| dc.description.abstract | Human platelets express tissue factor mRNA, synthesize the protein and store functionally silent
tissue factor (TF) which has procoagulant activity (PCA) after platelet activation. Platelet GPIbα plays
a major role in platelets adhesion and aggregation by signaling through the Scr family kinases. We
reported few years ago, that resting, non‐permeabilized human platelets express scarce surface TF,
which was strikingly enhanced and co‐localized with GPIbα after stimulation. We also observed that
platelet stimulation with Von Willebrand Factor (VWF) induces a faster procoagulant activity,
compared to other classical platelet agonists. The decryption surface TF involves both a
dithiol/disulfide switch and exposure of phosphatidylserine. GPIbα signaling elicits the exposure of
this phospholipid, although with a mild increase on intracellular calcium. Nevertheless, if the GPIbα
pathway is enough to induce the TF‐dependent PCA in platelets is unknown.
Aim: To investigate whether the increased TF‐PCA is mediated by the GPIbα downstream signaling.
Method: Washed platelets (WP), platelet rich plasma (PRP) or membrane platelets (MP) were
obtained from healthy adult donors. Platelet stimulation was done in aggregometer. Association
between TF and GPIbα was assessed by coprecipitation. Inhibitions studies using different blockers
were done to figure out the GPIbα signaling through PI3K, SKF, AKT, PKC, Syk and p38 MAPK. TF‐PCA
was evaluated by FXa generation measured by chromogenic assay.
Results: The inhibition of GPIbα downstream signaling pathways with PI3K (wortmannin), SKF
(SU6656 and PP2), AKT (AKT1/2) reduced significantly the TF‐PCA induced by VWF‐Ristocetin (Ris).
In contrast, inhibition of PKC (Gö6850) Syk (BAY61‐3606) and p38 MAPK (SB203580) had no
significant effect on FXa generation.
Total AKT co‐precipitates with TF, mostly in VWF‐R‐activated platelets and this association was
increasingly inhibited by AKT, PI3K and Lyn inhibitors, in this order. Agglutination of WP by VWF‐R
was not affected by SFK and PKC inhibitors. In order to analyze platelet PCA in a global test, PRP was
reconstituted with WP plus autologous platelet poor plasma and stimulation with Ris, followed by recalcification
and clotting time measurement. The accelerated PRP clotting time observed with Ris was
reversed when WP were pre‐incubated with α‐GPIbα, αTF mAbs or TFPI, but not by anti‐GPVI
antibody. In contrast, platelet AKT inhibition prior to reconstitution of PRP and Ris stimulation
prolonged significantly the clotting time as compared with no prior AKT inhibition.
Conclusion: Platelet TF‐PCA is specifically induced by GPIbα activation, which is dependent on
SFK/Lyn, PI3K and AKT activation. Platelet TF accelerates the clotting of PRP following GPIbα
activation, as well. | |
