Mayumi Fukuyama

Shear-induced platelet aggregation in cerebral ischemia.

Background and Purpose Recent evidence has suggested that shear-induced platelet aggregation is an important mechanism of thrombosis at arterial bifurcations or stenoses. We measured shear-induced platelet aggregation with a new apparatus in patients with cerebral ischemia and also studied correlations with other hemostatic parameters as well as the effect of antiplatelet agents. Methods The subjects were 75 patients with cerebral ischemia and 26 control subjects. Platelet aggregation was induced in titrated platelet-rich plasma by a high shear stress (108 dynes/cm2) that was applied by means of a cone-plate streaming chamber based on turbidimetry. We studied the correlation of test results with hemostatic parameters and also the effects of antiplatelet agents. Results Compared with the control subjects, an increase of shear induced platelet aggregation was observed in 21 patients with atherothrombotic stroke and 12 with transient ischemic attacks, but not in 11 with cardioembolic stroke or 31 with lacunar stroke. There was no significant correlation of shearinduced platelet aggregation with platelet count, agonistinduced platelet aggregation, fibrinogen level, or β-thromboglobulin level. The extent of shear-induced aggregation was not correlated with von Willebrand factor antigen levels but was significantly correlated with the amounts of larger von Willebrand factor multimers. Oral aspirin (81 mg/d) did not inhibit shear-induced platelet aggregation, whereas oral ticlopidine (200 mg/d) significantly inhibited it. Conclusions These results indicate that shear-induced platelet aggregation is increased in patients with atherothrombotic stroke and transient ischemic attacks, is correlated with the increase of larger von Willebrand factor multimers, and is corrected by ticlopidine but not by low-dose aspirin.

Epinephrine augments von Willebrand factor-dependent shear-induced platelet aggregation.

BackgroundShear-induced platelet aggregation (SIPA) is an important mechanism in thrombogenesis. von Willebrand factor (vWF) binding to platelet glycoprotein lb (GP Ib) has been found to be crucial for platelet aggregation under the high shear force probably generated in stenosed coronary artery. The physiological significance of vWF-dependent SIPA has not been clarified. Methods and ResultsBlood samples were collected from 23 normal volunteers. SIPA was continuously monitored using a modified cone-plate viscometer adapted for measuring the transmitted light intensity of the material. The effects of low concentrations of epinephrine, ADP, and collagen on SIPA under both low shear (12 dyne/cm2) and high shear (108 dyne/cm2) force were investigated. All agonists tested enhanced SIPA under low shear force, whereas only epinephrine augmented SIPA under high shear force. The maximum extents of SIPA under high shear force in the absence and presence of epinephrine (10 ng/ml) were 37.9 ± 11.5% and 59.7 ± 13.9%, respectively. The antagonist of the a2-adrenergic receptor yohimbine (1,μg/ml) antagonized the effects of epinephrine. The monoclonal antibody NMC-4 against vWF, which was shown to inhibit its binding to GP Ib, completely abolished SIPA under high shear force, even in the presence of epinephrine. However, this antibody only partially inhibited SIPA under low shear force. ConclusionsOur findings suggest that epinephrine is the agonist that enhances SIPA mediated by vWF through its specific receptor. This may be clinically important because occlusion of the coronary artery often occurs in stenosed atherosclerotic vessels under sympathetic stimulation.

Low shear stress can initiate von Willebrand factor-dependent platelet aggregation in patients with type IIB and platelet-type von Willebrand disease.

Platelets exposed to shear stress aggregate in the absence of exogenously added agonists, utilizing distinct platelet membrane receptors and ligands depending upon the level of shear stress applied. Using a modified cone and plate type viscometer, we previously demonstrated that, under low shear stress (18 dyn/cm2), aggregation is mediated by platelet membrane glycoprotein (GP) IIb-IIIa and fibrinogen, whereas aggregation induced by high shear stress (108 dyn/cm2) requires the binding of von Willebrand factor (vWF) to both GPIb-IX and GPIIb-IIIa (Ikeda, Y., M. Handa, K. Kawano, T. Kamata, M. Murata, Y. Araki, H. Anbo, Y. Kawai, K. Watanabe, I. Itagaki, et al. 1991. J. Clin. Invest. 87:1234-1240). Here we report that vWF-dependent aggregation occurs under low shear stress in citrated platelet-rich plasma (PRP) from two types of congenital bleeding disorders, platelet-type von Willebrand disease (vWD) and type IIB vWD, in both of which ristocetin-induced aggregation is known to be heightened. Aggregation induced by low shear stress was enhanced in both types of disorders compared to normal controls, and the enhancement was completely abolished by anti-vWF monoclonal antibody NMC-4, which blocks the GPIb-binding site on vWF. Under high shear stress, the extent of maximal aggregation was not different between controls and the patient groups although maximal aggregation was reached much more quickly in the latter. When citrated PRP was exposed to a gradient of shear stress (6 to 108 dyn/cm2 over a 5-min period), vWF-dependent aggregation, as judged from the inhibitory effect of NMC-4, first occurred at 14 dyn/cm2 in platelet-type vWD and at 10-12 dyn/cm2 in type IIB vWD, as compared with more than 81 +/- 20.1 dyn/cm2 in control platelets. These results suggest that an abnormality in either vWF or GPIb-IX triggers the aggregation-inducing interaction of the two molecules under low shear stress, which might explain the intravascular platelet clumping, that presumably underlies the thrombocytopenia observed in these bleeding disorders.

Sensitive enzyme-linked immunosorbent assays for the detection of bacterial superantigens and antibodies against them in human plasma.

Enzyme‐linked immunosorbent assays for the quantitation of bacterial superantigens, staphylococcal enterotoxins A, B and C, toxic‐shock syndrome toxin‐1 and streptococcal pyrogenic exotoxin A, were developed. The assays had sensitivity to quantitate these toxins to 1.4, 5.9, 16.3, 2.5 and 4.3 pg/ml, respectively, in a buffer including 50% human plasma. It takes only 150 min to complete the assays after plate preparation. Specificity of the assays agreed with those of reverse latex agglutination assay. We also developed enzyme‐linked immunosorbent assays to detect antibodies against these five superantigens. The assays are expected to be significant tools for the study of superantigens in several diseases.

Preparation of a superantigen-adsorbing device and its superantigen removal efficacies in vitro and in vivo

OBJECTIVE A new superantigen-adsorbing device (SAAD) was developed, and its characteristics and efficacy in septic animals were evaluated. METHODS The SAAD was prepared by stepwise chemical modification of a polystyrene-based composite fiber reinforced with polypropylene. Adsorption affinities for several factors and the biological effect of superantigen (SAg) removal were measured in vitro. Also, superantigen-infused rabbits were treated with SAAD, and the efficacy was evaluated in vivo. RESULTS When the SAAD was evaluated for its ability to adsorb SAg in human plasma (1 ng/mL each), the adsorption rates were 74%, 76% and 85% for staphylococcal enterotoxins A, B and C, respectively, and 80% and 72% for toxic shock syndrome toxin-1 (TSST-1) and streptococcal pyrogenic exotoxin A, respectively. In addition, the SAAD showed some affinity towards other molecules, such as streptococcal pyrogenic exotoxin B, beta2-microglobulin, and vancomycin. Residual activities in whole blood samples containing TSST-1 (1 ng/mL) after incubation with the SAAD were 125 pg/mL for tumor necrosis factor alpha (TNF-alpha) production, and 359 pg/mL for interleukin-8 (IL-8) production (the initial activities: 194 pg/mL for TNF-alpha production, and 1029 pg/mL for IL-8 production). When TSST-1/lipopolysaccharide (LPS)-infused rabbits were subjected to extracorporeal blood purification with a SAAD column, 50% of the animals survived for a 14-day period after the infusion. In contrast, all control animals died within 3 days after the infusion. CONCLUSION These results indicate that the SAg-adsorbing device may be useful in treating SAg-related diseases.

Physiological Response to Superantigen-Adsorbing Hemoperfusion in Toxin-Concentration-Controlled Septic Swine

Background/Aims: Superantigens are suspected of being potent initiators of gram-positive sepsis, and new therapies for superantigen elimination are required. The effects of hemoadsorption with a superantigen-adsorbing device (SAAD) were evaluated in septic swine. Methods: Toxic shock syndrome toxin-1 (TSST-1) was infused, and blood concentration was maintained at the clinical level for 6 h. Endotoxin was then infused to induce lethal shock. All animals were hemoperfused with SAAD or a control column for 8 h and changes in pathological parameters and mortality were examined. Results: Animals perfused with SAAD had a highly significant (p < 0.01) survival advantage compared with control groups at 24 h after initiation of the TSST-1 infusion. SAAD also suppressed the increase in the arteriovenous shunt ratio and decrease of partial arterial oxygen pressure at 6 h after TSST-1 infusion initiation. Conclusion: We suggest that there is a potential application of SAAD in treating superantigen-induced respiratory dysfunction and sepsis.

Mixed Bacterial Infection Model of Sepsis in Rabbits and Its Application to Evaluate Superantigen-Adsorbing Device

Background: Superantigens are suspected to be the potent and lethal pathogens of gram-positive sepsis, and a new therapy that targeted to superantigens are required. Methods: A mixed infection model was developed in rabbits by the cecal ligation and puncture associated with the intraperitoneal injection of Staphylococcus aureus, which produces toxic shock syndrome toxin 1 (TSST-1). Animals were also hemoperfused with a superantigen-adsorbing device (SAAD), or a control column. Results: The model animals revealed multiple organ failure and died 6–12 h after the injection of S. aureus. The plasma levels of TSST-1, but not of lipopolysaccharide (LPS), significantly (p < 0.01) and inversely correlated with mean arterial pressure (r = -0.63). Plasma TSST-1 level was significantly reduced and shock-onset time was significantly retarded in the SAAD treated group, although the survival time was not significantly affected. Conclusions: The animal model developed could serve as a model for sepsis. It is suggested that there is the potential application of SAAD in treating superantigen-related sepsis.