SummaryWe have investigated the mechanisms leading to platelet aggregation following thrombin interaction with the glycoprotein (GP) Ib-IX-V complex. We show that platelets desensitized for the two thrombin receptors, PAR-1 and PAR-4, are still able to aggregate in response to thrombin and that this aggregation can be inhibited by a monoclonal antibody (VM16d) that blocks thrombin binding to GPIbα, or by pretreatment of platelets with Mocarhagin, a protease that specifically cleaves GPIbα. The thrombin/GPIbα-initiated signaling cascade induces platelet shape change through activation of the Rho kinase p160ROCK, independent of calcium mobilization, transient MEK-1 phosphorylation as well as the cleavage of talin through a calcium-independent mechanism. This signaling cascade does not induce the exposure of high affinity αIIbβ3 integrin receptors, nor does it lead to µ-calpain cleavage of filamin or the integrin cytoplasmic tail. In contrast, we provide evidence that binding of thrombin to GPIbα induces fibrin binding to resting αIIbβ3 leading to fibrin-dependent platelet aggregation and clot retraction, that can be selectively inhibited by αIIbβ3 antagonists such as RGDS, the dodecapeptide or lamifiban, as well as by the fibrin polymerization inhibitor GPRP-amide.

Using a defined conversion factor, 4-weekly C.E.R.A. was efficacious in maintaining hemoglobin levels in pediatric patients with stable anemia of CKD undergoing hemodialysis, switching from maintenance treatment with epoetin alfa/beta or darbepoetin. Safety was consistent with the known C.E.R.A. safety profile in adults.

Integrin alphaIIbbeta3 plays a pivotal role in platelet aggregation by binding to fibrinogen. The beta3 cytoplasmic domain of alphaIIbbeta3 interacts with cytoskeletal and signaling proteins and is cleaved by micro -calpain, a calcium regulated cysteine protease. In the present study, we have investigated in more detail the cleavage of the beta3 cytoplasmic domain during platelet aggregation induced by thrombin, TRAP-1 and TRAP-4. Our data show that beta3 is cleaved in all three cases. The time course of beta3 cleavage and the amount of cleaved beta3 depends on the way platelets are activated and on the complete activation of micro -calpain, with a maximum of 90% of cleaved beta3 obtained when thrombin is used. Furthermore, our results also show that the cleaved alphaIIbbeta3 is mainly distributed in the Triton soluble fraction, indicating its inability to bind to the cytoskeleton. Interestingly, in the absence of GPIbalpha or following inhibition of thrombin binding to GPIbalpha, there is a reduction in the thrombin-induced calcium flux, beta3 cleavage and micro -calpain activation. These results suggest that cleavage of the beta3 cytoplasmic domain by micro -calpain might be an important step regulating the link between the cytoskeleton and alphaIIbbeta3 during platelet aggregation, and that GPIbalpha could function as a cofactor for the complete activation of platelets by thrombin.

We have investigated the role of the thrombin/GPIbalpha interaction in the adhesion of platelets to fibrin in a whole blood ex vivo perfusion model at a shear rate of 280 s(-1). Blood was perfused through parallel-plate chambers containing coverslips coated with cells expressing tissue factor, leading to the generation of thrombin and thus, deposition of fibrin onto the exposed cells. Adhesion of platelets to fibrin and thrombus growth were analyzed. Interestingly, when GPIbalpha was removed from the platelet surface by action of mocarhagin, platelet adhesion on fibrin was inhibited. Furthermore, a monoclonal antibody, VM16d, directed against the thrombin binding site on GPIbalpha also inhibited platelet adhesion on fibrin, showing the importance of the thrombin/GPIbalpha interaction.We then looked at the involvement of alphaIIbbeta3 and showed that platelet adhesion and thrombus growth on fibrin were inhibited by the dodecapeptide, whereas lamifiban only inhibited the growth of the platelet thrombus. These results indicated that binding of thrombin to GPIbalpha induced an intracellular signaling leading to the interaction of the platelet integrin alphaIIbbeta3 with the fibrin-dodecapeptide sequence.