Background-CD40L-CD40 interactions induce inflammatory signals in cells of the vascular wall. We evaluated the effects of glycoprotein (GP) IIb/IIIa (␣ IIb ␤ 3 ) 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-␣ v ␤ 3 (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 ␣ v ␤ 3 . 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 ␤ 3 -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.

Objective: Inflammatory conditions contribute to increased expression of various activity markers in platelets and endothelial cells, leading to atherosclerotic changes in the vascular wall. The objective of this study was to investigate possible protective effects of 1α,25-dihydroxyvitamin D₃ in an endothelial cell model. Methods: After a 24-hour incubation with 1α,25-dihydroxyvitamin D₃, human umbilical vein endothelial cells were stimulated with lipopolysaccharide (LPS) and incubated in direct contact with platelets. The expression of CD40L and CD62P in platelets, the expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1 (VCAM-1), the urokinase receptor uPAR and membrane type 1 matrix metalloproteinase (MT1-MMP) in endothelial cells and endothelial cell reactive oxygen species generation were measured by flow cytometry. Endothelial nitric oxide synthase was analyzed by Western blot. Results: The increased expression of VCAM-1 and MT1-MMP in endothelial cells by proinflammatory stimulation with LPS and by direct contact with activated platelets was significantly reduced through preincubation with 1α,25-dihydroxyvitamin D₃. Platelets in direct contact with preincubated endothelial cells showed significantly reduced CD62P expression when compared to platelets incubated with untreated endothelial cells. Conclusions: 1α,25-Dihydroxyvitamin D₃ attenuates platelet activation and the expression of VCAM-1 and MT1-MMP in human endothelial cells and could have early therapeutic relevance in atherosclerotic diseases.

Fibrinogen concentration influences mechanical and functional properties of the clot. The purpose of the present study was to identify threshold concentrations of fibrinogen resulting in relevant changes in whole blood clot elastic modulus and platelet contractile force, as well as plasma prothrombin time and activated partial thromboplastin time. We measured clot elastic modulus, platelet contractile force, and other hemostasis parameters in whole blood samples from 552 patients admitted to a surgical intensive care unit. Platelet contractile force and clot elastic modulus were measured using the Hemodyne apparatus. Fibrinogen levels were between less than 0.10 and 9.44 g/l, with a mean of 2.41 g/l. Mean platelet count was 203 x 10(9) l(-1), with a range of 16 x 10(9) l(-1) to 682 x 10(9) l(-1). High levels of fibrinogen result in improved mechanical stability and improved interaction of platelets with the fibrin network. Clot elastic modulus and platelet contractile force are correlated positively with plasma fibrinogen concentration. However, there was no threshold concentration or ceiling effect concerning the mechanical properties of the clots. In contrast, clotting time assays such as prothrombin time, thrombin time, or activated partial thromboplastin time are influenced by the fibrinogen concentration only at levels below 1 g/l. In linear regression analysis, clot elastic modulus was mainly influenced by fibrinogen concentration (F = 185.4, P < 0.0001), whereas platelet contractile force was influenced by fibrinogen (F = 197.0, P < 0.0001) and platelet count (F = 104.7, P < 0.0001). The present data show that 1 g/l is a threshold fibrinogen concentration for an effect on coagulation assays such as prothrombin time, thrombin time, or activated partial thromboplastin time, but increasing fibrinogen concentrations above this level results in further continuous improvement of mechanical properties of the whole blood clot.

In vivo administration of endotoxin leads to an activation of platelets and monocytes with an upregulation of proatherogenic CD40L on platelets. These findings underpin the role of inflammation in early atherogenesis through platelet and monocyte activation in an in vivo model.