Kuo-Jang Kao

Demonstration and characterization of specific binding sites for factor VIII/von Willebrand factor on human platelets.

The presence of specific Factor VIII/von Willebrand factor (FVIII/vWF) binding sites on human platelets has been demonstrated by using 125I-FVIII/vWF and washed human platelets. Binding is ristocetin-dependent and increases in proportion to the concentration of ristocetin from 0.2 to 1 mg/ml. Binding of 125I-FVIII/vWF to platelets can be competitively inhibited by unlabeled human or bovine FVIII/vWF, but not by human thrombin, fibrinogen, alpha 2-macroglobulin, equine collagen, or a lectin of Ricinus communis. Scatchard analysis of binding data indicated that the dissociation constant of FVIII/vWF receptors is 0.45--0.5 nM. There are 31,000 binding sites per platelet at 1 mg/ml of ristocetin concentration. The optimal pH range for binding is from 7.0 to 7.5. At a concentration of 2 mM, EGTA inhibits 86% of the binding; however, 20 mM of Ca++, Mg++, or EDTA have little effect. Binding sites for FVIII/vWF were found only on platelets, and no significant binding was detected with human erythrocytes or polymorphonuclear leukocytes.

In vitro effects of acetaminophen and its analogues on human platelet aggregation and thromboxane B2 synthesis.

We examined the effect of acetaminophen and the structural analogues 2,6-dimethylacetaminophen, 3,5-dimethylacetaminophen, and N-acetyl-p-benzoquinone imine on human platelet aggregation, 14C-serotonin secretion, and thromboxane B2 synthesis. Preincubation with 1 mM acetaminophen for 2 min completely inhibited arachidonic acid- and collagen-stimulated platelet aggregation. Thromboxane B2 production and 14C-serotonin secretion by arachidonic acid-stimulated platelets also were completely inhibited. Preincubation of platelets with 1 mM 3,5-dimethylacetaminophen inhibited collagen and arachidonic acid-induced aggregation and arachidonic acid-stimulated thromboxane B2 synthesis, while treatment with 2,6-dimethylacetaminophen did not inhibit aggregation and blocked thromboxane B2 formation to a much lesser degree. Preincubation with 1 mM N-acetyl-p-benzoquinone imine inhibited arachidonic acid-induced aggregation and 14C-serotonin secretion but had no effect on arachidonic acid-induced thromboxane B2 formation and collagen-induced platelet aggregation.

Membrane protein mediation of thromboxane A2 induced platelet aggregation

Summary Washed human platelets pretreated with chymotrypsin to remove membrane proteins retain full ability to convert 14 C-arachidonic acid to thromboxanes. However, the platelets no longer aggregate when treated with arachidonic acid or calcium ionophore A23187. While thromboxane A 2 may function as a calcium ionophore to mobilize intraplatelet Ca 2+ and to activate platelets for aggregation, the present studies indicate that thromboxane A 2 induced platelet aggregation is a process mediated by membrane proteins.

Lack of thromboxane A2 synthesis in platelet aggregation induced by factor VIII/von willebrand factor☆

Abstract Thromboxane A 2 (TXA 2 ) synthesis during factor VIII/von Willebrand factor (FVIII/vWF) induced-platelet aggregation was studied in human platelets prelabeled with (1- 14 C)-arachidonic acid ( 14 C-AA). Although previous studies demonstrate that thrombin, collagen or epinephrine induced platelet aggregation is associated with TXA 2 synthesis, no TXA 2 synthesis was detected in FVIII/vWF induced aggregation. Nevertheless, an increased release of 14 C-AA and its probable conversion into HETE was detected during platelet aggregation induced by FVIII/vWF in the presence of ristocetin. When thrombin was added to 14 C-AA labeled platelet suspensions, similar amounts of TXA 2 were synthesized with or without stirring, despite the fact that no platelet aggregation occurs without stirring. These results indicate that TXA 2 is not synthesized during FVIII/vWF induced platelet aggregation, and that aggregation of platelets is not a pre-requisite for TXA 2 synthesis.