[Congenital thrombotic thrombocytopenic purpura diagnosed in adulthood after repeated thrombocytopenia since neonatal period].

A 27-year-old woman was diagnosed with idiopathic thrombocytopenic purpura in the neonatal period, and was admitted to our hospital after presenting with impaired consciousness, purpura, nausea and vomiting, with a platelet count of 10×109/l. Congenital thrombotic thrombocytopenic purpura (cTTP) was suspected on the basis of recurrent thrombocytopenia and impaired consciousness, so tests for ADAMTS13 activity and inhibitor were performed. ADAMTS13 activity was severely decreased, ADAMTS13 inhibitor was negative, and platelet count increased after transfusion of fresh frozen plasma. These findings and the results of genetic testing done on all family members led to a diagnosis of cTTP. cTTP requires differential diagnosis even in adults. If a patient diagnosed with ITP in childhood has a history or findings that suggest cTTP during follow-up observation, it is necessary to actively consider ADAMTS13 testing.

von Willebrand factor Ristocetin co-factor activity to von Willebrand factor antigen level ratio for diagnosis of acquired von Willebrand syndrome caused by aortic stenosis.

Background: Severe aortic stenosis (AS) causes acquired von Willebrand syndrome by the excessive shear stress-dependent cleavage of high molecular weight multimers of von Willebrand factor (VWF). While the current standard diagnostic method is so-called VWF multimer analysis that is western blotting under nonreducing conditions, it remains unclear whether a ratio of VWF Ristocetin co-factor activity (VWF:RCo) to VWF antigen levels (VWF:Ag) of <0.7, which can be measured with an automated coagulation analyzer in clinical laboratories and is used for the diagnosis of hereditary von Willebrand disease. Objectives: To evaluated whether the VWF:RCo/VWF:Ag is useful for the diagnosis of AS-induced acquired von Willebrand syndrome. Methods: VWF:RCo and VWF:Ag were evaluated with the VWF large multimer index as a reference, which represents the percentage of a patient's VWF high molecular weight multimer ratio to that of standard plasma in the VWF multimer analysis. Results: We analyzed 382 patients with AS having transaortic valve maximal pressure gradients of >30 mmHg, 27 patients with peripheral artery disease, and 46 control patients free of cardiovascular disease with osteoarthritis, diabetes, and so on. We assumed a large multimer index of <80% as loss of VWF large multimers since 59.0% of patients with severe AS had the indices of <80%, while no control patients or patients with peripheral artery disease, except for 2 patients, exhibited the indices of <80%. The VWF:RCo/VWF:Ag ratios, measured using an automated blood coagulation analyzer, were correlated with the indices (rs = 0.470, P < .001). When the ratio of <0.7 was used as a cut-off point, the sensitivity and specificity to VWF large multimer indices of <80% were 0.437 and 0.826, respectively. Conclusion: VWF:RCo/VWF:Ag ratios of <0.7 may indicate loss of VWF large multimers with high specificity, but low sensitivity. VWF:RCo/VWF:Ag ratios in patients with AS having a ratio of <0.7 may be useful for monitoring the loss of VWF large multimers during their clinical courses.

Diagnostic and treatment guidelines for thrombotic thrombocytopenic purpura (TTP) in Japan 2023.

Thrombotic thrombocytopenic purpura (TTP) can rapidly become a life-threatening condition, and the importance of its appropriate diagnosis and treatment cannot be overstated. Until recently, TTP has mainly been diagnosed by clinical findings such as thrombocytopenia and hemolytic anemia. In addition to these clinical findings, however, reduced activity of a disintegrin-like and metalloprotease with thrombospondin type 1 motif 13 (ADAMTS13) below 10% has become internationally accepted as a diagnostic criterion for TTP. TTP is classified as immune-mediated TTP (iTTP) if the patient is positive for anti-ADAMTS13 autoantibodies, and as congenital TTP (cTTP) if ADAMTS13 gene abnormalities are detected. Fresh frozen plasma (FFP) transfusion is performed in patients with cTTP to supplement ADAMTS13. Plasma exchange therapy using FFP is conducted in patients with iTTP to supplement ADAMTS13 and to remove both anti-ADAMTS13 autoantibodies and unusually large von Willebrand factor (VWF) multimers. To suppress autoantibody production, corticosteroid therapy is administered in conjunction with plasma exchange. The monoclonal anti-CD-20 antibody rituximab is effective in patients with iTTP. In addition, caplacizumab, an anti-VWF A1 domain nanobody, has a novel mechanism of action, involving direct inhibition of platelet glycoprotein Ib-VWF binding. The recommended first-line treatments of iTTP in Japan are plasma exchange and corticosteroids, as well as caplacizumab.

Three Cases of Unprovoked Venous Thromboembolism with Prothrombin p.Arg596Gln Variant and a Literature Review of Antithrombin Resistance.

Antithrombin resistance (ATR) is a newly identified strong genetic predisposition to venous thromboembolism (VTE) caused by genetic variations in prothrombin with substitutions of Arg at position 596 with either Leu, Gln, or Trp. In the present report, we identified a missense variant p.Arg596Gln in 3 patients from 2 families with unprovoked VTE who each experienced their first VTE event at 19, 67, and 19 years old. The three patients did not show any positive markers for thrombophilia on routine testing, suggesting that patients with unprovoked VTE who have negative findings on thrombophilia tests may carry a prothrombin variant with ATR.

P2Y12 Reaction Units and Clinical Outcomes in Acute Large Artery Atherosclerotic Stroke: A Multicenter Prospective Study.

AIMS: We aimed to determine the association between acute platelet reactivity and clinical outcome in acute ischemic stroke (AIS) or transient ischemic attack (TIA) with large-artery atherosclerosis (LAA). METHODS: In this prospective, 16-multicenter study, we enrolled AIS/TIA patients with LAA receiving clopidogrel. We assessed the association of P2Y12 reaction units (PRU) 24 hours after initiation of antiplatelets with the CYP2C19 genotype and recurrent ischemic stroke within 90 days, and the difference between acute (≤ 7 days) and subacute (8-90 days) phases. RESULTS: Among the 230 AIS/TIA patients enrolled, 225 with complete outcome data and 194 with genetic results were analyzed. A higher PRU was significantly associated with recurrent ischemic stroke within 90 days (frequency, 16%), and within 7 days (10%). Twenty-nine patients (15%) belonged to a CYP2C19 poor metabolizer group (CYP2C19＊2/＊2, ＊2/＊3, or ＊3/＊3). Multivariable receiver-operating characteristic analysis showed a greater area-under-the-curve (AUC) in predicting recurrence within 7 days, compared to 8-90 days (AUC, 0.79 versus 0.64; p=0.07), with a cut-off PRU of 254. Multivariable analysis showed high PRU (≥ 254), which had a comparable predictive performance for recurrent ischemic stroke within 7 days (odds ratio, 6.82; 95% CI, 2.23-20.9; p＜0.001) to the CYP2C19 poor metabolizer genotype. The net reclassification improvement, calculated by adding high PRU (≥ 254) to a model including the CYP2C19 poor metabolizer genotype in the prediction of recurrence within 7 days, was 0.83 (p＜0.001). CONCLUSIONS: Acute PRU evaluation possesses predictive value for recurrent ischemic stroke, especially within 7 days in AIS/TIA with LAA.

Siglec-5 and Siglec-14 mediate the endocytosis of ADAMTS13.

BACKGROUND: The plasma metalloprotease ADAMTS13 regulates the thrombotic activity of the von Willebrand factor (VWF). ADAMTS13 is highly glycosylated and its carbohydrate chains are capped with sialic acid (SA). Thus, ADAMTS13 may interact with carbohydrate- and/or SA-binding plasma membrane receptors that are involved in the clearance of various plasma proteins. We have investigated ADAMTS13 endocytosis via Siglecs, which were originally identified as SA-binding immunoreceptor family proteins expressed on leukocytes and are also known as endocytic receptors. MATERIALS AND METHODS: Endocytic internalization of fluorescently labeled ADAMTS13 into HEK293 cells expressing Siglecs was examined via fluorescence microscopy. In vitro binding of ADAMTS13 to the extracellular region of Siglec-5 was examined. Plasma ADAMTS13 activity in human Siglec-5-expressing mice was measured. RESULTS AND CONCLUSIONS: Siglec-5- and Siglec-14-expressing cells internalized not only full-length ADAMTS13 (FL) but also the truncated form (MDTCS) at least partly in an SA-independent manner. Replacement of the V-set domain of Siglec-14 with that of Siglec-3 abrogated the internalization of ADAMTS13. ADAMTS13 directly bound to the extracellular region of Siglec-5 in vitro. Expression of Siglec-5 in the mouse liver resulted in a significant decrease in plasma ADAMTS13 activity. These results suggest that Siglec-5 and Siglec-14, which have nearly identical ligand-binding domains, may contribute to the regulation of plasma ADAMTS13 levels as endocytic receptors for ADAMTS13.

V-ATPase V0a1 promotes Weibel-Palade body biogenesis through the regulation of membrane fission.

Membrane fission, the division of a membrane-bound structure into two discrete compartments, is essential for diverse cellular events, such as endocytosis and vesicle/granule biogenesis; however, the process remains unclear. The hemostatic protein von Willebrand factor is produced in vascular endothelial cells and packaged into specialized secretory granules, Weibel-Palade bodies (WPBs) at the trans-Golgi network (TGN). Here, we reported that V0a1, a V-ATPase component, is required for the membrane fission of WPBs. We identified two V0a isoforms in distinct populations of WPBs in cultured endothelial cells, V0a1 and V0a2, on mature and nascent WPBs, respectively. Although WPB buds were formed, WPBs could not separate from the TGN in the absence of V0a1. Screening using dominant-negative forms of known membrane fission regulators revealed protein kinase D (PKD) as an essential factor in biogenesis of WPBs. Further, we showed that the induction of wild-type PKDs in V0a1-depleted cells does not support the segregation of WPBs from the TGN; suggesting a primary role of V0a1 in the membrane fission of WPBs. The identification of V0a1 as a new membrane fission regulator should facilitate the understanding of molecular events that enable membrane fission.

Commonly used anti-von Willebrand factor antibody for multimer analysis cross-reacts with fibronectin, which is difficult to distinguish from von Willebrand factor.

Von Willebrand factor (VWF) is a 500- to 15 000-kDa multimeric protein circulating in the blood. When VWF has a higher molecular weight, its hemostatic activity is greater. The size distribution of VWF multimers is usually analyzed by SDS-agarose gel electrophoresis followed by immunoblotting. We found that the most commonly used anti-VWF antibody cross-reacted with fibronectin in VWF multimer analysis. In addition, since the apparent molecular weights of VWF and fibronectin are almost identical, these molecules were difficult to distinguish by SDS-polyacrylamide gel electrophoresis followed by immunoblotting. Cross-reactivity between the anti-VWF antibody and fibronectin was inhibited by pretreating the antibody with fibronectin-coated plates. To obtain accurate data using anti-VWF antibodies, it is necessary to be aware of the possibility of cross-reactivity with fibronectin.

A novel homozygous variant of the thrombomodulin gene causes a hereditary bleeding disorder.

We report a 19-year-old Vietnamese woman who experienced several life-threatening bleeding events, including ovarian hemorrhage. Blood analysis revealed a decreased fibrinogen level with markedly elevated fibrinogen/fibrin degradation products and D-dimer levels. Despite hemostatic surgery and administration of several medications, such as nafamostat mesylate, tranexamic acid, and unfractionated heparin, the coagulation abnormalities were not corrected, and the patient experienced repeated hemorrhagic events. We found that administration of recombinant human thrombomodulin (rhTM) remarkably improved the patient's pathophysiology. Screening and sequencing of the TM gene (THBD) revealed a previously unreported homozygous variation: c.793T>A (p.Cys265Ser). Notably, the Cys265 residue forms 1 of 3 disulfide bonds in the epidermal growth factor (EGF)-like domain 1 of TM. Transient expression experiments using COS-1 cells demonstrated markedly reduced expression of TM-Cys265Ser on the plasma membrane relative to wild-type TM. The TM-Cys265Ser mutant was intracellularly degraded, probably because of EGF-like domain 1 misfolding. The reduced expression of TM on the endothelial cell membrane may be responsible for the disseminated intravascular-coagulation-like symptoms observed in the patient. In summary, we identified a novel TM variant, c.793T>A (p.Cys265Ser). Patients homozygous for this variant may present with severe bleeding events; rhTM should be considered a possible treatment option for these patients.

Arf GTPase-activating proteins SMAP1 and AGFG2 regulate the size of Weibel-Palade bodies and exocytosis of von Willebrand factor.

Arf GTPase-Activating proteins (ArfGAPs) mediate the hydrolysis of GTP bound to ADP-ribosylation factors (Arfs), which are critical to form transport intermediates. ArfGAPs have been thought to be negative regulators of Arfs; however, accumulating evidence indicates that ArfGAPs are important for cargo sorting and promote membrane traffic. Weibel-Palade bodies (WPBs) are cigar-shaped secretory granules in endothelial cells that contain von Willebrand factor (vWF) as their main cargo. WPB biogenesis at the Golgi was reported to be regulated by Arf and their regulators, but the role of ArfGAPs has been unknown. In this study, we performed siRNA screening of ArfGAPs to investigate the role of ArfGAPs in the biogenesis of WPBs. We found two ArfGAPs, SMAP1 and AGFG2, to be involved in WPB size and vWF exocytosis, respectively. SMAP1 depletion resulted in small-sized WPBs, and the lysosomal inhibitor leupeptin recovered the size of WPBs. The results indicate that SMAP1 functions in preventing the degradation of cigar-shaped WPBs. On the other hand, AGFG2 downregulation resulted in the inhibition of vWF secretion upon Phorbol 12-myristate 13-acetate (PMA) or histamine stimulation, suggesting that AGFG2 plays a role in vWF exocytosis. Our study revealed unexpected roles of ArfGAPs in vWF transport.

Current prophylactic plasma infusion protocols do not adequately prevent long-term cumulative organ damage in the Japanese congenital thrombotic thrombocytopenic purpura cohort.

Congenital thrombotic thrombocytopenic purpura (cTTP), known as Upshaw-Schulman syndrome, is an ultrarare thrombotic disorder caused by ADAMTS13 gene mutations; however, its long-term outcomes have not been widely studied. A questionnaire survey was administered to physicians of patients in the Japanese cTTP registry to characterise these outcomes. We analysed 55 patients in remission, with 41 cases receiving prophylactic fresh frozen plasma (FFP; median dosage: 13·2 ml/kg per month) and 14 receiving on-demand FFP. Patients receiving prophylactic FFP were considered as having a more severe form of the disease and had lower platelet counts and higher serum creatinine levels than those receiving on-demand FFP (median 138 × 109 /l vs. 243 × 109 /l, P = 0·003 and 0·71 mg/dl vs 0·58 mg/dl, P = 0·009, respectively). Patients who received prophylactic FFP more commonly developed organ damage, including renal impairment, cerebral infarctions, and cardiac hypofunction, than those who did not. Adverse FFP-related events were seen in 78% of the prophylactic FFP group, with allergic reactions being most common. Since current protocols for FFP administration to the prophylactic FFP group in Japan may be insufficient for preventing cumulative organ damage, a higher dosage of ADAMTS13 supply using recombinant ADAMTS13 agent is needed in these patients.

Carrier frequencies of antithrombin, protein C, and protein S deficiency variants estimated using a public database and expression experiments.

BACKGROUND: Genetic deficiencies of antithrombin (AT), protein C (PC), and protein S (PS) are risk factors for venous thromboembolism. In the general population, the prevalence of heterozygous deficiency of AT, PC, and PS are reported as approximately 0.02%-0.2%, 0.2%-0.4%, and 0.03%-0.5%, respectively. The Exome Aggregation Consortium (ExAC) provides a public database containing reference data for over 60 000 exomes. OBJECTIVE: This study aimed to determine the frequency of AT, PC, and PS deficiencies using the ExAC database and transient expression experiments. METHODS: In total, 133, 157, and 221 variants of SERPIN1 (encoding AT), PROC (PC), and PROS1 (PS), respectively, were registered as missense and putative loss-of-function variants in the ExAC database. Variants with relatively high allele frequencies were selected and randomly sampled. Recombinant proteins were expressed in human embryo kidney 293 cells and their secretion and anticoagulant activities examined. RESULTS AND CONCLUSION: We assessed 9 AT, 4 PC, and 14 PS variants with relatively high allele frequencies and randomly sampled 12 AT, 15 PC, and 19 PS missense variants. All 21 AT variants showed normal or mildly reduced secretion, and 6 showed reduced total activity (specific activity × antigen level). Of the 19 PC variants, 11 showed impaired total activity. All 33 PS variants showed normal or mildly reduced secretion, and 4 showed reduced total activity. Based on allele frequencies in the ExAC database, we calculated the frequencies of AT, PC, and PS genetic deficiency as 0.36%, 0.63%, and 0.39%, respectively.

Derlin-3 Is Required for Changes in ERAD Complex Formation under ER Stress.

Endoplasmic reticulum (ER)-associated protein degradation (ERAD) is a quality control system that induces the degradation of ER terminally misfolded proteins. The ERAD system consists of complexes of multiple ER membrane-associated and luminal proteins that function cooperatively. We aimed to reveal the role of Derlin-3 in the ERAD system using the liver, pancreas, and kidney obtained from different mouse genotypes. We performed coimmunoprecipitation and sucrose density gradient centrifugation to unravel the dynamic nature of ERAD complexes. We observed that Derlin-3 is exclusively expressed in the pancreas, and its deficiency leads to the destabilization of Herp and accumulation of ERAD substrates. Under normal conditions, Complex-1a predominantly contains Herp, Derlin-2, HRD1, and SEL1L, and under ER stress, Complex-1b contains Herp, Derlin-3 (instead of Derlin-2), HRD1, and SEL1L. Complex-2 is upregulated under ER stress and contains Derlin-1, Derlin-2, p97, and VIMP. Derlin-3 deficiency suppresses the transition of Derlin-2 from Complex-1a to Complex-2 under ER stress. In the pancreas, Derlin-3 deficiency blocks Derlin-2 transition. In conclusion, the composition of ERAD complexes is tissue-specific and changes in response to ER stress in a Derlin-3-dependent manner. Derlin-3 may play a key role in changing ERAD complex compositions to overcome ER stress.

Success and limitations of plasma treatment in pregnant women with congenital thrombotic thrombocytopenic purpura.

BACKGROUND: Congenital thrombotic thrombocytopenic purpura (cTTP), otherwise known as Upshaw-Schulman syndrome, is an extremely rare hereditary disease. Pregnancy is identified as a trigger for TTP episodes in patients with cTTP. OBJECTIVES: To investigate the ideal management of pregnant patients with cTTP. PATIENTS/METHODS: We identified 21 patients with a reproductive history (38 pregnancies) in a Japanese cTTP registry. Fetal outcomes were compared between two groups: group 1 (n = 12), pregnancy after diagnosis of confirmed cTTP by ADAMTS13 gene analysis; and group 2 (n = 26), pregnancy before diagnosis of confirmed cTTP. RESULTS: In group 1, ADAMTS13 activity was closely monitored until delivery in most cases. Among 10 pregnancies in group 1, prophylactic fresh frozen plasma (FFP) infusions during pregnancy were performed to replenish ADAMTS13. In group 2, prophylactic FFP infusions were not administrated in 23 pregnancies and FFP test infusions were performed in only three pregnancies. The live birth rate of group 1 was significantly higher than that of group 2 (91.7% vs 50.0%, respectively, P = .027). The fetal survival rates of women without FFP infusions were dramatically decreased after 20 weeks of gestation. The FFP infusion dosage in group 1 was generally higher than 5 mL/kg/wk by 20 weeks of gestation. CONCLUSIONS: Our results indicate that FFP infusions of more than 5 mL/kg/wk should be initiated as soon as patients become pregnant. However, even with these infusions, patients with repeated TTP episodes before pregnancy might have difficulty giving birth successfully. Recombinant ADAMTS13 products might be new treatment options for pregnant patients with cTTP.

Knock-in mice bearing constitutively active αIIb(R990W) mutation develop macrothrombocytopenia with severe platelet dysfunction.

BACKGROUND: To date, several mutations that induce constitutive activation of integrin αIIbß3 have been identified in congenital macrothrombocytopenia. Of these, αIIb(R995W) is the most prevalent mutation observed in Japanese patients with αIIbß3-related congenital macrothrombocytopenia. OBJECTIVE AND METHODS: The present study aimed to explore the effects of constitutive activation of the αIIb(R995W) mutation on platelet production, morphology, and function. We generated αIIb(R990W) knock-in (KI) mice corresponding to human αIIb(R995W). RESULTS: Platelet counts of heterozygous (hetero) and homozygous (homo) KI mice were decreased by ~10% and ~25% relative to those of wild-type (WT) mice, respectively, with increase in platelet size. Decrease in absolute reticulated platelet numbers in steady state, delayed recovery from thrombocytopenia induced by anti-platelet antibody and impaired response to exogenous thrombopoietin administration suggested impaired platelet production in KI mice. WT and KI mice showed no significant differences in the number of megakaryocytes and ploidy of megakaryocytes, whereas proplatelet formation was significantly impaired in homo mice. We observed a slight but significant reduction in platelet lifespan in homo mice. The homo mice showed dramatic reduction in αIIbß3 expression in platelets, which was accompanied by severe in vivo and in vitro platelet dysfunction. CONCLUSION: The αIIb(R990W) KI mice developed macrothrombocytopenia, which was primarily attributed to impaired proplatelet formation. In addition, homo KI mice showed marked downregulation in αIIbß3 expression in platelets with severe impaired platelet function, similar to Glanzmann thrombasthenia.