Role of glycoprotein Ib-von Willebrand Factor complex and of other modulators in platelet formation

Von Willebrand Factor (vWF) is a well-known mediator of platelet adhesion to the vessel wall and of platelet-platelet interactions under high shear-stress conditions. It is synthesized both in endothelial cells and megakaryocytes (MK) where it is stored in secretory organelles as large multimers. vWF has also been identified as a sensitive and distinct marker for early MKpoiesis and, more recently, exposure of human MK to vWF at high shear rates was reported to accelerate platelet production. Type 2B von Willebrand disease (vWD2B) results from a gain-offunction of vWF that has an increased affinity for platelet glycoprotein (GP) Ib alpha. Thrombocytopenia is often present in vWD2B patients and the presence of giant platelets has been reported. We recently demonstrated that primary CD34+ cells from healthy donors (HD) differentiated for 7 days in the presence of TPO and vWF released a higher number of platelets in a dose dependent manner, when compared to TPO alone. The promoting effects of vWF on platelet production are selective and related to GPIb α, as demonstrated by experiments performed with specific inhibitors. Figure 1A shows that the use of the antibody against GPIbalpha (LJIb1) can block vWF-dependent accelerated platelet production while LJIb10 MoAb against the thrombin-binding site of GPIb alpha was not effective. The lack of inhibition with tirofiban further shows that vWF is not mediating its promoting effect through GPIIbIIIa; in fact, fibronectin and fibrinogen, other ligands of GPIIbIIIa are not able to increase platelet production. vWF boosts CD34-derived platelet production from vWD2B patients as well, even if the total amount of platelet production remains low if compared to HD (Figure 1B). This observation suggests that interaction of exogenous vWF to membrane GPIb alpha is able to promote MK differentiation and that an increased vWF/GPIb interaction can accelerate the final step of platelet release. Such a difference might be due to the activation of specific signalling pathways downstream GPIb alpha. To this regard, it has been demonstrated that GPIb alpha, during MK differentiation, binds the signal transduction protein 14-3-3xi, down-regulating the Akt phosphorylation levels downstream TPO/c-mpl signalling. It has been established that PKCepsilon interacts with 14-3-3xi13 and we have demonstrated that it is a key molecule in the signalling pathways downstream TPO, being finely regulated during MK differentiation. 14 Figure 2A shows that PKCepsilon is induced in the early phases of TPO-mediated MK differentiation, while it is down-regulated from day 6 onward. As reported in Figure 2B-C, the over-expression of PKCepsilon   in TPO-induced primary CD34 cells from day 8 onward – using as negative control an inactive mutated PKCepsilon (PKCepsilon- m)15 – impairs: i) the platelet production (Figura 2B); ii) the expression of CD61, CD41 and CD42b surface markers (Figure 2C); iii) the expression of CD62p cytoplasmic maturation marker (Figure 2C). This is in agreement with the notion that PKCepsilon is not expressed in human platelets, although Crosby et al. once reported its presence. On the contrary, it is now well established that mouse platelets express PKCepsilon, where it plays a role in GPVI-mediated aggregation and dense granule release. To this respect it is worth noting that in human monocytes the adhesion to endothelial cells involves PKCepsilon signaling. Figure 3 shows that human platelet adhesion to collagen under controlled physiological shear rates is selectively increased by the transfection of the human recombinant PKCepsilon (rhPKCepsilon) protein. It would be important to unravel the crosstalk between GPIb alpha/vWF complex and PKCepsilon. To this respect, one could speculate that a slightly anticipated down-regulation of PKCepsilon might promote MK differentiation and platelet release while, on the contrary, an ectopic expression of PKCε in mature platelets might promote their activatory status.