Platelet attachment and spreading were monitored on glass and various protein coated glass, under shear with washed platelets, platelet rich plasma (PRP) and whole blood, using fluorescence Optimas imaging system and software. Results showed that the platelet adhesion and spreading were sensitive to the nature of precoated proteins and the type of medium used for introducing platelet suspension for the study. In general, the cell adhesion and spreading were higher with fibrinogen (Fg), fibronectin (Fn), von Willebrand Factor (vWF), and collagen precoated surfaces. In the presence of albumin on the surface, however, platelets could not attach and spread fully when using washed cells. But, the surface attachment and spreading of the cells were higher on albumin substrates on exposure to PRP or whole blood. This may be due to the replacement of precoated albumin by other plasma proteins, like Fg to facilitate the platelet-surface attachment. The composition of this layer determines the extent of platelet activation and the adhesive strength between platelets and polymer surface. These results indicate that multiple adhesion receptors can mediate platelet adhesion and spread to matrix proteins immobilized on surfaces. Further, these studies combined with some of our earlier observations and suggestions propose the need for developing in vitro tests that resemble in vivo conditions.