Soluble CLEC-2 is generated independently of ADAM10 and is increased in plasma in acute coronary syndrome: comparison with soluble GPVI.

Soluble forms of platelet membrane proteins are released upon platelet activation. We previously reported that soluble C-type lectin-like receptor 2 (sCLEC-2) is released as a shed fragment (Shed CLEC-2) or as a whole molecule associated with platelet microparticles (MP-CLEC-2). In contrast, soluble glycoprotein VI (sGPVI) is released as a shed fragment (Shed GPVI), but not as a microparticle-associated form (MP-GPVI). However, mechanism of sCLEC-2 generation or plasma sCLEC-2 has not been fully elucidated. Experiments using metalloproteinase inhibitors/stimulators revealed that ADAM10/17 induce GPVI shedding, but not CLEC-2 shedding, and that shed CLEC-2 was partially generated by MMP-2. Although MP-GPVI was not generated, it was generated in the presence of the ADAM10 inhibitor. Moreover, antibodies against the cytoplasmic or extracellular domain of GPVI revealed the presence of the GPVI cytoplasmic domain, but not the extracellular domain, in the microparticles. These findings suggest that most of the GPVI on microparticles are induced to shed by ADAM10; MP-GPVI is thus undetected. Plasma sCLEC-2 level was 1/32 of plasma sGPVI level in normal subjects, but both soluble proteins significantly increased in plasma of patients with acute coronary syndrome. Thus, sCLEC-2 and sGPVI are released by different mechanisms and released in vivo upon platelet activation.

Cobalt hematoporphyrin inhibits CLEC-2-podoplanin interaction, tumor metastasis, and arterial/venous thrombosis in mice.

The platelet activation receptor C-type lectin-like receptor 2 (CLEC-2) interacts with podoplanin on the surface of certain types of tumor cells, and this interaction facilitates tumor metastasis. CLEC-2 is also involved in thrombus formation and its stabilization. Because CLEC-2-depleted mice are protected from experimental lung metastasis and thrombus formation and do not show increased bleeding time, CLEC-2 may serve as a good target for antimetastatic or antithrombotic drugs. We screened 6770 compounds for their capability to inhibit CLEC-2-podoplanin binding using an enzyme-linked immunosorbent assay. In the first screening round, 63 compounds were identified and further evaluated by flow cytometry using CLEC-2-expressing cells. We identified protoporphyrin IX (H2-PP) as the most potent inhibitor and modified its hematoporphyrin moiety to be complexed with cobalt (cobalt hematoporphyrin [Co-HP]), which resulted in an inhibitory potency much stronger than that of H2-PP. Surface plasmon resonance analysis and molecular docking study showed that Co-HP binds directly to CLEC-2 at N120, N210, and K211, previously unknown podoplanin-binding sites; this binding was confirmed by analysis of CLEC-2 mutants with alterations in N120 and/or K211. Co-HP at a concentration of 1.53 µM inhibited platelet aggregation mediated through CLEC-2, but not that mediated through other receptors. IV administration of Co-HP to mice significantly inhibited hematogenous metastasis of podoplanin-expressing B16F10 cells to the lung as well as in vivo arterial and venous thrombosis, without a significant increase in tail-bleeding time. Thus, Co-HP may be a promising molecule for antimetastatic and antiplatelet treatment that does not cause bleeding tendency.

Podoplanin-positive periarteriolar stromal cells promote megakaryocyte growth and proplatelet formation in mice by CLEC-2.

Megakaryopoiesis is the hierarchical differentiation of hematopoietic stem cells into megakaryocytes. Differentiating megakaryocytes undergo maturation characterized by endomitosis and produce numerous platelets through proplatelet formation. C-type lectin-like receptor 2 (CLEC-2) is a podoplanin (PDPN) receptor mainly expressed on platelets and megakaryocytes. Deletion of platelet/megakaryocyte CLEC-2 causes thrombocytopenia in mice; however, its contribution to megakaryopoiesis remains unknown. Here, we show that megakaryopoiesis is promoted through the CLEC-2/PDPN interaction in the vicinity of arterioles in the bone marrow (BM). We have also identified PDPN-expressing BM arteriolar stromal cells, tentatively termed as BM fibroblastic reticular cell (FRC)-like cells. Platelet/megakaryocyte-specific CLEC-2 conditional knockout (cKO) mice showed a decrease in the number of immature megakaryocytes. CLEC-2 wild-type megakaryocyte expansion was augmented in vitro by the addition of recombinant PDPN, but not cKO megakaryocytes. Moreover, megakaryocyte colonies were colocalized with periarteriolar BM FRC-like cells in the BM. Coculture of megakaryocytes with BM FRC-like cells augmented megakaryocyte expansion, which was dependent upon the CLEC-2/PDPN interaction. Furthermore, we found that the CLEC-2/PDPN interaction induces BM FRC-like cells to secrete chemokine (C-C motif) ligand 5 (CCL5) to facilitate proplatelet formation. These observations indicate that a reciprocal interaction between CLEC-2 on megakaryocytes and PDPN on BM FRC-like cells contributes to the periarteriolar megakaryopoietic microenvironment in mouse BM.

Hot Water Extracts of the Royal Sun Mushroom, Agaricus brasiliensis (Higher Basidiomycetes), Inhibit Platelet Activation via the P2Y1 Receptor.

Hot water extracts of the medicinal mushroom Agaricus brasiliensis were investigated for their inhibition of platelet aggregation. The extracts significantly inhibited human platelet aggregation induced by adenosine 5'-diphosphate (ADP), but not by collagen or thrombin receptor-activating peptide. The extracts also had a significant inhibitory effect on shape change and intracellular calcium mobilization induced by ADP via inhibition of ADP binding to the P2Y1 receptor. In addition, oral administration of the extracts resulted in prolonged tail bleeding time in mice. The marked antiplatelet activity of the mushroom extracts involving the P2Y1 receptor suggests their potential therapeutic use against vascular disorders.

Bisphenol A exposure induces metabolic disorders and enhances atherosclerosis in hyperlipidemic rabbits.

Bisphenol A (BPA) is an artificial environmental endocrine disrupter. Excess exposure to BPA may induce many disorders in the metabolism and cardiovascular system. However, the underlying toxicological mechanisms remain largely unknown. In this study, we administered genetically hyperlipidemic Watanabe heritable hyperlipidemic (WHHL-MI) rabbits (male, 14 week old), which have more common features with humans than the mouse and rat especially in the metabolism and cardiovascular system, with BPA at 40 mg kg(-1) day(-1) for 8 weeks by gavage and compared their plasma lipids, glucose and insulin response with those of the vehicle group. All of the rabbits were sacrificed, and their pancreas, liver, adipose tissue, heart and aorta were analyzed using histological and morphometric methods. Furthermore, we treated human hepatoma HepG2 cells and human umbilical cord vein endothelial cells (HUVECs), with different doses of BPA based on the serum BPA levels in the WHHL rabbits for 6 h to investigate the possible molecular mechanisms. Our results showed that BPA-treated rabbits showed insulin resistance, prominent adipose accumulation and hepatic steatosis. Additionally, BPA exposure also caused myocardial injury and enhanced the development of atherosclerosis in the aortic arch with increased macrophage number (86%) and advanced lesion areas (69%). Increased expression of inflammatory genes found in the liver of BPA-treated rabbits along with the up-regulation of ER stress, lipid and glucose homeostasis and inflammatory genes in the cultured HepG2 cells and HUVECs suggest that BPA may induce metabolic disorders and enhance atherosclerosis through regulating above molecular pathways in the liver and endothelium.

Bisphenol A exposure enhances atherosclerosis in WHHL rabbits.

Bisphenol A (BPA) is an environmental endocrine disrupter. Excess exposure to BPA may increase susceptibility to many metabolic disorders, but it is unclear whether BPA exposure has any adverse effects on the development of atherosclerosis. To determine whether there are such effects, we investigated the response of Watanabe heritable hyperlipidemic (WHHL) rabbits to 400-µg/kg BPA per day, administered orally by gavage, over the course of 12 weeks and compared aortic and coronary atherosclerosis in these rabbits to the vehicle group using histological and morphometric methods. In addition, serum BPA, cytokines levels and plasma lipids as well as pathologic changes in liver, adipose and heart were analyzed. Moreover, we treated human umbilical cord vein endothelial cells (HUVECs) and rabbit aortic smooth muscle cells (SMCs) with different doses of BPA to investigate the underlying molecular mechanisms involved in BPA action(s). BPA treatment did not change the plasma lipids and body weights of the WHHL rabbits; however, the gross atherosclerotic lesion area in the aortic arch was increased by 57% compared to the vehicle group. Histological and immunohistochemical analyses revealed marked increases in advanced lesions (37%) accompanied by smooth muscle cells (60%) but no significant changes in the numbers of macrophages. With regard to coronary atherosclerosis, incidents of coronary stenosis increased by 11% and smooth muscle cells increased by 73% compared to the vehicle group. Furthermore, BPA-treated WHHL rabbits showed increased adipose accumulation and hepatic and myocardial injuries accompanied by up-regulation of endoplasmic reticulum (ER) stress and inflammatory and lipid metabolism markers in livers. Treatment with BPA also induced the expression of ER stress and inflammation related genes in cultured HUVECs. These results demonstrate for the first time that BPA exposure may increase susceptibility to atherosclerosis in WHHL rabbits.

Aspirin Half Maximal Inhibitory Concentration Value on Platelet Cyclooxygenase1 in Severe Type-2 Diabetes Mellitus is not Significantly Different from that of Healthy Individuals.

It is implicated that diabetic patients are more resistant to aspirin therapy than patients with other diseases or healthy individuals. We evaluated the inhibitory effects of aspirin on aggregation and the cyclooxygenase activity of platelets of 10 patients with severe type-2 diabetes mellitis (DM) and compared the results with those of healthy individuals. Although platelet aggregation had a tendency to be more resistant to aspirin with the DM group, there was no significant difference in half maximal inhibitory concentration 50 values of aspirin on the cyclooxygenase activity between the patients with DM and healthy individuals. Thus, the residual platelet aggregability uninhibited by aspirin appears to be independent of the cyclooxygenase activity. Since adenosine diphosphate (ADP) receptor blocking almost completely inhibited the residual platelet aggregability, it is suggested that hyperreactivity to ADP is more prevalent in patients with DM.

VacA, the vacuolating cytotoxin of Helicobacter pylori, binds to multimerin 1 on human platelets.

Platelets were activated under the infection with H. pylori in human and mice. We investigated the role of VacA, an exotoxin released by H. pylori in this context. Acid-activated VacA, but not heated VacA, induced platelet CD62P expression. However, VacA reacted with none of the alleged VacA receptors present on platelet membranes. We therefore analyzed VacA associated proteins obtained through VacA affinity chromatography, using MALDI-TOF-MS. Multimerin1 was detected in two consecutive experiments, as the binding protein for VacA. Plasmon resonance confirmed their binding, and dot blot analysis revealed that the peptide sequence AA 321-340 of multimerin 1 is the binding site for VacA. In conclusion, we propose a new interaction between multimerin1 and VacA , which may give another insight into H. pylori-induced platelet activations under H. pylori infection.

In-stent thrombosis after carotid artery stenting despite sufficient antiplatelet therapy in a bladder cancer patient.

In-stent thrombosis (IST) after carotid artery stenting (CAS) is a rare but potentially devastating complication. We present a case of early IST after CAS despite sufficient antiplatelet therapy in a patient with bladder cancer. A 77-year-old man under preventive triple antiplatelet therapy underwent CAS without any intra- or periprocedural complications. However, the patient developed a large asymptomatic IST 6 days after CAS. Anticoagulant therapy with argatroban was reintroduced to treat IST concomitant with antiplatelet agents. Subsequently, the IST shrank and disappeared without any thrombotic symptoms. Malignancy is regarded as an acquired thrombophilic condition associated with a significant risk of thrombosis. In the field of coronary stents, cancer is associated with a significant increasing risk of IST. The cause of IST in our case was possibly related in hypercoagulable state because of the patient's cancer. Attention for IST should be paid in CAS cases with these risk factors, and repeated examination is recommended.

Platelet aggregometry in the presence of PGE(1) provides a reliable method for cilostazol monitoring.

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.

Causes of thrombocytopenia in chronic hepatitis C viral infection.

We retrospectively studied 89 patients with chronic hepatitis C virus (HCV) infection, including 50 chronic hepatitis (CH) cases, 18 liver cirrhosis (LC) cases, and 21 LC with hepatocellular carcinoma (LC + HCC) cases, with regard to various factors related with thrombocytopenia. The platelet count decreased with the stage advancement of liver diseases. Multiple regression analysis revealed that splenomegaly and von Willebrand factor (vWF) were explanatory variables that correlated with thrombocytopenia. Splenomegaly appears to be the most responsible factor, although there are a considerable number of thrombocytopenic cases without splenomegaly, suggesting other factors may also be responsible. The vWF level is inversely correlated with the platelet count. Soluble thrombomodulin, a marker of endothelial dysfunction, increases with the advancement of liver fibrosis. It is positively correlated with vWF and inversely with the platelet count. Our present results imply that vascular endothelial dysfunction is also involved in thrombocytopenia during chronic HCV infection.

Measurement of the platelet retention rate in a column of collagen-coated beads is useful for the assessment of efficacy of antiplatelet therapy.

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.

Spontaneous platelet aggregation in normal subject assessed by a laser light scattering method: an attempt at standardization.

Platelet aggregometry by the laser light scattering (LS) method is sufficiently sensitive to detect small platelet aggregates that form spontaneously in vitro in the absence of agonists. Platelet aggregation without agonists is named spontaneous platelet aggregation (SPA). Since SPA has been suggested to be associated with various thrombotic diseases, it is essential to measure SPA and to establish a standard range of SPA values. In this study, we measured SPA in 167 healthy subjects by the LS method and attempted to clarify various factors influencing SPA, including the blood collection procedure. We also attempted to establish a tentative standard range of SPA values. SPA was quantitatively measured in terms of the maximum total LS intensity, which reflects small aggregates formed over 10 minutes (SMAX) and the area under the total LS intensity curve of small aggregates (SAUC). Since both the values of SMAX and SAUC were skewed and the log SMAX and log AUC values showed a normal distribution, the statistical analyses were performed using log SMAX and log SAUC. The log SMAX and log SAUC were significantly higher in the samples collected using a tourniquet and/or a 21 G needle, than in those collected without a tourniquet and/or with an 18 G needle. The log SAUC values were significantly lower in samples obtained with a syringe and/or 3.8% sodium citrate than in those obtained in vacuum sampling tubes and/or 3.13% or 3.14% sodium citrate. The Ht and plasma glucose concentration influenced the log SMAX values. We propose that to standardize SPA measurements, the measurements should be completed within two hours of blood sample collection and collected using the regular concentration of citrate. The standard range of SMAX values measured in samples obtained using a tourniquet and a 21 G needle was 2.0-23.99 (*10(3) mV*count). The standard range of SAUC values measured under same conditions was 0.58-9.12 (*10(6) mV*count*min). The standard range of SMAX values measured in samples obtained using a tourniquet, 21 G needle and a vacuum tube was 1.7-29.51 (*10(3) mV*count). The standard range of SAUC values measured under same conditions was 0.59-9.33 (*10(6) mV*count*min).

Effect of sarpogrelate, a 5-HT(2A) antagonist, on platelet aggregation in patients with ischemic stroke: clinical-pharmacological dose-response study.

BACKGROUND AND PURPOSE: It is widely accepted that antiplatelet therapy is effective for secondary prevention of atherosclerotic vascular diseases. We performed a double-blind, controlled clinical-pharmacological study to investigate the antiplatelet efficacy of sarpogrelate, a selective 5-hydroxytryptamine (5-HT(2A)) receptor antagonist, in patients with ischemic stroke, using a new assessment system employing combinations of 5-HT and epinephrine as agonists. METHODS: Forty-seven patients with ischemic stroke were randomly assigned to three groups: 15 patients received 25 mg sarpogrelate (group L), 16 patients received 50 mg (group M), and 15 patients received 100 mg (group H) orally, three times daily for 7 days. The effect was expressed as maximum intensity of platelet aggregation on the last day of medication. Two combinations of agonists, 0.5 micromol/l 5-HT plus 3 micromol/l epinephrine, and 1 micromol/l 5-HT plus 3 micromol/l epinephrine, were used to induce platelet aggregation. RESULTS: With both combinations of agonists, sarpogrelate treatment inhibited platelet aggregation dose-dependently (p < 0.025, Jonckheere test). In multiple-group comparison, the effect in group H was greater than that in group L or M (p < 0.025, Wilcoxon rank-sum test). CONCLUSION: Sarpogrelate treatment inhibited platelet aggregation dose-dependently in patients with ischemic stroke, as judged by a new assessment system employing combinations of 5-HT and epinephrine as agonists.

[Attempts for aspirin monitoring with a new assay system, Ultegra Rapid Platelet Function Assay (RPFA), based on turbidimetric platelet agglutination of whole blood samples].

Platelet aggregation measured by the optical density method has been applied for the assessment of platelet functions. However, as the method has to use platelet-rich plasma, it requires centrifugation of blood samples, which takes a considerable period of time. Using whole blood as samples has advantage because there is no pre-treatment of samples before measurement of platelet functions. Additionally, it would be desirable to have a bedside assay that reflects hyper-function of platelets and can monitor inhibitory effects of anti-platelet drugs. Rapid Platelet Function Assay (RPFA) is a qualitative test to aid the detection of platelet dysfunction due to anti-platelet drug ingestion, which uses citrated whole blood for sample in point of care or laboratory settings. The RPFA is a turbidimetric analysis, based on an optical detection system which measures platelet agglutination as an increase in light transmission. The aim of this study is to assess the accuracy and reproducibility of RPFA and to determine whether RPFA can monitor the effects of anti-platelet drugs. During the first 30 minutes after blood collection, Aspirin Reaction Units (ARU) determined with RPFA gradually increased, and reached its plateau after 30 minutes. The ARU values remained almost constant thereafter until 6 hours after blood collection (Fig. 3). These findings suggest that platelet function is unstable immediately after blood collection. Therefore, in this study ARU measurement was performed 60 minutes after blood collection. The reproducibility of ARU is very good both before and after aspirin intake. Seven days after daily uptake aspirin, ARU was decreased as compared with the control (440.8 +/- 39.4 vs. 663.4 +/- 2.4 ARU). RPFA measurement provides rapid information on platelet function that mirrors turbidimetric platelet agglutination and reflects COX1-depedent platelet activity.

Evaluation of platelet function under high shear condition in the small-sized collagen bead column.

We previously reported that platelet retention rates as measured with collagen-coated bead columns (the conventional column) reflect the processes of platelet adhesion and aggregation under low shear stress, and that this system could serve as an easy-to-use platelet aggregometry. With this column, platelet glycoprotein (GP) VI and GPIIb/IIIa, but not the GPIb-von Willebrand factor (VWF) interaction, play major roles in platelet activation. To develop a system that can better reflect the GPIb-VWF interaction under high shear stress, we designed a column containing small-sized beads (125-212 microm) coated with porcine collagen type I. As expected, the GPIb-VWF interaction played a crucial role in platelet retention rates at higher flow rates. Adenosine 5'-diphosphate, but not thromboxane A2, appears to support platelet activation in this system. The platelet retention rates among healthy individuals with the new columns are in the range wider than the conventional columns, and this diversity could be attributed to the broad range of the VWF antigen and/or its activity. It is suggested that this new column can serve as an easy-to-use method for evaluating the VWF antigen levels and its activity and for monitoring patients with thrombotic or bleeding disorders related to the VWF-GPIb interaction.

Sphingosine 1-phosphate-related metabolism in the blood vessel.

Sphingosine 1-phosphate (Sph-1-P) is a bioactive lipid released from activated platelets and plays an important role in vascular biology. In this study, we investigated Sph-1-P-related metabolism in the blood vessel, mainly using radio-labeled Sph and Sph-1-P. Sph was metabolically stable in the plasma, while it was converted into Sph-1-P in the presence of activated platelets. When the mixture of Sph-1-P and plasma was fractionated on a gel-filtration column, all Sph-1-P co-eluted with protein fractions that coincide with lipoproteins and albumin by agarose gel electrophoresis. When evaluated by polyacrylamide gel electrophoresis, 7.2 +/- 3.8%, 53.3 +/- 6.4%, and 39.5 +/- 7.9% of the radioactivity of Sph-1-P added to plasma was recovered in the low-density lipoprotein (LDL), high-density lipoprotein (HDL), and albumin fractions, respectively. On the other hand, 5.2 +/- 3.2%, 38.4 +/- 5.5%, and 56.3 +/- 5.7% of the radioactivity of Sph-1-P converted from Sph in collagen-stimulated platelets and released into the plasma was recovered in the LDL, HDL, and albumin fractions, respectively. When Sph-1-P release from activated platelets was examined, a stronger response was observed in the presence of albumin than lipoproteins, suggesting efficient Sph-1-P extraction from platelets by albumin. Finally, Sph-1-P, which is stable in the plasma, was markedly degraded by an ectophosphatase activity in the presence of vascular endothelial cells or in whole blood. Although Sph-1-P is stable in the plasma, it is likely that the level of this bioactive lipid is dynamically controlled by various factors including release from platelets, distribution among plasma proteins, and degradation by ectophosphatase.

Is the size of an abdominal aortic aneurysm associated with coagulopathy?

Abdominal aortic aneurysm (AAA) volume and intraluminal thrombi were analyzed with respect to the number and function of platelets, blood cells, and coagulation factors. A group of 43 patients who underwent repair of an AAA were enrolled in this study. The maximum diameter and volume of the AAA, and the volume of intraluminal thrombi and lumen were measured by computed tomography with planimetry. The platelet count and platelet function, prothrombin time, activated partial thromboplastin time, fibrinogen, plasminogen, antithrombin 3, fibrin degradation products (FDP), D-dimer, and blood cell counts were measured. Spontaneous platelet aggregation and the FDP, and D-dimer levels were elevated; all other factors remained within the normal range. Intraluminal thrombus volume was strongly correlated with the volume and diameter of the AAA. However, no correlation was observed between the size of the AAA and coagulating factors, including the number and aggregation value of platelets. AAAs are frequently associated with a coagulating disorder. However, its size and thrombus volume are not correlated with coagulation changes. Although an intraluminal thrombus increases along with fee enlargement of the AAA, the clinical manifestation of bleeding is rarely associated with an AAA. Therefore coagulopathy in patients with an AAA is not fully explained by its morphology.

Collagen-induced generation of platelet-derived microparticles in whole blood is dependent on ADP released from red blood cells and calcium ions.

We have evaluated the effects of different anti-coagulants or agonists on the generation of platelet-derived microparticles (PMPs) using flow cytometry. Twenty microg/ml of collagen induced significantly greater PMP formation in whole blood anti-coagulated with argatroban, a selective thrombin inhibitor, as compared with platelet-rich plasma, or whole blood anti-coagulated with citrate. Thus, whole blood kept at the physiological Ca2+ concentration provides an optimal condition for the formation of PMP. Convulxin, a GPVI-selective agonist, also induced PMP formation at the magnitude which far exceeds those of other agonists, such as thrombin receptor-activating peptide, ADP or epinephrine. These findings suggest that GPVI-mediated platelet activation plays a key role in the formation of PMP in the presence of physiological Ca2+ in whole blood. The addition of red blood cells to PRP potentiated PMP formation induced by collagen. Pretreatment of whole blood with the combination of creatine phosphate and creatine phosphokinase reduced PMP formation induced by collagen. Blockade of ADP receptors, P2Y12 with AR-C69931MX and P2Y1 with A3P5P, respectively, further suppressed collagen-induced PMP formation. We conclude that ADP released from red blood cells enhances PMP formation induced by collagen, and that both P2Y12 and P2Y1 contribute to ADP-potentiation of PMP generation induced by collagen.