Hirokazu Kondo

Biochemical characterization of the Rho GTPase-regulated actin assembly by diaphanous-related formins, mDia1 and Daam1, in platelets.

The diaphanous-related formins are actin nucleating and elongating factors. They are kept in an inactive state by an intramolecular interaction between the diaphanous inhibitory domain (DID) and the diaphanous-autoregulatory domain (DAD). It is considered that the dissociation of this autoinhibitory interaction upon binding of GTP-bound Rho to the GTPase binding domain next to DID induces exposure of the FH1-FH2 domains, which assemble actin filaments. Here, we isolated two diaphanous-related formins, mDia1 and Daam1, in platelet extracts by GTP-RhoA affinity column chromatography. We characterized them by a novel assay, where beads coated with the FH1-FH2-DAD domains of either mDia1 or Daam1 were incubated with platelet cytosol, and the assembled actin filaments were observed after staining with rhodamine-phalloidin. Both formins generated fluorescent filamentous structures on the beads. Quantification of the fluorescence intensity of the beads revealed that the initial velocity in the presence of mDia1 was more than 10 times faster than in the presence of Daam1. The actin assembly activities of both FH1-FH2-DADs were inhibited by adding cognate DID domains. GTP-RhoA, -RhoB, and -RhoC, but not GTP-Rac1 or -Cdc42, bound to both mDia1 and Daam1 and efficiently neutralized the inhibition by the DID domains. The association between RhoA and Daam1 was induced by thrombin stimulation in platelets, and RhoA-bound endogenous formins induced actin assembly, which was inhibited by the DID domains of Daam1 and mDia1. Thus, mDia1 and Daam1 are platelet actin assembly factors having distinct efficiencies, and they are directly regulated by Rho GTPases.

Constitutive GDP/GTP Exchange and Secretion-dependent GTP Hydrolysis Activity for Rab27 in Platelets

We have previously demonstrated that Rab27 regulates dense granule secretion in platelets. Here, we analyzed the activation status of Rab27 using the thin layer chromatography method analyzing nucleotides bound to immunoprecipitated Rab27 and the pull-down method quantifying Rab27 bound to the GTP-Rab27-binding domain (synaptotagmin-like protein (Slp)-homology domain) of its specific effector, Slac2-b. We found that Rab27 was predominantly present in the GTP-bound form in unstimulated platelets due to constitutive GDP/GTP exchange activity. The GTP-bound Rab27 level drastically decreased due to enhanced GTP hydrolysis activity upon granule secretion. In permeabilized platelets, increase of Ca2+ concentration induced dense granule secretion with concomitant decrease of GTP-Rab27, whereas in non-hydrolyzable GTP analogue GppNHp (β-γ-imidoguanosine 5′-triphosphate)-loaded permeabilized platelets, the GTP (GppNHp)-Rab27 level did not decrease upon the Ca2+-induced secretion. These data suggested that GTP hydrolysis of Rab27 was not necessary for inducing the secretion. Taken together, Rab27 is maintained in the active status in unstimulated platelets, which could function to keep dense granules in a preparative status for secretion.

Tuberous Sclerosis Tumor Suppressor Complex-like Complexes Act as GTPase-activating Proteins for Ral GTPases

The small GTPases RalA and RalB are multifunctional proteins regulating a variety of cellular processes. Like other GTPases, the activity of Ral is regulated by the opposing effects of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Although several RalGEFs have been identified and characterized, the molecular identity of RalGAP remains unknown. Here, we report the first molecular identification of RalGAPs, which we have named RalGAP1 and RalGAP2. They are large heterodimeric complexes, each consisting of a catalytic α1 or α2 subunit and a common β subunit. These RalGAP complexes share structural and catalytic similarities with the tuberous sclerosis tumor suppressor complex, which acts as a GAP for Rheb. In vitro GTPase assays revealed that recombinant RalGAP1 accelerates the GTP hydrolysis rate of RalA by 280,000-fold. Heterodimerization was required for this GAP activity. In PC12 cells, knockdown of the β subunit led to sustained Ral activation upon epidermal growth factor stimulation, indicating that the RalGAPs identified here are critical for efficient termination of Ral activation induced by extracellular stimuli. Our identification of RalGAPs will enable further understanding of Ral signaling in many biological and pathological processes.

Regulation of platelet dense granule secretion by the Ral GTPase-exocyst pathway.

Non-hydrolyzable GTP analogues, such as guanosine 5′-(β, γ-imido)triphosphate (GppNHp), induce granule secretion from permeabilized platelets in the absence of increased intracellular Ca2+. Here, we show that the GppNHp-induced dense granule secretion from permeabilized platelets occurred concomitantly with the activation of small GTPase Ral. This secretion was inhibited by the addition of GTP-Ral-binding domain (RBD) of Sec5, which is a component of the exocyst complex known to function as a tethering factor at the plasma membrane for vesicles. We generated an antibody against Sec5-RBD, which abolished the interaction between GTP-Ral and the exocyst complex in vitro. The addition of this antibody inhibited the GppNHp-induced secretion. These data indicate that Ral mediates the GppNHp-induced dense granule secretion from permeabilized platelets through interaction with its effector, the exocyst complex. Furthermore, GppNHp enhanced the Ca2+ sensitivity of dense granule secretion from permeabilized platelets, and this enhancement was inhibited by Sec5-RBD. In intact platelets, the association between Ral and the exocyst complex was induced by thrombin stimulation with a time course similar to that of dense granule secretion and Ral activation. Taken together, our results suggest that the Ral-exocyst pathway participates in the regulation of platelet dense granule secretion by enhancing the Ca2+ sensitivity of the secretion.

Unexpectedly High Prevalence of Acquired von Willebrand Syndrome in Patients with Severe Aortic Stenosis as Evaluated with a Novel Large Multimer Index

AIM Severe gastrointestinal bleeding sometimes occurs in patients with aortic stenosis (AS), known as Heydes syndrome. This syndrome is thought to be caused by acquired von Willebrand syndrome and is characterized by reduced large von Willebrand factor (vWF) multimers. However, the relationship between the severity of AS and loss of large vWF multimers is unclear. METHODS We examined 31 consecutive patients with severe AS. Quantitative evaluation for loss of large vWF multimers was performed using the conventional large vWF ratio and novel large vWF multimer index. This novel index was defined as the ratio of large multimers of patients to those of controls. RESULTS Loss of large vWF multimers, defined as the large vWF multimer index ＜80%, was detected in 21 patients (67.7%). The large vWF multimer ratio and the large vWF multimer index were inversely correlated with the peak aortic gradient (R = -0.58, p=0.0007, and R=-0.64, p＜0.0001, respectively). Anemia defined as hemoglobin ＜9.0 g/dl was observed in 12 patients (38.7%), who were regarded as Heydes syndrome. Aortic valve replacement was performed in 7 of these patients, resulting in the improvement of anemia in all patients from a hemoglobin concentration of 7.5±1.0 g/dl preoperatively to 12.4±1.3 g/dl postoperatively (p＜0.0001). CONCLUSIONS Acquired von Willebrand syndrome may be a differential diagnosis in patients with AS with anemia. The prevalence of AS-associated acquired von Willebrand syndrome is higher than anticipated.

Purification and functional analysis of a Rab27 effector munc 13-4 using a semi-intact platelet dense-granule secretion assay

We have demonstrated that small GTPase Rab27 regulates dense-granule secretion in platelets. Using Rab27a affinity chromatography, we purified Munc 13-4 as a novel Rab27a interacting protein from platelet cytosol. This chapter describes the purification of Munc 13-4 and an in vitro assay system analyzing the mechanism of dense-granule secretion in platelets. The activity of Munc 13-4 is tested in this assay.

Predictors of Rapid Progression and Clinical Outcome of Asymptomatic Severe Aortic Stenosis.

BACKGROUND The optimal timing of aortic valve replacement (AVR) is controversial in patients with asymptomatic severe aortic stenosis (AS) except when very severe. Prediction of progression of severe AS is helpful in deciding on the timing of AVR. The purpose of this study was to clarify the predictors of progression rate and clinical outcomes of severe AS. METHODSANDRESULTS We retrospectively investigated 140 consecutive patients with asymptomatic severe AS (aortic valve area [AVA], 0.75-1.0 cm(2)). First-year progression rate and annual progression rate of AVA and of aortic jet velocity (AV-Vel) were calculated. Cardiac events were examined and the predictors of rapid progression and cardiac events were analyzed. The median follow-up period was 36 months. The median annual progression rate was -0.05 cm(2)/year for AVA and 0.22 m/s/year for AV-Vel. Dyslipidemia, moderate-severe calcification, and first-year AV-Vel progression ≥0.22 m/s/year were independent predictors of cardiac events. Cardiac event-free rate was lower in patients with AV-Vel first-year progression rate ≥0.22 m/s/year than in those with a lower rate. Diabetes and moderate-severe calcification were related to first-year rapid progression. CONCLUSIONS The annual progression rate of severe AS was -0.05 cm(2)/year for AVA and 0.22 m/s/year for AV-Vel. Patients with first-year rapid progression or severely calcified aortic valve should be carefully observed while considering an early operation. (Circ J 2016; 80: 1863-1869).

Validating Utility of Dual Antiplatelet Therapy Score in a Large Pooled Cohort From 3 Japanese Percutaneous Coronary Intervention Studies

Background: The dual antiplatelet therapy (DAPT) score was developed to estimate ischemic and bleeding risks from the DAPT study. However, few studies validated its utility externally. We sought to validate the utility of the DAPT score in the Japanese population. Methods: In a pooled cohort of 3 studies conducted in Japan (the CREDO-Kyoto [Coronary Revascularization Demonstrating Outcome Study in Kyoto] Registry Cohort-2, RESET [Randomized Evaluation of Sirolimus-Eluting Versus Everolimus-Eluting Stent Trial], and NEXT [NOBORI Biolimus-Eluting Versus XIENCE/PROMUS Everolimus-Eluting Stent Trial]), we compared risks for ischemic and bleeding events from 13 to 36 months after percutaneous coronary intervention among patients with a DAPT score ≥2 (high DS) and a DAPT score <2 (low DS). Results: Among 12 223 patients receiving drug-eluting stents who were free from ischemic or bleeding events at 13 months after percutaneous coronary intervention, 3944 patients had high DS and 8279 had low DS. The cumulative incidence of primary ischemic end point (myocardial infarction/stent thrombosis) was significantly higher in high DS than in low DS (1.5% versus 0.9%, P=0.002), whereas the cumulative incidence of primary bleeding end point (GUSTO moderate/severe) tended to be lower in high DS than in low DS (2.1% versus 2.7%, P=0.07). The cumulative incidences of cardiac death, myocardial infarction, and stent thrombosis were also significantly higher in high DS than in low DS (2.0% versus 1.4%, P=0.03; 1.5% versus 0.8%, P=0.002; 0.7% versus 0.3%, P<0.001, respectively), whereas the cumulative incidences of noncardiac death and GUSTO severe bleeding were significantly lower in high DS than in low DS (2.4% versus 3.9%, P<0.001; 1.0% versus 1.6%, P=0.03, respectively). Conclusions: In the current population, the DAPT score successfully stratified ischemic and bleeding risks, although the ischemic event rate was remarkably low even in high DS. Further studies would be warranted to evaluate the utility of prolonged DAPT guided by the DAPT score.

Multiple Coronary Artery Aneurysms and Thoracic Aortitis Associated with IgG4-related Disease

A 60-year-old man was admitted due to the onset of right coronary artery (RCA) aneurysms. Coronary angiography showed two RCA aneurysms and focal stenosis with limitations in the blood flow. Balloon angioplasty was performed. However, the follow-up coronary angiography showed restenosis, an enlarged proximal aneurysm and a newly formed aneurysm. The serum immunoglobulin G4 level was elevated to 1,350 mg/dL and fluorodeoxyglucose positron emission tomography showed increased uptake in the ascending aorta, so the patient was diagnosed with immunoglobulin G4-related vascular disease. The prevention of further enlargement of the aneurysms and an improvement in the RCA flow were achieved with steroid therapy. Steroid therapy may therefore be effective for immunoglobulin G4-related vascular disease.

Relationship between plasma dabigatran concentration and activated partial thromboplastin time in Japanese patients with non-valvular atrial fibrillation

Activated partial thromboplastin time (aPTT) is recommended for monitoring anticoagulant activity in dabigatran‐treated patients; however, there are limited data in Japanese patients. To clarify the relationship between plasma dabigatran concentration and aPTT, we analyzed plasma dabigatran concentration and aPTT at various time points following administration of oral dabigatran in a Japanese hospital.