short report: Bleeding disorders in Lowe syndrome patients: evidence for a link between OCRL mutations and primary haemostasis disorders

Lowe syndrome (LS) is a rare X‐linked disorder caused by mutations in the oculocerebrorenal gene (OCRL), encoding OCRL, a phosphatidylinositol 5–phosphatase with a RhoGAP domain. An abnormal rate of haemorrhagic events was found in a retrospective clinical survey. Herein, we report the results of exploration of haemostasis in six LS patients. All patients had normal coagulation tests but prolonged closure times (CTs) in the PFA–100 system. Healthy donors’ blood samples incubated with a RhoA kinase inhibitor had prolonged CTs. This suggests that an aberrant RhoA pathway in platelets contributes to CT prolongation and primary haemostasis disorders in LS.     

Desmopressin in inherited disorders of platelet function

Summary.  Following the first clinical use in haemophilia and von Willebrand disease in 1977, the synthetic analogue of vasopressin 1-deamino-8- d -arginine vasopressin (DDAVP, desmopressin) was successfully employed for the management of a series of bleeding disorders, including congenital and acquired defects of platelet function. In this setting, few haemostatic approaches are available and, in particular for severe bleeding and major invasive procedures, the transfusion of platelet concentrates is the first-choice treatment. Therefore, DDAVP was (and remains) an attractive therapeutic alternative, being well tolerated, cost-saving, administrable at home (by the intranasal or subcutaneous concentrated formulations) and, in particular, enabling the avoidance of blood product exposition and the related risks (allergic reactions, transfusion transmitted infections). Despite three decades of clinical use, cellular mechanisms of haemostatic effects of DDAVP in platelet defects remain poorly known and the excellent results reported in some case series have not been strengthened by rigorous clinical trials, hampered by the rarity and the heterogeneity of these disorders. However, clinical experience more than evidence-based medicine reserved an established place to DDAVP in the management of inherited platelet disorders. This review will focus the available clinical data and the open issues of DDAVP in this setting.     

Platelet and red blood cell indices in Harris platelet syndrome

Inherited thrombocytopenias, including inherited giant platelet disorders (IGPD) or macro thrombocytopenias are relatively rare, but their prevalence is likely underestimated from complexities of diagnosis and a spectrum of subclinical phenotypes. Harris platelet syndrome (HPS) is the most common IGPD reported from the Indian subcontinent. Of note there are an increased number of hemoglobinopathies reported from the geographic location. We analysed red blood cell and platelet indices of blood donors with HPS from the north eastern part of India and compared them with blood indices of blood donors of south India. We found a statistically significant lower platelet count in blood donors with HPS (median, range) 132 (71–267) vs. 252 (160–478) as compared to donors from south India (P < 0.001). Mean platelet volume (MPV) was higher in donors with HPS 13.1, (range 12–21.9 fl) as compared to donors from south India 7.35 (range 6–9.2 fl) (P < 0.001). This study showed that blood donors with HPS had a low median platelet bio-mass 0.17 (0.10–0.38%) vs. 0.19 (0.13–0.28%) in donors from south India. The platelet distribution width (PDW) was 17.4 (14.9–19.6) in donors with HPS vs. 16.38 (15.2–18.5) in south Indian blood donors (P < 0.001). Thirty-three donors with HPS had a normal platelet count with MPV more than 12 fL. Only donors with HPS had giant platelets and thrombocytopenia on peripheral blood smear examination. None of these donors had Dohle body inclusion in their leukocytes. Compared to donors from south India, donors with HPS had a significantly lower hemoglobin 13.8 (12–16.3 gm/dL) vs. 14.8 (12–18) respectively (P < 0.001) while red distribution width (RDW) was higher in HPS 13.6 (11.5–16.7) vs. 12.8 (11.4–15.1). However we did not find any statistically significant difference in MCV, MCH, MCHC between the two groups. Peripheral blood smear did not show any obvious abnormal red blood cell morphology. In the blood donors with HPS we found a statistically higher MPV, RDW and a lower platelet count and platelet biomass. A population-based study will be helpful in determining the existence of any hemoglobinopathies among subjects with HPS.     

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).     

Characterization of a novel bleeding disorder with isolated prolonged bleeding time and deficiency of platelet microvesicle generation

Platelet prothrombinase activity and microvesicle (MV) generation were measured in four patients from three unrelated families with a life-long bleeding disorder associated with slightly prolonged bleeding time and isolated defective serum prothrombin consumption, without platelet function abnormality or von Willebrand factor defect. MV generation was reduced in all the patients either after thrombin plus collagen or A23187 calcium ionophore stimulation, whereas, at variance with Scott syndrome, prothrombinase activity was normal. This abnormality constitutes a new bleeding disorder, which provides new insights into the possible role of platelet microvesicles in health and disease. Furthermore, the results of this study suggest that MV generation should be investigated in patients with a bleeding history and apparently isolated prolonged bleeding time when prothrombin comsumption in serum is defective and all other investigations are normal.     

Guidelines for the laboratory investigation of heritable disorders of platelet function

The guideline writing group was selected to be representative of UK-based medical experts. MEDLINE was systematically searched for publications in English up to the Summer of 2010 using key words platelet, platelet function testing and platelet aggregometry. Relevant references generated from initial papers and published guidelines/reviews were also examined. Meeting abstracts were not included. The writing group produced the draft guideline, which was subsequently revised and agreed by consensus. Further comment was made by members of the Haemostasis and Thrombosis Task Force of the British Committee for Standards in Haematology. The guideline was then reviewed by a sounding board of approximately 40 UK haematologists, the British Committee for Standards in Haematology (BCSH) and the British Society for Haematology Committee and comments incorporated where appropriate. Criteria used to quote levels and grades of evidence are as outlined in appendix 7 of the Procedure for Guidelines Commissioned by the BCSH [http://www.bcshguidelines.com/BCSH_PROCESS/EVIDENCE_LEVELS_AND_GRADES_OF_RECOMMENDATION/43_GRADE.html]. The objective of this guideline is to provide healthcare professionals with clear guidance on platelet function testing in patients with suspected bleeding disorders. The guidance may not be appropriate to patients receiving antiplatelet therapy and in all cases individual patient circumstances may dictate an alternative approach.     

Genotype–phenotype relationship for six common polymorphisms in genes affecting platelet function from 286 healthy subjects and 160 patients with mucocutaneous bleeding of unknown cause

Polymorphisms affecting platelet receptors and intracellular proteins have been extensively studied in relation to their potential influence in thrombosis and haemorrhages. However, few reports have addressed their impact on platelet function, with contradictory results. Limitations of these studies include, among others, small number of patients, the platelet functional parameters analyzed and their known variability in the healthy population. We studied the effect of six polymorphisms [ ITGB3 1565T > C (HPA-1), GPIBA variable number tandem repeat and 524C > T (HPA-2), ITGA2 807C > T , ADRA2A 1780A > G , and TUBB1 Q43P ] on platelet function in 286 healthy subjects and their potential pathogenetic role in 160 patients with hereditary mucocutaneous bleeding of unknown cause. We found no effect of any of these polymorphisms on platelet aggregation, secretion, PFA-100^®, and thrombin generation in platelet rich plasma. Furthermore, patients and controls showed no significant differences in the frequency of any of these polymorphisms. Thus, our study demonstrated that polymorphisms in genes affecting platelet function do not influence significantly major platelet functions and appear irrelevant in the pathogenesis of bleeding disorders.     

A review of inherited platelet disorders with guidelines for their management on behalf of the UKHCDO

The inherited platelet disorders are an uncommon cause of symptomatic bleeding. They may be difficult to diagnose (and are likely to be under-diagnosed) and pose problems in management. This review discusses the inherited platelet disorders summarising the current state of the art with respect to investigation and diagnosis and suggests how to manage bleeding manifestations with particular attention to surgical interventions and the management of pregnancy.     

Investigation of platelet function and platelet disorders using flow cytometry

Patients with thrombocytopenia or platelet disorders are at risk of severe bleeding. We report the development and validation of flow cytometry assays to diagnose platelet disorders and to assess platelet function independently of platelet count. The assays were developed to measure glycoprotein levels (panel 1) and platelet function (panel 2) in sodium citrated blood. Twenty healthy volunteers and five patients diagnosed with different platelet disorders were included. Glycoprotein expression levels of the receptors Ia, Ib, IIb, IIIa and IX were measured and normalised with forward scatter (FS) as a measurement of platelet size. Platelet function was assessed by CD63, P-selectin and bound fibrinogen in response to arachidonic acid, adenosine diphosphate (ADP), collagen-related peptide, ristocetin and thrombin receptor-activation peptide-6. All patients except one with suspected δ-granule defect showed aberrant levels of glycoproteins in panel 1. Glanzmann's thrombasthenia and genetically verified Bernard–Soulier syndrome could be diagnosed using panel 1. All patients showed reduced platelet function according to at least one agonist. Using panel 2 it was possible to diagnose Bernard–Soulier syndrome, δ-granule defect and GPVI disorder. By combining the two assays, we were able to diagnose different platelet disorders and investigate platelet function independent of platelet count.     

Investigation of the contribution of an underlying platelet defect in women with unexplained heavy menstrual bleeding

Heavy menstrual bleeding (HMB) is often undiagnosed in women and can cause discomfort and distress. A haemostatic cause for excessive bleeding is often not routinely investigated and can lead to hysterectomy at an early age. A prospective cohort study was carried out to determine whether certain patients with unexplained HMB have an underlying platelet function defect (PFD). The Genotyping and Phenotyping of Platelets (GAPP) study recruited 175 women with HMB and 44 unrelated volunteers from 25 Haemophilia Centres across the UK, and a tertiary gynaecology service. Bleeding history was assessed using the International Society on Thrombosis and Haemostasis Bleeding Assessment Tool (ISTH-BAT). Platelet count, platelet size, haemoglobin and mean corpuscular volume were measured in whole blood using the Sysmex XN-1000 Haematology Analyzer. Platelet function testing using lumiaggregometry and flow cytometry was performed in patients included in this study. A PFD was identified in 47% (82/175) of patients with HMB. Cutaneous bleeding was the most frequent additional bleeding symptom (89% in PFD and 83% with no PFD). Whole blood platelet count was significantly lower (P < 0.0001) between the PFD group and no PFD group. The prevalence of anaemia did not differ between patients and healthy volunteers. Clinical evaluation alone is insufficient to determine presence of an underlying PFD in patients with HMB. Platelet function tests may be considered and clinical guidelines may include them in their algorithms. An appropriate diagnosis and subsequent tailored management of HMB may prevent unnecessary surgery and help manage future haemostatic challenges.     

Diagnostic evaluation of platelet function disorders

Platelet function disorders are inherited and acquired conditions that represent a common cause of bleeding. Their clinical findings are generally similar to von Willebrand disease. It is often challenging to diagnose common platelet function disorders due to heterogeneity in their features, uncertainties about their pathogenesis and genetic cause, variability in the procedures used to assess platelet function in diagnostic laboratories and the lack of diagnostic criterion. Some inherited platelet function disorders have been established to increase risks for bleeding and bleeding scores. However, bleeding history assessment tools are not validated for use in diagnosing platelet function disorders. Standardized tests that assess aggregation function, dense granule deficiency and dense granule secretion are useful for diagnosing common platelet function disorders, in addition to some rare conditions. Guidelines have emerged to improve and standardize the laboratory tests for diagnosing platelet function disorders, including how to interpret aggregometry findings. Nonetheless, there is need to further evaluate the features, pathogenesis and genetic cause of many platelet function disorders, including the inherited conditions that impair granule secretion.     

Prediction of bleeding and prophylactic platelet transfusions in cancer patients with thrombocytopenia

Studies on markers for bleeding risk among thrombocytopenic cancer patients are lacking. This prospective observational cohort study investigated whether platelet parameters and a standardised bleeding questionnaire predicted bleeding or prophylactic platelet transfusions in patients with cancer and thrombocytopenia. Admitted adult patients with cancer and platelet count <80 × 10⁹/L were enrolled, but excluded if they experienced surgery or trauma within 7 days or platelet transfusion within 14 days. Patients were interviewed, blood samples collected and, subsequently, spontaneous bleeding and prophylactic platelet transfusion within 30 days were registered.

Of 197 patients enrolled, 56 (28%) experienced bleeding. In multivariate analyses, predictors of bleeding were infection (adjusted odds ratio (OR) = 2.65 and 95% confidence interval (95% CI) 1.04–6.74); treatment with platelet inhibitors, heparin or warfarin OR = 2.34, 95% CI 1.23–4.48; urea nitrogen OR = 1.15, 95% CI 1.07–1.25; creatinine OR = 1.01, 95% CI 1.01–1.01; and haemoglobin OR = 0.62, 95% CI 0.41–0.93. Specific information regarding previous gastrointestinal bleeding OR = 3.33, 95% CI 1.19–9.34 and haematuria OR = 3.00, 95% CI 1.20–7.52 predicted bleeding whereas the standardised bleeding questionnaire did not.

Prophylactic platelet transfusions were administered to 97 patients. Predictors of prophylactic platelet transfusions were: platelet count OR = 0.96, 95% CI 0.94–0.97; fibrinogen OR = 0.88, 95% CI 0.83–0.95; mean platelet volume OR = 0.69, 95% CI 0.49–0.97; platelet aggregometry with OR = 2.48, 95% CI 1.09–5.64 for collagen-induced platelet aggregation within the lowest quartile; and albumin OR = 1.07, 95% CI 1.01–1.15.

In conclusion, except for immature platelet fraction (IPF), platelet parameters predicted prophylactic platelet transfusion but not bleeding. Bleeding risk factors were previous haematuria or gastrointestinal bleeding, infection, antiplatelet or anticoagulant treatment, high urea nitrogen, low haemoglobin or high creatinine.     

Analysis of 65 pregnancies in 34 women with five different forms of inherited platelet function disorders

This study evaluated 65 pregnancies in 34 women with five different inherited platelet function disorders. Gestation was similar to that of the general population. Severe bleeds requiring blood transfusions were observed in 50% of deliveries in Glanzmann thrombasthenia (GT), but not in the patients with delta storage pool disease, Hermansky‐Pudlak syndrome, P2Y12 defect or defect of thromboxane A2 receptor. Of note, severe haemorrhage also occurred in women with GT who had received prophylactic platelet transfusions, suggesting that better preventive treatments are required. Diagnosis and degree of spontaneous bleeding tendency before pregnancy were reliable parameters to predict the delivery‐related bleeding risk.     

Clinical utility of remote platelet function measurement using P-selectin: assessment of aspirin,clopidogrel, and prasugrel and bleeding disorders

Vascular diseases such as myocardial infarction and ischemic stroke are associated with increased platelet function whilst the risk of recurrence is reduced by antiplatelet agents such as aspirin, clopidogrel, and prasugrel. However, some patients exhibit high platelet reactivity, especially with clopidogrel. Existing platelet function tests may not be ideal in that they can be expensive, are often time consuming, and measurements must be made near to the patient and within a few hours of blood collection. Platelet activation leads to translocation of P-selectin from alpha-granules to the cell surface. Following activation with arachidonic acid (which is blocked by aspirin) or adenosine diphosphate (inhibited by clopidogrel) and fixation, samples may be stored or posted to a laboratory performing flow cytometric quantification of platelet P-selectin expression. Acute myocardial infarction and ischemic stroke are associated with high platelet reactivity on clopidogrel in 6–58% of patients when assessed with P-selectin expression, and high reactivity was associated with an increased risk of recurrence after myocardial infarction. Use of P-selectin expression tests may also be of relevance to surgical and veterinary practice and the diagnosis of mild bleeding disorders. The present review explores this topic in further detail.     

MicroRNAs as potential regulators of platelet function and bleeding diatheses

Although a growing number of studies suggest that microRNAs (miRNAs) play a relevant role in platelet biology, their implications in bleeding diatheses are starting to be investigated. Indeed, several studies have shown that alterations in the intracellular levels of highly expressed platelet miRNAs provoke a thrombotic phenotype. On the other hand, primary immune thrombocytopenia (ITP), which is considered the hallmark of acquired bleeding disorders, has been recently associated with altered levels of miRNAs in peripheral blood mononuclear cells, plasma, and platelets. In this review, we will focus on miRNAs that may affect the hemostatic and thrombotic functions of platelets, and we will discuss the different studies that have attempted to associate miRNAs with regulatory mechanisms of ITP.     

eNOS gene influences platelet phenotype in acute coronary syndrome patients on dual antiplatelet treatment

Nitric Oxide (NO) plays a relevant role in regulating platelet recruitment and eNOS is the major isoform known to be expressed in platelets. Polymorphisms in the eNOS gene with a reduced NO availability might affect platelet phenotype. The aim of our study was to evaluate the role of eNOS–786T > C, 894G > T and 4a/4b polymorphisms in modulating platelet phenotype in 1442 acute coronary syndrome (ACS) patients on dual antiplatelet therapy, previously investigated in relation to platelet function. Platelet aggregation on platelet-rich plasma after collagen (2 µg/mL), ADP (10 µM) and arachidonic acid (AA) (1 mM) stimuli and the genetic analysis of eNOS polymorphisms were assessed. In subjects carrying the eNOS 4a and -786C alleles a significantly higher maximal platelet aggregation value after AA was found (p = 0.02 and p = 0.047, respectively). eNOS 4a but not -786C allele weakly influenced platelet aggregation after collagen stimulus (p = 0.05). eNOS 4a allele significantly and independently influenced AA-induced platelet aggregation (p = 0.01). A significantly higher percentage of patients with AA-induced high residual platelet reactivity (RPR) was found in subjects carrying both eNOS 4a and -786C allele (p = 0.03 and p = 0.04, respectively). At logistic multivariate analysis, the eNOS 4a allele significantly influenced the AA-induced high residual platelet reactivity (p = 0.02). This study evidences a role for eNOS gene in moderately, but significantly, modulating platelet phenotype in a high-risk population on dual antiplatelet treatment.     

Cone and platelet analyser (CPA): a new test for the prediction of bleeding among thrombocytopenic patients

The risk of bleeding among thrombocytopenic patients was evaluated using our new cone and platelet analyser (CPA) test. Using this test, adherence of platelets was quantitated on extracellular matrix and expressed as percent of surface coverage (SC) and the average size (AS) of aggregates. 42 thrombocytopenic patients with ITP (n=23), post chemotherapy (n=12) and others (n=7) were tested over a total of 82 visits. On each visit, complete blood count and CPA tests were performed and patients were evaluated for evidence of bleeding (found in 40 visits). Bleeding patients had significantly lower platelet counts (27.4 ± 22.0 v 47.1 ± 21.0 × 10⁹/l), lower haematocrit values (30.2 ± 8.1 v 35.2 ± 6.6%), lower MPV (6.83 ± 1.89 v 8.98 ± 1.13 fl), and lower SC (4.87 ± 3.95 v 10.33 ± 5.48%) and AS (33.99 ± 14.94 v 52.9 ± 24.34 μm²). Univariate analysis yielded platelet count 20.0 × 10⁹/l, MPV 8fl, haematocrit <35%, SC < 5%, AS 40 μm² as significantly associated with bleeding, whereas only MPV and SC were associated with bleeding (OR 6.95, CI 2.25–21.46 and OR 4.27, CI 1.29–14.16, respectively) by multivariate analysis. When taken together, 21/22 of patients (95%) with both low SC (<5%) and low MPV (<8.0 fl) had bleeding symptoms, whereas only 9/43 (21%) patients with both these parameters above these values experienced bleeding symptoms. We conclude that the CPA test and the parameter SC (<5%) together with MPV (8 fl) might be used as independent predictors of bleeding in the management of thrombocytopenic patients.