Yohko Kawai

The role of von Willebrand factor and fibrinogen in platelet aggregation under varying shear stress.

Exposure of platelets to shear stress leads to aggregation in the absence of exogenous agonists. We have now found that different adhesive proteins and platelet membrane glycoproteins are involved in aggregation depending on the shear stress conditions and the concentration of divalent cations in the medium. When blood is collected with trisodium citrate as anticoagulant, which causes a decrease in the levels of external ionized calcium ([Ca2+]o), platelet aggregation can be induced under low shear force (12 dyn/cm2) and is mediated by fibrinogen binding to the glycoprotein IIb-IIIa complex. Aggregates formed under these conditions are not stable, and when shear force is increased to 68 dyn/cm2, disaggregation results. By contrast, platelets from blood collected with hirudin as anticoagulant, wherein [Ca2+]o is within normal plasma levels, do not undergo low shear-induced aggregation; however, after exposure to a shear force above 80 dyn/cm2, aggregation is observed but only when von Willebrand factor is present and can interact with both its platelet binding sites, glycoprotein Ib-IX and glycoprotein IIb-IIIa. Fibrinogen is not involved in high shear-induced aggregation which, in fact, occurs normally in patients with severe afibrinogenemia. Thus, von Willebrand factor in the absence of exogenous agonists can mediate platelet aggregation in experimental conditions that may mimic the hemorheological situation of partially occluded arteries. This pathway of platelet aggregation involving only one adhesive ligand and two membrane adhesion receptors may play a relevant role in thrombogenesis.

Soluble P‐selectin is present in normal circulation and its plasma level is elevated in patients with thrombotic thrombocytopenic purpura and haemolytic uraemic syndrome

Summary. P‐selectin is an integral membrane glycoprotein stored in the secretory granules of platelets and endothelial cells. To determine whether soluble P‐selectin may be present in the circulation of healthy humans, we used a sandwich immunoassay to assess citrated plasma from 50 subjects. P‐selectin was present in concentrations ranging from 19 to 521 ng/ml (mean ± SD = 121 ± 84 ng/ml). The apparent molecular weight of P‐selectin immunoisolated from platelet‐poor plasma was similar to that of the detergent‐soluble form isolated from platelet membrane. Plasma levels of P‐selectin were unaffected by the following procedures: (1) drawing of blood in the presence of protease inhibitors; (2) stimulation of platelet‐rich plasma with aggregating agents; (3) ultracentrifugation at 100000 g for 120 min at 4°C or filtration through a 0·22 μm membrane; or (4) preincubation of platelet‐poor plasma with immobilized anti‐platelet glycoprotein Ib monoclonal antibodies. It appeared that plasma P‐selectin did not result from the in vitro activation of platelets, nor was it derived from platelet microparticles. We also found that plasma P‐selectin levels were significantly elevated in patients with thrombotic thrombocytopenic purpura (12 patients, 332 ± 184 ng/ml, P<0.001) and haemolytic uraemic syndrome (17 patients, 297 ± 191 ng/ml, P < 0.0001), as compared to the normal levels. Thus, these data should facilitate the study of the pathophysiological significance of circulating P‐selectin.

Platelet-dependent thrombin generation in patients with diabetes mellitus: effects of glycemic control on coagulability in diabetes.

OBJECTIVES This study sought to assess the usefulness of platelet-dependent thrombin generation as an index of coagulability in diabetes and to determine the effect of glycemic control on coagulability in diabetes. BACKGROUND It is important to investigate the interaction of platelets and the coagulation factors to clarify the processes of the coagulation system in detail. METHODS Platelet-rich plasma (150 X 10(9)/liter), 0.5 ml, was prepared, and 40 mmol/liter of calcium chloride was added to initiate clotting. S-2238 was added to each sample in a microtiter plate every 10 min, and the absorbance of the released color product at 2 min was measured spectrophotometrically at a wavelength of 405 nm using a microtiter plate reader as thrombin generation. We measured the platelet-independent thrombin generation in patients with non-insulin-dependent diabetes mellitus grouped according to glycemic control. RESULTS Platelet-dependent thrombin generation at 30 min after calcium chloride addition was significantly higher in 23 patients with poorly glycemic-controlled non-insulin-dependent diabetes mellitus without complications, such as diabetic retinopathy, nephropathy and neuropathy (hemoglobin [Hb] A1c >/= 9.0%) than in 46 healthy normal subjects (448 +/- 75 vs. 165 +/- 28 mU/min, p < 0.001). Thrombin generation in 31 well controlled diabetic patients without complications (Hb A1c < 9.0%) was intermediate (240 +/- 72 mU/min) between those of the poorly controlled group and healthy normal subjects. Platelet-poor plasma from diabetic patients increased platelet-dependent thrombin generation in normal subjects. CONCLUSIONS Coagulability is evidently enhanced in patients with non-insulin-dependent diabetes mellitus compared with that in healthy normal subjects on the basis of assessments of the platelet-dependent thrombin generation, and good glycemic control may help to correct a hypercoagulable state in diabetic patients.

Platelet-Dependent Thrombin Generation in Patients With Hyperlipidemia

OBJECTIVES We evaluated coagulability as determined by platelet-dependent thrombin generation in hypercholesterolemic patients before and after treatment with pravastatin and in hypertriglyceridemic patients to investigate the usefulness of coagulability as an index of atherosclerosis and to determine the importance of treating hyperlipidemia. BACKGROUND An understanding of the interaction between platelets and the plasma coagulation system is important for clarifying the mechanism of the procoagulant process. METHODS We assessed coagulability in 58 patients with hypercholesterolemia (serum total cholesterol level > or = 220 mg/dl, age 56.5 +/- 1.5 years [mean +/- SEM]), 37 patients with hypertriglyceridemia (serum triglyceride level > or = 200 mg/dl, age 59.5 +/- 1.7 years), 13 patients with hypercholesterolemia plus hypertriglyceridemia (age 51.4 +/- 3.1 years) and 75 normal subjects (age 52.2 +/- 1.7 years). We also studied platelet-dependent thrombin generation in patients with hypercholesterolemia before and after treatment with pravastatin. Calcium chloride was added to 0.5 ml of platelet-rich plasma (150 x 10(9)/liter) to initiate coagulation. Ten microliters of the sample was transferred into 90 microliters of 3.8% sodium citrate at 10-min intervals for 30 min. A chromogenic substrate, S-2238, was added to each sample, and absorbance was measured spectrophotometrically at a wavelength of 405 nm to determine thrombin generation. RESULTS Platelet-dependent thrombin generation was increased in patients with hypercholesterolemia and patients with hypercholesterolemia plus hypertriglyceridemia (p < 0.01) compared with patients with hypertriglyceridemia and control subjects. Treatment with pravastatin normalized thrombin generation. CONCLUSIONS Hypercholesterolemia, but not hypertriglyceridemia, was associated with increased platelet-dependent thrombin generation. Pravastatin normalized the generation of thrombin.

Safety and Efficacy of Edoxaban, an Oral Factor Xa Inhibitor, Versus Enoxaparin for Thromboprophylaxis After Total Knee Arthroplasty: The STARS E-3 Trial

INTRODUCTION This phase 3 trial compared the safety and efficacy of edoxaban, an oral direct factor Xa inhibitor, with enoxaparin sodium (enoxaparin) for thromboprophylaxis after total knee arthroplasty (TKA) in patients in Japan and Taiwan. MATERIALS AND METHODS In this randomized, double-blind, double-dummy study, patients received oral edoxaban 30 mg once daily beginning 6 to 24 hours postsurgery or enoxaparin 2000 IU (equivalent to 20 mg) subcutaneously twice daily beginning 24 to 36 hours postsurgery for 11 to 14 days. The primary efficacy endpoint was the composite of symptomatic pulmonary embolism and symptomatic and asymptomatic deep vein thrombosis. Safety endpoints included the incidence of major bleeding, clinically relevant non-major (CRNM) bleeding, major bleeding or CRNM bleeding, all bleeding events, adverse events, and adverse drug reactions. RESULTS Of 716 patients enrolled, 360 and 356 were randomized to receive edoxaban or enoxaparin, respectively. The primary efficacy outcome occurred in 22/299 (7.4%) and 41/295 (13.9%) patients in the edoxaban and enoxaparin groups, respectively (relative risk reduction=46.8%), indicating non-inferiority (P <0.001) and superiority (P=0.010) of edoxaban versus enoxaparin. In the edoxaban and enoxaparin groups, major bleeding occurred in 4/354 (1.1%) versus 1/349 (0.3%) patients (P=0.373); major or CRNM bleeding occurred in 22/354 (6.2%) versus 13/349 (3.7%) patients (P=0.129), respectively. CONCLUSIONS Edoxaban 30 mg once daily was more effective for thromboprophylaxis than subcutaneous enoxaparin 2000 IU twice daily following TKA and demonstrated a similar incidence of bleeding events.

Safety and efficacy of edoxaban in patients undergoing hip fracture surgery

INTRODUCTION Edoxaban is an oral, direct, once-daily factor Xa inhibitor. This study evaluated the safety and efficacy of edoxaban compared to subcutaneous enoxaparin in Japanese patients undergoing hip fracture surgery. MATERIALS AND METHODS In this multicenter, randomized, open-label, active-comparator, phase 3 trial, 92 patients were randomized 2:1 to receive edoxaban 30mg once daily (n=62) or enoxaparin sodium (enoxaparin) 2000IU (equivalent to 20mg) twice daily (n=30) for 11 to 14days. The primary endpoints were the incidence of major or clinically relevant non-major (CRNM) bleeding and incidence of any bleeding events (major, CRNM, or minor bleeding). Secondary efficacy endpoints included the incidence of thromboembolic events, venous thromboembolism-related deaths, and all-cause deaths. Additional adverse events were recorded throughout the study. RESULTS In the edoxaban and enoxaparin treatment groups, the incidence of major or CRNM bleeding was 3.4% and 6.9%, respectively, while any bleeding event occurred in 25.4% and 17.2% of patients, respectively. The incidence of thromboembolic events was 6.5% in the edoxaban group and 3.7% in the enoxaparin group. All events were asymptomatic deep vein thrombosis. The incidence of adverse events was 72.9% and 82.8% in the edoxaban and enoxaparin groups, respectively. CONCLUSIONS Compared to subcutaneous enoxaparin 2000IU twice daily, oral edoxaban 30mg once daily demonstrated similar safety and efficacy in the prevention of thromboembolic events in Japanese patients undergoing hip fracture surgery. CLINICAL TRIALS REGISTRATION NUMBER NCT01181141.

Automated measurement of reticulated platelets in estimating thrombopoiesis

Abstract: We described a fully automated measurement of reticulated platelets using a fluorescent dye, auramine O, and a reticulocyte counter, the R‐3000, equipped with special software. Reproducibility and linearity were shown to be good. In the normal subjects studied (n = 60), the mean value for reticulated platelets was 0.98% ± 0.41% and the mean absolute count was 2.12 ± 0.69 × 109/l. The absolute count for reticulated platelets was significantly lower (p < 0.05) in patients with reduced thrombopoiesis as seen in acute myeloblastic leukemia, aplastic anemia or chemotherapy‐induced thrombocytopenia and it was elevated (p < 0.05) in essential thrombocythemia and in chronic myelocytic leukemia with thrombocytosis. All 20 patients with chronic idiopathic thrombocytopenic purpura had a high percentage of reticulated platelets. The percentage of reticulated platelets was significantly increased (p < 0.05) in patients with impaired thrombopoiesis despite the reduction in the absolute count. In 2 leukemic patients, an apparent rise was noticed in the percentage of reticulated platelets which preceded by several days a progressive increase in the platelet count at the recovery phase of thrombocytopenia. The results suggest that an automated measurement of reticulated platelets can be applied to routine laboratories for clinical use.

A monoclonal antibody-based enzyme immunoassay for human GMP-140/P-selectin

Two hybridoma cell lines producing monoclonal antibodies WGA-1 and PL7-6, reactive only with thrombin-stimulated human platelet have been established. Both these antibodies were investigated for their specific reactivity against GMP-140, based on the amino acid composition analysis of immunopurified antigen and N terminal amino acid sequencing of its protease fragments. A two-site enzyme immunoassay for quantification of human GMP-140 was developed using WGA-1 monoclonal antibody immobilized on 96-well microplates and horseradish peroxidase-labeled PL7-6 monoclonal antibody as detector. The assay was able to measure GMP-140 in serum and plasma with a sensitivity of about 5 ng/ml and a precision better than 10%. This assay will be useful for the detection of GMP-140 derived from platelets or endothelium in biological fluids and tissue extracts.

Evidence that plasma fibrinogen and platelet membrane GpIIb-IIIa are involved in the adhesion of platelets to an artificial surface exposed to plasma

We investigated the molecular mechanism(s) by which platelets adhere to an artificial surface exposed to plasma, using polystyrene microtiter plates pretreated with plasma. Washed platelets labelled with 51Cr were incubated with the plates under static conditions. Prostaglandin E1(PGE1) was added to the platelets to prevent platelet-platelet interactions. Adhesion required the presence of a divalent cation such as Mg++ or Ca++. Polyclonal anti-fibrinogen antibody inhibited adhesion by 70%. Polyclonal antibodies against fibronectin, vitronectin, von Willebrands Factor, and the Fc portion of human IgG, had no effect on adhesion. Platelets adhered normally to a surface pretreated with plasma from a patient with severe von Willebrands disease. No platelet adhesion occurred when the surface was pretreated with an afibrinogenemic plasma. Monoclonal antibodies against platelet membrane GPIIb-IIIa, potent inhibitors of ADP-induced fibrinogen binding to platelets, completely inhibited adhesion. Monoclonal antibodies against the GPIb alpha subunit and GPIc(VLA alpha 5) showed no inhibitory effects on adhesion. Platelets from a patient with Glanzmanns thrombasthenia (type I) did not adhere to the surface pretreated with normal plasma. These results suggest that plasma fibrinogen adsorbed onto the surface and that platelet membrane glycoprotein(GP)IIb-IIIa were responsible for adhesion in an activation-independent manner.

Safety and Efficacy of Edoxaban, an Oral Factor Xa Inhibitor, for Thromboprophylaxis After Total Hip Arthroplasty in Japan and Taiwan

Edoxaban, an oral direct factor Xa inhibitor, has proven antithrombotic efficacy. In a multicenter, phase II study, 264 total hip arthroplasty (THA) patients randomly received edoxaban 15 or 30 mg once daily or enoxaparin 2000IU (20-mg) twice daily for 11-14 days. Thromboembolic event incidences were 3.8% (3/78), 2.8% (2/72), and 4.1% (3/74) for edoxaban 15-mg, 30-mg, and enoxaparin, respectively (P=1.00). Edoxaban-induced prolongation of prothrombin time, international normalized ratio, and activated partial thromboplastin time were proportional to plasma edoxaban concentration. Major or clinically relevant non-major bleeding incidences were 2.2% (2/89), 1.2% (1/85), and 2.3% (2/87) for edoxaban 15-mg, 30-mg, and enoxaparin, respectively (P=1.00). Once-daily edoxaban showed similar efficacy and safety to enoxaparin for prevention of thromboembolic events in patients undergoing THA.