SummaryLow Density Lipoprotein (LDL) is known to sensitize platelets for physiological agonists. To clarify the basis of this sensitization, we investigated the involvement of p38MAP Kinase (p38MAPK). As dual phosphorylation on Thr180 and Tyr182 of p38MAPK is the trigger for activation of the kinase, p38MAPK-activity was measured with an antibody that recognizes the dual-phosphorylated sequence. LDL induced a rapid and dose dependent activation of p38MAPK. The activation was not inhibited by a wide variety of inhibitors of platelet signalling, including TxA2-formation, Phospholipase C-activation, Ca2+-mobilization and ERK 1/2-activation. Only a slight reduction in p38MAPK-activation was observed when protein kinase C was inhibited. Activation of p38MAPK was strongly inhibited by a rise in cAMP. Thus, p38MAPK-activation was upstream of most signalling pathways and close to the LDL-receptor. A number of platelet receptors was screened with the use of antibodies. Integrins αIIbβ3 and α2β1, as well as the FcγRII-receptor, CD36 (platelet glycoprotein IV), CD68 (gp110) and Low Density Lipoprotein-receptor related protein (LRP) were not implicated in LDL-induced p38MAPK-activation. Inhibition of LDL binding by modification of apo B100 lysines reduced p38MAPK-activation by 80 %. Activation of p38MAPK resulted in an increase in release of arachidonic acid, the precursor for thromboxane A2 synthesis. In conclusion, activation of p38MAPK might be the first step in platelet sensitization by LDL, leading to formation of arachidonate metabolites and increased aggregation and secretion responses to physiological agonists.

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 .

Low density lipoprotein (LDL) is known to sensitize platelets to agonists via integrin mediated outside-in signaling (Hackeng, C. M., Huigsloot, M., Pladet, M. W., Nieuwenhuis, H. K., Rijn, H. J. M. v., and Akkerman, J. W. N. (1999) Arterioscler. Thromb. Vasc. Biol., in press). As outside in signaling is associated with phosphorylation of p125(FAK), the effect of LDL on p125(FAK) phosphorylation in platelets was investigated. LDL induced p125(FAK) phosphorylation in a dose- and time- dependent manner. The phosphorylation was independent of ligand binding to integrin alphaIIbbeta3 and aggregation, such in contrast to alpha-thrombin-induced p125(FAK) phosphorylation, that critically depended on platelet aggregation. Platelets from patients with Glanzmann's thrombastenia showed the same LDL- induced phos- phorylation of p125(FAK) as control platelets, whereas alpha-thrombin completely failed to phosphorylate the kinase in the patients platelets. LDL signaling to p125(FAK) was independent of integrin alpha2 beta1, the FcgammaRII receptor, and the lysophosphatidic acid receptor and not affected by inhibitors of cyclooxygenase, protein kinase C, ERK1/2 or p38(MAPK). Phosphorylation of p125(FAK) by LDL was strongly inhibited by cyclic AMP. These observations indicate that LDL is a unique platelet agonist, as it phosphorylates p125(FAK) in platelet suspensions, under unstirred conditions and independent of integrin alphaIIb beta3.

Lipoprotein(a) (Lp(a)) is a LDL-like particle with an additional glycoprotein, apo(a), linked to apolipoprotein B-100. Apo(a) is highly homologous to parts of the plasminogen molecule, and numerous investigations have shown interference of Lp(a) with functions of plasminogen. In this report we studied the influence of apo(a) phenotype on the binding of Lp(a) to plasmin-modified immobilized des-AA-fibrinogen (desafib-X).Results indicate that Lp(a) binds to desafib-X in a specific and saturable way. There was a strongly significant negative correlation between apo(a) isoform length and maximal number of Lp(a) particles bound to the desafib-X matrix (N=18, r=0.84, p=0.002). There was no relation between apo(a) isoform length and K-d for the binding of Lp(a) to desafib-X. In two donors (10%) no specific binding to desafib-X was observed, as well as to lysine-sepharose.In conclusion, apo(a) isoform length influences the amount of Lp(a) binding to desafib-X. This implies that small isoforms apart from their usual higher plasma Lp(a) concentrations also are potentially more thrombogenic. The fact that Lp(a) from two donors did not bind to desafib-X, suggests that mutations may exist in the K4-10 domain of apo(a) abolishing its lysine-binding ability.