Under normal physiologic conditions the receptor for F. VIII/VWF on human platelets is not readily available unless the antibiotic, ristocetin, is added. Searching for a physiologic mechanism inducing the receptor for F. VIII/VWF on human platelets, we found that low doses of thrombin (0.01-0.05 U/ml) induce steady state binding of 125I- F. VIII/VWF to human platelets. The binding is saturable and specific. Specificity was established by demonstrating that 100-fold molar excess of unlabeled F. VIII/VWF inhibited the binding while an excess of fibrinogen or fibro- nectin, two common contaminants of F. VIII/VWF, did not inhibit binding of 125I-F. VIII/VWF. Furthermore, rabbit antibody against F. VIII inhibited the binding. Thrombin- induced binding to human platelets can be observed only with fresh, metabolically active platelets. Glutaralde- hyde-treated platelets did not bind 125I-F. VIII/VWF following their treatment with thrombin, although they exhibited good binding in the presence of ristocetin. Thrombin appears to induce binding by acting on platelets rather than on F. VIII/VWF itself. Prostacyclin (PGI2 10-9M) inhibited thrombin-induced binding of 125I-F. VIII/VWF to human platelets, while ritcocetin-induced binding remained unaffected. Strikingly, PGI2-induced inhibition of binding was observed not orly when PGI2 was added before thrombin but also when PGI2 (10-9 M) was used 30 minutes after thrombin treatment and addition of 125I-F. VIII/VWF. Thus, binding of F. VIII/VWF to platelet receptor is induced by low concentrations of thrombin and is inhibited by PGI2. Since both agents are generated in the vicinity of injured vessel wall they may contribute to platelet-F. VIII/VWF- vessel wall interaction in vivo by regulating “up and down” platelet receptor for F. VIII/VWF.
Anti-platelet antibody has been generally recognized as an important etiologic factor in idiopathic thrombocytopenic purpura (ITP) and the detection of platelet associated IgG (PAIgG) is essential to establish the diagnosis and also useful to predict the clinical course. Several methods to detect PAIgG have been proposed, most of which, however, require complicated manipulation and are not practical for clinical use. This paper describes a simple and rapid method to detect PAIgG.This method was devised, utilizing the ability of staphylococcal protein A (SpA) to bind specifically to human IgG. An adhesive percentage of sample platelets to sheep red blood cells coated with SpA was calculated by enumeration of sample platelet before and after treatment. It takes only several hours to complete the assay from the drawing of blood. In a total of 37 cases including 15 cases of ITP, PAIgG was studied, using this method and Fab, anti-Fab assay of Luiken. Binding Index (an adhesive percentage) was 9.3 ± 3.8% in mormal subjects and 26.5 ± 10% in ITP patients, which differnce was statistically significant (p<0.01). Binding Index was within normal range in patients with non-immune thrombocytopenia such as liver cirrhosis or splenomegaly. A good correlation was observed between Binding Index and platelet count in ITP (r= -0.7299, p<0.01) and also between Binding Index and values of PAIgG by Fab, anti-Fab assay (r=0.8309, p<0.01).These results suggest that this new method is reliable to detect PAIgG in addition to its simplicity and rapidity and practical in clinical use.
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