Search

Bleeding and thrombocytopenia are common referrals to the pediatric and adult hematology practice. The differential diagnosis encompasses a wide spectrum of entities that vary in acuity, severity, and etiology. Most will be acquired (especially in adult patients), but many can be inherited, and some may have manifestations affecting other organ systems. The first step: defining whether the symptoms and/or laboratory findings are clinically significant and warrant additional work-up, can be equally as challenging as reaching the diagnosis itself. How much bleeding is too much to be considered normal? How low of a platelet count is too low? Once the decision has been made to pursue additional studies, considering the increasing number of laboratory tests available, the diagnostic process can be complex. In this article, we outline a general approach for the evaluation of patients in whom an inherited platelet disorder is being considered. We present two clinical vignettes as introduction to the diagnostic approach to inherited platelet disorders. We describe the rationale for the different types of tests that are clinically available, their limitations, and finally the challenges that are frequently encountered in the interpretation of results. We also intend to provide some guidance on the expected phenotype in terms of severity of bleeding and/or thrombocytopenia according to the etiology of the inherited disorder. Our goal is to provide the practicing hematologist with a practical framework that is clinically applicable in their daily practice.

Essentials Patients with von Willebrand disease were enrolled in our study. Type 2 VWD diagnoses were based on original test results. Repeat evaluation resulted in many patients receiving a different type 2 diagnosis. Some genetic variants were particularly likely to move type 2 subcategories. ABSTRACT: Introduction Type 2 von Willebrand disease (VWD) refers to patients with a qualitative defect in von Willebrand factor. Accurate diagnosis of type 2 VWD subtypes can be challenging. Aim of the study To compare the historical diagnosis of type 2 VWD with current laboratory testing. Methods Subjects were enrolled in the Zimmerman Program either because of a preexisting diagnosis of VWD (retrospective cohort) or from evaluation for bleeding symptoms or suspected VWD (prospective cohort). Original diagnosis was assigned by the local center and central diagnosis was based on central laboratory testing. Results Two hundred and seventeen index cases in the retrospective cohort and 35 subjects in the prospective cohort carried a local diagnosis of type 2 VWD (29% and 6% of enrolled index cases, respectively). In the retrospective cohort, the diagnosis was confirmed in 66% of cases with a preexisting diagnosis of 2A, 77% 2B, 54% 2M, and 72% 2N. In the prospective cohort, 31% were confirmed 2A, 60% 2B, 23% 2M, and 100% 2N. Several genetic variants were repeatedly implicated in subjects with changed diagnosis: p.M1304R, p.R1315C, p.R1374C, and p.R1374H. Conclusions Both the prospective and retrospective cohorts demonstrated consistent variation in subjects whose diagnosis changed between 2A, 2B, and 2M. The importance of accurately diagnosing type 2 VWD may be most significant in the 2B subtype given potential concerns with the use of desmopressin in type 2B VWD. Some genetic variants appear in multiple types of VWD, making specific diagnoses challenging.

Background The variability in bleeding patterns among individuals with hemophilia A, who have similar factor VIII (FVIII) levels, is significant and the origins are unknown. Objective To use a previously validated mathematical model of flow-mediated coagulation as a screening tool to identify parameters that are most likely to enhance thrombin generation in the context of FVIII deficiency. Methods We performed a global sensitivity analysis (GSA) on our mathematical model to identify potential modifiers of thrombin generation. Candidates from the GSA were confirmed by calibrated automated thrombography (CAT) and flow assays on collagen-tissue factor (TF) surfaces at a shear rate of 100 per second. Results Simulations identified low-normal factor V (FV) (50%) as the strongest modifier, with additional thrombin enhancement when combined with high-normal prothrombin (150%). Low-normal FV levels or partial FV inhibition (60% activity) augmented thrombin generation in FVIII-inhibited or FVIII-deficient plasma in CAT. Partial FV inhibition (60%) boosted fibrin deposition in flow assays performed with whole blood from individuals with mild and moderate FVIII deficiencies. These effects were amplified by high-normal prothrombin levels in both experimental models. Conclusions These results show that low-normal FV levels can enhance thrombin generation in hemophilia A. Further explorations with the mathematical model suggest a potential mechanism: lowering FV reduces competition between FV and FVIII for factor Xa (FXa) on activated platelet surfaces (APS), which enhances FVIII activation and rescues thrombin generation in FVIII-deficient blood.

Essentials Signaling by Gas6 through Tyro3/Axl/Mer receptors is essential for stable platelet aggregation. UNC2025 is a small molecule inhibitor of the Mer tyrosine kinase. UNC2025 decreases platelet activation in vitro and thrombus formation in vivo. UNC2025's anti-platelet effect is synergistic with inhibition of the ADP receptor, P2Y12. SummaryBackground Growth arrest-specific protein 6 signals through the TAM (TYRO-3–AXL–MERTK) receptor family, mediating platelet activation and thrombus formation via activation of the aggregate-stabilizing αIIbβ3 integrin. Objective To describe the antithrombotic effects mediated by UNC2025, a small-molecule MERTK tyrosine kinase inhibitor. Methods MERTK phosphorylation and downstream signaling were assessed by immunoblotting. Light transmission aggregometry, flow cytometry and microfluidic analysis were used to evaluate the impact of MERTK inhibition on platelet activation and stability of aggregates in vitro. The effects of MERTK inhibition on arterial and venous thrombosis, platelet accumulation at microvascular injury sites and tail bleeding times were determined with murine models. The effects of combined treatment with ADP–P2Y1&12 pathway antagonists and UNC2025 were also evaluated. Results and Conclusions Treatment with UNC2025 inhibited MERTK phosphorylation and downstream activation of AKT and SRC, decreased platelet activation, and protected animals from pulmonary embolism and arterial thrombosis without increasing bleeding times. The antiplatelet effect of UNC2025 was enhanced in combination with ADP–P2Y1&12 pathway antagonists, and a greater than additive effect was observed when these two agents with different mechanisms of inhibition were coadministered. TAM kinase signaling represents a potential therapeutic target, as inhibition of this axis, especially in combination with ADP–P2Y pathway antagonism, mediates decreased platelet activation, aggregate stability, and thrombus formation, with less hemorrhagic potential than current treatment strategies. The data presented here also demonstrate antithrombotic activity mediated by UNC2025, a novel translational agent, and support the development of TAM kinase inhibitors for clinical applications.

Essentials Studies characterizing neutralizing antibodies (inhibitors) in hemophilia B (HB) are lacking. The current study describes anti–factor (F) IX antibody profiles in 37 patients who have HB. Anti-FIX IgG4 levels exhibited a strong positive correlation with Nijmegen–Bethesda results. These data will help to more clearly define, predict, and treat alloantibody formation in HB. SummaryBackground Hemophilia B (HB) is an inherited bleeding disorder caused by the absence or dysfunction of coagulation factor IX (FIX). A subset of patients who have HB develop neutralizing alloantibodies (inhibitors) against FIX after infusion therapy. HB prevalence and the proportion of patients who develop inhibitors are much lower than those for hemophilia A (HA), which makes studies of inhibitors in patients with HB challenging due to the limited availability of samples. As a result, there is a knowledge gap regarding HB inhibitors. Objective Evaluate the largest group of patients with inhibitor-positive HB studied to date to assess the relationship between anti-FIX antibody profiles and inhibitor formation. Methods A fluorescence immunoassay was used to detect anti-FIX antibodies in plasma samples from 37 patients with HB. Results Assessments of antibody profiles showed that anti-FIX IgG1-4, IgA, and IgE were detected significantly more often in patients with a positive Nijmegen–Bethesda assay (NBA). All NBA-positive samples were positive for IgG4. Anti-FIX IgG4 demonstrated a strong correlation with the NBA, while correlations were significant, yet more moderate, for anti-FIX IgG1-2 and IgA. Conclusions The anti-FIX antibody profile in HB patients who develop inhibitors is diverse and correlates well with the NBA across immunoglobulin (sub)class, and anti-FIX IgG4 is particularly relevant to functional inhibition. The anti-FIX fluorescence immunoassay may serve as a useful tool to confirm the presence of antibodies in patients who have low positive NBA results and to more clearly define, predict, and treat alloantibody formation against FIX.

Hemophilia A (HA) is an X-linked bleeding disorder caused by a deficiency in factor VIII (FVIII). von Willebrand disease (VWD) is characterized by a quantitative or qualitative defect in von Willebrand factor (VWF). Patients with VWD with severely low VWF or VWD Type 2N (VWD2N), a VWD subtype distinguished by defective VWF binding to FVIII, may have reduced FVIII levels secondary to their VWD. These patients superficially resemble patients with HA and pose a potential for misdiagnosis.To investigate the unexplained cause of bleeding in HA patients without known FVIII mutations by assessing plasma VWF antigen (VWF:Ag), FVIII binding capacities and VWF genotypes.Thirty-seven of 1027 patients with HA studied as part of the Hemophilia Inhibitor Research Study lacked identifiable F8 mutations. These patients (cases) and 73 patients with identified F8 mutations (controls) were evaluated for VWF:Ag, a patient's VWF capacity to bind FVIII (VWF:FVIIIB) and VWF sequence.Four cases had VWF:Ag3 IU dL(-1) and VWF mutations consistent with Type 3 VWD. Six cases and one control were heterozygous for mutations previously reported to cause Type 1 VWD (VWD1) (n = five cases and one control) or predicted to be deleterious by Polyphen2 and SIFT prediction tools (n = 1 case). One control had VWF:Ag30 IU dL(-1) and seven patients (four cases and three controls), including two cases who were heterozygous for a known VWD2N mutation, had reduced VWF:FVIIIB.These data emphasize that some patients diagnosed with HA require VWF assessments in order to achieve a comprehensive diagnosis and an optimal treatment strategy.

Essentials Idiopathic systemic capillary leak syndrome (SCLS) is characterized by episodes of vascular leakage. We present the case of a patient with SCLS who developed microangiopathic hemolytic anemia (MAHA). We propose that this anemia is the result of ADAMTS-13 loss in the third-space fluid. This suggests that MAHA can occur in patients with significant extravasation of proteins. Summary Idiopathic systemic capillary leak syndrome (SCLS) is a rare process characterized by acute and recurrent episodes of vascular leakage with severe hypotension, hypoalbuminemia, hemoconcentration and edema. Anemia and thrombocytopenia are not part of this syndrome, but here we present the case of a pediatric patient with a clinical presentation consistent with SCLS who subsequently developed microangiopathic hemolytic anemia at a time when she had significant fluid loss and anasarca. Based on serial ADAMTS-13 levels, we propose that the anemia in this patient developed as a result of ADAMTS-13 loss in the third-space fluid, a novel mechanism for acquired microangiopathic hemolytic anemia.

The development of neutralizing antibodies, referred to as inhibitors, against factor VIII is a major complication associated with FVIII infusion therapy for the treatment of hemophilia A (HA). Previous studies have shown that a subset of HA patients and a low percentage of healthy individuals harbor non-neutralizing anti-FVIII antibodies that do not elicit the clinical manifestations associated with inhibitor development.To assess HA patients' anti-FVIII antibody profiles as potential predictors of clinical outcomes.A fluorescence immunoassay (FLI) was used to detect anti-FVIII antibodies in 491 samples from 371 HA patients.Assessments of antibody profiles showed that the presence of anti-FVIII IgG1 , IgG2 or IgG4 correlated qualitatively and quantitatively with the presence of an FVIII inhibitor as determined with the Nijmegen-Bethesda assay (NBA). Forty-eight patients with a negative inhibitor history contributed serial samples to the study, including seven patients who had negative NBA titers initially and later converted to being NBA-positive. The FLI detected anti-FVIII IgG1 in five of those seven patients prior to their conversion to NBA-positive. Five of 15 serial-sample patients who had a negative inhibitor history and had anti-FVIII IgG1 later developed an inhibitor, as compared with two of 33 patients with a negative inhibitor history without anti-FVIII IgG1 .These data provide a rationale for future studies designed both to monitor the dynamics of anti-FVIII antibody profiles in HA patients as a potential predictor of future inhibitor development and to assess the value of the anti-FVIII FLI as a supplement to traditional inhibitor testing.

Genetic variants in the GP1BA and ITGA2 genes have been proposed as potential modifiers for arterial vascular disease and bleeding disorders. Since ancestry may play an important role in the prevalence of these variants, we sought to determine their allele frequency and linkage disequilibrium in a collection of 1064 DNA samples from 51 ethnic groups. We studied haplotypes of ITGA2 defined by single nucleotide substitutions at positions -52, 807, and 1648, and GP1BA variants defined by sequence changes in positions -5 (Kozak), 1018 (T145M, HPA-2) and 1285 (VNTR A, B, C and D). Frequency of haplotypes of ITGA2 showed considerable variation across the different groups, with a higher prevalence of the haplotype -52C or T/807C/1648A observed in African compared with caucasian and Asian populations. The haplotypes 52C/807T/1648A and -52T/807T/1648A were not observed in caucasians or South Americans. While relative frequencies of the GP1BA Kozak alleles were comparable across groups, the methionine allele (HPA-2b) showed a higher frequency in Africa (0.26) than in the other groups. We also observed a high prevalence of the VNTR B allele in the African and Israeli populations. Haplotype analysis revealed incomplete linkage disequilibrium between the HPA-2 and VNTR alleles. Incorporation of GP1BA variants into the set of SNPs already genotyped by the HapMap project disrupted the pre-existing haplotype block. These data provide a valuable resource for optimal selection of variants best tailored for association studies of vascular disease or bleeding disorders when examining individuals of different ancestral origins.