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.     

Efficacy and safety of anti-D for treatment of adults with immune thrombocytopenia

This study was planned to assess the response of anti-D in patients with immune thrombocytopenia (ITP). Twenty adults (8 males: 12 females) with a median age 33.5 years (19–59 years) were included. Nine patients had newly-diagnosed ITP, 6 had persistent ITP and 5 had chronic ITP. The overall response rate was 65%. Patients with newly diagnosed ITP showed response rates of 77% (7/9), persistent ITP had response rates 50% (3/6) and patients with chronic ITP had response rates of 60% (3/5). The median time to response was 3 days (1–11 days). There was no correlation of response with age, sex, severity of bleeding, presenting platelet counts, ABO blood group or prior steroid or IVIG response.     

The measurement and application of thrombin generation

It is the capacity to generate thrombin, and the enzymatic work that thrombin does, that determines blood coagulability. Therefore, measurement of the enzymatic work potential of thrombin provides a method for quantifying the composite effect of the multiple factors that determine coagulation capacity. The application of measurement of thrombin generation to clinical decision making has been hampered by numerous technical difficulties and pitfalls, many of which have now been overcome. Technical advances now permit rapid, reproducible measurement. A review of clinical studies performed to date indicates the need to appreciate the precise methodology used in each case and the need to consider standardisation in future studies. Applying thrombin generation measurement to clinical decision making will require up-to-date estimates of risks relating to the disorder and the intended therapeutic intervention. Ultimately management studies will be required if the clinical utility of measurement of thrombin generation is to be proven.     

Procoagulant profile in patients with immune thrombocytopenia

Despite their low platelet count some immune thrombocytopenia (ITP) patients seldom bleed, indicating the presence of factors to compensate thrombocytopenia. Moreover, ITP patients may have an increased risk for thrombosis. These facts suggest the presence of procoagulant mechanisms that have not been clarified yet. The aim of this study was to identify these possible factors. Moreover, the utility of rotational thromboelastometry (ROTEM^®) to test haemostasis in these patients was also evaluated. Patients with ITP presented a procoagulant profile due to an increased amount of platelet‐ and red cell‐microparticles, an increased resistance to protein C and the formation of a clot more resistant to fibrinolysis due to augmented levels of plasminogen activator inhibitor‐1, which might reflect an endothelial damage/activation in ITP patients. Despite increased maximum clot firmness and reduced lysis, ROTEM^® profiles showed a prolonged clotting time that might rely on the presence of anti‐platelet antibodies as suggested by the increased lagtime in thrombin generation test caused by plasma from ITP patients on platelets from healthy controls. These results indicate the need to individualize therapeutic treatment for ITP patients, considering their procoagulant profile and the presence of concomitant risk factors. Moreover, ROTEM^® appeared to be useful for evaluating haemostasis in ITP patients.     

High-dose factor VIIa increases initial thrombin generation and mediates faster platelet activation in thrombocytopenia-like conditions in a cell-based model system

Clinical experience has shown that high doses of recombinant factor VIIa (rFVIIa) may ensure haemostasis in thrombocytopenic patients. We have used a cell-based model system to mimic thrombocytopenia and analyse the effect of rFVIIa. Lowering the platelet density from 200 x 10(9)/l (reflecting normal conditions) to 100, 50, 20 and 10 x 10(9)/l revealed a platelet density-dependent decrease in the maximal rate of thrombin generation, a prolongation in the time to maximal thrombin activity and a lower maximal level of thrombin formed. The platelet activation, measured as the time to half-maximal P-selectin (CD62) exposure, was not significantly dependent on the platelet density in the range of 200 x 10(9)/l to 10 x 10(9)/l, although there was a tendency for slower platelet activation at 20 x 10(9) and 10 x 10(9) platelets/l than at the higher platelet densities. Addition of 50--500 nmol/l rFVIIa to samples with 20 x 10(9) or 10 x 10(9) platelets/l shortened the lag phase of thrombin generation as well as the time to half-maximal platelet activation. Our data indicate that high doses of rFVIIa may help to provide haemostasis in thrombocytopenic patients by increasing the initial thrombin generation, resulting in faster platelet activation and thereby compensating for the lower number of platelets present.     

Markers of platelet activation and thrombin generation

The laboratory assessment of platelet biology and coagulation has traditionally focused on abnormalities, congenital or acquired, that provide insights for understanding hemostatic defects, unexplained bleeding, and pharmacologic titration of compounds designed to attenuate thrombotic potential. Because atherosclerotic vascular disease is associated with thrombotic events and there exists a clear link between atherosclerosis, inflammation, and thrombosis, the development of markers that have pathobiologic, prognostic, and treatment-related relevance has taken on a role of considerable importance.     

Retrospective evaluation of bleeding tendency and simultaneous thrombin and plasmin generation in patients with rare bleeding disorders

Rare bleeding disorders (RBDs) are a heterogeneous group of diseases with varying bleeding tendency, only partially explained by their laboratory phenotype. We analysed the separate groups of RBD abnormalities, and we investigated retrospectively whether the novel haemostasis assay (NHA) was able to differentiate between bleeding tendency. We have performed simultaneous thrombin generation (TG) and plasmin generation (PG) measurements in 41 patients affected with deficiencies in prothrombin, factor (F) V, FVII, FX, FXIII and fibrinogen. Parameters of the NHA were classified based on (major or minor) bleeding tendency. Patients with deficiencies in coagulation propagation (FII, FV and FX) and major type of bleedings had diminished TG (expressed as AUC) below 20% of control. FVII deficient patients only had prolonged thrombin lag-time ratio of 1.6 ± 0.2 (P < 0.05) and normal AUC (92-125%). Afibrinogenemic patients demonstrated PG of 2-29% of normal and appeared to correlate with the type of mutation. Thrombin peak-height (57 ± 16%) was reduced (not significant) in these patients and AUC was comparable to the reference (102 ± 27%). FXIII-deficient plasmas resulted in a reduced thrombin peak-height of 59 ± 13% (P < 0.05) and normal AUC (90 ± 14%). Thrombin peak-height (P < 0.01) and plasmin potential (P < 0.05) were lower in the major bleeders compared with the minor bleeders. These results provided distinct TG and PG curves for each individual abnormality and differentiation of bleeding tendency was observed for thrombin and PG parameters. Prospective studies are warranted to confirm these retrospective results.     

Surface 12-lead electrocardiographic findings and plasma markers of thrombin activity and generation in patients with myocardial ischemia at rest

Background: Myocardial ischemia at rest is typically associated with atherosclerotic coronary artery disease, atheromatous plaque rupture, and intracoronary thrombosis. In areas of advanced disease and vascular injury, the extent of thrombus is influenced largely by a delicate balance of procoagulant factors, favoring thrombus initiation, growth, and development, and anticoagulant factors, attempting to limit potentially flow-limiting coronary thrombosis. Thrombin, a 308 amino acid serine protease, is considered the most potent procoagulant factor in the setting of acute vessel wall injury, playing an essential role in the conversion of fibrinogen to fibrin, accelerating the prothrombinase complex, activating platelets, and stabilizing fibrin polymers. The purpose of this study was to determine the relationship between electrocardiographic abnormalities and markers of thrombin activity and generation among patients with unstable angina and non-Q-wave myocardial infarction.Methods and Results: In a study of 36 patients (59.1 ± 11.0 years) with myocardial ischemia at rest participating in the Thrombolysis in Myocardial Ischemia (TIMI) IIIB trial, thrombin activity in plasma, as determined by fibrinopeptide A (FPA), prothrombin fragment 1.2 (F 1.2), and thrombin-antithrombin III complexes (TAT) concentrations, were found to be increased significantly when compared with healthy volunteers (p < 0.004). Thrombin generation was also increased modestly compared with age-matched patients with stable coronary artery disease undergoing elective cardiac catheterization. Given that the surface 12-lead electrocardiogram (ECG) is frequently abnormal in patients with ischemic chest pain at rest and represents a readily available, first-line diagnostic test for assessing disease activity and treatment response, we investigated whether ECG abnormalities and thrombin activity/ generation in plasma were correlated. Twenty-six patients (72%) had ECG changes compatible with myocardial ischemia at the time of study entry, including 18 (50%) with newly inverted T waves (or pseudonormalization), 14 (39%) with reversible ST-segment depression, and 4 (11%) with transient (<30 minutes) ST-segment elevation. Within the predefined ECG groups there were no differences in plasma thrombin activity between patients with and those without confirmed abnormalities. Similarly, there were no differences in either plasma thrombin activity or generation between the predefined ECG groups.Conclusion: Although ECG abnormalities supporting the presence of myocardial ischemia occur commonly in patients with chest pain at rest, they do not correlate closely with markers of thrombin activity and generation in plasma. The diagnostic and prognostic capabilities of these diagnostic tools, considered either alone or together, require further investigation.Presented in part at the American College of Cardiology 42nd annual Scientific Session, Anaheim, CA, March 14–18, 1993.     

Use of thromboelastography and thrombin generation assay to predict clinical phenotype in patients with severe FVII deficiency

Bleeding tendency is weakly correlated with the activity of factor VII (FVII) in the plasma of patients with FVII deficiency. A laboratory method for predicting bleeding risk in patients with this coagulation disorder is lacking. We investigated whether global coagulation assays, specifically thromboelastography (TEG) and thrombin generation assay (TGA), could be used to predict bleeding risk. We also sought to identify factors that may explain the differences in bleeding phenotype observed among individuals with severe FVII deficiency. The study comprised 12 patients with severe FVII deficiency (FVII activity <1%). Eleven patients were homozygous for the Gln100Arg mutation and one patient was compound heterozygous. Clinically, 10 patients had increased haemorrhagic diathesis, whereas two patients were asymptomatic. Blood sampling was performed at baseline for TEG and TGA analyses. The platelet aggregation assay was performed and the plasma level of anticoagulation inhibitors and thrombophilic risk factors assessed. No difference in the TEG and TGA results was observed in all FVII‐deficient individuals. The level of free tissue factor pathway inhibitor was within the normal range and similar in symptomatic and asymptomatic subjects. None of the participants had the FV Leiden mutation, prothrombin gene mutation, or abnormal anticoagulant inhibitor levels. Asymptomatic subjects showed normal platelet aggregation. These data suggested that TEG and TGA were not suitable methods for predicting the clinical phenotype in FVII‐deficient subjects.     

Evaluating the impact of thrombopoietin receptor agonist medications on patient outcomes and quality of life in paediatric immune thrombocytopenia through semi-structured interviews

Over the last decade, treatment of immune thrombocytopenia (ITP) in children has advanced to include thrombopoietin receptor agonist (TPO-RA) medications. Concurrently, there has been an increased emphasis on patient-reported outcomes—especially quality of life—to guide treatment. Assessing the impact of TPO-RAs on quality of life in paediatric ITP is therefore a priority. In this single-centre integrative mixed-methods study, a cohort of children with ITP prescribed a TPO-RA was identified. These children and/or their caregivers were invited to participate in semi-structured interviews focussed on quality-of-life measures. Independently, a retrospective chart review collected ITP-related data (platelet count, bleeding events) and TPO-RA data (dosing, side effects). Among the 23 eligible patients, 20 were represented in interviews. On chart review, 11/20 patients responded to TPO-RA by meeting platelet count criteria of ≥50 × 10⁹/L for six or more weeks in the absence of rescue therapy. In interviews with these children and/or their parents, 19/20 expressed the TPO-RA had ‘worked’, with 11/20 reporting benefit to mood and 11/20 reporting increased participation in activities/sports. Concerns were raised in interviews about TPO-RA medication cost (17/20), medication administration (10/20) and potential side effects (10/20). In conclusion, this study suggests that TPO-RA use in children with ITP improves quality of life.     

Beyond the platelet count: immature platelet fraction and thromboelastometry correlate with bleeding in patients with immune thrombocytopenia

Platelet counts (PC) estimate bleeding risk in Immune Thrombocytopenia (ITP). We investigated whether measures of thromboelastometry and absolute immature platelet fraction (A‐IPF) would correlate better with acute bleeding score (ABS) than PC or mean platelet volume (MPV). Simultaneous determination of ABS, complete blood count and thromboelastometry was performed in 141 ITP patients; 112 underwent A‐IPF testing. Subgroup analyses were performed for paediatric subjects, PC <60 × 10⁹/l and <30 × 10⁹/l. PC significantly inversely correlated with ABS in all subjects, PC <30 × 10⁹/l and total paediatric cohort. MPV did not correlate with ABS in any subgroup. Thromboelastometry measures of clot firmness, but not PC, significantly correlated with ABS in all subjects with PC <60 × 10⁹/l, and children with PC <60 × 10⁹/l and <30 × 10⁹/l. A‐IPF demonstrated stronger correlation with ABS than did PC among all subjects, those with PC <60 × 10⁹/l, all children and children with PC <30 × 10⁹/l (r = ?0·37; r = ?0·34; r = ?0·44; r = ?0·60) versus ABS with PC (r = ?0·36; ns; r = ?0·32; ns). Stronger correlations of both thromboelastometry measures of clot firmness and A‐IPF than PC with ABS suggest factors beyond PC, i.e. related to platelet function, contribute to ITP bleeding pathophysiology. Thromboelastometry, A‐IPF and ABS can be incorporated into routine or acute visits.     

Blood product support for delivery in severe factor X deficiency: the use of thrombin generation to guide therapy

Background

Severe FX deficiency is a rare disorder with a variable bleeding tendency but spontaneous life threatening haemorrhage can occur. Treatment for invasive procedures and spontaneous bleeding is with prothrombin complex concentrates (PCC). When used in large or repetitive doses these are associated with a thrombotic tendency. FX:C levels of 0.15 – 0.30 IU/ mL are thought to be haemostatic during surgery . There is only limited information on the outcome and management of pregnancy in severe FX deficiency. Caesarean section is suggested as delivery mode to reduce the risk of intracranial/abdominal neonatal haemorrhage, but successful vaginal deliveries are also described. The calibrated automated thrombin generation assay (CAT) is a global coagulation test that measures the time course of thrombin generation. It has been reported to correlate with prothrombotic states and the severity of bleeding in rare coagulation disorders. The variability in phenotype, the uncertainty of the minimal haemostatic FX:C concentration and the association of PCC’s with thrombosis make thrombin generation of interest in the management of FX deficient patients.

Patient

We describe the use of CAT as a possible means to monitor treatment with PCC (Beriplex^® ) in a patient with severe FX deficiency (FX:C < 0.01 IU/mL) during successful vaginal delivery and epidural anaesthesia.

Results

Thrombin generation was normal at FX:C 0.80 IU/mL but only borderline normal at FX:C 0.25 IU/mL. Repetitive doses over 3 days increased thrombin generation to the upper limit of normal at FX:C 0.25 IU/mL consistent with a prothrombotic tendency after multiple doses. The increase in thrombin generation was not related to prothrombin levels.

Conclusion

The data suggest that CAT may be used to monitor treatment with PCC in FX deficiency. Higher levels than previously thought may be needed to normalize thrombin generation. Further studies into the correlation with bleeding or thrombosis are needed before the approach can be accepted in clinical practice.     

Effect of thrombopoietin receptor agonists on markers of coagulation and P-selectin in patients with immune thrombocytopenia

Thrombopoietin-receptor-agonists (TPO-RA) are effective treatments of immune thrombocytopenia (ITP). Previous long-term TPO-RA clinical trials have shown that thrombotic events occurred in 6% of TPO-RA-treated ITP patients. To explore the increased risk of thrombosis, the effects of TPO-RA on markers of coagulation and P-selectin were studied.

The study comprised two ITP cohorts and controls. Cohort 1 included 26 patients with sequential samples acquired before and during treatment with TPO-RA. Cohort 2 included a single sample in 18 patients on TPO-RA for more than one year. Thrombin generation (endogenous thrombin potential (ETP)) prothrombin fragments 1 + 2 (F₁₊₂), D-dimer, and plasminogen-activator-inhibitor-1 (PAI-1) were measured as well as soluble P-selectin (sP-selectin). Sequential expression of encoding genes for P-selectin (SELP) and PAI-1 (SERPINE1) was determined in four patients in cohort 1.

Significantly higher levels of F₁₊₂, D-dimer, and PAI-1 were found in ITP patients before TPO-RA treatment and in patients on long-term TPO-RA treatment than in controls. Pre-treatment levels of sP-selectin did not differ from controls. Analysis of longitudinal trends showed an increase in platelet count, sP-selectin, and PAI-1 after initiation of TPO-RA, followed by gradual decline. Platelet count and sP-selectin remained at higher levels throughout the study, whereas PAI-1 did not. Levels of other studied parameters did not show significant changes after initiation of treatment. Expression of SELP was up-regulated after initiation of TPO-RA, while the expression of SERPINE1 showed no significant changes.

In conclusion, elevated pre-treatment levels of F₁₊₂, D-dimer and PAI-1 are compatible with ITP being an intrinsically pro-thrombotic condition. After TPO-RA treatment, there were no significant changes in markers of coagulation activation or fibrinolysis, except for an initial increase in PAI-1 and a significant increase in sP-selectin both of which may contribute to increased thrombotic risk associated with TPO-RA treatment in ITP.     

Fatigue in adult patients with primary immune thrombocytopenia

Background: Patients with primary immune thrombocytopenia (ITP) commonly describe symptoms of fatigue. However, hematologists rarely consider fatigue a manifestation of ITP. Objectives: To document the prevalence of fatigue among patients with ITP and to determine the patient characteristics that are associated with fatigue. Methods: Using a cross‐sectional design, we surveyed 1871 members of the UK ITP Support Association [585 (31%) responded], and 93 patients enrolled in the Oklahoma (US) ITP Registry [68 (73%) responded] with questions about their ITP and with validated symptom assessment scales for fatigue, daytime sleepiness, and orthostatic symptoms. Results: The prevalence of fatigue among both UK (39%) and US (22%) patients was significantly greater than expected compared with normal subjects (P < 0.0001 and P < 0.0001 respectively). In univariate analysis of the combined cohorts, fatigue was associated with a platelet count <100 000/μL, treatment with steroids, bleeding symptoms, presence of other medical conditions, daytime sleepiness, and orthostatic symptoms. Fatigue was not associated with age, gender, duration of ITP, or splenectomy status. Multivariate analysis of the combined cohorts was stratified for the presence or absence of bleeding symptoms. Among 107 patients with bleeding symptoms, fatigue was independently associated with a platelet count <100 000/μL and female gender. Among 491 patients without bleeding symptoms, fatigue was independently associated with a platelet count <30 000/μL, presence of other medical conditions, daytime sleepiness, and orthostatic symptoms. Conclusions: Fatigue is a common symptom among patients with ITP. These data provide the basis for future studies to define the clinical importance of fatigue in ITP.