Engagement of Glycoprotein IIb/IIIa (α IIb β 3 ) on Platelets Upregulates CD40L and Triggers CD40L-Dependent Matrix Degradation by Endothelial Cells

BACKGROUND: CD40L-CD40 interactions induce inflammatory signals in cells of the vascular wall. We evaluated the effects of glycoprotein (GP) IIb/IIIa (alpha(IIb)beta3) engagement that occurs during platelet-endothelium interactions on CD40L surface exposure on platelets and initiation of proteolytic activity in human umbilical vein endothelial cells (HUVECs). METHODS AND RESULTS: Transient (60-minute) adhesion of thrombin-prestimulated platelets enhanced HUVEC expression of urokinase-type plasminogen activator receptor and membrane type-1 matrix metalloproteinase (MT1-MMP) (reverse transcriptase-polymerase chain reaction, flow cytometry) and secretion of urokinase-type plasminogen activator, tissue-type plasminogen activator, and MMP-1 (ELISA) and induced proteolytic activity via MMP-2 and MMP-9 (gelatin zymography). These effects were abrogated by hindrance of physical platelet-endothelial contacts using transwell systems or inhibited by GRGDSP, mAbs anti-GP IIb/IIIa (7E3), anti-alpha(v)beta3 (LM609), or anti-CD40L (TRAP1). In addition, MMP-2 and MMP-9 were inhibited by specific GP IIb/IIIa antagonists tirofiban, lamifiban, or integrelin. On endothelial cells, induction of proteolytic activity by activated platelets was mimicked by CD40 engagement using soluble CD40L but not affected by antibody clustering of alpha(v)beta3. On platelets, CD40L and CD62P exposure was enhanced on adhesion to HUVECs or immobilized fibrinogen and was abrogated by GRGDSP or LM609. In suspension, cross-linking of GP IIb/IIIa by fibrinogen plus secondary mAb upregulated CD40L surface exposure. Consistently, bivalent mAb 7E3 upregulated CD40L, whereas ligation of GP IIb/IIIa by soluble fibrinogen alone or monovalent Fab-fragment c7E3 had no effect. CONCLUSIONS: Platelet adhesion via GP IIb/IIIa upregulates CD40L and CD62P surface exposure. Proteolytic activity of HUVEC is induced by the concerted action of beta3-integrin-mediated platelet adhesion and subsequent CD40L-induced signals in HUVECs. Effective anti-GP IIb/IIIa or anti-CD40L strategies might, therefore, contribute to plaque stabilization.