Secondary signals mediated by GPIIb/IIIa in thrombin-activated platelets

We have previously found that stimulation of aequorin-loaded platelets by thrombin produced a two-peaked increase in intracellular free calcium concentration ([Ca2+]i), and the development of the second peak of [Ca2+]i was closely related with the aggregation. In this report, we studied the interrelationship between the GPIIb/IIIa complex, aggregation, cytoskeletons and [Ca2+]i of platelets. The pretreatment of the platelets with dihydrocytochalasin B (4 microM), an actin polymerization inhibitor, did not inhibit aggregation and TXB2 production, but did inhibit both actin polymerization and the second peak of [Ca2+]i increase induced by thrombin, suggesting that actin polymerization and the second peak of [Ca2+]i are interrelated. GRGDSP (100 microM), a synthetic anti-adhesive peptide, has already been reported to inhibit platelet aggregation and the second peak of [Ca2+]i induced by thrombin. It also inhibited actin polymerization and TXB2 production, suggesting that aggregation was important for not only the generation of the second peak of [Ca2+]i but also for actin polymerization and TXB2 production. PGI2 (5 nM) did not abolish but only delayed aggregation, TXB2 production, actin polymerization and the second peak of [Ca2+]i increase. These findings suggest that the secondary signals are caused by aggregation (fibrinogen-binding to the GPIIb/IIIa) in thrombin-aggregated platelets, which results in the TXA2 production and the secondary peak of [Ca2+]i increase, and the latter was dependent on actin polymerization.     

Phosphoinositide turnover in human platelets is stimulated by albumin.

The effect of human serum albumin (HSA) on the hydrolysis of phosphatidylinositides in human platelets labeled with myo(3H)inositol was studied. Incubation of platelets with HSA (4 gm/dl) for 10 seconds increased IP2, and IP3, by 169% and 217% respectively. 93% of IP3 accumulated within the first 10 seconds. This effect was also shared by bovine serum albumin, although no changes in IP3 levels occurred with ovalbumin. All albumin species used induced 45Ca+2 release from platelets irrespective of its effect on IP3 accumulation. These findings indicate that albumin may function in biological systems by inducing intracellular signaling.     

Diglyceride/monoglyceride lipases pathway is not essential for arachidonate release in thrombin-activated human platelets

Human platelets prelabeled with arachidonate exhibited a rapid and transient rise in arachidonoyl monoglyceride in addition to arachidonoyl diglyceride following thrombin stimulation. Substantial release of arachidonate and its metabolites also occurred at the early phase. Preincubation of labeled platelets with RHC 80267, a potent inhibitor of diglyceride lipase, prior to thrombin stimulation abolished the transient rise in monoglyceride but not the increase in diglyceride and the release of arachidonate and its metabolites. These results suggest that diglyceride does metabolize to monoglyceride and release arachidonate in intact platelets. However, the diglyceride/monoglyceride lipases pathway does not appear to be essential in releasing arachidonate during thrombin stimulation.     

Activated platelets form protected zones of adhesion on fibrinogen and fibronectin-coated surfaces

Leukocytes form zones of close apposition when they adhere to ligand- coated surfaces. Because plasma proteins are excluded from these contact zones, we have termed them protected zones of adhesion. To determine whether platelets form similar protected zones of adhesion, gel-filtered platelets stimulated with thrombin or ADP were allowed to adhere to fibrinogen- or fibronectin-coated surfaces. The protein- coated surfaces with platelets attached were stained with either fluorochrome-conjugated goat anti-human fibrinogen or anti-human fibronectin antibodies, or with rhodamine-conjugated polyethylene glycol polymers. Fluorescence microscopy revealed that F(ab')2 anti- fibrinogen (100 kD) did not penetrate into the contact zones between stimulated platelets and the underlying fibrinogen-coated surface, while Fab antifibrinogen (50 kD) and 10 kD polyethylene glycol readily penetrated and stained the substrate beneath the platelets. Thrombin- or ADP-stimulated platelets also formed protected zones of adhesion on fibronectin-coated surfaces. F(ab')2 anti-fibronectin and 10 kD polyethylene glycol were excluded from these adhesion zones, indicating that they are much less permeable than those formed by platelets on fibrinogen-coated surfaces. The permeability properties of protected zones of adhesion formed by stimulated platelets on surfaces coated with both fibrinogen and fibronectin were similar to the zones of adhesion formed on fibronectin alone. mAb 7E3, directed against the alpha IIb beta 3 integrin blocked the formation of protected adhesion zones between thrombin-stimulated platelets and fibrinogen or fibronectin coated surfaces. mAb C13 is directed against the alpha 5 beta 1 integrin on platelets. Stimulated platelets treated with this mAb formed protected zones of adhesion on surfaces coated with fibronectin. These protected zones were impermeable to F(ab')2 antifibronectin but were permeable to 10 kD polyethylene glycol. These results show that activated platelets form protected zones of adhesion and that the size of molecules excluded from these zones depends upon the composition of the matrix proteins to which the platelets adhere. They also show that formation of protected zones of adhesion by platelets requires alpha IIb beta 3 integrins while the permeability properties of these zones of adhesion are regulated by both alpha IIb beta 3 and alpha 5 beta 1 integrins.     

Integrin-dependent translocation of phosphoinositide 3-kinase to the cytoskeleton of thrombin-activated platelets involves specific interactions of p85 alpha with actin filaments and focal adhesion kinase

Thrombin-induced accumulation of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) but not of PtdIns(3,4,5,)P3 is strongly correlated with the relocation to the cytoskeleton of 29% of the p85 alpha regulatory subunit of phosphoinositide 3-kinase (PtdIns 3-kinase) and is accompanied by a significant increase in PtdIns 3-kinase activity in this subcellular fraction. Actually, PtdIns(3,4)P2 accumulation and PtdIns 3-kinase, pp60c-src, and p125FAK translocations as well as aggregation were concomitant events occurring with a distinct lag after actin polymerization. The accumulation of PtdIns(3,4)P2 and the relocalization of PtdIns 3-kinase to the cytoskeleton were both dependent on tyrosine phosphorylation, integrin signaling, and aggregation. Furthermore, although p85 alpha was detected in anti- phosphotyrosine immunoprecipitates obtained from the cytoskeleton of thrombin-activated platelets, we failed to demonstrate tyrosine phosphorylation of cytoskeletal p85 alpha. Tyrphostin treatment clearly reduced its presence in this subcellular fraction, suggesting a physical interaction of p85 alpha with a phosphotyrosyl protein. These data led us to investigate the proteins that are able to interact with PtdIns 3-kinase in the cytoskeleton. We found an association of this enzyme with actin filaments: this interaction was spontaneously restored after one cycle of actin depolymerization-repolymerization in vitro. This association with F-actin appeared to be at least partly indirect, since we demonstrated a thrombin-dependent interaction of p85 alpha with a proline-rich sequence of the tyrosine-phosphorylated cytoskeletal focal adhesion kinase, p125FAK. In addition, we show that PtdIns 3-kinase is significantly activated by the p125FAK proline-rich sequence binding to the src homology 3 domain of p85 alpha subunit. This interaction may represent a new mechanism for PtdIns 3-kinase activation at very specific areas of the cell and indicates that the focal contact-like areas linked to the actin filaments play a critical role in signaling events that occur upon ligand engagement of alpha IIb/beta 3 integrin and platelet aggregation evoked by thrombin.     

Dopamine-induced toxicity is synergistically potentiated by simultaneous HSP-90 and Akt inhibition in oligodendrocyte progenitors

Oligodendrocyte progenitors are highly susceptible to various insults. Their limited antioxidant defenses and high levels of apoptotic factors, such as Bax and pro-caspase-3 contribute to their sensitivity. We previously showed that dopamine (DA) is toxic to oligodendrocyte progenitors by inducing superoxide generation, lowering glutathione levels and promoting apoptosis through caspase-3 activation. In contrast, factors that contribute to cell survival and defense against dopamine (DA) toxicity are less studied. Here, we explored the role of two molecules which play important roles in cell survival, namely the heat shock protein 90 (HSP-90) and the protein kinase Akt, using the selective inhibitors, 17-AAG and Akt inhibitor III, respectively. The HSP-90 inhibitor caused a decrease in P-Akt level, induced caspase-3 activation, increased nuclear condensation and caused a loss in cell viability. Furthermore, 17-AAG potentiated DA-induced apoptosis by enhancing caspase-3 activation. In addition, the Akt inhibitor alone exacerbated DA toxicity and in combination with 17-AAG caused synergistic potentiation of DA toxicity by enhancing caspase-3 activation. Together, these results indicate that HSP-90 is essential for oligodendrocyte progenitor survival. Both HSP-90 and Akt play important roles in concert in the defense against DA-induced apoptosis.     

Protein kinase C phosphorylation of syntaxin 4 in thrombin-activated human platelets

We postulated that the syntaxins, because of their key role in SNARE complex formation and exocytosis, could be important targets for signaling by intracellular kinases involved in secretion. We found that syntaxin 4 was phosphorylated in human platelets treated with a physiologic agent that induces secretion (thrombin) but not when they were treated with an agent that prevents secretion (prostacyclin). Syntaxin 4 phosphorylation was blocked by inhibitors of activated protein kinase C (PKC), and, in parallel assays, PKC inhibitors also blocked secretion from thrombin-activated platelets. In platelets, cellular activation by thrombin or phorbol 12-myristate 13-acetate decreased the binding of syntaxin 4 with SNAP-23, another platelet t-SNARE. Phosphatase inhibitors increased syntaxin 4 phosphorylation and further decreased syntaxin 4-SNAP-23 binding induced by cell activation. Conversely, a PKC inhibitor blocked syntaxin 4 phosphorylation and returned binding of syntaxin 4-SNAP-23 to that seen in nonstimulated platelets. In vitro, PKC directly phosphorylated platelet syntaxin 4 and recombinant syntaxin 4. PKC phosphorylation in vitro inhibited (71 +/- 8%) the binding of syntaxin 4 to SNAP-23. These results provide evidence that extracellular activation can be coupled through intracellular PKC signaling so as to modulate SNARE protein interactions involved in platelet exocytosis.     

Measurements of growth cone adhesion to culture surfaces by micromanipulation

Neurons were grown on plastic surfaces that were untreated, or treated with polylysine, laminin, or L1 and their growth cones were detached from their culture surface by applying known forces with calibrated glass needles. This detachment force was taken as a measure of the force of adhesion of the growth cone. We find that on all surfaces, lamellipodial growth cones require significantly greater detachment force than filopodial growth cones, but this differences is, in general, due to the greater area of lamellipodial growth cones compared to filopodial growth cones. That is, the stress (force/unit area) required for detachment was similar for growth cones of lamellipodial and filopodial morphology on all surfaces, with the exception of lamellipodial growth cones on L1-treated surfaces, which had a significantly lower stress of detachment than on other surfaces. Surprisingly, the forces required for detachment (760-3,340 mudynes) were three to 15 times greater than the typical resting axonal tension, the force exerted by advancing growth cones, or the forces of retraction previously measured by essentially the same method. Nor did we observe significant differences in detachment force among growth cones of similar morphology on different culture surfaces, with the exception of lamellipodial growth cones on L1-treated surfaces. These data argue against the differential adhesion mechanism for growth cone guidance preferences in culture. Our micromanipulations revealed that the most mechanically resistant regions of growth cone attachment were confined to quite small regions typically located at the ends of filopodia and lamellipodia. Detached growth cones remained connected to the substratum at these regions by highly elastic retraction fibers. The closeness of contact of growth cones to the substratum as revealed by interference reflection microscopy (IRM) did not correlate with our mechanical measurements of adhesion, suggesting that IRM cannot be used as a reliable estimator of growth cone adhesion.     

Cholecystokinin-induced hypophagia is not potentiated by cancer anorexia

The interaction of cholecystokinin-induced hypophagia with cancer anorexia was investigated within both acute (Walker 256 carcinosarcoma) and chronic (methylcholanthrene-induced sarcoma) animal models of cancer anorexia. Cholecystokinin octapeptide (CCK8) effectively reduced feeding for at least one hour in both groups of rats. However, this peptide was no more effective in inducing hypophagia in tumor-bearing rats than in nontumor-bearing control rats when tested at a variety of doses (0.5, 5.0 and 50.0 microgram/kg; IP) both prior to and after the development of anorexia. Therefore, these data do not support a role of cholecystokinin in the mediation of experimental cancer anorexia, since no synergism of CCK8-induced hypophagia with the anorexia was observed.     

The effect of plasma von Willebrand factor on the binding of human factor VIII to thrombin-activated human platelets

The binding of 35S-labeled recombinant human Factor VIII to activated human platelets was studied in the presence and absence of exogenous plasma von Willebrand factor. In the absence of added von Willebrand Factor, platelets bound 210 molecules of Factor VIII/platelet when the unbound Factor VIII concentration was 2.0 nM (Kd = 2.9 nM). As the von Willebrand factor concentration was increased, the number of Factor VIII molecules bound/platelet decreased to 10 molecules of Factor VIII bound/platelet at 24 micrograms/ml of added vWF. Addition of an anti-vWF monoclonal antibody that inhibits the vWF-Factor VIII interaction attenuated the ability of vWF to inhibit binding of Factor VIII to platelets. In contrast, addition of a control anti-vWF antibody that does not block the vWF-Factor VIII interaction did not affect the ability of vWF to inhibit Factor VIII binding to platelets. From the vWF concentration dependence of inhibition of Factor VIII-platelet binding, a dissociation constant for the Factor VIII-vWF interaction was calculated (Kd = 0.44 nM). To further elucidate the role that vWF may play in preventing the interaction of Factor VIII with platelets, the platelet binding properties of a Factor VIII deletion mutant (90-73) which lacks the primary vWF-binding site was studied. The binding of this mutant was unaffected by added exogenous vWF. These observations demonstrate that Factor VIII can interact with platelets in a manner independent of vWF but that excess vWF in plasma can effectively compete with platelets for the binding of Factor VIII. In addition, since cleavage of Factor VIII by thrombin separates a vWF-binding domain from Factor VIIIa, we propose that activation of Factor VIII by thrombin may elicit release of activated Factor VIII from vWF and thereby make it fully available for platelet binding.     

Photoreversible surfaces to regulate cell adhesion

We report the development of a photoreversible cell culture substrate. We demonstrate the capacity to modify the adhesivity of the substrate using light, altering its capacity to support cell growth. Polyelectrolyte multilayers (PEMs) were used to produce tunable substrates of different thickness and matrix stiffness, which have different intrinsic capacities to support cell adhesion and survival. Surfaces were top-coated with a poly(acrylic acid)-poly(allylamine hydrochloride) polyelectrolyte bilayer functionalized with a small fraction (<1%) of an azobenzene-based photoswitchable sidegroup, which included the cell-adhesive three-amino-acid peptide RGD. Irradiation with light-induced geometric switching of the azo bond, resulting in changes to RGD exposure and consequently to cell adhesion and survival, was investigated on a variety of surfaces of different thickness and stiffness. Substrate stiffness, as modified by the thickness, had a significant influence on the adhesion of NIH 3T3 cells, consistent with previous studies. However, by disrupting the isomerization state of the azobenzene-linked RGD and exposing it to the surface, cell adhesion and survival could be enhanced up to 40% when the positioning of the RGD peptide was manipulated on the softest substrates. These findings identify permissive, yet less-than-optimal, cell culture substrate conditions that can be substantially enhanced using noninvasive modification of the substrate triggered by light. Indeed, where cell adhesion was tuned to be suboptimal under baseline conditions, the light-induced triggers displayed the most enhanced effect, and identification of this 'Goldilocks zone' was key to enabling light triggering.     

Cytochalasin inhibition of hexose transport by platelets

Previously we described a two-transporter model (T1, T2) for galactose uptake by platelets (Horne, M.K. and Hart, J.S. (1986) Biochim. Biophys. Acta 856, 448-456). In the current work we have sought corroborative evidence for this model by studying the effects of cytochalasins on this transport system. Of the various cytochalasins tested, cytochalasin B was the most potent inhibitor (I) of galactose transport, whereas cytochalasin A was less inhibitory and dihydrocytochalasin B and cytochalasin E had no inhibitory effect. The same order of potency was observed for the inhibition of L-glucose diffusion into platelets. The mechanism of cytochalasin B inhibition was investigated in detail. Inhibition of T1 was competitive and required a higher concentration of cytochalasin B (Ki1 approximately 1.7 microM) than inhibition of T2, which was of a mixed type (Ki2 approximately 0.8 microM). The effect of cytochalasin B on T2 could be accounted for by a membrane alteration which enhanced the affinity of the transporter for galactose while simultaneously preventing passage of the TSI complex into the cell. Since a similar effect on membrane permeability would also explain cytochalasin B inhibition of L-glucose diffusion, it is hypothesized that cytochalasin B binds to a membrane structure shared by T2 and the passage for L-glucose. The differences in cytochalasin B sensitivity and mechanism of inhibition manifested by T1 and T2 support our original hypothesis that galactose is indeed transported by kinetically distinct agencies and suggest that these may be physically distinct as well.     

Lysis of Escherichia coli by glycine is potentiated by pyridoxine starvation

Pyridoxineless mutants of Escherichia coli are lysed in a few hours when starved for pyridoxine in a glucose minimal medium containing glycine at 10 mM. The lysis is prevented equally well by l-alanine and by d-alanine when either is present at 0.1 mM. The lysis is potentiated by 0.5 mM l-methionine. The peculiar susceptibility of E. coli B to glycine-mediated lysis during starvation for pyridoxine suggests that the starvation reduces the availability of some normal antagonist of glycine, presumably alanine.     

Evidence for the selective association of a subpopulation of GPIIb-IIIa with the actin cytoskeletons of thrombin-activated platelets

Activation of blood platelets triggers a series of responses leading to the formation and retraction of blood clots. Among these responses is the establishment of integrin-mediated transmembrane connections between extracellular matrix components and the actin cytoskeleton of the platelet. Here we report that a specific subpopulation of the major platelet integrin, glycoprotein IIb-IIIa (GPIIb-IIIa) (also referred to as alpha IIb beta 3 integrin), becomes incorporated into the detergent- insoluble actin cytoskeleton of platelets during the platelet activation response. The cytoskeletal association of GPIIb-IIIa is independent of platelet aggregation and fibrin sedimentation and is sensitive to cytochalasin D treatment. As determined by Western immunoblot analysis, approximately 22% of the total cellular GPIIb-IIIa becomes associated with the actin cytoskeleton upon thrombin activation in a manner that is independent of the detection of talin, alpha- actinin, or vinculin in the complex. We found that the cytoskeleton- associated GPIIb-IIIa is derived from an intracellular source since it is not available for lactoperoxidase-catalyzed radioiodination before platelet activation. Two intracellular sources of GPIIb-IIIa are present in resting platelets: GPIIb-IIIa associated with the alpha- granule secretory compartment as well as surface-inaccessible domains of the surface-connected canalicular system. Interestingly, alpha- granule secretion, which occurs in thrombin-activated platelets and results in the translocation of intracellular GPIIb-IIIa to the plasma membrane, appears to be required for the cytoskeleton incorporation of GPIIb-IIIa that we observe. Collectively, our data provide evidence that a subpopulation of GPIIb-IIIa derived from an intracellular source is selectively linked to the actin cytoskeleton of platelets upon thrombin activation in the absence of platelet aggregation.     

Evidence for coupling of phospatidic acid formation and calcium influx in thrombin-activated human platelets

The time-sequential relationship between Ca²⁺ flux, phospholipid metabolism and platelet activation have been examined. Thrombin-activation caused a marked enhancement in ⁴⁵Ca²⁺ influx and a decrease in extracellular Ca²⁺ concentration measured by murexide dye, which occurred in parallel with the conversion of 1,2-diacylglycerol (DG) to phosphatidic acid (PA). The incorporated ⁴⁵Ca²⁺ was located mainly in cytosolic fraction. The influx of Ca²⁺ was observed to commence prior to the onset of lysophospholipids formation and subsequent liberation of arachidonic acid. These data provide evidence which indicates a coupling between the rapid PI-turnover and the active Ca²⁺ influx, in which phosphatidic acid (PA) may serve as a Ca²⁺ ionophore.     

A cardiac specific analog of angiotensin I potentiated by converting enzyme inhibition

[1-Sarcosine, 7-Alanine] angiotensin I [( 1-Sar, 7-Ala] AI) and closely related analogs were tested for inotropic activity in the isolated cat heart, and for pressor activity in the intact conscious sheep both before and during converting enzyme inhibition (CEI). [1-Sar, 7-Ala] AI exhibited potent inotropic activity but was only weakly pressor. [1-Sar] AI, [1-Sar, 5-Val] AI, [1-Sar, 7-alpha MeAla] AI [1-Sar, 5-Val, 7-NMeAla] AI and [1-Sar, 5-Val, 7-Sar] were all potent agonists in both preparations. The action of [1-Sar, 7-Ala] AI was potentiated by CEI in both the isolated heart and the intact sheep. The activity of the remaining analogs was either partially or completely blocked by CEI. The activity of all analogs was inhibited by AII receptor blockade. These data indicate that the nature of the substitution in position 7 determines the affinity of the analog for converting enzyme. The [7-Ala] substitution appears to decrease the effect of the analog upon vascular receptors.     

MPP(+)-induced mitochondrial dysfunction is potentiated by dopamine

MPP(+), the major metabolite of the Parkinsonism-inducing compound MPTP, responsible for the destruction of the nigrostriatal pathway in primates and rodents, has been assayed in isolated rat liver mitochondria in the presence of physiological concentrations of dopamine or analogous concentrations of melanin-dopamine. 5 microM MPP(+) in the presence of 70 microM dopamine or melanin-dopamine, but not alone, decreased the heat production and oxygen consumption of a mitochondrial suspension activated with succinate and ADP. Both dopamine and oxidized dopamine plus MPP(+) also decreased the mitochondrial reductive power measured with MTT. Mitochondrial swelling was observed, associated with an increase in membrane mitochondrial potential, as a synergistic effect between low concentrations of MPP(+) and dopamine. It is suggested that cytosolic dopamine, by itself or via its autooxidation products, may play a relevant role in the mitochondrial toxicity of MPP(+). A failure in the regulation of the storage/release of dopamine could aggravate a mitochondrial damage and trigger the neurodegenerative process underlying MPTP toxicity and Parkinson's disease.     

Synthesis of phosphatidylinositol 3,4-bisphosphate but not phosphatidylinositol 3,4,5-trisphosphate is closely correlated with protein-tyrosine phosphorylation in thrombin-activated human platelets.

Synthesis of D-3-phosphorylated phosphoinositides and its correlation with protein-tyrosine phosphorylation were examined using human platelets. Thrombin stimulation of platelets resulted in time- and dose-dependent production of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2), which is absent from resting platelets. In contrast, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) was detected in resting platelets, but remained unaffected by thrombin treatment. The production of PtdIns(3,4)P2 but not PtdIns(3,4,5)P3 was inhibited by pretreatment with staurosporine or dibutyryl cyclic adenosine monophosphate (dbcAMP). Protein-tyrosine phosphorylation, which is reportedly involved in generation of 3-phosphorylated phosphoinositides, was elicited in thrombin-activated platelets. The tyrosine phosphorylation was suppressed by pretreatment with staurosporine or dbcAMP. These observations suggest that synthesis of PtdIns(3,4)P2 but not PtdIns(3,4,5) P3 is closely correlated with protein-tyrosine phosphorylation in human platelets.