| There are many platelet molecules that participate in thrombosis. The platelet specific integrin alphaIIbbeta3 is essential for both platelet adhesion and aggregation. When platelets come in contact with an exposed subendothelium they initially tether to and roll along the surface with approximately 5% forming an immediate stable attachment to the developing thrombus and the remainder translocating and/or detaching. The stable adhesion of platelets to the exposed matrix is mediated by alpha IIbbeta3 and may reflect the structural state of the integrin. Previous studies show that protein phosphorylation/dephosphorylation reactions modulate U-IJ3 function. We hypothesize that transient threonyl phosphorylation of (33 maintains alphaIIbbeta3 in a low affinity state allowing only a small percentage of platelets that come in contact with the growing thrombus to adhere. Using an antibody specific for phosphorylated threonine-753 on the beta3 cytoplasmic tail, we have shown that a subset of alphaIIbbeta3, in platelets treated with 0.2 units/ml thrombin, undergo a transient threonine-753 phosphorylation that peaks at approximately 15 to 30 sec and then rapidly dephosphorylates by 60 sec. The amount of beta3 that becomes phosphorylated was calculated to be less than 10% and was found to be expressed on the surface of the platelet. To further examine the role of this phosphorylated residue in integrin function, we transfected Chinese Hamster Ovary (CHO) cells with wild type beta3, beta3 with a threonine-753 to alanine mutation, and beta3 with a threonine-753 to aspartic acid mutation, to mimic a phosphorylated residue. Using these cells, we found that the cells adhere to and spread on a fibrinogen matrix, and beta3 localizes to focal adhesions regardless of the amino acid present at residue-753. On the contrary, cells with a threonine-753 to aspartic acid mutation migrate on fibrinogen at a faster rate than either the wild type or alanine mutation cells. It is believed this may be due to a decreased adherence strength of the integrity with either the cytoskeleton or the extracellular matrix, allowing the cell to release attachments at a faster rate and thereby migrate more quickly. Together, these data suggest that transient phosphorylation of beta 3 on threonine-753 may be a physiological regulatory event in the platelet activation cascade which modulates thrombus formation. |
