Pro-atherogenic effect of interleukin-4 in endothelial cells: modulation of oxidative stress, nitric oxide and monocyte chemoattractant protein-1 expression.

BACKGROUND: Although considered as an anti-inflammatory cytokine, interleukin-4 (IL-4) has been shown to be pro-atherogenic in mice models of atherosclerosis. OBJECTIVES: In order to elucidate this paradox, we have investigated the effects of IL-4 on characteristic atherogenic parameters in human umbilical vein endothelial cells (HUVECs): production of reactive oxygen species, expression of monocyte chemoattractant protein-1 (MCP-1) and nitric oxide (NO) bioavailability. RESULTS: Incubation of HUVECs with IL-4 resulted in an increased production of reactive oxygen species and extracellular O(2)(-)(*) measured using fluorogenic probes and Cytochrome c that was inhibited by superoxide dismutase or gp91ds-tat, a selective NADPH oxidase inhibitor. The latter also inhibited IL-4 induced over-expression of MCP-1 mRNA measured by classical and real time RT-PCR. Incubation of HUVECs with IL-4 reduced thrombin-induced NO release, detected by electrochemistry, an effect which was reversed by incubation with superoxide dismutase. Both production of reactive oxygen species and MCP-1 mRNA over-expression induced by IL-4 were fully inhibited by selective inhibitors of phosphatidyl inositol 3-kinase. CONCLUSION: The data demonstrate that IL-4 up-regulates the expression of MCP-1 and decreases NO bioavailability through activation of NADPH oxidase in endothelial cells. These results are in favor of a pro-inflammatory and pro-atherogenic effect of IL-4 in vascular tissues.

Thrombomodulin prolongs thrombin-induced extracellular signal-regulated kinase phosphorylation and nuclear retention in endothelial cells.

On endothelial cells, thrombin binds to thrombomodulin (TM), an integral membrane-bound glycoprotein, and to protease-activated receptors (PARs). Thrombin binding to TM modulates endothelial cell and smooth muscle cell proliferation mediated through PAR1. We studied the phosphorylation and nuclear translocation of extracellular signal-regulated kinases (ERKs) 1 and 2 in human umbilical vein endothelial cells activated by thrombin. Thrombin and thrombin receptor-activating peptide (TRAP)-induced DNA synthesis were significantly inhibited by PD98059, an inhibitor of ERK phosphorylation. Immunoblots of phosphorylated ERKs (pERKs) and immunocytochemical studies of pERK localization revealed differences in the signal generated by thrombin and TRAP. After a short activation (15 minutes), the phosphorylation and the intracellular localization of pERKs were the same with the 2 agonists. After 4 hours, however, pERKs were visualized in the nuclei of thrombin-activated cells but barely detectable in TRAP-activated cells. Moreover, after 4 hours, the pERKs were visualized in the nuclei of cells stimulated by TRAP in the presence of a thrombin mutant that bound to TM, whereas they were around the nuclei in cells stimulated by thrombin in the presence of a monoclonal antibody preventing thrombin binding to TM. The results demonstrate that ERKs are involved in human umbilical vein endothelial cell DNA synthesis mediated by PAR agonists, that the duration of pERK nuclear retention is in inverse ratio to the mitogenic response, and that in addition to its role in the regulation of blood coagulation, TM acts as a thrombin receptor that modulates the duration of pERK nuclear retention and cell proliferation in response to thrombin.

Change in the physical state of platelet plasma membranes upon ionophore A23187 activation. A fluorescence polarization study.

Human platelets were isolated and fluorescence-labelled by 1,6-diphenylhexatriene. Diphenylhexatriene was essentially localized in the plasma membrane, as indicated by trinitrobenzenesulfonate-quenching experiments. A decrease of the fluorescence polarization of diphenylhexatriene was observed upon ionophore A23187 addition in the absence of aggregation. 0.3 microM ionophore allowed to reach the maximum rate of the decrease of fluorescence polarization; it also maximally stimulated the light transmission change, the serotonin release and the thromboxane B2 synthesis. The amplitude of the fluorescence polarization decrease was maximum at platelet concentrations between 4 X 10(7) and 7 X 10(7)/ml. The presence of Ca2+ in the medium increased the rate constant of the polarization change. Chlorpromazine (60 microM) completely inhibited this transition, but at 30 microM its inhibitory effect was reversed by Ca2+. The membrane events implied in platelet activation very likely lead to fluidization of the plasma membrane, perhaps by its fusion with the membranes of internal granules which are relatively depleted of cholesterol. Ca2+ plays a central role in the triggering of the observed effects at the membrane level.

Mechanisms involved in platelet activation induced by a monoclonal antibody anti glycoprotein IIb-IIIa: inositol phosphate production is not the primary event.

The mechanisms involved in platelet aggregation by a monoclonal antibody (mAb) P256 specific for the GPIIb-IIIa complex was investigated following metabolic 32P labelling of platelets. When compared with thrombin, inositol phosphates (InsP) production during P256-induced activation was delayed and no apparent peak, but a small and sustained production of [32P]-Ins(1,4,5)P3 and [32P]-Ins(1,3,4,5)P4, was observed between 20 and 90 s. [32P]-Ins(1,3,4)P3 was also produced with a maximum after 90 s. Addition of the ADP scavenger creatinine phosphate/creatine phosphokinase (CP/CPK) and of the cyclooxygenase inhibitor aspirin together with P256 almost totally abolished InsP formation, whereas platelet aggregation and protein phosphorylation were partially inhibited. F(ab')2 fragments of P256 also aggregated platelets but to a smaller extent than IgG, and without any measurable InsPs. To characterize further P256-induced activation, the phosphorylation of p43, the main substrate of protein kinase C (PKC) and the phosphorylation of tyrosine protein (P-Tyr) was also studied. PKC activation was smaller with P256-IgG than with thrombin but both thrombin and P265-IgG induced a similar profile of P-Tyr involving seven major bands, whereas P256-F(ab')2 only occasionally activated PKC but always significantly phosphorylated a 64,000 molecular weight P-Tyr. The data indicate that the binding of P256 to GPIIb-IIIa, in contrast with thrombin, does not initially lead directly to the activation of the phosphoinositide phospholipase C to produce InsP's but rather involves the activation of protein kinases and also both fragments F(ab')2 and Fc play a specific role in the platelet responses to the mAb. Only the crosstalk between the two pathways evoked by F(ab')2 and Fc respectively allows the activation of all platelet activation systems.

Tyrosine phosphorylation and association of FcgammaRII and p72(Syk) are not limited to the FcgammaRII signalling pathway.

The tyrosine kinase p72(Syk) plays a critical role in platelet signal transduction. It associates with the platelet receptor for the Fc domain of IgGs, FcgammaRII, following stimulation by FcgammaRII cross-linking. Here, we show that p72(Syk) and FcgammaRII tyrosine phosphorylation and association occured following platelet stimulation by: (1) two monoclonal antibodies, which form a bridge between a target antigen and FcgammaRII, and (2) the G-protein-coupled receptor agonist thrombin. The kinetics of the p72(Syk)/FcgammaRII association depended on the signalling pathway (i.e., the antigen targeted or the thrombin receptor). We established a direct relationship between the level of FcgammaRII phosphorylation and the detection of its association with p72(Syk). Inhibition of p72(Syk) by piceatannol resulted in partial or total inhibition of FcgammaRII phosphorylation, after immunological activation or addition of thrombin, respectively, suggesting that p72(Syk) participates in FcgammaRII phosphorylation. The results provide evidence that p72(Syk)/FcgammaRII association is not restricted to immunological activation.

Calcium mobilisation controls tyrosine protein phosphorylation independently of the activation of protein kinase C in human platelets.

We have investigated the regulation of tyrosine proteins phosphorylation by intracellular Ca2+ level ([Ca2+]i) and protein kinase C (PKC) during platelet stimulation. We found that chelation of extracellular calcium completely prevented phosphorylation of tyrosine proteins induced by thapsigargin and phorbol 12-myristate 13-acetate (PMA), whereas, when induced by thrombin, it prevented a subset of tyrosine proteins. The selective inhibition of PKC by GF 109203X did not abolish tyrosine protein phosphorylation when induced by thrombin and thapsigargin. The results suggest that in human platelets tyrosine protein phosphorylation is dependent on [Ca2+]i, although direct PKC activation can also induce phosphorylation of tyrosine proteins.

Association of the protein tyrosine phosphatase PTP1C with the protein tyrosine kinase c-Src in human platelets.

Protein tyrosine phosphatase 1C (PTP1C), highly expressed in hematopoietic cells, is a soluble protein tyrosine phosphatase containing two Src homology 2 (SH2) domains at the N-terminus and two putative sites of tyrosine phosphorylation at the C-terminus. This paper reports that PTP1C and c-Src could be coimmunoprecipitated during thrombin-induced platelet activation. Moreover, association between the two signalling proteins occurred only after PTP1C had been tyrosine phosphorylated. In in vitro experiments, PTP1C bound to the SH2 domain of c-Src, suggesting that association between tyrosine phosphorylated PTP1C and c-Src was mediated by the SH2 domain of c-Src. Finally, in resting platelets, PTP1C was mainly found in the Nonidet P-40 soluble fraction whereas following thrombin-induced activation, around 17% of PTP1C was associated with the insoluble fraction.

Calpain controls the balance between protein tyrosine kinase and tyrosine phosphatase activities during platelet activation.

Protein phosphorylation was studied during platelet stimulation in two ranges of ionized [Ca2+]. At ionized [Ca2+]i< or = 1 microM, proteins were phosphorylated. At ionized [Ca2+]i > or = 4 microM, phosphoproteins disappeared. Protein dephosphorylation was prevented by the combined action of calpeptin and phosphatase inhibitors. Protein tyrosine phosphatase activity was stimulated regardless of the ionized [Ca2+] level. Protein tyrosine kinase activity was stimulated at ionized [Ca2+]i < or =1 microM, whereas at ionized [Ca2+]i > or =4 microM, no protein tyrosine kinase activity was observed except in the presence of calpeptin. Thus, the massive tyrosine phosphoprotein disappearance observed at a high ionized [Ca2+]i resulted not only in protein tyrosine phosphatase activation, but also in calpain-induced protein tyrosine kinase inactivation.

Signal transduction in normal and pathological thrombin-stimulated human platelets.

Human blood platelets stimulated by thrombin undergo very rapid morphological changes, the most characteristic of which are pseudopod formation and granule centralization. These early changes in shape are accompanied by a transient decrease (30%) in phosphatidyl inositol 4,5-bisphosphate (PIP2) which occurs in the first 10 s after thrombin addition. Transient decreases in phosphatidyl inositol 4-phosphate (PIP) and phosphatidyl inositol (PI) occur later (20-30 s). These events lead to the formation of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DG) and hence phosphatidate (PA). Two polypeptides are phosphorylated during the same time span: the myosin light chain (P20) and a 43 kDa protein (P43). Concomitant with these molecular changes, platelet 'release reaction' occurs, i.e., liberation of the different granule constituents into the external medium: the earliest concerns dense bodies which liberate adenine nucleotides, calcium and serotonin; alpha-granules then liberate adhesive and specific proteins and are followed by lysosomes which liberate hydrolases. Pathological platelets from patients with inherited disorders, presenting well-characterized and specific defects of either the platelet membrane (GT) or storage granules (GPS and HPS), have also been studied. The results obtained lead to the following conclusions: (1) the transducing system is normal in platelets unable to aggregate; (2) phosphorylation of P20 and P43 proteins can be complete with impaired release; and (3) when platelets lack alpha-granules the transducing system as well as the release of other granule populations are impaired. These results evidence the relationship between the absence of intraplatelet components and metabolic events.

Further characterization of wheat germ agglutinin interaction with human platelets: exposure of fibrinogen receptors.

It was previously shown that Wheat germ agglutinin, (WGA)-induced platelet activation occurred when only 17% of the lectin binding sites were occupied on the platelet surface and WGA caused the release of a platelet constituent which in turn participates in the observed effect. We now further define the platelet activation induced by WGA: the lectin induces a binding of fibrinogen to specific surface receptors. 125I-fibrinogen binding increases with the WGA concentration from 5 to 15 micrograms/ml. Binding occurs without addition of exogenous calcium; its analysis demonstrated 54,000 sites with a Ka = 0.8 X 10(6) M-1. Addition of 1 mM Ca2+ enhances the 125I-fibrinogen binding and reveals a second class of sites with higher affinity (9200 sites, Ka = 0.17 X 10(8) M-1). This 125I-fibrinogen binding is totally abolished by EDTA, ATP and arginine, and inhibited by 75% by CP/CPK; cyclooxygenase inhibitors and PGE1 also reduce the fibrinogen binding. Thus the WGA-induced fibrinogen binding is release-dependent and responsible for the aggregation process but not for the agglutinating effect of the lectin.

Different abilities of thrombin receptor activating peptide and thrombin to induce platelet calcium rise and full release reaction.

Synthetic peptides (TRAP or Thrombin Receptor Activating Peptide) corresponding to at least the first five aminoacids of the new N-terminal tail generated after thrombin proteolysis of its receptor are effective to mimic thrombin. We have studied two different TRAPs (SFLLR, and SFLLRN) in their effectiveness to induce the different platelet responses in comparison with thrombin. Using Indo-l/AM-labelled platelets, the maximum rise in cytoplasmic ionized calcium was lower with TRAPs than with thrombin. At threshold concentrations allowing maximal aggregation (50 microM SFLLR, 5 microM SFLLRN and 1 nM thrombin) the TRAPs-induced release reaction was about the same level as with thrombin, except when external calcium was removed by addition of 1 mM EDTA. In these conditions, the dense granule release induced by TRAPs was reduced by over 60%, that of lysosome release by 75%, compared to only 15% of reduction in the presence of thrombin. Thus calcium influx was more important for TRAPs-induced release than for thrombin-induced release. At strong concentrations giving maximal aggregation and release in the absence of secondary mediators (by pretreatment with ADP scavengers plus aspirin), SFLLRN mobilized less calcium, with a fast return towards the basal level and induced smaller lysosome release than did thrombin. The results further demonstrate the essential role of external calcium in triggering sustained and full platelet responses, and emphasize the major difference between TRAP and thrombin in mobilizing [Ca2+]i. Thus, apart from the proteolysis of the seven transmembrane receptor, another thrombin binding site or thrombin receptor interaction is required to obtain full and complete responses.

Role of Fc gamma RIIA gene polymorphism in human platelet activation by monoclonal antibodies.

The aim of this study was to determine if there is a correlation between the activity of a MoAb as an agonist and its ability to bind to the Fc platelet receptor, Fc gamma RIIa. A polymorphism at amino acid 131 [arginine (Arg) or histidine (His)] of Fc gamma RIIa was first shown to be determinant for MoAb-IgG1 binding on monocytes. To clarify the role of this polymorphism in platelet activation by MoAb-IgG1 we (i) established the Fc gamma RIIa polymorphism at the gene level by adapting the denaturating gradient gel electrophoresis method, (ii) analyzed the binding affinity of the MoAbs to Fc gamma RIIa on platelets from homozygous Arg, homozygous His, and heterozygous Arg/His donors, and (iii) characterized the different reactivities of platelets according to the Fc gamma RIIA polymorphism. Among 167 caucasian donors we found 46% heterozygous Arg/His, 36% homozygous His and 18% homozygous Arg. ALB6, and anti CD9, P256 an anti GPIIb-IIIa, and AP3 an anti-GPIIIa were chosen according to their ability (ALB6, P256) or not (AP3) to activate platelets. These 3 MoAbs-IgG1 bind to Fc gamma RIIa with a stronger affinity for the Arg-form of Fc gamma RIIa, a result which was confirmed with the use of diverse MoAbs directed against various antigens. The different abilities of MoAbs to bind to the two Fc gamma RIIa forms were well correlated to the different platelet responses induced by ALB6 and P256. However, low concentrations of ALB6, which allow full activation of platelets from homozygous Arg donors, as did P256, did not induce any activation of platelets from homozygous His donors, whereas P256 is able to induce a low aggregation. The results further define the respective roles of the antigen and the Fc receptor, depending on the MoAb, and the role of the Fc gamma RIIa polymorphism in platelet activation induced by MoAbs. In addition, the results obtained with MoAbs unable to induce platelet activation provided evidence that the binding of a MoAb on Fc gamma RIIa does not predict its ability to activate platelets.

Relationship between mepacrine-labelled dense body number, platelet capacity to accumulate 14C-5-HT and platelet density in the Bernard-Soulier and Hermansky-Pudlak syndromes.

We have used the mepacrine-labelling procedure to measure the dense body (serotonin storage organelle) content of the platelets of 2 hereditary disorders where abnormalities in dense body number were suspected. The platelets were incubated with mepacrine and examined by fluorescence microscopy. A mean number of 5.4 +/- 0.8 (SD) dense bodies per platelet was calculated from the data obtained using platelets isolated from 40 normal human subjects. In contrast the platelets of 2 patients with the Bernard-Soulier syndrome contained an average of 14 and 17 labelled granules. This increase was associated with a much greater capacity of the platelets to accumulate 14C-5-HT. The opposite result was obtained using the platelets from 2 patients with the Hermansky-Pudlak syndrome which contained few granules labelled by mepacrine and took up less 14C-5-HT than normal human platelets. Centrifugation of the patients' platelets on discontinuous sucrose gradients showed that the platelets of the 2 Bernard-Soulier patients were much denser than normal whereas a high proportion of low density platelets was observed in the Hermansky-Pudlak syndrome. These results further define the platelet abnormalities in the two syndromes and suggest that dense body number may be one of the factors governing platelet density.

Importance of the FcgammaRIIa-Arg/His-131 polymorphism in heparin-induced thrombocytopenia diagnosis.

Heparin-induced thrombocytopenia (HIT) involves heparin-dependent antibodies which induce platelet activation. In the present study, we searched for a relationship between the polymorphism of the Fc receptor (FcgammaRIIa) and the development of HIT. In this purpose, all the donors were genotyped for their FcgammaRIIA and HIT patients were selected on the basis of at least one positive answer by 14C-serotonin release assay (SRA). The frequency distribution of the FcgammaRIIa polymorphism in the HIT patient group was similar to that observed in the healthy control group. Moreover, a statistical analysis taking into account our results and those of 3 previously published studies, suggested at most only a weak association between HIT and the FcgammaRIIa-131 polymorphism. Laboratory tests used to diagnose HIT rely on the activation of normal donor platelets but fail to detect every HIT positive patient. We determined the role of FcgammaRIIa-131 polymorphism on the reactivity of control platelets to HIT plasmas. When control platelet FcgammaRIIa-131 was of Arg/Arg form, only 47% of the HIT plasmas were positive by SRA, compared to 81% and 74% for His/His or His/Arg forms, respectively. We also compared the level of anti PF4/heparin antibodies in the HIT plasmas with the response obtained by SRA. The mean anti PF4/heparin antibodies level in HIT plasma was significantly lower in negative SRA than in positive tests when using control platelets from FcgammaRIIa-Arg/Arg 131 and heterozygous donors. Thus, the variability of control platelets to respond to HIT plasmas in the SRA test is related to both the FcgammaRIIa-131 polymorphism, and to the amount of anti PF4/heparin antibodies.

Thrombomodulin modulates the mitogenic response to thrombin of human umbilical vein endothelial cells.

Thrombin interacts with its receptor and thrombomodulin on endothelial cells. We evaluated the respective roles of these two proteins on human umbilical vein endothelial cell (HUVEC) growth by comparing thrombin, S195A (a mutant thrombin in which the serine of the charge stabilizing system had been replaced by alanine), and the receptor activating peptide (TRAP). Thrombin and TRAP induced DNA synthesis (half maximal cell proliferation with 5 nM and 25 microM, respectively), whereas S195A thrombin was inactive, inferring that growth is mediated through the thrombin receptor. Surprisingly, cells stimulated by TRAP exhibited a maximal proliferation twice greater than that obtained with thrombin. Combination of thrombin and TRAP resulted in a mitogenic response higher than by thrombin alone, but lower than by TRAP alone. The role of thrombomodulin was evaluated by adding an anti-thrombomodulin antibody, which prevents formation of the thrombin-thrombomodulin complex. Antibody did not interfere with cell proliferation induced by TRAP, but enhanced that induced by thrombin. We conclude that formation of the thrombin-thrombomodulin complex restrains HUVEC proliferation mediated through the thrombin receptor.

A new approach in the study of the molecular and cellular events implicated in heparin-induced thrombocytopenia. Formation of leukocyte-platelet aggregates.

Heparin-induced thrombocytopenia (HIT), a relatively common complication of heparin therapy, results of platelet activation, via the receptor for the Fc domain of IgG (FcgammaRIIa), by heparin-dependent-antibodies, commonly directed against the heparin-platelet factor 4 (H-PF4) antigenic complex. Our strategy was to use whole blood allowing the study of leukocyte-platelet interactions. Experiments were performed with blood from healthy donors incubated with HIT patients' plasma and different concentrations of heparin. We showed that 75% of the HIT patients' plasma induced the formation of leukocyte-platelet-aggregates in a heparin-dependent-manner. The formation of leukocyte-platelet-aggregates induced by HIT plasma in the presence of heparin was (i) independent of the healthy blood donor FcgammaRIIa polymorphism, (ii) correlated with the levels of anti H-PF4 IgG antibodies contained in the patients' plasma, and to a lesser extent to anti H-PF4 IgM antibodies, and (iii) was mediated by P-selectin. This report opens new prospects in the study of the molecular and cellular events implicated in HIT.

Thiosulfinates inhibit platelet aggregation and microparticle shedding at a calpain-dependent step.

Thiosulfinates (TSs) are sulfur compounds generated through the processing of different Allium species with antiplatelet property. To further define this platelet inhibitory effect we studied diallyl-TS (Al2TS), dipropyl-TS (Pr2TS). and dimethyl-TS (Me2TS) on platelet responses. The three TSs inhibited dose-dependent platelet aggregation, with IC50 values of 15+/-2, 19+/-2, and 9+/-1 microM for Al2TS, Pr2TS and Me2TS, respectively. TSs had no effect on the expression of a platelet procoagulant surface, measured by flow cytometry as the binding of annexin V-FITC. They inhibited the microparticle shedding and clot retraction. Since the microparticle shedding is a calpain-activation dependent step, we assessed calpain activation by analysis of autoproteolysis in shorter active forms and by talin proteolysis in the presence of TSs. Calpain activation was inhibited by TSs independently of fibrinogen binding. Thus, TSs represent a new category of platelet inhibitors, acting on calpain-induced events.

Lonomia obliqua caterpillar spicules trigger human blood coagulation via activation of factor X and prothrombin.

In southern Brazil, envenomation by larvae of the moth Lonomia obliqua (Walker) may result in blood clotting factor depletion, leading to disseminated intravascular coagulation with subsequent haemorrhage and acute renal failure which may prove fatal. We have examined the effect of a crude extract of spicules from these caterpillars on in vitro hemostasis. The extract alone did not aggregate platelets and had no detectable effect on purified fibrinogen, suggesting that extract induces clot formation by triggering activation of the clotting cascade. In agreement with the presence of thrombin-mediated activity, hirudin prevented clot formation. The extract was found to activate both prothrombin and factor X, suggesting that the depletion of blood clotting factors results from the steady activation of factor X and prothrombin. Heating and diisopropylfluorophosphate abolished the procoagulant activity of the extract, indicating that the active component involved is a protein that may belong to the serine protease family of enzymes. The ability of hirudin to inhibit this coagulant activity suggests that this inhibitor could be beneficial in the treatment of patients envenomed by L. obliqua caterpillars.

The effect of aspirin on 5-hydroxytryptamine uptake and release by human platelets.

The influence of aspirin on 5-hydroxytryptamine (5-HT) uptake and storage by human blood platelets has been investigated. Uptake of 5-HT was strongly inhibited. In 30 min aspirin released 50% of the 5-HT that had been incorporated into the platelets prior to the addition of the aspirin. These results are discussed in terms of possible interference with a 5-HT membrane receptor and the impairment of 5-HT storage in the dense granules.

Role of chondroitin 4-sulphate as a receptor for polycation induced human platelet aggregation.

1. Proteoglycans provide negatively charged sites on the surface of platelets, leukocytes and endothelial cells. Since chondroitin 4-sulphate is the main proteoglycan present on the platelet surface, the role of this molecule in mediating the activation of human platelets by polylysine was studied. 2. Platelets were desensitized with phorbol 12-myristate 13-acetate (PMA, 10 nM) 5 min before the addition of polylysine to platelet-rich plasma (PRP). Changes in the intracellular Ca2+ concentration were measured in fura2-am (2 microM) loaded platelets and protein phosphorylation was assessed by autoradiography of the electrophoretic profile obtained from [32P]-phosphate labelled platelets. The release of dense granule contents was measured in [14C]-5-hydroxytryptamine loaded platelets and the synthesis of thromboxane (TXA2) was assessed by radioimmunoassay. Surface chondroitin 4-sulphate proteoglycan was degraded by incubating platelets with different concentrations of chondroitinase AC (3 min, 37 degrees C). The amount of chondroitin 4-sulphate remaining in the platelets was then quantified after proteolysis and agarose gel electrophoresis. 3. The addition of PMA to PRP before polylysine inhibited the aggregation by 88 +/- 18% (n = 3). Staurosporine (1 microM, 5 min) prevented the PMA-induced inhibition. Chondroitinase AC (4 pu ml-1 to 400 muu ml-1, 3 min) abolished the polylysine-induced aggregation in PRP but caused only a discrete inhibition of ADP-induced aggregation. The concentration of chrondroitin 4-sulphate in PRP (0.96 +/- 0.2 microgram/10(8) platelets, n = 3) and in washed platelets (WP; 0.35 +/- 0.1 microgram/10(8) platelets, n = 3) was significantly reduced following incubation with chondroitinase AC (PRP = 0.63 +/- 0.1 microgram/10(8) platelets and WP = 0.08 +/- 0.06 microgram/10(8) platelets). 4. Washed platelets had a significantly lower concentration of chondroitin 4-sulphate than platelets in PRP. The addition of polylysine to WP induced a rapid increase in light transmission which was not accompanied by TXA2 synthesis or the release of dense granule contents. This effect was not inhibited by sodium nitroprusside (SNP), iloprost, EDTA or the peptide RGDS. This event was accompanied by the discrete phosphorylation of plekstrin and myosin light chain, which were inhibited by staurosporine (10 microM, 10 min). The hydrolysis of platelet surface chondroitin 4-sulphate strongly reduced the polylysine-induced phosphorylation. 5. Our results indicate that polylysine activates platelets through a specific receptor which could be the proteoglycan chondroitin 4-sulphate present on the platelet membrane.