Abstract
Soluble fibrinogen binding to the glycoprotein IIb-IIIa complex (integrin alpha IIb beta 3) requires platelet activation. The intracellular mediator(s) that convert glycoprotein IIb-IIIa into an active fibrinogen receptor have not been identified. Because the lipid composition of the platelet plasma membrane undergoes changes during activation, we investigated the effects of lipids on the fibrinogen binding properties of purified glycoprotein IIb-IIIa. Anion exchange chromatography of lipids extracted from platelets exposed to thrombin or other platelet agonists resolved an activity that increased fibrinogen binding to glycoprotein IIb-IIIa. A monoester phosphate was important for activity, and phosphatidic acid coeluted with the peak of activity. Purified phosphatidic acid dose-dependently promoted a specific interaction between glycoprotein IIb-IIIa and fibrinogen which possessed many but not all of the properties of fibrinogen binding to activated platelets. Phosphatidic acid appeared to increase the proportion of fibrinogen binding-competent glycoprotein IIb-IIIa complexes without altering their affinity for fibrinogen. The effects of phosphatidic acid were a result of specific structural properties of the lipid and were not mimicked by other phospholipids. Lysophosphatidic acid, however, was a potent inducer of fibrinogen binding to glycoprotein IIb-IIIa. These results demonstrate that specific lipids can affect fibrinogen binding to purified glycoprotein IIb-IIIa and suggest that the lipid environment has the potential to influence fibrinogen binding to its receptor.
Highlights
From the $Department of Pharmacology, Center for Thrombosis and Hemostasis and the §Departments of Medicine and Pediatrics, Uniuersity of North Carolina, Chapel Hill,North Carolina 27599
We have found that fractionated lipids from agonist-treated platelets as well as purified phosphatidic acid (PA) and lyso-PA can increase fibrinogen binding to mAbimmobilized GP IIb-IIIa
We used a microtiter plate assay in which GP IIb-IIIa was immobilized by the GP IIIa-specific monoclonal antibodies (mAb) AP3
Summary
Fibrinogen Binding Properties of Ab-immobilized G P ZIbIIIa-The goal of thesestudies was to determine whether lipids can modulate the fibrinogen binding activity of purified GP IIb-IIIa. Effects of Lipid Extracts from Resting and Activated Platelets on Fibrinogen Binding to Purified G P IIb-IIIa-Lipids were extracted from washed platelets treated with platelet activators (thrombin, the thromboxane Ap-mimetic U46619, or PMA) or a platelet activation-inhibitor(PG12)and tested for their effects on fibrinogen binding to GPIIb-IIIa either before or after fractionation of the extracts by anion exchange chromatography. DEAE-chromatography resolved an activity from lipid extracts of U46619- and PMA-treated platelets, which increased the lZ51-fibrinogenbinding by 3and 3.5-fold, respectively (data not shown). Several fractions (e.g. fractions 1 and 2, Fig. 1)appeared to lower fibrinogen binding This effect was probably nonspecific since these fractions lowered fibrinogen binding to wells lacking GP IIbIIIa aswell as towells containing GP IIb-IIIa. The activity in the peak fraction from thrombin-stimulated plateletscontained a critical monoester phosphate, as evidenced by the ability of alkaline phosphatase treatment (1 unit/ml) to reduce the fibrinogen binding activity by 58%. Similar resultswere obtained with lipids extracted from platelets treated with the thromboxane APmimetic U46619 or PMA
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