Evidence that platelet buoyant density,but not size,correlates with platelet age in man

Following infusion of ⁵¹Cr-labeled autologous platelets into normal subjects, high-density (HD) and low-density (LD) platelet cohorts were isolated by prolonged centrifugation in isosmotic arabino-galactan (Stractan). Specific radioactivity of LD platelets declined rapidly post-infusion (T_1/2 = 1.5 days), but specific radioactivity of HD platelets remained constant or increased over a 3–4-day period and gradually declined for 6–7 days thereafter. These differences were exaggerated when platelet cohorts enriched in LD or HD cells by slow centrifugation in high-density albumin were labeled and transfused. Mean survival of a platelet cohort enriched with HD cells was significantly (P < 0.02) shorter (7.73 days) than that of a cohort enriched with LD cells (9.33 days). In normal subjects treated with aspirin, capacity for thromboxane synthesis was regained more rapidly (P < 0.05) in LD than in HD platelets. HD and LD platelets differed only slightly in mean volume (HD platelets = 7.57 μ³, LD platelets = 6.87 μ³, 0.05 < P < 0.01). We believe the most logical interpretation of these findings is that under normal conditions in man, newly formed platelets are less dense on the average than total platelets and become more dense as they age in the circulation. Thus, specific radioactivity of LD platelets declines rapidly as these platelets move into a more dense compartment and are replaced by newly formed, un-labelled cells; specific radioactivity of HD platelets remains constant or increases as labelled platelets enter this compartment in numbers equal to or greater than the number leaving it at the end of their life span. The similarity in mean volumes of LD and HD platelets suggests that platelet size is unrelated to platelet age under normal conditions.     

Kinetics of platelet density subpopulations in splenectomized mongrel dogs

Autologous ⁵¹Cr-platelet kinetic studies were performed in splenectomized mongrel dogs. Mean survival time of PRP-platelets was 5.4 ± 1.5 (SD) days (n = 6). The curves, though slightly curvilinear, showed mostly a linear type of decay, denoting that platelet removal from the circulation is mainly determined by aging of the cells. High-density (HD) and low-density (LD) platelet cohorts were isolated in Stractan gradients from samples drawn daily after infusion of labeled platelets. Specific radioactivity in HD cohorts declined rapidly postinfusion (T1/2 = 1.3 days), but specific radioactivity in LD platelets increased for 2 days and steadily declined for 4 days thereafter (n = 6). Labeled HD platelets, comprising 11.7% of the total population, lived significantly longer in circulation than LD platelets (19.1% of the total population) (n = 3). The patterns of decay of the radioactivity, however, do not have all the characteristics of pure age-cohort survival curves; 3.7 days after the infusion of labeled HD platelets, the specific radioactivity in LD cohorts was six times higher than on day 1, but attained only 20% of the initial specific radioactivity in HD platelets. After the infusion of labeled LD platelets no radioactivity was recovered in circulating HD cohorts. These findings indicate that mongrel dog platelets decrease in density with aging, but also that platelet density heterogeneity is in part determined during the thrombopoietic process. These data are consistent with those of other authors in rabbits and rhesus monkeys, but contrast with the observations that platelets in humans, baboons, and Macaca fasicularis monkeys increase in density with age, suggesting that the displacement of platelets toward compartments of either higher or lower density depends on the species under study.     

A double antibody radioassay for the detection of platelet associated IgG

A double antibody radioimmunoassay has been developed for the detection of platelet associated IgG (PAIgG). The assay employs conventional radioassay technology and, as such, readily may be adopted by laboratories familiar with radioassay techniques. The assay is sensitive to 0.6 ng IgG and can be undertaken on 10⁶-10⁷ platelets, or fewer in the case of thrombocytopenia with raised PAIgG. The assay can be completed within 2 working days. Normal PAIgG levels in EDTA blood were 0.8 to 5.7 μg IgG/10⁹ platelets. However, normal levels depended upon the anticoagulant used for blood collection and were on average five-fold higher for blood taken into EDTA than for blood taken into ACD; normal range 0.1–1.2 μg/IgG 10⁹ platelets. PAIgG was assayed on blood taken simultaneously into ACD and EDTA from 15 patients with chronic idiopathic thrombocytopenia (ITP). PAIgG was raised in 14 out of 15 EDTA samples, but in only 11 of 15 ACD samples. EDTA was therefore regarded as the anticoagulant of choice. A total of 20 of 23 (87%) patients with chronic ITP had PAIgG values above the normal range. The PAIgG in chronic ITP was weakly related to platelet count, weakly and inversely related to platelet volume, but was independent of serum IgG concentration.     

Platelet apoptosis in paediatric immune thrombocytopenia is ameliorated by intravenous immunoglobulin

To evaluate the role of intravenous immunoglobulin (IVIg) in platelet apoptosis in paediatric immune thrombocytopenia, we investigated the platelets of 20 paediatric patients with acute immune thrombocytopenia (ITP), before and after IVIg treatment. Healthy children with platelet counts in the normal range and children with thrombocytopenia due to chemotherapy were enrolled as controls. All ITP patients presented with platelet counts <20 × 10⁹/l and bleeding symptoms. Markers of apoptosis, including activated caspase‐3, ‐8 and ‐9, phosphatidylserine (PS) exposure, mitochondrial inner membrane potential (ΔΨm), as well as platelet‐derived microparticle formation, were analysed by flow cytometry. After IVIg treatment, platelet counts increased to >20 × 10⁹/l in all patients. ITP patients had significantly increased proportions of platelets with activated caspase‐3, ‐8 and ‐9, with PS exposure, and with decreased ΔΨm, and demonstrated increased microparticle formation. Except for ΔΨm, these markers for apoptosis were reduced by IVIg treatment. Platelets of children with thrombocytopenia after chemotherapy also demonstrated increased microparticle formation and decreased ΔΨm, but no activation of caspases 3, 8 and 9 or PS exposure. In conclusion, in acute paediatric ITP, enhanced platelet apoptosis is seen at diagnosis that normalizes after IVIg treatment.     

Complement activation on platelets correlates with a decrease in circulating immature platelets in patients with immune thrombocytopenic purpura

The role of the complement system in immune thrombocytopenic purpura (ITP) is not well defined. We examined plasma from 79 patients with ITP, 50 healthy volunteers, and 25 patients with non‐immune mediated thrombocytopenia, to investigate their complement activation/fixation capacity (CAC) on immobilized heterologous platelets. Enhanced CAC was found in 46 plasma samples (59%) from patients with ITP, but no samples from patients with non‐immune mediated thrombocytopenia. Plasma from healthy volunteers was used for comparison. In patients with ITP, an enhanced plasma CAC was associated with a decreased circulating absolute immature platelet fraction (A‐IPF) (<15 × 10⁹/l) (P = 0·027) and thrombocytopenia (platelet count < 100 × 10⁹/l) (P = 0·024). The positive predictive value of an enhanced CAC for a low A‐IPF was 93%, with a specificity of 77%. The specificity and positive predictive values increased to 100% when plasma CAC was defined strictly by enhanced C1q and/or C4d deposition on test platelets. Although no statistically significant correlation emerged between CAC and response to different pharmacological therapies, an enhanced response to splenectomy was noted (P < 0·063). Thus, complement fixation may contribute to the thrombocytopenia of ITP by enhancing clearance of opsonized platelets from the circulation, and/or directly damaging platelets and megakaryocytes.     

Two human monoclonal antiplatelet autoantibodies established from patients with chronic idiopathic thrombocytopenic purpura

Two human hybridomas secreting antiplatelet autoantibodies were established by somatic cell fusion using splenocytes from two patients with chronic idiopathic thrombocytopenic purpura (ITP). These monoclonal antibodies, HT7F and HT8C, were of the IgM isotype and reacted with autologous and allogeneic platelets fixed with paraformaldehyde (PFA). They also bound to fresh platelets. An elution study showed that eluates from allogeneic platelets reacted with autologous platelets. These results indicated that HT7F and HT8C were autoantibodies recognizing a site on the platelet surface. Both monoclonal antibodies were able to induce complement activation in vitro. HT7F was demonstrated to bind to a platelet protein having a molecular mass of 105 kDa under both nonreducing and reducing conditions. No human hybridoma synthesizing antibody against 105 kDa platelet protein has been reported to date. These antibodies may play a role in the pathogenesis of thrombocytopenia in some ITP patients.     

Evaluation of platelet function in thrombocytopenia

Whole blood aggregometry is a functional assay for determination of platelet function. Until now, whole blood aggregometry has not been considered feasible at low platelet counts. Hence, the objectives of the present study were to explore platelet function in thrombocytopenia using a novel index of impedance aggregometry adjusted for platelet count and evaluate the association to platelet function assessed by flow cytometry. Hirudin anticoagulated blood was collected from 20 healthy volunteers, 20 patients with primary immune thrombocytopenia (ITP), and 17 hematological cancer patients. Platelet function was analyzed by impedance aggregometry and by flow cytometry. Collagen, adenosine diphosphate, thrombin receptor agonist peptide-6, and ristocetin were used as agonists for both analyses. Thrombocytopenia in healthy whole blood was induced in vitro employing a recently published method. Platelet aggregation of thrombocytopenic patients was evaluated relative to the aggregation of healthy volunteers at the same platelet count. In flow cytometry, platelet function was described as expression of the platelet surface glycoproteins: bound fibrinogen, CD63, and P-selectin. Similar platelet counts were obtained in the patient groups (p = 0.69) (range: 13–129 × 10⁹/l). Aggregation adjusted for platelet count was significantly increased in ITP patients compared to healthy platelets across all agonists. The platelet aggregation was high in the 95% prediction interval, with 18 ITP patients above the prediction interval in at least two agonists. In contrast, the platelet aggregation was low in the prediction interval in cancer patients, and three cancer patients with platelet aggregation below the prediction interval in at least one agonist. ITP patients displayed increased expression of bound fibrinogen and CD63 following activation, compared with particularly cancer patients, but also compared with healthy platelets. This study demonstrated the feasibility of a novel approach to perform platelet function analyses in thrombocytopenia using impedance aggregometry adjusted for platelet count.     

Platelet calmodulin correlates with platelet turnover

We measured the calmodulin content in platelets in 13 normal persons and in 62 patients with hematological diseases. The level of platelet calmodulin was higher in patients with idiopathic thrombocytopenic purpura (ITP), systemic lupus erythematosus, myeloproliferative disorders, acute leukemia in a recovery phase, aplastic anemia, thrombosis and hypersplenism as compared to the controls. Among the patients with ITP, calmodulin was lower in responders than in nonresponders and those at the initial diagnosis. We also measured the volume, life-span and aggregation of the platelets and demonstrated a significant relationship between the calmodulin level and the platelet volume, and a negative relationship between the calmodulin level and platelet life-span, there was no correlation between the calmodulin level and platelet aggregation. We thus conclude that platelet calmodulin is inversely correlated with platelet turnover.     

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.     

Correlation of membrane anisotropy with function in subpopulations of human blood platelets*

Summary. Human blood platelets were fractionated on a discontinuous Percoll gradient into high density (HD), intermediate (ID), and low density (LD) platelets. The subpopulations were characterized with regard to [¹⁴C]serotonin uptake and release, cAMP content, aggregation, and membrane anisotropy, which is inversely related to membrane fluidity. Membrane anisotropy, which was high in LD platelets, was found to decrease with increasing density (LD> ID > HD). LD platelets showed significant lower cAMP levels and [¹⁴C]serotonin uptake than the total platelet population (TPP) and ID and HD platelets. Upon ADP and serotonin stimulation the cAMP content was reduced in all platelet populations with the exception of HD platelets in which cAMP was unchanged. Upon thrombin stimulation the cAMP content was reduced only in TPP and LD platelet population and it was increased in HD platelet population. Thrombin activation changed the anisotropy only in LD platelets. Thrombin at a concentration of 0·001 U/ml reduced whereas 0·01 and 0·05 U thrombin/ml increased the membrane anisotropy significantly. As compared with TPP and the other subpopulations, LD platelets were most sensitive upon ADP and thrombin stimulated [¹⁴C]serotonin release as well as upon ADP, serotonin and thrombin induced aggregation. The findings suggest that the differing functional abilities of the platelet subpopulations are correlated to the various membrane anisotropies observed in these fractions.     

Total sialic acid in human and canine platelets does not change with the platelet age.

Total platelet sialic acid (SA) was measured in three experimental conditions: (1) human and canine platelet density subpopulations obtained by centrifugation in arabinogalactan gradients, (2) circulating canine platelets during recovery from experimental immune and mechanical thrombocytopenias, and (3) platelets obtained from a patient with chronic immune thrombocytopenic purpura before and after splenectomy. The density of human and canine platelets is, in part, determined by their age. We found no significant differences in total SA between high-density (HD) and low-density (LD) platelets (9.32 +/- 2.0 vs. 9.55 +/- 1.3 micrograms/mg of platelet protein in dogs and 9.02 +/- 2.3 vs. 9.10 +/- 2.9 micrograms/mg in humans). In the human and canine thrombocytopenic models, the entrance of new platelets from the bone marrow is followed by their aging in the circulation. In these models, no significant changes in total SA content were detected in sequential measurements during the recovery of the thrombocytopenia. Accordingly, we conclude that total SA in human and canine platelets is unrelated to their age in circulation. These results do not support the notion that the loss of SA from membrane glycoproteins determines the recognition and removal of platelets from the circulation.     

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.     

Protein A immunoadsorption in chronic refractory ITP reverses increased platelet activation but fails to achieve sustained clinical benefit

Adults with chronic relapsing ITP present a difficult therapeutic challenge. The ongoing antibody- mediated platelet destruction in this group might be expected to be associated with increased expression of platelet surface membrane activation antigens. We have studied a group of 10 patients with refractory ITP and 35 healthy controls. Using an immediate, sensitive, unfixed, whole blood, flow cytometric method to detect platelet surface P-selectin and GP53, we have detected markedly increased platelet activation in the ITP group compared with the controls (P-selectin; patient median 24.5% v control median 2.0%, GP53 median 6.5% v 2.1%, P < 0.01 for both). Five patients underwent protein A immunoadsorption therapy. The effect of protein A immunoadsorption on platelet activation before, during and after 18 treatments in these patients was studied and patients were followed-up to assess clinical outcome. Platelet-associated immunoglobulin measurements were made before and at the end of six treatments. Platelet activation decreased after immunoadsorption. P-selectin expression fell significantly; pre- and post-treatment median values differed by 15.5%, P < 0.01, for GP53 the difference was 2.5.%, P = NS. A reduction in both platelet-associated IgG (median reduction of 11.8 ng/10⁶ platelets, P = 0.08) and IgM (7.6 ng/10⁶ platelets, P = 0.06) was recorded.     

A diagnostic approach that may help to discriminate inherited thrombocytopenia from chronic immune thrombocytopenia in adult patients

Inherited thrombocytopenia (IT) is a heterogeneous group of rare diseases that are often confused with immune thrombocytopenia (ITP). The objective of this study was to supply clinicobiological elements that allow a distinction to be drawn between IT and chronic ITP. We then compared 23 adult patients with IT and 9 patients with chronic ITP. Our study revealed six discriminating criteria: (i) an age of discovery <34 years: positive predictive value (PPV) = 88.2% [63.6; 98.5], (ii) a family history of thrombocytopenia: PPV = 100.0% [82.4; 100.0], (iii) a personal history of bleeding: PPV = 100% [76.8; 100.0], (iv) a mean platelet volume >11 fL: PPV = 93.3% [68.1; 99.8], (v) an excess of giant platelets on blood smear: 100.0% [76.8; 100.0], and (vi) a percentage >44% of platelets with a surface area >4 µm² in electron microscopy: PPV = 83.3% [58.6; 96.4]. If at least three of these criteria were combined, it was possible to distinguish IT from chronic ITP with 91.3% [72.0; 98.9] sensitivity and PPV = 100.0% [66.4; 100.0] specificity. The secondary objective of this study was to assess the prevalence of potential IT diagnosis in patients with chronic thrombocytopenia of uncertain origin. Applying our diagnostic approach to a series of 20 cases allowed us to estimate that 40% of them could be suffering from IT. Finally, our diagnostic approach may help to correctly distinguish IT from chronic ITP, particularly in the context of macrothrombocytopenia.     

Glycocalicin in the diagnosis and management of immune thrombocytopenia

Abstract: We studied glycocalicin (GC), expressed as plasma GC concentration and as GC index (ratio to platelet count), in 129 thrombocytopenic patients (platelet count <100×10⁹/1) and 60 sex- and age-matched controls. Seventy-two patients had idiopathic immune thrombocytopenia, 32 secondary immune thrombocytopenia, 8 microangiopathic thrombocytopenia and 17 thrombocytopenia secondary to bone marrow aplasia. Patients with immune thrombocytopenia (ITP) were also subclassified, according to their clinical behaviour, as having active disease or being in spontaneous or therapy-induced partial remission. A significant correlation was found between glycocalicin levels and platelet count both in normals and in patients with bone marrow aplasia (r = 0.75). ITP patients showed a GC index significantly higher than controls (6.02±7.87 vs. 0.9±0.2, p<0.001). When ITP patients with similar platelet count (30–50×10⁹/l) were studied, the mean level of GC and the GC index were significantly higher in those patients with active disease than in those in remission (0.97±0.38 vs. 0.58±0.17 μg/ml, p<0.05; 6.41±2.64 vs. 3.44±0.94, p<0.05, respectively). A longitudinal study performed in 10 patients with different subtypes of ITP suggested a positive correlation between GC index and the activity of the disease. The GC value and GC index were significantly higher in patients with microangiopathic thrombocytopenia than in controls (1.44±0.73 vs. 0.8±0.16 μg/ml, p<0.01; and 18.77±22.23 vs. 0.9±0.2, p<0.001, respectively). The GC value was significantly lower in bone marrow failure (0.15±0.04 μg/ml, p<0.01) compared to controls, while no difference was observed in the GC index. Our data confirm that the GC index is helpful in differentiating thrombocytopenia due to increased platelet destruction from the one due to impaired production. In addition, the assay has been proven useful in the differential diagnosis of different ITP subtypes and their follow-up.     

A fatal case of transplantation-mediated alloimmune thrombocytopenia following liver transplantation

Objective and importance: Transplantation-mediated alloimmune thrombocytopenia (TMAT) occurs when leukocytes transferred in a donor organ from a patient with immune thrombocytopenia (ITP), mount a response against recipient platelets. We present the first fatal case of TMAT following liver transplantation and review its aetiology and treatment.

Clinical presentation: The liver donor had ITP and died from an intracranial haemorrhage. The recipient platelet count fell to 2?×?10⁹/l on post-operative day 2. Treatment refractory thrombocytopenia resulted in pulmonary haemorrhage and death. TMAT did not occur in a kidney recipient from the same ITP donor.

Intervention: Extramedullary haematopoiesis was identified in the donor liver biopsy. Antibodies against platelet GPIb/IX were demonstrated in both donor and recipient. The thrombocytopenia was refractory to platelet transfusions, intravenous immunoglobulin, methylprednisolone, rituximab, romiplostim, plasmapheresis, vincristine and splenic artery embolization. On review of the literature, severe thrombocytopenia (<10?×?10⁹/l) has started within 3 days of transplantation in all reported TMAT cases. Serious non-fatal bleeding was observed in 3/5 previously reported cases. The optimal treatment is unclear. TMAT should resolve as donor lymphocytes are eliminated but re-transplantation may be required in severe refractory cases. TMAT has been reported in recipients of a liver but not kidney or heart transplant from ITP donors, probably because of the greater burden of co-transplanted lymphoid tissue.

Conclusion: Before using the liver of an ITP donor, the recipient’s fully informed consent is required. However, the risk of TMAT from an ITP donor is currently unknown and systematic review of donor registries is needed.     

A rapid quantitation of platelet-associated IgG by nephelometry

Platelet-associated IgG (PAIgG) was measured by a simple rapid nephelometric technique using washed solubilized platelets and commercially available, prestandardized reagents. Normal subjects with normal platelet counts had PAIgG levels of 2.1-6.7 fg/platelet. Subjects with idiopathic immune thrombocytopenic purpura (ITP) had levels of 7.2-43.3 fg/platelet. Ninety percent of ITP patients had values exceeding 2 SD units of the mean of normal subjects. Elevated values were also found in 17% of patients with recovered ITP, patients with SLE with and without thrombocytopenia, patients with thrombocytopenia occurring during septicemia, and patients with IGg myeloma. Results can be obtained within several hours of receipt of blood specimen, and are similar to the reports that used more complex techniques.     

Flow cytometric analysis of platelet cyclooxygenase-1 and -2 and surface glycoproteins in patients with immune thrombocytopenia and healthy individuals

Immature platelets may contain more platelet enzymes such as cyclooxygenase (COX)-1 and COX-2 than mature platelets. Patients with immune thrombocytopenia (ITP) have a higher fraction of immature platelets and can therefore be utilized as a biological model for investigating COX-1 and COX-2 platelet expression. The aims were to develop flow cytometric assays for platelet COX-1 and COX-2 and to investigate the COX-1 and COX-2 platelet expression, platelet turnover, and platelet glycoproteins in ITP patients (n = 10) compared with healthy individuals (n = 30). Platelet count and platelet turnover parameters (mean platelet volume (MPV), immature platelet fraction (IPF), and immature platelet count (IPC)) were measured by flow cytometry (Sysmex XE-5000). Platelet COX-1, COX-2, and the glycoproteins (GP)IIb, IX, Ib, Ia, and IIIa were all analyzed by flow cytometry (Navios) and expressed as median fluorescence intensity. COX analyses were performed in both whole blood and platelet rich plasma (PRP), whereas platelet glycoproteins were analyzed in whole blood only. ITP patients had significantly lower platelet count (55 × 10⁹/L) than healthy individuals (240 × 10⁹/L, p < 0.01), but a higher MPV (p = 0.03) and IPF (p < 0.01). IPC was similar for the two groups (p = 0.74). PRP had significantly lower MPV (p < 0.01) and significantly higher platelet count and IPC (both p-values <0.03) when compared with whole blood. IPF was similar for PRP and whole blood (p = 0.18). COX-1 expression was 10 times higher and COX-2 expression was 50% higher in PRP than in whole blood (p_COX-1 < 0.01, p_COX-2 < 0.01). Platelet COX-1 expression was higher in ITP patients than healthy individuals using whole blood (p_COX-1 < 0.01) and PRP, though this was nonsignificant in PRP (p_COX-1 = 0.17). In ITP patients, positive correlations were found between platelet turnover and COX-1 expression (all p-values <0.01, rho = 0.80–0.94), whereas healthy individuals showed significant though weaker correlations between platelet turnover and COX-1 and COX-2 expressions (all p-values <0.03, rho = 0.44–0.71). GPIIb, IX, and Ib expression was increased in ITP patients compared with healthy individuals (all p-values < 0.03). GPIIb, IX, Ib, and IIIa showed positive correlations with platelet turnover in ITP patients (all p-values <0.02, rho = 0.71–0.94), but weak and nonsignificant correlations in healthy individuals (all p-values >0.14, rho = 0.11–0.28). In conclusion, ITP patients expressed higher COX-1 and platelet glycoprotein levels than healthy individuals. COX-1 and platelet glycoproteins demonstrated positive correlations with platelet turnover in ITP patients. In healthy individuals, COX-1 and COX-2 expression correlated positively with platelet turnover. PRP was more sensitive compared with whole blood as regards determination of COX. Therefore, PRP is the recommended matrix for investigating COX-1 and COX-2 in platelets.     

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.     

Thrombocytopenia model with minimal manipulation of blood cells allowing whole blood assessment of platelet function

In vitro models of thrombocytopenia are useful research tools. Previously published models have shortcomings altering properties of platelets and other blood components. The aim of the present study was to develop a whole blood method to induce thrombocytopenia with minimal manipulation, and to describe platelet function in induced thrombocytopenia in individuals with healthy platelets. Hirudin anticoagulated blood was obtained from 20 healthy volunteers. One part of the blood was gently centrifuged at 130g for 15 minutes. The platelet-rich plasma was replaced with phosphate-buffered saline to establish thrombocytopenia. Various levels of thrombocytopenia were achieved by combining different volumes of baseline whole blood and thrombocytopenic blood. Platelet counts were measured by flow cytometry (Navios, Beckman Coulter) and routine haematological analyser (Sysmex XE-5000). Platelet function was analysed by impedance aggregometry (Multiplate® Analyzer, Roche) and by flow cytometry (Navios, Beckman Coulter) using collagen, adenosine diphosphate, thrombin receptor activating peptide-6 and ristocetin as agonists. Median baseline platelet count was 227×10⁹/l. The in vitro model yielded median platelet counts at 51×10⁹/l (range 26–93×10⁹/l). We observed minor, yet significant, changes in platelet size and maturity from baseline to modelled thrombocytopenia. In the thrombocytopenic samples, significant and positive linear associations were found between platelet count and platelet aggregation across all agonists (all p-values<0.001). Platelet function assessed by flow cytometry showed minimal alterations in the thrombocytopenic samples. A new whole blood-based model of thrombocytopenia was established and validated. This new model serves as a useful future tool, particularly to explore platelet function in patients with thrombocytopenia.