Previous studies have demonstrated that a high level of shear stress can produce platelet aggregation without the addition of any agonist. We investigated whether high shear stress could cause both platelet aggregation and shedding of microparticles from the platelet plasma membrane. A coneplate viscometer was used to apply shear stress and microparticle formation was measured by flow cytometry. It was found that microparticle formation increased as the duration of shear stress increased. Both microparticles and the remnant platelets showed the exposure of procoagulant activity on their surfaces. Investigation of the mechanisms involved in shear-dependent microparticle generation showed that binding of von Willebrand factor (vWF) to platelet glycoprotein lb, influx of extracellular calcium, and activation of platelet calpain were required to generate microparticles under high shear stress conditions. Activation of protein kinase C (PKC) promoted shear-dependent microparticle formation. Epinephrine did not influence microparticle formation, although it enhanced platelet aggregation by high shear stress. These findings suggest the possibility that local generation of microparticles in atherosclerotic arteries, the site that pathologically high shear stress could occur, may contribute to arterial thrombosis by providing and expanding a catalytic surface for the coagulation cascade.
Platelet-activating factor (PAF) acetylhydrolase has been recognized as an enzyme that inactivates PAF. We developed a convenient and reproducible method for determining human serum PAF acetyihydrolase activity. The assay was based on measurement of 1I4Clacetate produced from 1-O-alkyl-2-1'4C]-acetyl-sn-glycero-3-phosphocholine upon precipitation of the complex of radioactive substrate and albumin with TCA. The apparent Km value of PAF acetylhydrolase (near the physiological concentration of serum protein) was 1.5 X 10-4 M PAF. 32 subjects with serum PAF acetylhydrolase deficiency were found among 816 healthy Japanese adults. The low PAF acetylhydrolase activity in the deficient serum might not be due to the presence of enzyme inhibitor. Both the sensitivity to PAF and the metabolism of PAF in platelets from PAF acetylhydrolase-deficient subjects were almost the same as those of normal subjects. Deficiency in serum PAF acetylhydrolase appeared to be transmitted by autosomal recessive heredity among five Japanese families. Among healthy adults, healthy children, and asthmatic children, who were grouped into five classes on the basis of respiratory symptoms (remission, wheezy, mild, moderate, and severe groups), the probability of PAF acetylhydrolase deficiency was significantly higher in groups with severe symptoms (moderate and severe) (P < 0.01). These results suggest that deficiency of serum PAF acetylhydrolase might be one of the factors leading to severe respiratory symptoms in asthmatic children.
SummaryWe investigated the association of amyloid β-protein precursor (APP) and platelet derived microparticles in 20 normal controls and 91 patients with various diseases causing a thrombotic tendency. Compared with the controls, the mean percentage of APP-positive microparticles was significantly greater in the patients with cerebral infarction (39.1 ± 17.7%, p <0.001), diabetes (31.1 ± 12.6%, p <0.001), and uremia (30.1 ± 14.7%, p <0.01), but not in those with hypertension (8.2 ±6.3%, p = NS). Sixteen patients with cerebral infarction, 20 with diabetes, and 11 with uremia had microparticles with very high APP levels. In normal controls, 7.2 ± 3.7% of the microparticles were positive for P-selectin, while the percentage in cerebral infarction, diabetes, uremia, and hypertension was respectively 43.5 ± 15.1%, 40.0 ± 12.8%, 31.8 ±12.2%, and 11.6 ±7.3%. There was a significant correlation between P-selectin and APP positivity of microparticles. Our results suggest that microparticle APP may have a regulatory influence on coagulation abnormalities.
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