R-Ras contains a proline-rich site that binds to SH3 domains and is required for integrin activation by R-Ras

R-Ras contains a proline-rich motif that resembles SH3 domain-binding sites but that has escaped notice previously. We show here that this site in R-Ras is capable of binding SH3 domains and that the SH3 domain binding may be important for R-Ras function. A fusion protein containing the SH3 domains of the adaptor protein Nck interacted strongly with the R-Ras proline-rich sequence and with the intact protein. The binding was independent of whether R-Ras was in its GDP or GTP form. The Nck binding, which was mediated by the second of the three SH3 domains of Nck, was obliterated by mutations in the proline-rich sequence of R-Ras. The interaction of Nck with R-Ras could also be shown in yeast two-hybrid assays and by co-immunoprecipitation in human cells transfected with Nck and R-Ras. Previous results have shown that the expression of a constitutively active R-Ras mutant, R-Ras(38V), converts mouse 32D monocytic cells into highly adherent cells. Introducing the proline mutations into R-Ras(38V) suppressed the effect of R-Ras on 32D cell adhesion while not affecting GTP binding. These results reveal an unexpected regulatory pathway that controls R-Ras through an SH3 domain interaction. This pathway appears to be important for the ability of R-Ras to control cell adhesion.     

The beta chain of chicken fibrinogen contains an atypical thrombin cleavage site

A cDNA corresponding to almost the entire coding region of the mRNA for the beta chain of chicken fibrinogen was sequenced. At the protein level, significant homology to the beta subunits of other vertebrate fibrinogens was found, with the highest degree of amino acid identity localized in the C-terminal region. In general, features conserved in the fibrinogens from other species also characterize the chicken sequence, including the cysteine motifs bordering an alpha-helical permissive region of fixed length and a single glycosylation site in the C-terminal region. However, the site of thrombin-catalyzed cleavage, which in other species consists of an Arg-Gly peptide bond, is instead an Arg-Ala bond in the chicken beta chain. The Ala was confirmed directly from a sequencing analysis of the purified beta chain of chicken fibrin. This finding may explain the observed slow clotting time of chicken fibrinogen relative to that of other species.     

Evidence that activation of platelets and endothelium by thrombin involves distinct sites of interaction. Studies with the dysthrombin, Thrombin Quick I

Previous results indicate extensive similarity of the active site regions of thrombin (EC 3.4.21.5) and Thrombin Quick, a congenital dysthrombin. A binding defect of Thrombin Quick toward fibrinogen is indicated by an increased KI when fibrinogen is present as a competitive inhibitor in the hydrolysis of tosyl-Gly-Pro-Arg-p-nitroanilide. In the present study, Thrombin Quick I is shown to have an activity of 1.3 and 34%, respectively, toward fibrinogen and prothrombin. Like the activity observed in prothrombin hydrolysis, Thrombin Quick I was 30% as effective as thrombin in stimulating release of thromboxane from platelets. Thrombin Quick was 1.7 and 2.4%, as effective as thrombin in stimulating platelet aggregation and prostacyclin production, respectively. Based on the activity of Thrombin Quick I in the reactions investigated, it is concluded that 1) the three cellular responses studied are initiated by proteolytic action of thrombin, 2) thrombin stimulation of aggregation and thromboxane release from platelets occurs via two different receptors, 3) the thrombin cellular interaction resulting in platelet aggregation and prostacyclin release must involve the thrombin active site as well as a secondary binding site required for optimal interaction with fibrinogen, and 4) the release of thromboxane from platelets does not involve the interaction of thrombin at the extrinsic binding site.     

Membrane dynamic alterations associated with activation of human platelets by thrombin

Two fluorescent probes, N-carboxymethylisatoic anhydride, which binds to membrane proteins, and 1,6-diphenyl-1,3,5-hexatriene, a lipophilic label, have been used to follow membrane microenvironmental changes. Activation of human platelets by thrombin resulted in a simultaneous increase in values of fluorescence polarization (P) of both probes during the stages of shape change and secretion, which further increased during platelet aggregation. The similar pattern of changes in P for both probes indicates the interdependence of lipids and proteins in the activated platelet membrane.     

Membrane microenvironmental changes during activation of human blood platelets by thrombin. A study with a fluorescent probe.

Membrane microenvironmental changes associated with thrombin-induced platelet activation were followed by fluorescence intensity and polarization studies of 1,6-diphenyl-1,3,5-hexatriene (DPH)-labeled human platelets. The labeling of washed platelets with DPH did not alter platelet intactness and morphology. In response to thrombin, DPH-labeled platelets exhibited reduced serotonin release, yet aggregation was barely inhibited. Shape change induced by thrombin or ADP was indistinguishable in control and in DPH-labeled platelets. During platelet aggregation induced by thrombin, fluorescence intensity increased by about 14%, which may indicate a more hydrophobic exposure of the probe. However, no change in fluorescence was detected during platelet shape change, induced either by thrombin in presence of EDTA or by ADP. Thrombin-activated platelets exhibited an increase in values of fluorescence polarization (P) during the stages of shape change and secretion, which further increased during aggregation. A similar pattern of increase in P values characterized platelet shape changes, caused either by thrombin in the presence of EDTA or by ADP. Changes in individual platelets are discernible from the alterations of the aggregating cells. These results may indicate that platelet activation is accompanied by an increase in rigidity of the membrane lipids. Functionally, the elevated "microviscosity" may reflect a primary role of membrane lipids in modulating the process of platelet activation or secondary transitions in lipids due to membrane events mediated by proteins.     

The thrombin high-affinity binding site on platelets is a negative regulator of thrombin-induced platelet activation. Structure-function studies using two mutant thrombins, Quick I and Quick II.

To elucidate the thrombin domains required for high-affinity binding and platelet activation, the platelet binding properties of thrombin and two mutant thrombins, thrombin Quick I and Quick II, were compared to their agonist effects in elevating intraplatelet [Ca2+]. In Quick I, a mutation within the fibrinogen binding groove results in decreased clotting and platelet aggregating activities, whereas in Quick II, a mutation in the primary substrate binding pocket abolishes both activities. Dysthrombin binding was decreased compared to thrombin. The fibrinogen binding groove appeared more important than the primary substrate pocket for high-affinity binding since Quick I showed drastically reduced, and Quick II only slightly reduced, binding affinity (Kd approximately 200 and approximately 10 nM, respectively). The deduced interaction of thrombin with its high-affinity binding site indicated that the thrombin catalytic site is directed toward the platelet surface and therefore, when bound, is proteolytically inactive. Quick I (0.5-5 nM) elicited intraplatelet [Ca2+] fluxes at concentrations where high-affinity binding was undetectable. Saturation of high-affinity binding sites with active-site-modified thrombin did not affect thrombin-induced (0.5 nM) or Quick I-induced (5 nM) responses. In contrast, addition of D-Phe-Pro-Arg chloromethyl ketone (FPRCK) subsequent to thrombin or Quick I stimulation of platelets abolished agonist-induced responses. Since Quick I was only 10-17% as effective as thrombin in increasing intraplatelet [Ca2+], our data support a model in which thrombin acts enzymatically on a platelet membrane "substrate", through an interaction mediated in part by the fibrinogen binding groove of thrombin. This conclusion is consistent with the inhibition observed with high concentrations (greater than 100 nM) of Quick II and FPRCK-modified thrombin (FPR-thrombin) in platelets stimulated with low concentrations of thrombin (less than 0.5 nM) or Quick I (less than 2 nM), consistent with inhibition by substrate depletion. In contrast, concentrations of FPR-thrombin or Quick II (less than 100 nM), which saturated predominantly the high-affinity binding sites, enhanced the platelet responses induced by thrombin (less than 0.5 nM). Thus, occupation of the high-affinity sites with inactive thrombin increased the concentration of active thrombin available for substrate interaction. Quick I-induced responses were not enhanced, consistent with its inability to interact with the high-affinity site. Since thrombin bound to the high-affinity site is proteolytically inactive, we hypothesize that the thrombin high-affinity binding site on platelets functions to alter thrombin activity and platelet activation.     

The high-affinity binding of laminin to cells. Assignation of a major cell-binding site to the long arm of laminin and of a latent cell-binding site to its short arms

The laminin proteolytic fragments 1 (derived from the intersection of the short arms of the cruciform laminin molecule) and 8 (derived from the laminin long arm) bind to distinct receptors on HT-1080 human fibrosarcoma cells; both fragments are shown here to inhibit the high-affinity binding of laminin to these cells. Inhibition of binding between fragment 8 and laminin was competitive, whereas that between fragment 1 and laminin was noncompetitive. This indicates that laminin and fragment 8 most probably share the same cellular receptors, whereas laminin and fragment 1 bind to distinct receptors, inhibition being due to steric hindrance. Surprisingly, fragment 1-4 (corresponding to the complete short arms of laminin) neither bound to HT-1080 cells nor inhibited the binding of laminin or fragment 1. After treatment of fragment 1-4 with pepsin, however, the smaller subfragment 1 was liberated, which could then bind to the cells, and so was shown to block the binding of laminin and fragment 1. We conclude that native laminin bound to HT-1080 cells via the fragment-8-binding site near the end of its long arm. Although these cells also have distinct receptors for the short arm fragment 1, this receptor-binding site was not used as it appeared to be latent within the native laminin molecule.     

The occupancy of glycoprotein IIb-IIIa complex modulates thrombin activation of human platelets

Platelet membrane glycoprotein (GP IIb-IIIa), besides its activity as adhesive protein receptor, displays a number of properties supporting its involvement in the mechanisms of transduction of the activation signal. Recently we have observed that GP IIb-IIIa ligands, mostly fibrinogen, inhibit Ca2+ movement and cytoskeleton reorganization caused by mild platelet activation. These findings led us to investigate the effect of GP IIb-IIIa ligands on agonist-induced platelet responses, with particular attention to the two major messenger generating systems, involving the activation of phospholipase C and the inhibition of cAMP production. In this paper we demonstrate that the occupancy of the major adhesive protein receptor on the platelet surface modulates the phosphatidylinositol cycle decreasing the amount of IP3, IP2 and IP produced after mild platelet activation as well as the pattern of protein phosphorylation. The platelet cAMP content of activated platelets was also affected and kept higher when evaluated under the same experimental conditions. Our data provide evidence for a role of fibrinogen binding in regulating the degree of activation of circulating platelets.     

Spacial isolation of protein kinase C activation in thrombin stimulated human platelets

Thrombin stimulation of human platelets is associated with turnover of inositol phospholipids, mobilization of intracellular Ca2+ stores, and activation of protein kinase C. However, within 5 minutes, the thrombin receptor desensitizes, but can be re-coupled to its effectors by stimulation of alpha 2-adrenergic receptors (Crouch and Lapetina, J. Biol. Chem. 263, 3363-3371, 1988). This effect of epinephrine was found to be inhibited by preincubation of platelets with phorbol ester, suggesting that protein kinase C was inhibitory. However, since thrombin also activated protein kinase C and epinephrine was active following thrombin stimulation of platelets, this implied that thrombin activation of protein kinase C may have been spacially isolated near the thrombin receptor and could not inactivate alpha 2-receptor activity. In the present paper, we have tested this possibility, and we present evidence which strongly favours the possibility that protein kinase C activation by receptors induces its local translocation to the cell membrane.     

Acetylated prothrombin as a substrate in the measurement of the procoagulant activity of platelets: elimination of the feedback activation of platelets by thrombin.

Human prothrombin was acetylated to produce a modified prothrombin that upon activation by platelet-bound prothrombinase generates a form of thrombin that does not activate platelets but retains its amidolytic activity on a chromogenic peptide substrate. If normal prothrombin is used in such an assay, the thrombin that is generated activates the platelets in a feedback manner, accelerating the rate of thrombin generation and thereby preventing accurate measurement of the initial platelet procoagulant activity. Acetylation of prothrombin was carried out over a range of concentrations of sulfo-N-succinimidyl acetate (SNSA). Acetylation by 3 mM SNSA at room temperature for 30 min at pH 8.2 in the absence of metal ions produced a modified prothrombin that has <0.1% clotting activity (by specific prothrombin clotting assay), but it is activated by factor Xa (in the presence of either activated platelets or factor Va + anionic phospholipid) to produce thrombin activity that is measurable with a chromogenic substrate. Because the feedback action on the platelets is blocked, thrombin generation is linear, allowing quantitative measurement of the initial platelet activation state.     

The plasma membrane Ca2+ pump contains a site that interacts with its calmodulin-binding domain.

A synthetic, 28-residue peptide derived from the calmodulin-binding sequence of the plasma membrane Ca2+ pump (C28W) inhibits the ATPase activity of a calpain-produced, truncated fragment of the enzyme. The fragment, which has lost the calmodulin-binding domain, has a molecular mass of 124 kDa and is fully active in the absence of calmodulin. Replacement of Trp-8 in the peptide by an Ala decreases the overall inhibitory activity, while replacement with a Tyr increases it. However, at very low peptide concentrations the effect of Tyr replacement disappears. The synthetic peptide has been made photoactivatable by replacing Phe in position 9 with a synthetic phenylalanine analogue containing a diazirine group and was radioactively labeled by coupling a [3H]acetyl function to its N terminus. After cross-linking with the derivatized peptide, the 124-kDa fragment has been proteolyzed with either Lys-C, Asp-N, or V8 proteases, and the fragment(s) have been separated. Partial sequencing of the cross-linked, radioactive peptides has identified a site of the pump located C terminally to the phosphoenzyme-forming aspartic acid, spanning residues 537-544 of the hPMCA4 isoform of the enzyme. It is concluded that this sequence is part of a site which binds the calmodulin-binding domain of the pump.     

Vitronectin is phosphorylated by a cAMP-dependent protein kinase released by activation of human platelets with thrombin

Activation of freshly isolated human platelets with a physiological stimulant (thrombin) causes them to release a cAMP-dependent protein kinase which specifically phosphorylates one plasma protein (Mr 75000). This protein is immunochemically and biochemically identified as vitronectin (also know as S protein), which was previously implicated in blood clotting, complement function and cell adhesion.     

Differential hydrolysis of phospholipid molecular species during activation of human platelets with thrombin and collagen

Mass changes in the various molecular species of phospholipids were determined after stimulation of human platelets with thrombin and collagen. Upon stimulation, every molecular species of phosphatidylinositol and phosphatidylserine was equally hydrolyzed, whereas the molecular species of phosphatidylcholine and diacyl- and alkenylacylphosphatidylethanolamine containing arachidonic acid were selectively hydrolyzed. At low Ca2+ concentrations, which result from mobilization of intracellular Ca2+ stores, phosphatidylinositol, phosphatidylcholine, and diacylphosphatidylethanolamine were hydrolyzed after stimulation with thrombin, whereas only phosphatidylinositol was hydrolyzed with production of thromboxane B2 after stimulation with collagen. At high Ca2+ concentrations, phosphatidylcholine and diacylphosphatidylethanolamine were hydrolyzed after stimulation with collagen, and phosphatidylserine and alkenylacylphosphatidylethanolamine were degraded after stimulation with both thrombin and collagen. [1-14C]Arachidonic acid was heterogeneously incorporated into the individual molecular species of the various phospholipid classes, indicating that the determination of mass is essential for an accurate picture of phospholipid hydrolysis. The data reported here indicate that the Ca2+ concentration affects the differential degradation of phospholipid molecular species in activated human platelets.     

The promoter of the tgt/sec operon in Escherichia coli is preceded by an upstream activation sequence that contains a high affinity FIS binding site.

The tgt/sec operon in E. coli consists of five genes: queA, tgt, ORF12, secD, and secF. QueA and Tgt participate in the biosynthesis of the hypermodified t-RNA nucleoside Queuosine, whereas SecD and SecF are involved in protein secretion. Examination of the promoter region of the operon showed structural similarity to promoter regions of the rrn-operons. An upstream activation sequence (UAS) containing a potential binding site for the factor of inversion stimulation (FIS) was found. Gel retardation assays and DNaseI footprinting indicated, that FIS binds specifically and with high affinity to a site centred at position -58. Binding of FIS caused bending of the DNA, as deduced from circular permutation analysis. Various 5' deletion mutants of the promoter region were constructed and fused to a lacZ reporter gene to determine the influence of the UAS element on the promoter strength. An approximately two-fold activation of the promoter by the UAS element was observed.     

Nitric oxide inhibits thrombin receptor-activating peptide-induced phosphoinositide 3-kinase activity in human platelets.

Although nitric oxide (NO) has potent antiplatelet actions, the signaling pathways affected by NO in the platelet are poorly understood. Since NO can induce platelet disaggregation and phosphoinositide 3-kinase (PI3-kinase) activation renders aggregation irreversible, we tested the hypothesis that NO exerts its antiplatelet effects at least in part by inhibiting PI3-kinase. The results demonstrate that the NO donor S-nitrosoglutathione (S-NO-glutathione) inhibits the stimulation of PI3-kinase associated with tyrosine-phosphorylated proteins and of p85/PI3-kinase associated with the SRC family kinase member LYN following the exposure of platelets to thrombin receptor-activating peptide. The activation of LYN-associated PI3-kinase was unrelated to changes in the amount of PI3-kinase physically associated with LYN signaling complexes but did require the activation of LYN and other tyrosine kinases. The cyclic GMP-dependent kinase activator 8-bromo-cyclic GMP had similar effects on PI3-kinase activity, consistent with a model in which the cyclic nucleotide mediates the effects of NO. Additional studies showed that wortmannin and S-NO-glutathione have additive inhibitory effects on thrombin receptor-activating peptide-induced platelet aggregation and the surface expression of platelet activation markers. These data provide evidence of a distinct and novel mechanism for the inhibitory effects of NO on platelet function.     

Characteristics of a thrombin inhibitor secreted by activated platelets

A 77-kDa complex of thrombin and a protein secreted by activated platelets had little if any thrombin amidolytic activity, indicating that the secreted protein is an inhibitor. The molecular weight of the inhibitor before reaction with thrombin was approximately 50,000. The apparent second-order rate constant for complex formation was estimated to be 1.3 x 10(6) M-1 s-1 (mean of four measurements); it was not affected by heparin or heparinase. These properties distinguish this inhibitor from other protease inhibitors secreted by platelets. The inhibitor reacted with trypsin and possibly with urokinase but not with factor Xa.     

Biphasic kinetics of activation and signaling for PAR1 and PAR4 thrombin receptors in platelets

Thrombin activates platelets in an ordered sequence of events that includes shape change, increase in cytoplasmic Ca(2+), activation of the alphaIIbbeta3 integrin, granule secretion, aggregation, and formation of a stable hemostatic plug. Activation of this process has also been implicated in the pathogenesis of atherosclerosis, stroke, and thrombosis. There are two identified thrombin-activated receptors on the surface of human platelets. PAR1 is a high-affinity thrombin receptor, and PAR4 is a low apparent affinity thrombin receptor of uncertain function. The goal of these studies is to determine the kinetics of thrombin activation of PAR1 and PAR4 and to relate the individual inputs from each receptor to platelet Ca(2+) signaling, secondary autocrine stimulation, and aggregation. Using a combination of PAR-specific peptide ligands and anti-PAR1 reagents, we separated the biphasic thrombin Ca(2+) response of platelets into two discrete components-a rapid spike response caused by PAR1, followed by a slower prolonged response from PAR4. Despite having a 20-70-fold slower rate of activation, PAR4 produces the majority of the integrated Ca(2+) signal that is sustained by the continuous presence of catalytically active thrombin. Surprisingly, PAR4 activation is much more effective than PAR1 activation in mounting secondary autocrine Ca(2+) signals from secreted ADP. The strong ADP response due to activated PAR4, however, requires prior activation of PAR1 as would normally occur during treatment of platelets with thrombin. Thus, the late signal generated by activated PAR4 is not redundant with the early signal from PAR1 and instead serves to greatly extend the high intracellular Ca(2+) levels that support the late phase of the platelet aggregation process.