Akira Suehiro

Enhancing effect of advanced glycation end products on serotonin-induced platelet aggregation in patients with diabetes mellitus.

Advanced glycation end products (AGEs) are thought to be responsible for some complications of diabetes mellitus (DM), including microangiopathy. Plasma serotonin is increased in diabetes mellitus patients, and this increase is related, at least in part, to platelet hyperfunction. In order to clarify the relationship between advanced glycation end products, serotonin, and thrombotic complications in diabetes mellitus patients, we examined the effect of advanced glycation end products on serotonin-induced platelet aggregation. In diabetic patients, although serotonin-induced platelet aggregation was enhanced with an increase in serum-advanced glycation end products, there was no correlation between platelet aggregation and either hemoglobin A1c or fasting blood sugar. To examine the direct effect of advanced glycation end products on platelet aggregation, we prepared advanced glycation end products by in vitro incubation of human albumin with glucose (250 mM) at 37 degrees C for 8 weeks. Serotonin-induced platelet aggregation was dose-dependently increased by advanced glycation end products. Adenosine diphosphate-induced platelet aggregation also was increased by advanced glycation end products, but this increment was diminished by addition of sarpogrelate, a selective serotonin receptor antagonist. These results suggest that advanced glycation end products enhance platelet aggregation through the serotonin receptor, and perhaps influencing the development of thrombotic complications in diabetic patients.

Extracellular pH Affects Platelet Aggregation Associated with Modulation of Store-Operated Ca2+ Entry

The pH dependence of store-operated Ca(2+) influx (SOCI) into human platelets, as well as its physiological consequence, aggregation, was studied. In Ca(2+)-free medium, thapsigargin (1 microM) induced a small increase in intracellular free-Ca(2+) ([Ca(2+)](i)), which was not affected by changes in extracellular pH. The addition of Ca(2+) (0.5-3 mM) after Ca(2+) store depletion caused by thapsigargin resulted in concentration-dependent increases in [Ca(2+)](i) (SOCI), which were strongly inhibited by SKF-96365 (100 microM), an inhibitor of receptor-mediated Ca(2+) entry. SOCI was inhibited by acidosis (pH 6.9) and augmented by alkalosis (pH 7.9). The addition of Ca(2+) (0.5-3 mM) to platelets, which were kept in Ca(2+)-free medium, slightly but significantly increased [Ca(2+)](i). This Ca(2+) leak entry was also decreased and increased by extracellular acidosis (pH 6.9) and alkalosis (pH 7.9), respectively, but not affected by SKF-96365. Neither thapsigargin (1 microM) stimulation in Ca(2+)-free solution nor elevation of extracellular Ca(2+) alone was sufficient to induce platelet aggregation. In contrast, the addition of Ca(2+) (1 mM) to platelets activated by thapsigargin resulted in aggregation, which was markedly inhibited by SKF-96365 (100 microM). Platelet aggregation associated with SOCI was also inhibited by extracellular acidosis (pH 6.9) and augmented by extracellular alkalosis (pH 7.9). These results suggest that acidosis-induced inhibition, as well as alkalosis-induced promotion of platelet aggregation, involve pH effects on SOCI.

Mechanism of Hematuria in Glomerular Disease

From an electron-microscopic study in a case of diffuse membranous glomerulonephritis, we would like to propose a possible mechanism of hematuria in glomerular disease. There are several factors we sh

Clinical significance of new coagulation and fibrinolytic markers in ischemic stroke patients.

Background and Purpose: We investigated plasma levels of D-dimer products of crosslinked fibrin degradation products, thrombin-antithrombin III complex, and plasmin-α2-antiplasmin complex for detecting coagulation system activation in ischemic stroke patients to determine the possible effect of age on these marker levels. Methods: We measured plasma levels of these three markers in 54 acute ischemic stroke patients within 5 days of stroke onset, in 44 chronic ischemic stroke patients over 3 months from onset, and in 50 age-matched healthy subjects. We divided the stroke patients into two subgroups, those with visible occlusion and those with nonvisible occlusion having obstruction of the major cerebral artery. Results: The plasma levels of these three markers were significantly (p<0.01) higher in the stroke patients than in controls. Significant differences did not exist at any level between the patients and controls in the younger-aged subjects (≤64 years of age), but did exist in the older-aged subjects (≥75 years of age). An age-related increase of the marker levels was noted between stroke patients and controls. No significant difference in the three markers was found among any of the stroke patients. Conclusions: Increased levels of these markers in stroke patients seem to be related mostly to age.

Clinical significance of circulating hepatocyte growth factor, a new risk marker of carotid atherosclerosis in patients with Type 2 diabetes

Aims  Recent studies have provided increasing evidence that hepatocyte growth factor (HGF) has a pathophysiological role in the development of diabetic complications. We set out to determine the relationship between serum HGF and risk factors for macroangiopathy including carotid atherosclerosis. Carotid atherosclerosis is an established and important risk factor for both cerebral and coronary artery diseases.

Inhibitory effect of vitamin E (α -tocopherol) on spontaneous platelet aggregation in whole blood

Abstract Vitamin E (D-α-tocopherol) inhibited spontaneous human platelet aggregation in whole blood in the 20–200 μ g/ml range. When α -tocopherol (20 μ g/ml) and aspirin (0.5 mM), or α -tocopherol and the mixture of phosphocreatine (1.5 mM) and creatine phosphokinase (50 U/ml) (CP/CPK) were added to this reaction system, a synergic inhibitory effect on aggregation was observed. On the other hand, when both α -tocopherol and the specific inhibitor of platelet activating factor (CV-3988 : 0.38 mM) were added to this system, the inhibition was the same as that caused by the addition of CV-3988 alone, suggesting there was no synergism, i.e., that the effect of α -tocopherol is related to the inhibition of platelet activating factor (PAF)-induced platelet aggregation in whole blood. However, α -tocopherol (20 or 50 μ g/ml) did not inhibit PAF (10 nM) induced platelet aggregation in platelet rich plasma (PRP). These results suggest that the inhibition of platelet aggregation in whole blood by α -tocopherol is due to the inhibition of PAF synthesis, and is unrelated to adenosine diphosphate (ADP) or thromboxane A2.

Exogenous nitric oxide inhibits platelet activation in whole blood.

We examined the effect of NO on collagen-induced whole blood aggregation and platelet activation in whole blood by using impedance aggregometry and flow cytometry. For the extracellular generation of NO, we chose sodium nitroprusside dihydrate (SNP), and as intracellular generators of NO, L-arginine and isosorbide dinitrate (ISDN). The latter two significantly inhibited whole blood aggregation, whereas SNP had no such effect. The inhibitory effect of ISDN was diminished by addition of methylene blue (MB) or 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-l-oxyl 3-oxide (carboxyl-PTIO), and the inhibitory effect of L-arginine was diminished by addition of N(G)-monomethyl-L-arginine monoacetate (L-NMMA). Although the addition of ISDN increased the cyclic guanosine monophosphate (cGMP) level in whole blood and in the suspension of platelets and white blood cells (PLTs + WBCs), no increase was found in platelet-rich plasma (PRP). The P-selectin expression on the platelet surface in whole blood was reduced by ISDN and L-arginine. These findings suggest that the intracellular generation of NO inhibits whole blood aggregation, and this mechanism may play an important role in its antithrombotic effect in whole blood.

The role of platelet hyperfunction in thrombus formation in hyperlipidemia

The mechanism of thrombus formation in hyperlipidemia was studied. Attempts at artificial creation of an arterial thrombus in control rabbits stenosing the femoral artery by ligature were not successful unless ellagic acid was administered by injection. However, in rabbits with hyperlipidemia, mere creation of stenosis in the femoral artery resulted in a high percentage of thrombus formation. In rabbits with hyperlipidemia, both thromboxane (Tx) A2 biosynthesis in platelets and prostacyclin (PGI2) biosynthesis in the aorta were increased and these changes were noted at the level of cyclooxygenase in the arachidonic acid metabolic pathway. Therefore, these results suggest that thrombi are likely to be formed in hyperlipidemia and that such thrombus formation is due largely to platelet hyperfunction.

Suitability of four polymorphic DNA markers for indirect genetic diagnosis of haemophilia A in Japanese subject

Indirect genetic diagnosis using polymorphic DNA markers can detect carriers of haemophilia A (HA). These markers include CA repeat polymorphisms at intron 13 (CA-13) and CT-AG at intron 22 of the coagulation factor VIII (FVIII) gene, and also certain restriction fragment length polymorphisms (RFLPs) such as HindIII at intron 19 and BclI at intron 18. Recently, CA repeat polymorphism at intron 6 (CA-6) was also reported. We compared usefulness of the BclI RFLP, the HindIII RFLP, and CA-13 to that of CA-6. Heterozygosity of markers was examined in 282 X chromosomes obtained from 205 subjects including HA patients. Expected heterozygosity of the HindIII and BclI RFLPs was 30.0% and 27.9%, while observed heterozygosity was 28.0% and 26.5%. Expected heterozygosity of CA-13 was 45.6%, and observed heterozygosity was 40.0%. CA-6 showed two alleles with repeat numbers of 13 and 14, expected and observed heterozygosity were low (1.4% and 2.6%, respectively). When we used these markers in a HA lineage where the mother was a carrier according to coagulation factor assays, carrier diagnosis was possible using CA-13, the HindIII RFLP, and the BclI RFLP. This was not true for CA-6, for which the mother was homozygous. Although CA-13, and the HindIII and BclI RFLPs were useful for indirect genetic diagnosis of HA, CA-6 proved less useful because of low heterozygosity.

Effects of gliclazide on platelet aggregation and the plasminogen activator inhibitor type 1 level in patients with type 2 diabetes mellitus

Vascular complications are a common factor determining morbidity and mortality of diabetic patients. In vitro studies have revealed that gliclazide has antiplatelet activities. To clinically assess this action, we measured the effects of gliclazide on platelet activities and abnormal fibrinolysis in patients with type 2 diabetes mellitus. We studied 14 patients aged 38 to 72 years (9 men and 5 women) with type 2 diabetes mellitus who have been treated with glibenclamide in our hospital for more than 6 months. We switched from glibenclamide to gliclazide using the average ratio of the respective doses, 2.5 vs 40 mg. We titrated the dose of gliclazide to keep the glycemic control at the same level as the previous (glibenclamide) treatment. We measured 10 micromol/L serotonin-induced or 0.5 micromol/L adenosine diphosphate (ADP)-induced platelet aggregate formation by particle counting using light scattering at baseline and up to 6 months after the switch. After switching to gliclazide, platelet aggregate formation induced by serotonin was significantly reduced (P < .05, compared with the levels observed after glibenclamide treatment). The body mass index, fasting plasma glucose, immunoreactive insulin, homeostasis model assessment of insulin resistance, hemoglobin A(1c) (HbA(1c)), total cholesterol, triglycerides, high-density lipoprotein cholesterol, prothrombin time, activated partial thromboplastin time, fibrinogen, thrombin-antithrombin III complex, plasmin-alpha2-plasmin inhibitor complex, and plasma plasminogen activator inhibitor type 1 (PAI-1) were not changed. In the group with improved HbA(1c) (n = 5), ADP-induced platelet aggregate formation and plasma PAI-1 level were significantly reduced (P < .05, compared with the group with aggravated HbA(1c), n = 9). Multiple regression analysis showed that percentage change of ADP-induced platelet aggregate formation (standardized beta = 0.540, P < .05) was independently associated with percentage change of plasma PAI-1 level in addition to percentage change of HbA(1c) (standardized beta = 0.657, P < .05) (R = 0.939, P < .05) after switching to gliclazide. The other independent variants, like the final dose of gliclazide, homeostasis model assessment of insulin resistance, percentage change of prothrombin time, activated partial thromboplastin time, and total cholesterol, were not significantly associated with the percentage change of plasma PAI-1 level. These results indicate that gliclazide inhibits platelet aggregation via the serotonin pathway, independently of the metabolic control per se. Furthermore, in the patients with improved glycemic control, gliclazide could inhibit ADP-induced platelet aggregation and reduce PAI-I level. Taken together, the results show that gliclazide may be more useful for the prevention of diabetic vascular complications than glibenclamide.