Response: Is there a physiologically relevant experimental model system to study GP1b-mediated signaling in platelets?

Jackson et al argue that the results obtained from the suspension-based GPIb-mediated signaling model should be confirmed in adhesion-based assays.^1,2  When GPIb is engaged by VWF under physiological conditions, it is known that GPIb causes platelet stimulation. In the suspension-based experimental system, ristocetin or botrocetin together with VWF initiates agglutination through GPIb under stirring conditions, which leads to platelet aggregation through fibrinogen receptor activation. The suspension-based system provides a chief advantage of comparing signaling events in stimulated and unstimulated platelets. This model system is well established, and numerous investigators have used this system to elucidate signaling mechanisms initiated by GPIb, for example.^2-6  On the other hand, immobilized VWF under flow conditions was also used by several investigators for this purpose.^7-9  There are, however, some differences and some similarities in the findings when these 2 systems are used, and it is debatable whether one system mirrors the physiological events more accurately than the other.

For example, the conclusion that GPIb-mediated fibrinogen receptor activation depends on secreted ADP^1-5  is supported by the studies of Mazzucato et al⁸  using the immobilized VWF system. To the contrary, Kasirer-Friede et al⁷  found that GPIb activates fibrinogen receptor independently of secreted ADP in the adhesion assays. Such variance in the results is attributable to the sensitivities of the techniques used (for example, crude aggregate detection versus PAC-1 binding). Moreover, numerous studies in the past have suggested that agglutination-elicited signaling is different from adhesion- and shear-elicited GP1b signaling.^5,10-12  A more plausible explanation is that these 3 experimental systems represent 3 separate phases of VWF-GP1b engagement. According to this hypothesis, the signaling events are unlikely to be identical in each of these model systems, as the underlying physiological events in those phases may be distinct.

It should be recognized that any model system will have certain limitations. Flow systems on immobilized surfaces are no exception. It is possible that the VWF used is contaminated with other plasma proteins or agents. In addition, it is possible that in this immobilized conformation, VWF could activate other platelet receptors (either directly or through contaminants). A recent study suggests that VWF can induce lamellipodia formation through distinct platelet receptors.¹³  Hence, caution must be exercised for all the experimental models in use.

We can agree only that the conclusions are limited to the experimental conditions. Hence, we cannot absolutely favor one experimental system over the other until there is a consensus in the community that one system is more physiologically relevant than the other.