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Introduction: Cilostazol has been shown to be effective for prevention and treatment of cerebral infarction. However, there appears to be no widely accepted method appropriate for monitoring cilostazol. We attempted to establish an assay system for cilostazol monitoring, using platelet aggregation induced by arachidonic acid (AA) in the presence of PGE(1) which upregulates intracellular cyclic AMP., Methods: Blood was drawn from stroke patients before and after cilostazol intake. AA-induced platelet aggregation after pretreatment with 0~30nM PGE(1) for 2minutes was measured by light transmittance aggregometry., Results: AA-induced platelet aggregation was 73.1±2.2% in the absence of PGE(1), and pretreatment with 30nM PGE(1) had virtually no inhibitory effect on platelet aggregation prior to cilostazol intake. In contrast, after cilostazol intake, 30nM PGE(1) significantly inhibited platelet aggregation to 12.7±4.5% (p=7.8×10(-11)) , while in the absence of PGE(1) platelet aggregation remained similar to that of prior-to-cilostazol value (70.6±3.5%). The plasma concentration of cilostazol ranged from 0.55 to 3.51μM. In the presence of 30nM PGE(1), all the patients with cilostazol concentrations exceeding 1μM had their platelet aggregation inhibited almost completely. ROC analysis suggests that AA-induced platelet aggregation in the presence of 30nM PGE(1) had the excellent sensitivity (90.5%) and specificity (88.4%) for monitoring cilostazol., Conclusions: AA-induced platelet aggregation in the presence of 30nM PGE(1) could give good estimate on plasma concentrations of cilostazol. It is suggested that this system is a good tool for monitoring cilostazol., (Copyright © 2012. Published by Elsevier Ltd.)

Thrombus formation on disrupted atherosclerotic lesion is a key mechanism of cardiovascular events. Podoplanin (Aggrus), expressed on the surface of several tumor cells, is an endogenous ligand for C-type lectin-like receptor 2 (CLEC-2), and is involved in tumor cell-induced platelet aggregation and its malignant potency. Podoplanin, which is also expressed in lymphatic endothelial cells, facilitates blood/lymphatic vessel separation. However, podoplanin expression in atherosclerotic lesion has not been investigated. To clarify podoplanin expression in atherosclerotic lesion and to assess its importance for the onset of cardiovascular events, we examined podoplanin expression in abdominal aortas obtained from 31 autopsy cases. Immunohistochemical analysis indicated that podoplanin was localized to smooth muscle cells and macrophages. Moreover, podoplanin immunoreactivity was increased in advanced atherosclerotic lesions containing necrotic core, many macrophages and smooth muscle cells, compared with early lesions composed of smooth muscle cells and small numbers of macrophages. Furthermore, Western-blot and real time-PCR analyses showed that podoplanin expression was significantly enhanced in advanced atherosclerotic lesions, compared with early lesions. These results suggest that podoplanin contributes to thrombotic property of advanced stages of atherosclerosis and that it might be a novel molecular target for an anti-thrombus drug., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Introduction: A simple, validated method to measure platelet function is unavailable for bedside use. Measurement of platelet retention rate using a column of collagen-coated beads and whole blood is a new, simple assay that reflects platelet aggregation. This study was aimed to examine the utility of this assay to assess efficacy of antiplatelet drug therapy., Methods: Citrated whole blood (1.5 ml) in a syringe was passed through a polyvinyl tube packed with collagen-coated beads for 40 seconds using a syringe pump. Platelet retention rate in the column was calculated from platelet counts in blood before and after passage. An increase in the retention rate reflects an increase in platelet activity. This new platelet retention assay and the traditional optical aggregometry assay were performed in 331 patients with stable coronary artery disease (CAD)., Results: The retention rate was significantly reduced in patients taking dual antiplatelet therapy (aspirin plus clopidogrel or ticlopidine) compared with aspirin alone. There was a significant linear correlation between the platelet retention rate and platelet aggregability measured by the traditional method (r=0.44, p<0.001). In multivariate Cox proportional hazards analysis, higher platelet retention rate was an independent predictor of future cardiovascular events in patients on dual antiplatelet therapy (hazard ratio 3.9, 95% CI 1.6 to 9.5, p=0.003)., Conclusions: Measurement of the platelet retention rate in a column of collagen-coated beads may be useful for monitoring the efficacy of antiplatelet drug therapy in patients with CAD.

Microparticles released from cells (MPs) may play a role in the normal hemostatic response to vascular injury and a role in clinical diseases because they express phospholipids, which function as procoagulants. Although flow cytometry is the most widely used method for studying MPs, some novel assays such as tissue factor-dependent procoagulant assay or the ELISA method have been reported. However, the use of MP quantification as a clinical tool is still a matter of debate. Elevated platelet-derived MP, endothelial cell-derived MP, and monocyte-derived MP concentrations are documented in almost all thrombotic diseases occurring in both venous and arterial beds. However, the clear significance of MPs in various clinical conditions remains controversial. For example, it is not known if MPs found in peripheral blood vessels cause thrombosis, or whether they are the result of thrombosis. On the other hand, numerous studies have shown that not only the quantity but also the cellular origin and composition of circulating MPs are dependent on the type of disease, the disease state and medical treatment. In addition, many different functions have also been attributed to MPs. Thus, the number and type of clinical disorders associated with elevated MPs is currently increasing.

The integrin alpha(IIb)beta(3) plays a critical role in mediating clot retraction by platelets which is important in vivo in consolidating thrombus formation. Actin-myosin interaction is essential for clot retraction. In the present study, we demonstrate that the structurally distinct Src kinase inhibitors, PP2 and PD173952, significantly reduced the rate of clot retraction, but did not prevent it reaching completion. This effect was accompanied by abolition of alpha(IIb)beta(3)-dependent protein tyrosine phosphorylation, including PLCgamma2. A role for PLCgamma2 in mediating clot retraction was demonstrated using PLCgamma2-deficient murine platelets. Furthermore, platelet adhesion to fibrinogen leads to MLC phosphorylation through a pathway that is inhibited by PP2 and by the PLC inhibitor, U73122. These results demonstrate a partial role for Src kinase-dependent activation of PLCgamma2 and MLC phosphorylation in mediating clot retraction downstream of integrin alpha(IIb)beta(3).

Introduction: Peripheral arterial disease is one of the systemic atherosclerotic diseases, and patients with the disorder are classified in the high risk group of coronary artery disease. A lower ankle brachial index is a frequent finding in peripheral arterial disease. While platelet microaggregates are a significant predictor of adverse clinical outcome in coronary artery disease, the significance of platelet aggregability in peripheral arterial disease has not been elucidated., Materials and Methods: Small platelet aggregates measured using laser-light scattering and ankle brachial index were determined in 42 patients with both coronary artery disease and peripheral arterial disease (peripheral group), 56 patients with only coronary artery disease (coronary group) and 32 patients without both (control group)., Results: The level of small platelet aggregates was increased significantly in the peripheral group (4.3 x 10(4) [range 2.2 x 10(4) to 7.4 x 10(4)]) compared with both the coronary (1.1 x 10(4) [range 0.3 x 10(4) to 5.0 x 10(4)]) and control groups (0.5 x 10(4) [range 0.1 x 10(4) to 0.9 x 10(4)]). There was a significant inverse correlation between log small platelet aggregates and ankle brachial index (n=130, r=-0.422, p<0.001). Multivariate logistic regression analysis revealed that a lower ankle brachial index (<0.90) was an independent determinant of increased levels of small platelet aggregates., Conclusions: Platelet aggregability was increased in patients with peripheral arterial disease with the degree of platelet aggregation being closely associated with ankle brachial index. It is possible that this change in platelet activity may be one mechanism to explain why a lower ankle brachial index is a predictor of poor prognosis in patients with peripheral arterial disease.

A recently developed platelet aggregometer using a laser light scattering method is capable of monitoring the increase in size of small-sized platelet aggregates (diameter 9-25 microm), which cannot be detected with the conventional methods. Whether coronary spasm can cause platelet aggregation in the coronary circulation is unknown. We investigated platelet aggregation, especially small-sized platelet aggregates, simultaneously in the coronary sinus and the aortic root in 18 patients with coronary spastic angina before and after a left coronary artery spasm induced by intracoronary injection of acetylcholine, and in 15 patients with stable exertional angina before and after acute myocardial ischemia induced by rapid right atrial pacing. Platelet aggregation in 12 patients with chest pain syndrome was also examined before and after coronary spasms provoked by acetylcholine. The number of small-sized platelet aggregates increased significantly in the coronary sinus [2.0+/-0.6 x 104 to 4.1+/-1.0 x 104 (V), P<.01] and in the aortic root [1.7+/-0.6 x 104 to 3.2+/-0.6 x 104 (V), P<.05], and the coronary sinus-arterial difference in the number of small-sized platelet aggregates [2.3+/-1.9 x 103 to 1.1+/-0.4 x 104 (V), P<.01] increased significantly after attacks in the coronary spastic angina group, but remained the same in the stable exertional angina group after attacks and in the chest pain syndrome group after the administration of acetylcholine. Therefore, we can conclude that acute myocardial ischemia induced by coronary spasm causes platelet aggregation in the coronary circulation.

The effect of extracellular Na+ on Ca++ influx in activated human platelets was evaluated with two methods. One method employed computer-aided subtraction of the time course of intracellular Ca++ changes in the absence of extracellular Ca++ from that observed in the presence of extracellular Ca++. The other utilized intracellular Ca++ elevation following the addition of Ca++ to a platelet suspension already activated by thrombin in the absence of extracellular Ca++. Both methods demonstrated that depletion of extracellular Na+ significantly attenuated Ca++ influx in activated platelets. That extracellular Na+ modifies the opening state of Ca++ channels was further substantiated with an experiment with Sr++ instead of Ca++. A flow cytometric study confirmed the validity of these experiments. Neither Na+/H+ exchange nor changes in the membrane potential appeared to be related to the effect of extracellular Na+ on Ca++ influx, and the mechanism by which extracellular Na+ permits Ca++ mobilization remains to be elucidated.

A Ca++-ionophore, ionomycin, increased the volume of human platelets suspended in a Ca++-containing buffer. This change in cell volume was dependent upon ionomycin and extracellular Ca++ concentrations, suggesting that the volume change occurs when the intracellular Ca++ reaches a certain level (greater than uM as determined by aequorin method). The ionomycin-induced volume increase was suppressed by replacement of extracellular Na+ with membrane-impermeable N-methyl-D-glucamine or Cs+, but not with Li+, K+, or Rb+. Ethylisopropylamiloride, a potent inhibitor of the Na+/H+ exchanger, had only weak inhibitory effect, and the apparent Km for Na+ was approximately 350 mM, which is much larger than that of the Na+/H+ exchanger. It is suggested that certain mechanisms other than the Na+/H+ exchanger are responsible for ionomycin-induced volume increase.

The presence of extracellular bicarbonate potentiated platelet intracellular pH rises induced by thrombin. The effect was most remarkable in sodium-depleted buffers. This effect of bicarbonate was dose-dependent and was inhibited by anion channel blockers. Bicarbonate also potentiated the pHi recovery after acid loading, but had no effect on alkali loading. It was suggested that anion exchange, most probably that of bicarbonate/chloride, contributes to the regulation of intracellular pH in human platelets.