Diagnosis of platelet dysfunction in children: clinical predictors and test methods

Blood Vessels Thrombosis & Hemostasis · 2025-12-15 · 1 citations

= .026). Neither mucocutaneous bleeding (most common presenting indication) nor bleeding severity scores correlated with increased odds of PFD diagnosis. Both LTA and WBILA showed sensitivity of >91% and specificity of >84% for PFD diagnosis. A multivariable model identified younger age, male sex, thrombocytopenia, genetic disorders, and gastrointestinal bleeding with PFD diagnosis in the WBILA cohort. In conclusion, younger, male patients have a higher incidence of PFD. These data support that WBILA can effectively rule out PFD in a pediatric population.

HTRS2025.O8B.1 Abstract Travel Award

Research and Practice in Thrombosis and Haemostasis · 2025-11-01

Use of CD19-targeted immune modulation to eradicate AAV-neutralizing antibodies

Molecular Therapy · 2025-03-08 · 6 citations

Neutralizing antibodies (NAbs) against adeno-associated virus (AAV) represent a significant obstacle to the efficacy of systemic recombinant AAV vector administration or re-administration. While there are some promising preclinical immunomodulation strategies in development, insights into which B cell subsets and compartments maintain persistent AAV NAb may define the optimal eradication strategy. Given the limited success of CD20-directed monotherapy in previous studies, we hypothesized that CD19-directed approaches that extend targeting into the plasma cell compartments may improve AAV NAb eradication. We tested this approach in mice using chimeric antigen receptor T (CAR-T) cells or monoclonal antibodies (mAbs). We observed that combination mAbs targeting CD19, CD22, CD20, or B220 in mice did not eliminate tissue-resident B cells and, correspondingly, did not deplete pre-existing high titer AAV8 NAb. In contrast, CD19 CAR-T therapy eliminated peripheral and tissue-resident B cells and plasma cells and resulted in a marked reduction or eradication of high titer AAV8 NAb that permitted successful transgene expression following systemic AAV8 re-administration in mice. This successful therapeutic approach in mice identifies the population and location of B cells necessary to reduce or eradicate AAV NAb sufficiently to permit successful transgene expression with systemic AAV vector administration.

Rates of inhibitor development and immune tolerance in children with severe Hemophilia A on emicizumab prophylaxis

Blood · 2025-11-03

Abstract Introduction: Congenital hemophilia A (HA) is a genetically inherited bleeding disorder caused by pathologic variants in the F8 gene, resulting in absent or decreased levels of coagulation factor VIII (FVIII) protein and impairment of physiologic hemostasis. The absence of FVIII results in both trauma-induced and spontaneous bleeding in people with HA (PwHA), which can be life-threatening. Treatment or prevention of bleeding with exogenous FVIII protein infusions can lead to the development of neutralizing anti-FVIII antibodies (“inhibitors”), which render FVIII treatment ineffective. The use of emicizumab, a bispecific antibody which mimics the activity of FVIII, circumvents the need for FVIII prophylactic infusions. In real-world and clinical trial data, bleeding rates and FVIII infusion utilization have decreased in PwHA on emicizumab prophylaxis. Furthermore, historically PwHA with inhibitors were treated with immune tolerance induction (ITI) to eradicate FVIII inhibitors, however, the necessity of ITI in the era of emicizumab is an open question. The rates of inhibitor development and uptake of ITI in this current era of treatment are unknown. Objectives: This study aims to compare PwHA whose initial prophylactic regimen was FVIII compared to those receiving emicizumab as initial prophylaxis. The primary objective was to compare FVIII exposure days (EDs) and inhibitor development between PwHA on FVIII or emicizumab prophylaxis. The secondary objective is to understand the use of ITI treatment in PwHA on either treatment. Methods: A retrospective chart review was performed at the Children's Healthcare of Atlanta (CHOA) Hemophilia Treatment Center under an IRB-approved protocol. PwHA born between 2014 and 2023 with severe disease were included. Demographic data and clinical data (hemophilia mutation as well as inhibitor, ITI, and FVIII ED history) were extracted from the clinical chart. Rates of inhibitor development and ITI usage were compared before and after licensure of emicizumab for all PwHA (2018). Results:A total of 53 males with severe Hemophilia A born between 2014 and 2023 were included in the study. Initial prophylaxis was FVIII in 30 (56.6%) and emicizumab in 23 (43.4%). The study population was 43.6% Black, 41.8% Caucasian, 3.6% Asian, 3.6% multiracial, 7.3% of unknown race, and 25.5% identified as Hispanic. There were no differences in race by initial prophylactic regimen or FVIII inhibitor status. A total of 22 participants developed inhibitors, 15 (68.8%) from the FVIII prophylaxis group and 7 (31.8%) from the emicizumab group. At data cutoff, the incidence of inhibitors was 50% in FVIII group compared to 30.4% in the emicizumab group (odds ratio 2.14, 95% CI 0.69-6.16) at median of 17 (range 3-90) versus 10 (range 4-15) FVIII EDs, respectively (p=0.032, t-test). The median cumulative FVIII EDs in the FVIII group was >150 (range 5 to > 150) and in the emicizumab group was 6 (range 2-112). Of note, 15/23 PwHA with emicizumab prescribed as their initial prophylactic regimen had FVIII exposures prior to emicizumab initiation with a median of 4 FVIII EDs (range 1-13). Consequently, the median age at ED1 was similar between the FVIII and emicizumab groups (9.5 vs 8.6 mo) but median age at ED5 was delayed in the emicizumab group (13.5 vs 17.8 mo, p = 0.011, Mantel-Cox time to event analysis). As expected, emicizumab delayed FVIII EDs as demonstrated by only 1/22 participants in the emicizumab group having surpassed ED20 compared to all 26/30 in the FVIII group (p<0.001, Fisher's exact test) at the last data cutoff. The inhibitor rates in PwHA born before or after 2018 (emicizumab licensure year) were similar at 40.7% and 39.3%, respectively. The rate of ITI usage in PwHA with inhibitors has decreased from 76.9% before emicizumab licensure to 22.2% after. Conclusion:Our data suggest that FVIII inhibitor rates may be lower in PwHA initially started on emicizumab prophylaxis, though these PwHA may not have yet reached sufficient FVIII EDs. In addition, the elevated number of Black study participants adds critical data given racial disparities in research on inhibitor development. Ongoing analyses will include a comparison of the timing of FVIII EDs and inhibitor development in PwHA on emicizumab prophylaxis to determine if inhibitor rates upon reaching 50 FVIII EDs are different than for those on FVIII. Lastly, initiation of ITI appears to be decreased or delayed since the licensure of emicizumab.

Response to TFPI-inhibition in severe factor v deficiency with a novel compound heterozygous mutation

Blood · 2025-11-03

Abstract Introduction Coagulation factor V (FV) plays a central role in blood clot formation. FV has both procoagulant and anticoagulant properties making it a key regulator of thrombin generation. Congenital deficiency of FV is rare (1 in 1 million births) and presents with mild to severe bleeding complications in the affected individuals. Low to undetectable antigen levels and low functional activity is a hallmark of homozygous or compound heterozygous mutations in the F5 gene. Heterozygous individuals have about 50% normal FV antigen expression and are asymptomatic. Prior studies have shown that plasma tissue factor pathway inhibitor-alpha (TFPIα) levels correlate with plasma FV. It is thought in FV deficiency reduced levels of TFPIα may blunt the bleeding phenotype making it an important and unique modifier of FV deficiency. Patient history Here, we describe a case of a 14 year old male with severe FV deficiency (<1% FV activity). He had intracranial hemorrhage during infancy and has been admitted multiple (8) times for bleeding episodes and treated with plasma transfusions. Bleeds were either from sports participation or spontaneous joint and muscle bleeds. His mother is clinically asymptomatic with no bleeds despite past caesarean births and has FV level of ~50%. Methods Genomic DNA from the consented patient was isolated from peripheral blood and then outsourced to PreventionGenetics (USA) for a F5 gene testing and analyses using Next-Gen sequencing. FV activity was determined with standard one-stage clotting assay. Plasma and platelet FV antigens were measured and visualized by immunoassays and western blotting analyses using FV-specific polyclonal and monoclonal (mAb) antibodies, respectively. Free plasma protein S was measured with a commercial ELISA kit (Asserachrom®, Stago). Thrombin generation assays (TGA) were triggered with low tissue-factor concentration and thrombin generation assessed with anti-TFPIα mAbs and in mixing studies with pooled normal plasma. Results Genetic analyses revealed compound heterozygous mutations in the F5 gene: one in exon 3 (c254T>G) resulting in Leu-57 to Arg (Leu57Arg; legacy numbering) change and a second in exon 13 (A>C/T) leading to a nonsense mutation (Leu906*; legacy numbering). Plasma prothrombin time was 46.6 (reference range: 11.1-13.5 sec) and activated partial thromboplastin time was 156.2 (reference range: 25.0-37.0 sec). Plasma FV antigen was 3.3% of normal measured by ELISA and undetectable via western blotting analyses. Since TFPI is a known modifier of FV, we observed that total plasma, and platelet TFPIα levels were 62%, and 46%, of normal, respectively, and free protein S was normal (130.3 ± 0.3 nM). It is established that inhibiting TFPI's major function enhances thrombin generation in the context of bleeding. To assess this, we first blocked TFPI function using an anti-K2 mAb in a TGA. We found that the effect on peak thrombin was undetectable and similar to the unmodified patient plasma. However, adding a cocktail of anti-TFPI mAbs targeting each of its domains (K1, K2, K3 and C-term domains) to the patient's plasma shortened the lag time and enhanced peak thrombin by ~30% of normal suggesting blocking all molecular interactions of TFPI in the context of reduced FV antigen has a detectable procoagulant effect. Furthermore, in mixing studies assessed by TGA, 10% pooled normal plasma was enough to restore both peak thrombin and lag time in the patient's plasma to levels seen in normal healthy control plasma. Conclusions We identified a compound heterozygous mutation in a severe FV deficiency patient including a previously reported Leu57Arg variant and a novel Leu906* nonsense variant that is not reported in the clinical F5 gene database. In silco analyses suggest that a hydrophobic to basic amino acid substitution at position 57 may alter protein folding and function. A termination at Leu906* leads to nonsense mediated decay of mRNA and no translation consistent with reduced FV antigen in the patient. Further, we demonstrated in this study that complete inhibition of TFPI function, an important modifier of FV, promotes thrombin generation at low plasma FV levels in severe FV deficiency.

Analysis of Angiographic Patterns in Acute Coronary Syndrome Patients With Diabetes Mellitus: Correlation With HbA1c Levels

Cureus · 2024-10-21

Background The relation of diabetes mellitus with cardiovascular diseases is well-known, even acute coronary syndrome (ACS). High levels of glycated haemoglobin (HbA1c) are used is a sign of long-term glycemic control and may be associated with severity in coronary artery disease (CAD). In this particular setting, the aim of this investigation was to examine the relationship between the HbA1c values and the angiographic patterns of patients who were admitted with an ACS diagnosis. Methodology A cross-sectional study included 120 patients diagnosed with ACS. The criteria for eligibility included patients with ST-elevation myocardial infarction, unstable angina, and non-ST elevation myocardial infarction with history of documented diabetes mellitus. Every patient had a suitable clinical examination, had their HbA1c checked, and had coronary angiography to determine the severity of their CAD. Descriptive statistics and ANOVA were performed for statistical analyses to determine the connection between angiographic patterns and HbA1c. Results Patients with elevated HbA1c levels demonstrated a strong association with severe coronary artery disease. Notably, those with HbA1c exceeding 10.5% exhibited significant triple vessel disease and Type C lesions, indicative of advanced coronary artery disease. Statistical analyses revealed a marked difference in angiographic patterns across various HbA1c categories (p < 0.05). Conclusion The findings of this study suggest that maintaining optimal HbA1c levels is essential for mitigating the severity of coronary artery disease in patients with ACS. Moreover, effective glycemic control may be protective against advanced coronary atherosclerosis and subsequent cardiovascular complications in both diabetic and non-diabetic individuals.

Enhanced procoagulant activity of select hemophilia B causing factor IX variants with emicizumab

Blood · 2024-07-10 · 8 citations

ABSTRACT: Emicizumab improves the procoagulant activity of select loss-of-function factor IX (FIX) variants with likely dysfunctional assembly of the intrinsic Xase complex, resulting in hemophilia B (HB). FVIII mimetics may represent an alternative nonfactor therapy for select patients with HB.

RNAing toward a new therapy for hemophilia

Blood · 2024-05-30 · 1 citations

In this issue of Blood, Kenet et al 1 report phase 3 trial results demonstrating safety and efficacy of once monthly fitusiran vs factor or bypassing agent (BPA) prophylaxis in patients with hemophilia without and with inhibitors, respectively.

Success of Immune Tolerance Induction after AAV Gene Therapy in High-Responding Inhibitor Hemophilia a Dogs May be <i>F8</i> Genotype Dependent

Blood · 2024-11-05 · 1 citations

Background The development of neutralizing alloantibodies (inhibitors) remains the major side effect of factor VIII (FVIII) replacement therapy for hemophilia A (HA). Inhibitors significantly increase morbidity and mortality. Though the advent of non-factor therapies for HA has dramatically improved hemostatic prophylaxis for inhibitor patients, inhibitor eradication and immune tolerance induction (ITI) remain essential therapeutic goals. Recombinant adeno-associated viral (rAAV) gene therapy for HA has almost completely excluded patients with current or a history of inhibitors. We have previously published that rAAV gene therapy with canine (c) FVIII induced immune tolerance in 5 HA dogs with inhibitors (Finn et al 2010, Doshi et al 2024). HA dogs are an outbred, long-lived, naturally occurring, large animal model that both recapitulates the HA bleeding phenotype and consistently develops inhibitors in a species-specific manner. These 5 previously described dogs all had F8 intron 22 inversion (F8-INV22), which is the most common genotype in severe HA patients and dogs. Both their inhibitor titer at the time of rAAV administration and their peak titer prior to rAAV was ≤21 Bethesda Units (BU). The success rate of ITI in inhibitor patients with large gene deletions is &amp;lt;20% (Coppola et al 2009), threefold lower than the success rate in patients with F8-INV22 and similar translocations. Here, we report results of an ongoing preclinical study evaluating the hypothesis that rAAV gene therapy can induce immune tolerance in these more challenging clinical scenarios, including higher historical inhibitor titers and large F8 gene deletions. Methods To test this hypothesis, we developed 2 novel HA canine models with high-responding inhibitors: a) 2 male littermates with F8-INV22 (Duck Toller/Spaniel cross) with high-responding inhibitors; b) an HA dog (Mountain Cur) with a previously undescribed large F8 multi-exon deletion (exons 22-25) and high-responding inhibitors. All studies are approved by the UNC Institutional Animal Care and Use Committee. rAAV vectors were serotype 8 with a previously reported high-expressing cFVIII variant (Doshi et al 2024). Results High-Responding F8-INV22: T01 and T02 received 9E12 vg/kg rAAV when their inhibitor titers were &amp;gt;80 BU. Within 1 year, both exhibited inhibitor eradication and anti-cFVIII IgG2 normalization. This inhibitor eradication was consistent with stringent immune tolerance, as evidenced by the absence of inhibitor recurrence despite repeated challenges with cFVIII protein. cFVIII activity has been between 2-7% normal with significant reduction in bleeds for &amp;gt;8 years (ongoing study). F8 multi-exon deletion: This model's proband S38 received 1E13 vg/kg rAAV when his inhibitor titer was 600 BU. He had initial detectable spike of cFVIII antigen level, but his course was prolonged by an anamnestic response (peak 745 BU) that occurred after a life-threatening bleed (hemoglobin 3 g/dl) requiring whole blood transfusion. Subsequently, he demonstrated steadily declining inhibiter titer and anti-cFVIII IgG2. However, he was euthanized for humane reasons after another life-threating hemorrhage; at the time of euthanasia (2 years after rAAV), his inhibitor titer and declined &amp;gt;90% its peak value. 5 puppies produced by cloning of S38 were challenged weekly with cFVIII 25 U/kg. All 5 developed inhibitors &amp;gt; 100 BU after the 3rd dose. One (Z10) received 6E12 vg/kg rAAV when his inhibitor titer was 438 BU. He demonstrated &amp;gt;50% reduction in his inhibitor titer until day 73 when he had a life-threatening bleed requiring whole blood transfusion. He then had an anamnestic response to &amp;gt;1000 BU that by day 147 had fallen to 254 BU in this ongoing study. Conclusions rAAV gene therapy with a single-administration can provide HA dogs with F8-INV22 genotypes-even with preexisting high-responding, high-titer inhibitors: 1) inhibitor eradication, 2) sustained immune tolerance, and 3) continuous FVIII expression and phenotypic amelioration. However, as is observed with current protein-based ITI approaches, rAAV-based ITI in HA dogs with large F8 deletions remains very challenging. These data may inform ongoing clinical trials evaluating rAAV gene therapy for HA inhibitor patients.

Intron 22 inversions: cross-reactive material or no cross-reactive material?

Journal of Thrombosis and Haemostasis · 2024-11-28