukemia, the bleeding tendency was similar in both types of leukemia and at all platelet counts (below 20 X 10(9)/l). Fever, not associated with sepsis, augmented the bleeding severity of patients with ANLL. Stable or rising counts of platelets were associated with significantly lower bleeding tendency above 10 X 10(9)/l only in ANLL patients. The decision for prophylactic platelet administration at counts below 20 X 10(9)/l should be guided by the type of the leukemia (ALL vs. ANLL), the cause of thrombocytopenia (chemotherapy vs. leukemia per se), the trend of the platelet counts, presence of fever and patient's age (below or above 18 years).(ABSTRACT TRUNCATED AT 250 WORDS)     

Platelets in idiopathic thrombocytopenic purpura are increased in size but are of normal density

To determine whether platelet size and volume are related to one another or to platelet age, subpopulations of platelets from patients with idiopathic thrombocytopenic purpura (ITP) have been produced on the basis of density using Percoll gradients. The density distribution of platelets from patients with ITP and from patients with other forms of thrombocytopenia (thought to be nonimmune in nature) was the same as in normal controls. However, the platelets in each density subpopulation from ITP patients were increased in size. beta-thromboglobulin (beta TG) content of platelets from each patient group and the normals increased with density and tended to be higher in ITP than in normal controls. beta TG concentration per unit platelet volume and its level in plasma were similar in ITP patients and in normal controls. This suggests that the apparently normal density of ITP platelets was not a result of degranulation of large, dense platelets. Thus platelet size and density are independently determined and the increased size of platelets in immune thrombocytopenia may be the result of abnormalities in their production.     

Relationship between size and thiazole orange fluorescence of platelets in patients undergoing high-dose chemotherapy.

The reticulated platelet count relies upon the assumption that newly formed platelets contain a residual amount of RNA which selectively binds the dye thiazole orange (TO) and greatly enhances its fluorescence signal. It has, however, recently been shown that almost half of the platelet TO-signal is derived from the labelling of dense-granule nucleotides. It is therefore possible that the higher TO fluorescence of young platelets partially derives from the higher granule content due to their larger volume. To investigate the relationship between platelet size and TO fluorescence we studied 13 patients with high-risk breast cancer undergoing high-dose chemotherapy. Mean platelet volume, platelet distribution width, platelet-large cell ratio, membrane content of glycoprotein Ib and IIb-IIIa and platelet aggregation were significantly greater during resolution than during development of thrombocytopenia, suggesting a prevalence of young and old platelets respectively. Mean TO fluorescence per cell was higher in the platelet population enriched in young cells than in that enriched in old cells, but this difference was no longer observed when the ratio TO signal/platelet size was examined. Moreover, RNase treatment and platelet degranulation reduced TO fluorescence to a similar extent in platelet populations enriched in young or old cells. Therefore our data suggest that the higher TO signal of young platelets is derived, to a significant extent, from their larger volume and granule content.     

Effects of anagrelide on in vivo megakaryocyte proliferation and maturation in essential thrombocythemia

To define the mechanism by which anagrelide normalizes the platelet count in essential thrombocythemia, we studied in vivo megakaryocytopoiesis in 10 newly diagnosed patients prior to and while on anagrelide therapy. Using flow cytometric analysis of aspirated marrow, megakaryocytopoiesis was quantified and correlated with the autologous platelet production rate. Megakaryocytes were identified by CD41a expression and enumerated in relation to the nucleated marrow erythroid precursors. Megakaryocyte diameters were directly measured by time-of-flight technique, and cell ploidy was measured by DNA staining. Two to 3 thousand megakaryocytes were analyzed in each sample. In the 10 patients, the platelet count was 1063 +/- 419 x 10(9) platelets/L (mean +/- 1 SD) with markedly increased production (237 +/- 74 x 10(9) platelets/L per day versus 43.1 +/- 8.4 x 10(9) platelets/L per day in healthy individuals). The platelet survival was 8.2 +/- 1.1 days versus 9.0 +/- 0.5 days in healthy controls (P >.05). Megakaryocyte diameter was increased to 46 microm (versus 37 microm in controls; range, 21 microm for 2N to 56 microm for 64N cells). The volume increased to 48 x 10(3) microm(3) versus 26 x 10(3) microm(3) in controls, and the number increased to 14 x 10(6)/kg (versus 7 x 10(6)/kg in controls), resulting in 3.7-fold increase in megakaryocyte mass (66 x 10(10) microm(3)/kg versus 18 x 10(10) microm(3)/kg). Cell ploidy was enhanced showing a modal ploidy of 32N (versus 16N in healthy controls) with marked increase in 64N and 128N cells (P <.05). Anagrelide therapy reduced the platelet counts to 361 +/- 53 x 10(9) platelets/L and the turnover rate to 81 x 10(9) platelets/L per day. The platelet survival was unchanged. Following therapy, megakaryocyte number decreased to 8 x 10(6)/kg, diameter to 40 microm, and volume to 34 x 10(3) microm(3) with a normalized modal ploidy of 16N, resulting in a megakaryocyte mass reduced by 60% (28 x 10(10) microm(3)/kg; P <.05). This reduction in cell mass closely correlated with the reduction in platelet count and production rate by 66% (r = 0.96). The present data indicate that in essential thrombocythemia anagrelide therapy decreases circulating platelets by reducing both megakaryocyte hyperproliferation and differentiation.     

IVIg treatment increases thrombin activation of platelets and thrombin generation in paediatric patients with immune thrombocytopenia

Clinical manifestations and laboratory parameters of haemostasis were investigated in 23 children with newly diagnosed immune thrombocytopenia (ITP) before and after intravenous immunoglobulin (IVIg) treatment. ITP patients with platelet counts of less than 20 × 10⁹/L and mild bleeding symptoms, graded by a standardized bleeding score (BS), were compared with healthy children with normal platelet counts and children with chemotherapy-related thrombocytopenia. Markers of platelet activation and platelet apoptosis in the absence and presence of platelet activators were analysed by flow cytometry; thrombin generation in plasma was determined. ITP patients at diagnosis presented with increased proportions of platelets expressing CD62P and CD63 and activated caspases, and with decreased thrombin generation. Thrombin-induced activation of platelets was reduced in ITP compared with controls, while increased proportions of platelets with activated caspases were observed. Children with a higher BS had lower proportions of CD62P-expressing platelets compared with those with a lower BS. IVIg treatment increased the number of reticulated platelets, the platelet count to more than 20 × 10⁹/L and improved bleeding in all patients. Decreased thrombin-induced platelet activation, as well as thrombin generation, were ameliorated. Our results indicate that IVIg treatment helps to counteract diminished platelet function and coagulation in children with newly diagnosed ITP.     

Detection and Quantification of Platelet-bound Antibodies with Immunoperoxidase

The use of immunocytochemical techniques enables one to quantify antibodies bound to cell membranes. We have tested the platelets of subjects with immune thrombocytopenic purpura by this method. The washed platelets were incubated in the presence of antiglobulin conjugated with an equimolar amount of peroxidase. The fixed peroxidase was revealed and quantified by an enzymatic reaction using orthodianizidine-H2O2. From the results the number of immunoglobulin molecules per platelet was calculated. The test was calibrated so as to eliminate non-specific fixation of the reagent. Under these conditions the platelets of subjects with idiopathic thrombocytopenic purpura bound a much large amount of detectable immunoglobulin (800-21 000 molecules per platelet) than those of normal subjects (235 +/- 74 sites per platelet) or subjects with non-immune thrombocytopenia (395 +/- 203 molecules per platelet).     

Familial Thrombopathic Thrombocytopenia

A family with hereditary thrombopathic thrombocytopenia is described, with eight affected members in three generations. The haemorrhagic diathesis manifests itself by bleeding from mucous membranes and after operative procedures. The platelet count varies from 20,000 to 80,000 per cu.mm. The number of megakaryocytes in the bone marrow has been examined in two affected members, and the platelet survival in one, and both are normal.
The thrombopathy consists in a deficiency of platelet factor 3 and in morphological anomalies. Electron microscopy has revealed a low content of α-granules in platelets from two cases. The heredity shows an autosomal dominant pattern. A linkage of this genetic defect with blood group O seems probable.