Development of new antiatherosclerotic and antithrombotic drugs utilizing F11 receptor (F11R/JAM-A) peptides.

Peptides with enhanced resistance to proteolysis, based on the amino acid sequence of the F11 receptor molecule (F11R, aka JAM-A/Junctional adhesion molecule-A), were designed, prepared, and examined as potential candidates for the development of anti-atherosclerotic and anti-thrombotic therapeutic drugs. A sequence at the N-terminal of F11R together with another sequence located in the first Ig-loop of this protein, were identified to form a steric active-site operating in the F11R-dependent adhesion between cells that express F11R molecules on their external surface. In silico modeling of the complex between two polypeptide chains with the sequences positioned in the active-site was used to generate peptide-candidates designed to inhibit homophilic interactions between surface-located F11R molecules. The two lead F11R peptides were modified with D-Arg and D-Lys at selective sites, for attaining higher stability to proteolysis in vivo. Using molecular docking experiments we tested different conformational states and the putative binding affinity between two selected D-Arg and D-Lys-modified F11R peptides and the proposed binding pocket. The inhibitory effects of the F11R peptide 2HN-(dK)-SVT-(dR)-EDTGTYTC-CONH2 on antibody-induced platelet aggregation and on the adhesion of platelets to cytokine-inflammed endothelial cells are reported in detail, and the results point out the significant potential utilization of F11R peptides for the prevention and treatment of atherosclerotic plaques and associated thrombotic events.

Genomic structure, organization and promoter analysis of the human F11R/F11 receptor/junctional adhesion molecule-1/JAM-A.

The F11-receptor (F11R) (a.k.a. JAM-1, JAM-A, CD321) is a cell adhesion molecule of the immunoglobulin superfamily involved in platelet adhesion, secretion and aggregation. In addition, the F11R plays a critical role in the function of endothelial cells and in platelet adhesion to inflamed endothelium. In the present study, we used partial sequences of the human F11R gene, F11R cDNAs, and information in unannotated human genome databases, to delineate the F11R gene. We found that the F11R gene is composed of 13 exons (E1a, 1b, 1c, E1-E10) encoding two groups of mRNAs differing in length and sequence at their 5' UTRs, referred to as type 1 and type 2 messages. Type 1 cDNAs are shorter at the 5' end and contain a region not found within type 2 messages. Type 1 mRNAs are present in endothelial cells (EC), platelets, white blood cells and in the cell lines CMK, HeLa, K562, HOG and A549, while type 2 messages are limited to EC. Type 1 messages contain exons E1-E10 whereas type 2 messages usually contain exons E1a, 1c, part of E1 and E2-E10. The translation start site is localized in the 3' end of E1, common for both type 1 and type 2 messages. Expression of these messages is regulated by two alternative promoters, P1 and P2. P1 is a TATA-less promoter containing an initiator element, multiple transcription start sites, several GC and CCAAT boxes, and GATA, NF-kappaB and ets consensus sequences. The cloned P1 drives efficient expression of the luciferase reporter gene. A high level of similarity between human P1 and its rat and mouse counterparts was observed. Promoter P2, located upstream of P1, contains a TATA box, GC boxes, a CCAAT box and GATA and ets consensus sequences. 3' RACE provided evidence for variability in the 3' UTR due to the presence of two polyadenylation signals. The finding of multiple regulatory sites in the promoters supplements the biochemical evidence that the F11R has several different roles in the functional repertoire of endothelial cells, platelets and other cells. In particular, the presence of NF-kappaB provides additional evidence to the significance of the F11R function in the initiation of inflammatory thrombosis.

Platelet-activating factor in infants at risk for necrotizing enterocolitis.

BACKGROUND: Platelet-activating factor (PAF) is a heterogeneous phospholipid that has been implicated as participating in a number of perinatal disease processes including necrotizing enterocolitis (NEC). METHODS: Baseline blood levels of PAF and related lipids (PAF-LL) were measured for 164 infants at risk for NEC from 3 neonatal intensive care units. Serial levels were obtained from the 11 infants in whom NEC developed. RESULTS: The mean peak PAF-LL in the infants without NEC was 2.03 +/- 1.96 ng/mL. Infants with stage II (n = 6) and III (n = 5) NEC had elevated peak PAF-LL values (mean peak value 13.6 +/- 6.9 ng/mL). No PAF-LL measurements obtained from infants during stage II or III NEC were <2.03 ng/mL. Three infants had PAF-LL elevations before the development of any clinical or radiographic evidence of NEC. PAF-LL level increased as the severity of NEC increased and decreased with its resolution. Setting a PAF-LL level of 10.2 ng/mL as a cutoff for NEC had a positive predictive value of 100%. CONCLUSIONS: PAF-LL determinations can complement clinical and radiographic studies to diagnose and follow the progression of NEC. PAF-LL may have a role in the evolution of NEC.

Identification of ecto-PKC on surface of human platelets: role in maintenance of latent fibrinogen receptors.

Human platelets express a protein phosphorylation system on their surface. A specific protein kinase C (PKC) antibody, monoclonal antibody (MAb) 1.9, which binds to the catalytic domain of PKC and inhibits its activity, causes the aggregation of intact platelets while inhibiting the phosphorylation of platelet surface proteins. Photoaffinity labeling with 100 nM 8-azido-[alpha(32)P]ATP identified this ecto-PKC as a single surface protein of 43 kDa sensitive to proteolysis by extracellular 0.0005% trypsin. Inhibition of the binding of 8-azido-[alpha(32)P]ATP to the 43-kDa surface protein by MAb 1.9 identified this site as the active domain of ecto-PKC. Covalent binding of the azido-ATP molecule to the 43-kDa surface protein inhibited the phosphorylative activity of the platelet ecto-PKC. Furthermore, PKC pseudosubstrate inhibitory peptides directly induced the aggregation of platelets and inhibited azido-ATP binding to the 43-kDa protein. Platelet aggregation induced by MAb 1.9 and by PKC inhibitory peptides required the presence of fibrinogen and resulted in an increase in the level of intracellular free calcium concentration. This increase in intracellular free calcium concentration induced by MAb 1.9 was found to be dependent on the binding of fibrinogen to activated GPIIb/IIIa integrins, suggesting that MAb 1.9 causes Ca(2+) flux through the fibrinogen receptor complex. We conclude that a decrease in the state of phosphorylation of platelet surface proteins caused by inhibition of ecto-PKC results in membrane rearrangements that can induce the activation of latent fibrinogen receptors, leading to platelet aggregation. Accordingly, the maintenance of a physiological steady state of phosphorylation of proteins on the platelet surface by ecto-PKC activity appears to be one of the homeostatic mechanisms that maintain fibrinogen receptors of circulating platelets in a latent state that cannot bind fibrinogen.

Cloning of the human platelet F11 receptor: a cell adhesion molecule member of the immunoglobulin superfamily involved in platelet aggregation.

This study demonstrates that the human platelet F11 receptor (F11R) functions as an adhesion molecule, and this finding is confirmed by the structure of the protein as revealed by molecular cloning. The F11R is a 32-/35-kd protein duplex that serves as the binding site through which a stimulatory monoclonal antibody causes platelet aggregation and granule secretion. A physiological role for the F11R protein was demonstrated by its phosphorylation after the stimulation of platelets by thrombin and collagen. A pathophysiological role for the F11R was revealed by demonstrating the presence of F11R-antibodies in patients with thrombocytopenia. Adhesion of platelets through the F11R resulted in events characteristic of the action of cell adhesion molecules (CAMs). To determine the structure of this protein, we cloned the F11R cDNA from human platelets. The predicted amino acid sequence demonstrated that it is an integral membrane protein and an immunoglobulin superfamily member containing 2 extracellular C2-type domains. The structure of the F11R as a member of a CAM family of proteins and its activity in mediating adhesion confirm each another. We conclude that the F11R is a platelet-membrane protein involved in 2 distinct processes initiated on the platelet surface. The first is antibody-induced platelet aggregation and secretion that are dependent on both the FcgammaRII and the GPIIb/IIIa integrin and that may be involved in pathophysiological processes associated with certain thrombocytopenias. The second is an F11R-mediated platelet adhesion that is not dependent on either the FcgammaRII or the fibrinogen receptor and that appears to play a role in physiological processes associated with platelet adhesion and aggregation. (Blood. 2000;95:2600-2609)

Enhancement of human platelet aggregation and secretion induced by rapamycin.

BACKGROUND: Rapamycin is a new immunosuppressive drug of the macrolide type. Despite binding to one of the FK-binding proteins as the initial step in intracellular action, further effects differ from those of the other fungally derived macrolides, cyclosporine and tacrolimus. We have previously demonstrated an enhancement of agonist-mediated platelet activation by cyclosporine and tacrolimus which was associated with increased phosphorylation of two intracellular platelet proteins, p20 and p40. Because rapamycin utilizes the same class of binding proteins as tacrolimus, but its action is not associated with the inhibition of calcineurin, we postulated that if the stimulatory effect of cyclosporine or tacrolimus was due to calcineurin inhibition, rapamycin should not affect platelets in a similar fashion. METHODS: Normal, washed human platelets were treated with various concentrations of rapamycin (from ng to microg/ml), and pre-incubated at 37 degrees C with rapamycin for various periods (1-30 min). Several platelet functional parameters were measured in samples treated with rapamycin and these parameters were compared with control platelet samples treated with the vehicle for the same period. Platelet aggregations following exposure to ADP or to the thrombin equivalent, TRAP-6, were measured as changes in optical transmission in a Chronolog lumi-aggregometer. Each experiment was repeated at three or more times and the mean results were used for statistical comparison. RESULTS: Rapamycin-treated platelets demonstrated an increase in their dose- and time-dependent sensitivity to ADP, resulting in a significantly enhanced primary wave of ADP-induced platelet aggregation followed by a secondary wave of aggregation, indicative of granule secretion. Furthermore, rapamycin-treated platelets showed significantly enhanced sensitivity to TRAP-6 as demonstrated by an increase in the initial velocity of aggregation, an increase in their maximal extent of aggregation and an enhancement of granular ATP secretion. Concentrations of rapamycin in the ng range, as well as short pre-incubation times (within min), were sufficient to cause significant enhancement of agonist-induced platelet aggregation and secretion (P < 0.001) as compared with their vehicle controls. CONCLUSIONS: Rapamycin significantly potentiates agonist-induced platelet aggregation in a time- and dose-dependent manner. As these findings are similar to those observed with the other fungal macrolides, we hypothesize that inhibition of calcineurin may not be necessary for the increase in intracellular protein phosphorylation observed following exposure of platelets to cyclosporine or tacrolimus. Whether the rapamycin-induced enhancement of sensitivity to agonists and platelet hyperaggregability explains the thrombocytopenia observed in patients when high doses of rapamycin are administered in the clinical setting, and whether these effects are synergistic with cyclosporine, are questions which remain to be investigated.

Activation of human platelets by protein kinase C antibody: role for surface phosphorylation in homeostasis.

A monoclonal antibody that inhibits protein kinase C (PKC) activity, as well as PKC pseudosubstrate inhibitory peptides, was found to cause aggregation of human platelets followed by granular secretion. Binding of this antibody to the platelet surface was demonstrated directly by flow cytometry and immunofluorescence microscopy. Assays of ecto-protein kinase activity revealed that this antibody inhibits the phosphorylation of five proteins on the platelet surface. The platelet aggregation induced by extracellular PKC inhibitors could be blocked by the addition of the membrane-impermeable phosphatase inhibitor, microcystin. Thus the inhibition of surface protein phosphorylation together with continuous dephosphorylation, namely, a decrease in the phosphorylation state of surface proteins, causes the activation of platelets. The aggregation caused by decreased surface phosphorylation appears to be initiated by the exposure of active fibrinogen-binding sites on the platelet surface, as demonstrated by the formation of fibrinogen-dependent microaggregates, as the first step in this process. We conclude that the phosphorylation of surface proteins by a platelet ecto-protein kinase C protects platelets from spontaneous aggregation and thus can play an important role in homeostatic mechanisms that maintain circulating platelets in a resting state.

Platelet activating factor (PAF) in memory formation: role as a retrograde messenger in long-term potentiation.

Long-term potentiation (LTP) is a neurophysiological process that has been implicated in memory formation. The elevation of intracellular Ca2+ levels in postsynaptic neurons, an essential step in the induction of LTP in the hippocampus, can lead to activation of the enzyme acetyl-CoA:lyso-PAF acetyltransferase that is required for PAF synthesis in neurons. Thus, during the induction of LTP, stimulation of Ca2+ influx by glutamate receptors would lead to a postsynaptic increase in PAF biosynthesis. A main target for PAF action in neurons is the stimulation of neurotransmitter release via Ca(2+)-dependent vesicular exocytosis, a process that occurs presynaptically. In this article we describe the evidence obtained to-date for the pre- and postsynaptic events outlined, above, and demonstrate for the first time that during the induction of LTP by high-frequency stimulation (HFS) a 9-fold increase in PAF release to the extracellular environment occurs within 60 min following HFS. This finding provides the evidence that PAF can diffuse from postsynaptic sites of synthesis to presynaptic sites of action, and thus function as a retrograde messenger in the induction of LTP. Based on these data, we present a scheme in which postsynaptic glutamate receptors cooperate with presynaptic PAF receptors in a reverberating cycle that can amplify the transmission in a Hebbian synapse.

Surface protein phosphorylation by ecto-protein kinase is required for the maintenance of hippocampal long-term potentiation.

During the induction of long-term potentiation (LTP) in hippocampal slices adenosine triphosphate (ATP) is secreted into the synaptic cleft, and a 48 kDa/50 kDa protein duplex becomes phosphorylated by extracellular ATP. All the criteria required as evidence that these two proteins serve as principal substrates of ecto-protein kinase activity on the surface of hippocampal pyramidal neurons have been fulfilled. This phosphorylation activity was detected on the surface of pyramidal neurons assayed after synaptogenesis, but not in immature neurons nor in glial cells. Addition to the extracellular medium of a monoclonal antibody termed mAb 1.9, directed to the catalytic domain of protein kinase C (PKC), inhibited selectively this surface protein phosphorylation activity and blocked the stabilization of LTP induced by high frequency stimulation (HFS) in hippocampal slices. This antibody did not interfere with routine synaptic transmission nor prevent the initial enhancement of synaptic responses observed during the 1-5 min period immediately after the application of HFS (the induction phase of LTP). However, the initial increase in the slope of excitatory postsynaptic potentials, as well as the elevated amplitude of the population spike induced by HFS, both declined gradually and returned to prestimulus values within 30-40 min after HFS was applied in the presence of mAb 1.9. A control antibody that binds to PKC but does not inhibit its activity had no effect on LTP. The selective inhibitory effects observed with mAb 1.9 provide the first direct evidence of a causal role for ecto-PK in the maintenance of stable LTP, an event implicated in the process of learning and the formation of memory in the brain.

Surface phosphorylation by ecto-protein kinase C in brain neurons: a target for Alzheimer's beta-amyloid peptides.

The powerful regulatory machinery of protein phosphorylation operates in the extracellular environment of the brain. Enzymatic activity with the catalytic specificity of protein kinase C (PKC) was detected on the surface of brain neurons, where it can serve as a direct target for neurotrophic and neurotoxic substances that control neuronal development and cause neurodegeneration. This activity fulfilled all the criteria required of an ecto-protein kinase (ecto-PK). Detailed analysis of surface protein phosphorylation in cultured brain neurons using specific exogenous substrates (casein, histones, and myelin basic protein), inhibitors (PKC-pseudosubstrate 19-36; K252b) and antibodies (anti-PKC catalytic region M.Ab.1.9, antibodies to the carboxy-terminus of eight PKC isozymes) revealed several types of ecto-PK activity, among them ecto-PKs with catalytic specificity of the PKC isozymes zeta and delta. The activity of the neuronal ecto-PKC is constitutive and not stimulated by phorbol esters. the phosphorylation of a 12K/13K surface protein duplex by ecto-PKC-delta was found to be developmentally regulated, with peak activity occurring during the onset of neuritogenesis. Alzheimer's amyloid peptides beta 1-40 and beta 25-35 applied at neurotrophic concentrations stimulated the phosphorylation of endogenous substrates of ecto-PKC activity in brain neurons but inhibited specifically this surface phosphorylation activity with the same dose-response relationships that cause neurodegeneration. As may be expected from a relevant pathophysiological activity, beta-amyloid peptide 1-28 did not inhibit this surface phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulatory antibody-induced activation and selective translocation of protein kinase C isoenzymes in human platelets.

A novel stimulatory monoclonal antibody (Mab) termed Mab.F11 induces granular secretion and subsequent aggregation of human platelets. Mab.F11 recognizes a unique 32 and 35 kDa protein duplex on the platelet membrane surface, called the F11 receptor; binding of Mab.F11 to its receptor results in increased intracellular phosphorylation of P47, the known protein kinase C (PKC) substrate pleckstrin. In order to determine whether the mechanism of action of Mab.F11 involves direct activation of PKC, two types of functional assays for measuring PKC activity were performed. Measurement of PKC activity in digitonin-permeabilized platelets revealed that Mab.F11 produced a rapid, 2-3 fold increase in the control value in the phosphorylation of the PKC peptide substrate, PKC(19-31) Ser25. The increase in PKC activity induced by Mab.F11 was found to be associated with the platelet membrane; a 1.6-fold control value increase in membrane PKC activity occurred rapidly, within 10 s of the addition of Mab.F11. The translocation from the cytoplasm to the membrane induced by Mab.F11 in PKC isoenzymes alpha and zeta was reversible, whereas translocation of the PKC isoenzymes delta, beta, eta' and theta was irreversible, with PKC levels remaining elevated in the membrane for at least 15 min. Taken together, our results demonstrate that in the initial stages of platelet activation by this stimulatory antibody, the enhanced membrane PKC activity reflects the presence of all six isoenzymes. At later stages, PKC activity is reflective of four isoenzymes. These results demonstrate that separate groups of PKC isoenzymes must be involved in different aspects of platelet activation. The long lag period and prolonged activation time of platelets by Mab.F11 renders this agonist most suitable for identifying the isoenzymes and their specific endogenous protein substrates involved in platelet secretion and aggregation induced by platelet membrane protein antibodies.

Mechanisms of platelet activation by a stimulatory antibody: cross-linking of a novel platelet receptor for monoclonal antibody F11 with the Fc gamma RII receptor.

The mechanisms by which a stimulatory monoclonal antibody (mAb), called mAb F11, induces granular secretion and aggregation in human platelets have been characterized. Fab fragments of mAb F11, as well as an mAb directed against the platelet Fc gamma RII receptor (mAb IV.3) were found to inhibit mAb F11-induced platelet secretion and aggregation, indicating that the mAb F11 IgG molecule interacts with the Fc gamma RII receptor through its Fc domain and with its own antigen through its Fab domain. The mAb F11 recognized two platelet proteins of 32 and 35 kDa on the platelet membrane surface, as identified by Western blot analysis. We purified both proteins from human platelet membranes using DEAE-Sepharose chromatography followed by mAb F11 affinity chromatography. When added to platelet-rich plasma, the purified proteins dose-dependently inhibited mAb F11-induced platelet aggregation. The purified protein preparation also competitively inhibited the binding of 125I-labelled mAb F11 to intact platelets. The N-terminal 26 amino acid sequences of both the 32 and 35 kDa proteins were identical and contained a single unblocked serine in the N-terminal position. When digested with N-glycanase, the 32 and 35 kDa proteins were converted into a single approximately 29 kDa protein, indicating that these two proteins are derived from the same core protein but differ in their degree of glycosylation. Internal amino acid sequence analysis of the F11 antigen provided information concerning 68 amino acids and suggested two consensus phosphorylation sites for protein kinase C (PKC). The phosphorylation by PKC of the isolated F11 antigen was observed following stimulation by phorbol 12-myristate 13-acetate. Databank analysis of the N-terminal and internal amino acid sequences of the F11 antigen indicated that the N-terminal sequence exhibited the highest degree of similarity to the variable region of the alpha-chain of human T-cell receptors (TCR). In contrast, the F11 internal sequences did not exhibit any similarity to the TCR. Our results demonstrate that the F11 antigen is a novel platelet membrane surface glycoprotein which becomes cross-linked with the Fc gamma RII receptor when platelets are activated by the stimulatory mAb F11. These mechanisms may be relevant to the production of immune thrombocytopenia by platelet-activating antibodies.

Long-term potentiation in the hippocampus induced by platelet-activating factor.

Platelet-activating factor (PAF) is an ether phospholipid that serves as an autacoid in a wide range of biological processes. We found that when PAF was added to hippocampal slices, it induced a stable and concentration-dependent increase in excitatory postsynaptic potential and population spike recordings (long-term potentiation [LTP]). The PAF effect was blocked by the PAF receptor antagonists BN 52021 and WEB 2086 and the N-methyl-D-aspartate receptor antagonists MK 801 and 2-amino-5-phosphonovalerate. However, these PAF receptor antagonists did not block LTP induced by high frequency stimulation. The facilitation induced by PAF could not be reversed by PAF receptor antagonists. Induction of either PAF-or tetanus-triggered facilitation occluded the subsequent expression of the other, suggesting a common pathway. LTP is a type of synaptic plasticity associated with the formation of memory, and PAF may play an important modulatory role in this process.

A new protein kinase C, nPKC eta', and nPKC theta are expressed in human platelets: involvement of nPKC eta' and nPKC theta in signal transduction stimulated by PAF.

We have detected in human platelets two protein kinase C isozymes that have not been reported previously. Using an anti-nPKC theta antibody and Western blotting, we calculated the molecular weight of platelet nPKC theta as 79K. This molecular weight is identical to that described for nPKC theta in skeletal muscle and in COS cells transfected with the nPKC theta-cDNA. Using an anti-nPKC eta antibody, we determined the molecular weight of an immunoreactive protein, which we called nPKC eta', to be 95K. This molecular weight is higher than that of nPKC eta found in lung and skin tissue of 82K and 78K, and it is higher than nPKC eta of COS cells transfected with the nPKC eta-cDNA expression plasmid. Together with previous reports, these findings make the total number of PKC isozymes in human platelets equal to six. These are the PKC isozymes: alpha, beta, delta and zeta, which have been previously described, and eta' and theta which we describe here. To assess the functionality of these new PKC isoforms, we stimulated platelets with PAF. We found a 200% and 175% increase in the levels of membrane-bound nPKC eta' and nPKC theta, respectively, in human platelets stimulated by PAF. A concomitant decrease in the level of these isoforms in the cytoplasm was observed. This PAF-induced translocation was time-dependent, and it reached its peak after a 1 minute incubation of human platelets with PAF for nPKC theta and 30 seconds for nPKC eta'.

Ecto-protein kinase and surface protein phosphorylation in PC12 cells: interactions with nerve growth factor.

The phosphorylation of surface proteins by ecto-protein kinase has been proposed to play a role in mechanisms underlying neuronal differentiation and their responsiveness to nerve growth factor (NGF). PC12 clones represent an optimal model for investigating the mode of action of NGF in a homogeneous cell population. In the present study we obtained evidence that PC12 cells possess ecto-protein kinase and characterized the endogenous phosphorylation of its surface protein substrates. PC12 cells maintained in a chemically defined medium exhibited phosphorylation of proteins by [gamma-32P]ATP added to the medium at time points preceding the intracellular phosphorylation of proteins in cells labeled with 32Pi. This activity was abolished by adding apyrase or trypsin to the medium but was not sensitive to addition of an excess of unlabeled Pi. As also expected from ecto-protein kinase activity, PC12 cells catalyzed the phosphorylation of an exogenous protein substrate added to the medium, dephospho-alpha-casein, and this activity competed with the endogenous phosphorylation for extracellular ATP. Based on these criteria, three protein components migrating in sodium dodecyl sulfate gels with apparent molecular weights of 105K, 39K, and 20K were identified as exclusive substrates of ecto-protein kinase in PC12 cells. Of the phosphate incorporated into these proteins from extracellular ATP, 75-87% was found in phosphothreonine. The phosphorylation of the 39K protein by ecto-protein kinase did not require Mg2+, implicating this activity in the previously demonstrated regulation of Ca(2+)-dependent, high-affinity norepinephrine uptake in PC12 cells by extracellular ATP. The protein kinase inhibitor K-252a inhibited both intra- and extracellular protein phosphorylation in intact PC12 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclosporine A enhances agonist-induced aggregation of human platelets by stimulating protein phosphorylation.

Use of the immunosuppressant drug cyclosporine A (CSA) has resulted in improved renal graft survival. However, an increased incidence of arterial and venous thrombotic diseases, hemolytic-uremic type syndrome, and findings resembling vasculitis in the kidneys of patients with CSA nephrotoxicity and accelerated atherogenesis have been reported. These disorders may be related to CSA-induced abnormalities in platelet function. We report here that CSA causes increased ADP-stimulated aggregation in isolated platelet suspensions indicating that CSA has a direct effect on platelet function, independent of CSA interactions with plasma factors. Maximal hyperaggregability of ADP-stimulated platelets occurred following a 1 h preincubation period with CSA. Hyperaggregability of platelets due to the presence of CSA was dose-dependent and approached plateau between 200-500 ng/ml CSA. We determined that CSA exerted its effects through a signal transduction pathway involving the phosphorylation of two intracellular proteins, a 40 kD substrate of PKC (p47) and the 20 kD light chain of myosin (p20), a substrate of calcium/calmodulin dependent kinase. Preincubation with CSA resulted in a 200% increase in the phosphorylation of these proteins in platelets stimulated with ADP. We conclude that CSA enhances ADP-induced platelet aggregation and secretion, in part, by potentiating the phosphorylative response of specific intracellular proteins to stimulation by agonists. This process may be responsible for the increased thrombosis and atherogenesis observed in CSA-treated patients.