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Abstract
—LDL is known to increase the sensitivity of human platelets for agonists and to induce
aggregation and secretion independently at high concentrations, but its mechanism
of action is largely obscure. To clarify how LDL increases platelet sensitivity, cells
were incubated in lipoprotein-poor plasma and treated with collagen at a concentration
that induced ≈20% secretion of 14 C-serotonin. Preincubation with LDL (30 minutes
at 37°C) enhanced secretion in a dose-dependent manner to 60±14% at a concentration
of 2 g LDL protein/L. Similar stimulation by LDL was seen when secretion was induced
by the thrombin receptor–activating peptide. This enhancement was strongly reduced
(1) in the presence of monoclonal antibody PAC1 against activated α IIb β 3 ,
a polyclonal antibody against α IIb , and in the presence of the fibrinogen peptides
GRGDS and HHLGGAKQAGDV; (2) in α IIb β 3 -deficient platelets; and (3) after dissociation
of α IIb β 3 . In contrast, binding of 125 I-LDL to normal platelets in the
presence of PAC1, anti-α IIb , GRGDS, and HHLGGAKQAGDV, and to α IIb β 3 -deficient
platelets was normal. LDL increased the surface expression of fibrinogen in lipoprotein-poor
plasma and fibrinogen-free medium, suggesting that extracellular and granular fibrinogen
bind to α IIb β 3 after platelet-LDL interaction. Platelets deficient in fibrinogen
(<0.5% of normal) or von Willebrand Factor (<1% of normal) but containing normal amounts
of other ligands for α IIb β 3 preserved responsiveness to LDL, indicating that
occupancy of α IIb β 3 was not restricted to fibrinogen. Inhibition of protein
kinase C (bisindolylmaleimide) diminished fibrinogen binding and sensitization by
LDL; inhibition of tyrosine kinases (herbimycin A) left fibrinogen binding unchanged
but diminished sensitization by LDL. We conclude that an increased concentration of
LDL, such as observed in homozygous familial hypercholesterolemia, sensitizes platelets
to stimulation by collagen and thrombin receptor–activating peptide via ligand-induced
outside-in signaling through integrin-α IIb β 3 .
Staurosporine is the most potent inhibitor of protein kinase C (PKC) described in the literature with a half-maximal inhibitory concentration (IC50) of 10 nM. Nevertheless, this natural product is poorly selective when assayed against other protein kinases. In order to obtain specific PKC inhibitors, a series of bisindolylmaleimides has been synthesized. Structure-activity relationship studies allowed the determination of the substructure responsible for conferring high potency and lack of selectivity in the staurosporine molecule. Several aminoalkyl bisindolylmaleimides were found to be potent and selective PKC inhibitors (IC50 values from 5 to 70 nM). Among these compounds GF 109203X has been chosen for further studies aiming at the characterization of this chemical family. GF 109203X was a competitive inhibitor with respect to ATP (Ki = 14 +/- 3 NM) and displayed high selectivity for PKC as compared to five different protein kinases. We further determined the potency and specificity of GF 109203X in two cellular models: human platelets and Swiss 3T3 fibroblasts. GF 109203X efficiently prevented PKC-mediated phosphorylations of an Mr = 47,000 protein in platelets and of an Mr = 80,000 protein in Swiss 3T3 cells. In contrast, in the same models, the PKC inhibitor failed to prevent PKC-independent phosphorylations. GF 109203X inhibited collagen- and alpha-thrombin-induced platelet aggregation as well as collagen-triggered ATP secretion. However, ADP-dependent reversible aggregation was not modified. In Swiss 3T3 fibroblasts, GF 109203X reversed the inhibition of epidermal growth factor binding induced by phorbol 12,13-dibutyrate and prevented [3H] thymidine incorporation into DNA, only when this was elicited by growth promoting agents which activate PKC. Our results illustrate the potential of GF 109203X as a tool for studying the involvement of PKC in signal transduction pathways.
[1
]From the Departments of Clinical Chemistry (C.M.H., M.H., M.W.P., H.J.M.v.R.) and
Haematology (C.M.H., M.H., H.K.N., J.-W.N.A.), University Hospital Utrecht, and Institute
for Biomembranes, Utrecht University, Utrecht, The Netherlands.