Targeting Tissue Factor Pathway Inhibitor with concizumab to improve haemostasis in patients with Glanzmann thrombasthenia: an in vitro study.

BACKGROUND: Glanzmann thrombasthenia (GT) is caused by an inherited defect of platelet αIIbß3 integrin. Concizumab,a monoclonal antibody specific for Tissue Factor Pathway Inhibitor (TFPI), abolishes its anticoagulant effect. OBJECTIVES: To evaluate the in vitro ability of concizumab to improve haemostasis in GT. PATIENTS/METHODS: The effects of concizumab were evaluated in whole blood or platelet-rich plasma (PRP) from GT patients (n=5-9) using a thrombin generation assay (TGA), rotational thromboelastometry (ROTEM), a global fibrinolytic capacity assay and a flow-chamber assay (T-TAS). Washed platelets (WP) and 20 nM recombinant activated factor VIIa (rFVIIa) were included for comparison. RESULTS: The lag time in the TGA was significantly longer (+85%, p<0.0001) in GT patients than in controls. WP, rFVIIa and concizumab each significantly improved thrombin generation profiles. The ROTEM clotting time was significantly longer in GT patients than in controls (677 s vs 523 s; p=0.03). However, CT improved after adding WP, rFVIIa or concizumab. Under flow, occlusive thrombi were present in all healthy controls after 10 min, whereas platelet-fibrin depositions were not seen in GT patients. Sub-occlusive or occlusive thrombi formed when GT blood was mixed with WP, rFVIIa or concizumab. Clots in GT PRP were more susceptible to fibrinolysis and were improved by WP, rFVIIa or concizumab. CONCLUSIONS: Concizumab enhanced thrombin generation, decreased the ROTEM CT, improved thrombus formation under flow and reduced clot lysis. Our results demonstrate the potential of concizumab for subcutaneous prophylaxis in GT patients.

Association of laboratory test results with the bleeding history in patients with inherited platelet function disorders (the Bleeding Assesment Tool - LABoratory tests substudy): communication from the Platelet Physiology ISTH-SSC.

Background: In hemophilia and von Willebrand disease, the degree of alteration of laboratory assays correlates with bleeding manifestations. Few studies have assessed the predictive value for bleeding of laboratory assays in patients with inherited platelet function disorders (IPFDs). Objectives: To assess whether there is an association between platelet function assay results and bleeding history, as evaluated by the International Society on Thrombosis and Haemostasis (ISTH) bleeding assessment tool (BAT). Methods: Centers participating in the international ISTH-BAT validation study were asked to provide results of the diagnostic assays employed for the patients they enrolled, and the association with the individual patients' bleeding score (BS) was assessed. Results: Sixty-eight patients with 14 different IPFDs were included. Maximal amplitude of platelet aggregation was significantly lower in patients with a pathologic BS and correlated inversely with the BS, a finding largely driven by the subgroup of patients with Glanzmann thrombasthenia and CalDAG-GEFI deficiency; after their exclusion, TRAP-induced aggregation remained significantly lower in patients with a pathologic BS. Bleeding time was significantly more prolonged in patients with a high BS than in those with a normal BS (27.1 ± 6.2 minutes vs 15.1 ± 10.6 minutes; P < .01). Reduced α-granule content was significantly more common among patients with a pathologic BS than among those with a normal BS (80% vs 20%; P < .05). Receiver operating characteristic curve analysis revealed a significant discriminative ability of all the aforementioned tests for pathologic BS (P < .001), also after exclusion of patients with Glanzmann thrombasthenia and CalDAG-GEFI deficiency. Conclusion: This study shows that altered platelet laboratory assay results are associated with an abnormal ISTH-BAT BS in IPFD.

In vitro characterization of rare anti-αIIbß3 isoantibodies produced by patients with Glanzmann thrombasthenia that severely block fibrinogen binding and generate procoagulant platelets via complement activation.

Background: Glanzmann thrombasthenia (GT) is a rare bleeding disorder caused by inherited defects of the platelet αIIbß3 integrin. Platelet transfusions can be followed by an immune response that can block integrin function by interfering with fibrinogen binding. Objectives: In this study, we aimed to determine the prevalence of such isoantibodies and better characterize their pathogenic properties. Methods: Twelve patients with GT were evaluated for anti-αIIbß3 isoantibodies. Sera from patients with GT with or without anti-αIIbß3 isoantibodies were then used to study their in vitro effect on platelets from healthy donors. We used several approaches (IgG purification, immunofluorescence staining, and inhibition of signaling pathways) to characterize the pathogenic properties of the anti-αIIbß3 isoantibodies. Results: Only 2 samples were able to severely block integrin function. We observed that these 2 sera caused a reduction in platelet size similar to that observed when platelets become procoagulant. Mixing healthy donor platelets with patients' sera or purified IgGs led to microvesiculation, phosphatidylserine exposure, and induction of calcium influx. This was associated with an increase in procoagulant platelets. Pore formation and calcium entry were associated with complement activation, leading to the constitution of a membrane attack complex (MAC) with enhanced complement protein C5b-9 formation. This process was inhibited by the complement 5 inhibitor eculizumab and reduced by polyvalent human immunoglobulins. Conclusion: Our data suggest that complement activation induced by rare blocking anti-αIIbß3 isoantibodies may lead to the formation of a MAC with subsequent pore formation, resulting in calcium influx and procoagulant platelet phenotype.

Emergency management of patients with Glanzmann thrombasthenia: consensus recommendations from the French reference center for inherited platelet disorders.

Glanzmann thrombasthenia (GT) is a genetic bleeding disorder characterised by severely reduced/absent platelet aggregation in response to multiple physiological agonists. The severity of bleeding in GT varies markedly, as does the emergency situations and complications encountered in patients. A number of emergency situations may occur in the context of GT, including spontaneous or provoked bleeding, such as surgery or childbirth. While general management principles apply in each of these settings, specific considerations are essential for the management of GT to avoid escalating minor bleeding events. These recommendations have been developed from a literature review and consensus from experts of the French Network for Inherited Platelet Disorders, the French Society of Emergency Medicine, representatives of patients' associations, and Orphanet to aid decision making and optimise clinical care by non-GT expert health professionals who encounter emergency situations in patients with GT.

Multicenter evaluation of light transmission platelet aggregation reagents: communication from the ISTH SSC Subcommittee on Platelet Physiology.

BACKGROUND: Light transmission aggregation (LTA) is used widely by the clinical and research communities. Although it is a gold standard, there is a lack of interlaboratory harmonization. OBJECTIVES: The primary objective was to assess whether sources of activators (mainly adenosine diphosphate [ADP], collagen, arachidonic acid, epinephrine, and thrombin receptor activating peptide6) and ristocetin contribute to poor LTA reproducibility. The secondary objective was to evaluate interindividual variability of results to appreciate the distribution of normal values and consequently better interpret pathologic results. METHODS: An international multicenter study involving 28 laboratories in which we compared LTA results obtained with center-specific activators and a comparator that we supplied. RESULTS: We report variability in the potency (P) of activators in comparison with the comparator. Thrombin receptor activating peptide 6 (P, 1.32-2.68), arachidonic acid (P, 0.87-1.43), and epinephrine (P, 0.97-1.34) showed the greatest variability. ADP (P, 1.04-1.20) and ristocetin (P, 0.98-1.07) were the most consistent. The data highlighted clear interindividual variability, notably for ADP and epinephrine. Four profiles of responses were observed with ADP from high-responders, intermediate-responders, and low-responders. A fifth profile corresponding to nonresponders (5% of the individuals) was observed with epinephrine. CONCLUSION: Based on these data, the establishment and adoption of simple standardization principles should mitigate variability due to activator sources. The observation of huge interindividual variability for certain concentrations of activators should lead to a cautious interpretation before reporting a result as abnormal. Confidence can be taken from the fact that difference between sources is not exacerbated in patients treated with antiplatelet agents.

Combined Platelet and Red Blood Cell Recovery during On-pump Cardiac Surgery Using same™ by i-SEP Autotransfusion Device: A First-in-human Noncomparative Study (i-TRANSEP Study).

BACKGROUND: Centrifugation-based autotransfusion devices only salvage red blood cells while platelets are removed. The same™ device (Smart Autotransfusion for ME; i-SEP, France) is an innovative filtration-based autotransfusion device able to salvage both red blood cells and platelets. The authors tested the hypothesis that this new device could allow a red blood cell recovery exceeding 80% with a posttreatment hematocrit exceeding 40%, and would remove more than 90% of heparin and 75% of free hemoglobin. METHODS: Adults undergoing on-pump elective cardiac surgery were included in a noncomparative multicenter trial. The device was used intraoperatively to treat shed and residual cardiopulmonary bypass blood. The primary outcome was a composite of cell recovery performance, assessed in the device by red blood cell recovery and posttreatment hematocrit, and of biologic safety assessed in the device by the washout of heparin and free hemoglobin expressed as removal ratios. Secondary outcomes included platelet recovery and function and adverse events (clinical and device-related adverse events) up to 30 days after surgery. RESULTS: The study included 50 patients, of whom 18 (35%) underwent isolated coronary artery bypass graft, 26 (52%) valve surgery, and 6 (12%) aortic root surgery. The median red blood cell recovery per cycle was 86.1% (25th percentile to 75th percentile interquartile range, 80.8 to 91.6) with posttreatment hematocrit of 41.8% (39.7 to 44.2). Removal ratios for heparin and free hemoglobin were 98.9% (98.2 to 99.7) and 94.6% (92.7 to 96.6), respectively. No adverse device effect was reported. Median platelet recovery was 52.4% (44.2 to 60.1), with a posttreatment concentration of 116 (93 to 146) · 109/l. Platelet activation state and function, evaluated by flow cytometry, were found to be unaltered by the device. CONCLUSIONS: In this first-in-human study, the same™ device was able to simultaneously recover and wash both platelets and red blood cells. Compared with preclinical evaluations, the device achieved a higher platelet recovery of 52% with minimal platelet activation while maintaining platelet ability to be activated in vitro.

Obstetrical complications in hereditary fibrinogen disorders: the Fibrinogest study.

BACKGROUND: Women with hereditary fibrinogen disorders (HFDs) seem to be at an increased risk of adverse obstetrical outcomes, but epidemiologic data are limited. OBJECTIVES: We aimed to determine the prevalence of pregnancy complications; the modalities and management of delivery; and the postpartum events in women with hypofibrinogenemia, dysfibrinogenemia, and hypodysfibrinogenemia. METHODS: We conducted a retrospective and prospective multicentric international study. RESULTS: A total of 425 pregnancies were investigated from 159 women (49, 95, and 15 cases of hypofibrinogenemia, dysfibrinogenemia, and hypodysfibrinogenemia, respectively). Overall, only 55 (12.9%) pregnancies resulted in an early miscarriage, 3 (0.7%) resulted in a late miscarriage, and 4 (0.9%) resulted in an intrauterine fetal death. The prevalence of live birth was similar among the types of HFDs (P = .31). Obstetrical complications were observed in 54 (17.3%) live birth pregnancies, including vaginal bleeding (14, 4.4%), retroplacental hematoma (13, 4.1%), and thrombosis (4, 1.3%). Most deliveries were spontaneous (218, 74.1%) with a vaginal noninstrumental delivery (195, 63.3%). A neuraxial anesthesia was performed in 116 (40.4%) pregnancies, whereas general or no anesthesia was performed in 71 (16.6%) and 129 (44.9%) pregnancies, respectively. A fibrinogen infusion was administered in 28 (8.9%) deliveries. Postpartum hemorrhages were observed in 62 (19.9%) pregnancies. Postpartum venous thrombotic events occurred in 5 (1.6%) pregnancies. Women with hypofibrinogenemia were at an increased risk of bleeding during the pregnancy (P = .04). CONCLUSION: Compared with European epidemiologic data, we did not observe a greater frequency of miscarriage, while retroplacental hematoma, postpartum hemorrhage, and thrombosis were more frequent. Delivery was often performed without locoregional anesthesia. Our findings highlight the urgent need for guidance on the management of pregnancy in HFDs.

Platelet function studies in myeloproliferative neoplasms patients with Calreticulin or JAK2 V617F mutation.

Background: JAK2 V617F and Calreticulin (CALR) mutations are the most frequent molecular causes of Phi-negative myeloproliferative neoplasms (MPN). Patients with CALR mutations are at lower risk of thrombosis than patients with JAK2 V617F. We hypothesized that CALR-mutated blood platelets would have platelet function defects that might explain why these patients are at lower risk of thrombosis. Objectives: Our main objective was to explore and compare platelet function depending on the MPN molecular marker. Methods: We analyzed platelet function in 16 patients with MPN with CALR mutations and 17 patients with JAK2 V617F mutation and compared them with healthy controls. None of these patients was taking antiplatelet therapy. We performed an extensive analysis of platelet function and measured plasmatic soluble P-selectin and CD40L levels. Results: We observed significant defects in platelet aggregation, surface glycoprotein expression, fibrinogen binding, and granule content in platelets from patients with MPN compared with that in controls. Moreover, soluble CD40L and P-selectin levels were elevated in patients with MPN compared with that in controls, suggesting an in vivo platelet preactivation. Comparison of platelet function between patients with CALR and JAK2 V617F MPN revealed only minor differences in platelets from patients with CALR. However, these results need to be interpreted within the context of absence of an inflammatory environment that could impact platelet function during MPN. Conclusions: These results do not support the hypothesis that calreticulin-mutated platelets have platelet function defects that could explain the lower thrombotic risk of patients with CALR.

High Rates of Anti-αIIbß3 Antibodies Produced by a Glanzmann Thrombasthenia Patient after First and Unique Red Blood Cells Administration.

Immunization against the platelet αIIbß3 glycoprotein due to blood transfusion represents one of the most severe complications in Glanzmann thrombasthenia (GT) disease. Anti-αIIbß3 isoantibodies development may lead to ineffective platelet transfusion and can, in case of pregnancy, cross the placenta leading to fetal thrombocytopenia. We describe here the case of a girl with type I GT who developed high rates of anti-αIIbß3 isoantibodies after first and unique blood transfusion. Surprisingly, this patient had only received red blood cell concentrates and immunization was presumably stimulated by the residual presence of platelets in concentrates. This study emphasizes the need for regular anti-αIIbß3 antibodies screening in GT, even though patients have never been previously transfused with platelet concentrates.

HemosIL VWF:GPIbR Assay Has a Greater Sensitivity than VWF:RCo Technique to Detect Acquired von Willebrand Syndrome in Myeloproliferative Neoplasms.

BACKGROUND: Acquired von Willebrand syndrome (AVWS) is frequent in patients with myeloproliferative neoplasms (MPNs). For von Willebrand factor (VWF) functional evaluation, ristocetin cofactor activity by aggregometry (VWF:RCo) is considered the gold standard but has limitations, and automated activity measurement has been developed such as the HemosIL VWF:RCo Werfen with particle agglutination (VWF:GPIbR). OBJECTIVES: To evaluate the performance of VWF:GPIbR with HemosIL VWF:RCo Werfen (VWF:GPIbR) versus VWF:RCo in patients with thrombocytosis in the context of MPNs (T-MPNs) and in patients with secondary thrombocytosis (ST). METHODS: MPN patients with thrombocytosis >450 G/L (T-MPNs) were compared with patients with ST due to inflammation or iron deficiency. VWF activity (VWF:Act) was analyzed using VWF:RCo or VWF:GPIbR. VWF analysis was completed by analysis of VWF multimers and VWF collagen binding (CB) assay (VWF:CB). RESULTS: A total of 33 T-MPNs and 18 ST patients were included. Compared with aggregometry, evaluation of VWF:Act by VWF:GPIbR led to lower values in T-MPN patients, but also in ST patients. Interestingly, although the VWF:RCo/VWF:Ag ratio did not reveal differences between T-MPNs and ST patients, the VWF:GPIbR/VWF:Ag ratio analysis allowed us to suspect AVWS only in T-MPN patients. Using the distribution of VWF multimer analysis and VWF:CB, we here demonstrated that VWF:GPIbR allows AVWS diagnosis in nine T-MPNs as opposed to aggregometry. CONCLUSION: Evaluation of VWF:Act using VWF:GPIbR has a greater sensitivity compared with aggregometry to detect AVWS in T-MPN patients.

How I manage pregnancy in women with Glanzmann thrombasthenia.

Glanzmann thrombasthenia (GT) is a rare inherited platelet function disorder caused by a quantitative and/or qualitative defect of the αIIbß3 integrin. Pregnancy and delivery are recognized risk periods for bleeding in women with GT. The newborn may also be affected by fetal and neonatal immune thrombocytopenia induced by the transplacental passage of maternal anti-αIIbß3 antibodies, which can lead to severe hemorrhage and fetal loss. Pregnancy in women with GT thus requires a multidisciplinary approach, including prepregnancy counseling and a treatment plan for delivery for both the mother and child. In this article, we summarize the current knowledge on pregnancy in women with GT and describe how we manage this severe platelet disorder in our clinical practice.

A mutation in SLC37A4 causes a dominantly inherited congenital disorder of glycosylation characterized by liver dysfunction.

SLC37A4 encodes an endoplasmic reticulum (ER)-localized multitransmembrane protein required for transporting glucose-6-phosphate (Glc-6P) into the ER. Once transported into the ER, Glc-6P is subsequently hydrolyzed by tissue-specific phosphatases to glucose and inorganic phosphate during times of glucose depletion. Pathogenic variants in SLC37A4 cause an established recessive disorder known as glycogen storage disorder 1b characterized by liver and kidney dysfunction with neutropenia. We report seven individuals who presented with liver dysfunction multifactorial coagulation deficiency and cardiac issues and were heterozygous for the same variant, c.1267C>T (p.Arg423∗), in SLC37A4; the affected individuals were from four unrelated families. Serum samples from affected individuals showed profound accumulation of both high mannose and hybrid type N-glycans, while N-glycans in fibroblasts and undifferentiated iPSC were normal. Due to the liver-specific nature of this disorder, we generated a CRISPR base-edited hepatoma cell line harboring the c.1267C>T (p.Arg423∗) variant. These cells replicated the secreted abnormalities seen in serum N-glycosylation, and a portion of the mutant protein appears to relocate to a distinct, non-Golgi compartment, possibly ER exit sites. These cells also show a gene dosage-dependent alteration in the Golgi morphology and reduced intraluminal pH that may account for the altered glycosylation. In summary, we identify a recurrent mutation in SLC37A4 that causes a dominantly inherited congenital disorder of glycosylation characterized by coagulopathy and liver dysfunction with abnormal serum N-glycans.

Comparison of different activators of coagulation by turbidity analysis of hereditary dysfibrinogenemia and controls.

Turbidity analysis is widely used as a quantitative technique in hereditary dysfibrinogenemia. We aimed to compare several coagulation triggers in hereditary dysfibrinogenemia and control plasmas. We included 20 patients with hereditary dysfibrinogenemia, 19 with hotspot mutations Aα Arg35His (n = 9), Aα Arg35Cys (n = 2), γ Arg301His (n = 6), γ Arg301Cys (n = 2), and one with Aα Phe27Tyr, and a commercial pooled normal plasma. Fibrin polymerization was activated by bovine or human thrombin or tissue factor (TF), in the presence or absence of tissue type plasminogen activator. The lag time (min), slope (mOD/s), maximum absorbance (MaxAbs, mOD), and area under the curve (AUCp, OD s) were calculated from the fibrin polymerization curves and the time for 50% clot degradation (T50, min), AUCf (OD s) and the overall fibrinolytic potential from fibrinolysis curves. The lag time was significantly shorter and AUC increased in Aα Arg35His patients with bovine thrombin as compared with human thrombin. The MaxAbs and AUCp were significantly higher in γArg301His patients with bovine thrombin compared with human thrombin. Fibrin polymerization parameters of patients' samples were closer to those of control when assessed with TF compared with both human and bovine thrombin. T50 and overall fibrinolytic potential were similar in all samples regardless of the coagulation trigger used, however, with TF the AUCf of Aα Arg35His and γ Arg301His groups were significantly decreased compared with control. Bovine and human thrombin cannot be used equally for studying fibrin polymerization in hotspot hereditary dysfibrinogenemia or control plasmas.

A new case with Hermansky-Pudlak syndrome type 9, a rare cause of syndromic albinism with severe defect of platelets dense bodies.

Hermansky-Pudlak syndrome (HPS) is a rare form of syndromic oculocutaneous albinism caused by disorders in lysosome-related organelles. Ten genes are associated with different forms of HPS. HPS type 9 (HPS-9) is caused by biallelic variants of BLOC1S6. To date, only three patients with HPS-9 have been reported. We described one patient presenting with ocular features of albinism. Genetic analysis revealed two compound heterozygous variants in the BLOC1S6 gene. Extended hematological studies confirmed the platelet storage pool disease with absence of dense granules and abnormal platelet aggregation. By reviewing the previous published cases we confirm the phenotype of HPS-9 patients. This patient is the only one described with dextrocardia and abnormal psychomotor development.

Immunization against αIIb ß3 and αv ß3 in Glanzmann thrombasthenia patients carrying the French Gypsy mutation.

Essentials The c.1544+1G>A mutation was identified in Gypsy Glanzmann thrombasthenia (GT) patients. Gypsy GT patients express normal αv ß3 carrying HPA-1b epitopes. To demonstrate HPA-1a alloimmunization by modified antigen capture assays. Gypsy GT patients could develop anti-HPA-1a alloantibodies against ß3 and αv ß3 . ABSTRACT: Background Glanzmann thrombasthenia (GT) is a rare bleeding disorder caused by the absence or the dysfunction of the platelet αIIb ß3 integrin. A founder mutation in the ITGA2B gene was previously identified in French Gypsy patients. Interestingly, this mutation was strongly linked to the human platelet antigen-1b (HPA-1b). The HPA-1bb Gypsy patients are at risk of isoimmunization against αIIb ß3 , as this complex is not expressed at their platelet surface. Tentatively, they would, however, not have an increased risk of developing anti-HPA-1a alloantibodies by exposure of αIIb ß3 on platelets from random platelet transfusions. However, the ß3 chain can also associate with the αv subunit expressed at the platelet surface. Because Gypsy GT patients express normal αv ß3 carrying HPA-1b epitopes, these patients might develop anti-HPA-1a alloantibodies reacting with αv ß3 and/or ß3 . Objectives/Patients/Methods To demonstrate this hypothesis, sera from HPA-1bb (n = 5) and HPA-1ab (n = 1) Gypsy GT patients were investigated by modified antigen capture assay using platelets or stable transfected cells. Furthermore, stable transfected cells expressing either αIIb ß3 or αv ß3 together with soluble monomeric chimeric ß3 (as absorbent) were used to differentiate anti-ß3 and anti-αv ß3 reactivity. Results Only HPA-1bb patients developed alloantibodies reacting with HPA-1a cells. Further analysis showed that HPA-1bb patients developed anti-HPA-1a alloantibodies reacting with ß3 and/or αv ß3 . Conclusion In this study, we found that HPA-1bb patients who failed to express αIIb ß3 on the platelet surface can develop alloantibodies against HPA-1a reacting with ß3 as well as αv ß3 . This is of particular importance as anti-HPA-1a alloantibodies might cause fetal neonatal alloimmune thrombocytopenia and/or platelet transfusion refractoriness.

BLOC1S5 pathogenic variants cause a new type of Hermansky-Pudlak syndrome.

PURPOSE: Hermansky-Pudlak syndrome (HPS) is characterized by oculocutaneous albinism, excessive bleeding, and often additional symptoms. Variants in ten different genes have been involved in HPS. However, some patients lack variants in these genes. We aimed to identify new genes involved in nonsyndromic or syndromic forms of albinism. METHODS: Two hundred thirty albinism patients lacking a molecular diagnosis of albinism were screened for pathogenic variants in candidate genes with known links to pigmentation or HPS pathophysiology. RESULTS: We identified two unrelated patients with distinct homozygous variants of the BLOC1S5 gene. Patients had mild oculocutaneous albinism, moderate bleeding diathesis, platelet aggregation deficit, and a dramatically decreased number of platelet dense granules, all signs compatible with HPS. Functional tests performed on platelets of one patient displayed an absence of the obligate multisubunit complex BLOC-1, showing that the variant disrupts BLOC1S5 function and impairs BLOC-1 assembly. Expression of the patient-derived BLOC1S5 deletion in nonpigmented murine Bloc1s5-/- melan-mu melanocytes failed to rescue pigmentation, the assembly of a functional BLOC-1 complex, and melanosome cargo trafficking, unlike the wild-type allele. CONCLUSION: Mutation of BLOC1S5 is disease-causing, and we propose that BLOC1S5 is the gene for a new form of Hermansky-Pudlak syndrome, HPS-11.