Takanori Moriki

Effects of VerifyNow P2Y12 test and CYP2C19*2 testing on clinical outcomes of patients with cardiovascular disease: A systematic review and meta-analysis

The VerifyNow P2Y12 test is a whole-blood, light transmission-based optical detection assay that measures adenosine diphosphate-induced platelet aggregation in a cartridge containing fibrinogen-coated beads. The usefulness of this device to assess the effect of clopidogrel on platelet function was recently highlighted for predicting cardiovascular (CV) events. We therefore performed a meta-analysis to analyze whether adverse clinical outcomes of patients with CV disease increase in association with high on-treatment platelet reactivity (HPR) using the VerifyNow P2Y12 test. We also investigated the effect of the CYP2C19*2 polymorphism on the occurrence of CV events among clopidogrel-treated patients. Relevant studies were searched for in the PubMed database and Cochrane Central Register of Controlled Trials database, and selected if they included data about platelet function assessed using the VerifyNow P2Y12 test or CYP2C19*2 polymorphism in clopidogrel-treated patients with CV disease, and the occurrence of CV events within 6–12 months. In our meta-analysis, 4817 subjects from eight studies and 5307 subjects from seven studies were included for review of platelet function and CYP2C19*2 polymorphism, respectively. Overall, 2237 (46.4%) patients had HPR and these patients had significantly higher odds of CV events compared to patients without HPR (odds ratio(OR) = 3.05, 95% confidence interval: 2.33–3.98, p < 0.001). Heterogeneity among studies was not significant (Cochrans Q-test, p = 0.80 and I2 = 0%). In the secondary investigation, 1926 (36.3%) patients had at least one CYP2C19*2 allele. Heterogeneity in the OR of the CV events between the CYP2C19*2 allele carriers and non-carriers was observed among the seven studies (Cochrans Q-test, p = 0.01 and I2 = 56.5%), and the range of the ORs among the seven studies was 0.58–16.6. In conclusion, HPR assessed by VerifyNow P2Y12 test was associated with increased adverse clinical outcomes of clopidogrel-treated patients with CV disease.

Epitope analysis of autoantibodies to ADAMTS13 in patients with acquired thrombotic thrombocytopenic purpura

INTRODUCTION Autoantibodies to ADAMTS13 have a pivotal role in the pathogenesis of acquired thrombotic thrombocytopenic purpura (TTP). By decreasing the function of ADAMTS13, autoantibodies impair the cleavage of ultra-large von Willebrand factor (UL-VWF) multimers into smaller sizes, leading to lethal platelet-VWF thrombi in the microcirculation. We therefore aimed to determine the sites of autoantibody recognition on ADAMTS13. MATERIALS AND METHODS In this study, IgG purified from 13 acquired TTP patients were examined to determine their binding sites on ADAMTS13. Immobilized IgG on microtiter plate or proteinG beads was screened by phage library expressing various peptides of ADAMTS13. RESULTS In screening, diverse peptide sequences were obtained from almost all of the ADAMTS13 domains, including the spacer domain, which is considered a major binding site. In particular, we detected an identical amino-acid sequence in the C-terminus of the spacer domain from Gly662 to Val687 that was recognized by autoantibodies from 5 TTP patients. The specific autoantibody was expected to be associated with the plasma levels of the ADAMTS13 antigen or activity, and with the quantity of ADAMTS13 autoantibodies or the inhibitory autoantibody titer in TTP patient plasma. These measurements, however, did not seem to be related to the presence or absence of the specific autoantibody. CONCLUSIONS These findings indicate that the specific autoantibody might be a feature of acquired TTP, although its clinical significance remains to be elucidated.

Studies of a microchip flow-chamber system to characterize whole blood thrombogenicity in healthy individuals

INTRODUCTION A whole blood flow-chamber system, the Total Thrombus-formation Analysis System (T-TAS), was developed for quantitative analysis of platelet thrombus formation (PTF) using microchips with thrombogenic surfaces (collagen, PL chip; collagen plus tissue thromboplastin, AR chip) under shear stress conditions. We evaluated the usefulness of the T-TAS for assessing individual thrombogenicity compared with other platelet function tests. MATERIALS AND METHODS Blood samples from 31 healthy volunteers were applied to the T-TAS to measure PTF starting time (T10: time to reach 10 kPa), occlusion time (T60 for PL chip; T80 for AR chip), and area under the curve (AUC10, area under curve until 10 min for PL chip; AUC30, 30 min for AR chip) under various shear rates (1000, 1500, 2000s(-1) for PL chip; 300 s(-1) for AR chip). Platelet functions were also tested using platelet aggregometry, the PFA-100 (collagen and epinephrine [C/EPI], collagen and adenosine diphosphate [C/ADP]), and the VerifyNow P2Y12 assay. RESULTS Individual pressure waveforms, including PTF starting and ending points, varied among healthy subjects. In the PL chip, T10 and AUC10 showed a shear-dependent correlation with C/EPI or C/ADP. VerifyNow P2Y12 values were not significantly associated with the parameters of the T-TAS. Platelet counts were correlated with all AR measurements, and mostly with PL measurements. CONCLUSION The results of the T-TAS were associated with those of the PFA-100 in many respects, indicating that its characteristics are related to shear-induced PTF. The T-TAS showed few correlations with platelet aggregometry and the VerifyNow P2Y12 assay. The T-TAS may allow for the measurement of comprehensive parameters of individual thrombogenicity under whole blood flow conditions.

A family with hereditary factor X deficiency with a point mutation Gla32 to Gln in the Gla domain (factor X Tokyo).

We report a new family with hereditary factor X deficiency. The propositus had a markedly prolonged prothrombin time, a mild prolongation of activated partial thromboplastin time and a clotting time activated by Russells viper venom. Factor X activity in plasma was 3 u/dl (normal range 56–138 u/dl). Factor X antigen level was 61 u/dl. Molecular analysis revealed a homozygous mutation, Glu (GAG) to Gln (CAG) at residue 32 which normally undergoes γ‐carboxylation within the γ‐carboxyglutamic acid rich domain. The genotypes of family members completely correlated with their factor X activities. It is suggested that the Glu32 to Gln mutation is the molecular basis for the abnormal factor X in this family.

Identification of epitopes on ADAMTS13 recognized by a panel of monoclonal antibodies with functional or non-functional effects on catalytic activity

INTRODUCTION von Willebrand factor (VWF) cleavage by ADAMTS13 is mediated by multi-step interactions between their multi-domain structures. To clarify the relationship between inhibitory effects of monoclonal antibodies and epitopes on each ADAMTS13 domain, we analyzed how each ADAMTS13 domain contributes to catalyze VWF using a mouse anti-ADAMTS13 monoclonal antibody panel. MATERIALS AND METHODS FRETS-VWF73 assay was used to examine the effects of 14 anti-ADAMTS13 monoclonal antibodies on the catalytic activity of plasma ADAMTS13. Epitope mapping was performed using phage surface display. Libraries expressing peptide fragments of ADAMTS13 were screened with the monoclonal antibodies. RESULTS Eleven epitopes of 14 monoclonal antibodies were successfully defined. Three monoclonal antibodies recognizing metalloprotease or disintegrin-like domains strongly inhibited the catalytic activity and their epitopes were on Gln159-Asp166, Tyr 305-Glu327, and Asn308-Glu376. Five monoclonal antibodies recognizing TSP1-3 to -7 repeats showed weak inhibitory effects, and their epitopes were on Pro744-Ala806, Pro856-Cys864, Gln892-Gly940, Cys1007-Cys1072, and Gln1163-Asn1185. Four monoclonal antibodies recognizing the TSP1-1, TSP1-2, CUB1 or CUB2 domains had no inhibitory effects, and their epitopes, except that for TSP1-1, were Pro682-Cys742, Thr1200-Cys1213, and Gln1409-Glu1414. Two monoclonal antibodies recognizing cysteine-rich and spacer domains showed moderate inhibitory effects, but their epitopes were not determined. CONCLUSIONS We revealed the epitopes of 11 monoclonal anti-ADAMTS13 antibodies on each of the domains and clarified their association with inhibitory effects on VWF catalysis under static conditions. Catalytic activity correlated strongly with the epitopes on metalloprotease and disintegrin-like domains, weakly with those on TSP1-3 to -7 repeats, and negatively with those on TSP1-1, -2, and CUB domains.

Identification of ADAMTS13 peptide sequences binding to von Willebrand factor

ADAMTS13 cleaves multimeric von Willebrand factor (VWF) to regulate VWF-mediated thrombus formation. To search ADAMTS13 peptide sequences binding to VWF, a lambda-phage library expressing various peptides of ADAMTS13 on the surface was screened using VWF either immobilized or in solution under static condition. By the first screening, peptides sharing the C-terminus of spacer domain from Arg(670) to Gln(684) (epitope-A) were selected. To explore additional sites, peptide sequences from the first screening were synthesized and added to the second screening. Consequently, Pro(618) to Glu(641) (epitope-B) in the middle of spacer domain was obtained from immobilized VWF condition. Synthetic epitope-B peptide inhibited the cleavage of VWF by ADAMTS13, while the synthetic epitope-A peptide did not as efficiently as epitope-B. Elimination of four amino acids from either sides of epitope-B terminus markedly reduced the inhibitory effect. These two sites in the spacer domain may play significant roles in binding to VWF.

Genetic analysis of hereditary factor X deficiency in a French patient of Sri Lankan ancestry: In vitro expression study identified Gly366Ser substitution as the molecular basis of the dysfunctional factor X

We investigated a new family with cross-reactive material-positive factor X (FX) deficiency. The proband was an 11-year-old French girl from Sri Lanka with a tendency towards severe bleeding. The FX antigen level was 67%, although the activity with extrinsic pathway was 1 U/dl. The complete nucleotide sequences of all exons and exon/intron junctions of the patients genomic DNA revealed a homozygous G ← A substitution in exon 8, which would result in replacement of Gly366 with Ser. The proband is the first reported case of homozygote for the FX Gly366Ser mutation. Heterozygosity for Gly366Ser substitution was previously reported in a Japanese patient (FX Nagoya 2). We studied the functional consequences by expressing mutant FX Gly366Ser protein in HEK293 cells. FX Gly366Ser was secreted into the culture media at levels similar to wild-type FX; however, mutant FX activities were only 0.04, 1.05, and 0.75% of wild-type FX upon activation by the extrinsic system, the intrinsic system, and Russells viper venom, respectively. Moreover, the activity of FX Gly366Ser was undetectable when analyzed with chromogenic-activated FX and thrombin generation assays. These data suggest that the Gly366Ser substitution would cause a major defect in function of the FX molecule.

Characterization of the gene encoding mouse platelet glycoprotein Ibβ

Abstract Platelet glycoprotein (GP) Ib/IX/V complex is a major receptor for von Willebrand factor (vWF), which mediates platelet adhesion and aggregation under high shear stress conditions. It is composed of GPIbα, GPIbβ, GPIX, and GPV. All subunits for the human receptor have been cloned and characterized. However, the function of GPIbβ is still elusive. To obtain further information of GPIbβ, we have determined the genomic sequence of mouse GPIbβ (1466 bp). The deduced amino acid sequence (206aa) was 88% identical to the human GPIbβ protein. All cysteine residues, putative N-linked glycosylation site (Asn41), and putative phosphorylation site (Ser166) were conserved. The promoter region contained putative GATA and ets binding motif implicated in megakaryocytic expression. Mouse GPIbβ also contained a leucine-rich glycoprotein (LRG) sequence of 24 amino acids same as human GPIbβ.

Diagnostic and treatment guidelines for thrombotic thrombocytopenic purpura (TTP) 2017 in Japan

Thrombotic thrombocytopenic purpura (TTP) can rapidly progress into a life-threatening condition, thus the importance of appropriate diagnosis and treatment cannot be overstated. Until recently, TTP has mainly been diagnosed by clinical findings such as thrombocytopenia and non-immune 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 been accepted internationally as a diagnostic criterion for TTP. In the present guidelines, we have taken all of these criteria into consideration. TTP is classified as acquired if the patient is positive for anti-ADAMTS13 autoantibodies, and as congenital if ADAMTS13 gene abnormalities are detected. Fresh-frozen plasma (FFP) transfusion is performed in patients with congenital TTP to supplement ADAMTS13. Plasma exchange therapy using FFP is conducted in patients with acquired TTP to supplement ADAMTS13 and remove anti-ADAMTS13 autoantibodies. To suppress autoantibody production, corticosteroid therapy may be administered in conjunction with plasma exchange. Recent reports show that the monoclonal anti-CD-20 antibody rituximab is effective in patients with refractory or relapsed TTP.