Diacylglycerol elevations in control platelets are unaccompanied by pleckstrin phosphorylation. Implications for the role of diacylglycerol in platelet activation

Several laboratories have reported that diacylglycerol levels in human platelets (approximately 100 pmol/10(9) platelets) increased severalfold in response to 0.5-1 U/ml thrombin. We report here fluctuations in diacylglycerol mass in control platelets, the magnitude of which were 60-90% of that measured in platelets treated with 0.2-0.5 U/ml of thrombin. These control platelets were not activated by such criteria as absence of aggregation, secretion, phosphatidic acid production and phosphorylation of the protein kinase C substrate, pleckstrin. Thrombin treatment evoked all of the above responses. Analysis of the diacylglycerol molecular species by reverse-phase HPLC of the dimethylated, phosphorylated derivatives showed that all of the molecular species that were present in control platelets were also present in thrombin-treated platelets. Most of the species appeared to fluctuate at random in control platelets with the exception of 1-stearoyl-2-arachidonoyl-sn-glycerol which was more or less stable and increased severalfold over control values only upon thrombin treatment. Furthermore, only this species accumulated as [32P]phosphorylated PtdOH in thrombin-treated platelets prelabelled with [32P]Pi. Our findings show that, in platelets, elevation of diacylglycerol molecular species other than the 1-stearoyl-2-arachidonoyl species occurs, but these changes are not necessarily linked to activation of protein kinase C as measured by pleckstrin phosphorylation which was observed only upon elevation of 1-stearoyl-2-arachidonoyl-sn-glycerol.     

Structure/function analyses of recombinant variants of human factor Xa: factor Xa incorporation into prothrombinase on the thrombin-activated platelet surface is not mimicked by synthetic phospholipid vesicles

This report describes the expression, purification, and characterization of a series of recombinant factor Xa variants bearing aspartate substitutions for each of the glutamate residues which normally undergo gamma-carboxylation. Factor X was expressed in human embryonic kidney cells and purified from conditioned media by immunoaffinity and hydroxylapatite chromatography. Factor X was activated with Russell's viper venom factor X activator, and single-chain unactivated factor X was removed from activated factor X by size-exclusion chromatography. Recombinant wild-type factor Xa had normal activity in a clotting assay, and mutants with aspartate substitutions for glas residues 16, 26, and 29 had no detectable clotting activity. In purified component assays, these gla variants had essentially no detectable activity in the prothrombinase complex assembled on synthetic phospholipid vesicles but had significant activity when the prothrombinase was assembled on thrombin-activated platelets. In addition, the gla 32 variant had normal activity in the platelet prothrombinase but diminished activity in prothrombinase assembled on synthetic PSPC vesicles. These differences were not accounted for by the total phospholipid composition of the thrombin-activated platelet membrane. We have produced fully active recombinant human factor Xa and demonstrated that gla residues 16, 26, and 29 are critical for normal activity of factor Xa. More importantly, this study provides an extensive characterization of macromolecular enzyme complex formation with gla variants of a vitamin K-dependent coagulation protein and provides evidence that prothrombinase complex assembly on thrombin-activated platelets is not equivalent to assembly on synthetic phospholipid vesicles. The data suggest that thrombin-activated platelets possess some element(s) (other than 30% phosphatidyl serine or factor Va), presumably either protein or phospholipid, that serves as a component of the factor Xa binding site.     

Targeting of the Yersinia pestis YopM protein into HeLa cells and intracellular trafficking to the nucleus

The YopM virulence protein of Yersinia pestis has been described as binding human alpha-thrombin and inhibiting thrombin-induced platelet aggregation in vitro. However, recent studies have shown that a YopM-CyaA fusion protein could be targeted vectorially into eukaryotic cells through the Yersinia type III secretion system. In this study, our objective was to characterize YopM's fate in more detail. We followed YopM in the culture medium and inside infected HeLa cells. We confirmed that the native YopM is targeted into HeLa cells, where it is insensitive to exogenous trypsin. The bacteria must be surface located to target YopM, and YopB and YopD are necessary, whereas the LcrE protein (called also YopN) makes this process more efficient. Immunofluorescence localization revealed that YopM, in contrast to YopE, is not only targeted to the cytoplasm but also trafficks to the cell's nucleus by means of a vesicle-associated pathway that is strongly inhibited by brefeldin A, perturbed by monensin or bafilomycin A1 and dependent upon microtubules (decreased by colchicine and nocodazole). These findings revealed a novel interaction of Yersinia pestis with its eukaryotic host.     

Heterogeneous platelet-activating factor (PAF) receptors and calcium increase in platelets and macrophages

We used the increase in cytosolic Ca2+ levels, [Ca2+]i, as a way to characterize PAF (platelet-activating factor, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) receptors in human platelets and rat and human macrophages. [Ca2+] was measured by means of the fluorescent probe fura-2/acetoxymethylester. PAF recognized heterogeneous receptors in human macrophages only (curve slope <1). The PAF antagonist SCH 37370 (1-acetyl-4(8-chloro-5,6-dihydro-11H-benzo[5.6]cyclohepta[1,2-b]pyridine -11-ylidine)piperidine) abolished [Ca2+]i elevation in human platelets, while in rat and human macrophages the maximal inhibition was 76% and 85%, respectively. On the contrary, the antagonist WEB 2086 (3-[4-(2-chlorophenyl)-9-methyl-6Hthieno[3,2-f] [1,2,4]triazolo-[4,3-a] [1,4]-diazepin-2-yl]-1-(4-morpholiny)-1-propanon, apafant) totally inhibited the effect of PAF in both platelets and macrophages. The WEB 2086 concentration-response curves had a slope <1 in the three cell types, indicating interaction with heterogeneous receptors. Accordingly, 3H-WEB 2086 bound to two different classes of sites. Both phases of [Ca2+]i elevation (influx or release) were equally affected by the antagonists. These data support the notions that: 1) PAF receptors are heterogeneous; 2) the two antagonists have a different selectivity toward the receptor subtypes: WEB 2086 recognizes two different receptors both in platelets and in macrophages, while SCH 37370 does not discriminate between receptor subtypes in platelets, and only interacts with one subtype in macrophages; and 3) both SCH 37370 and WEB 2086 display different potencies in rat and human macrophages.     

The assembly of the prothrombinase complex on adherent platelets

Prothrombinase complex assembly, in real time, on platelets adherent to immobilized von Willebrand Factor (vWf) was examined by total internal reflection fluorescence spectroscopy (TIRFS). Electron microscopy showed that the platelets adhered to vWf in a largely unactivated state and could be activated by thrombin. Antibody binding to glycoprotein (GP) Ib and functional GPIIb-IIIa receptor molecules on adherent platelet membranes monitored by TIRFS also indicated that platelets adhered in a largely unactivated state. Maximal expression of the receptor form of GPIIb-IIIa detected by antibody binding was seen only after thrombin stimulation of the adherent platelets. Antibody binding to GPIb was detected on adherent platelets. A reduction in antibody binding was observed after thrombin stimulation of the platelets, indicating a change in GPIb as a consequence of thrombin stimulation of the platelets. The binding of the protein components of the prothrombinase complex to adherent and thrombin-stimulated adherent platelets was then studied individually. Factor Va bound to adherent and thrombin-stimulated adherent platelets was then studied individually. Factor Va bound to adherent and thrombin-stimulated adherent platelets with an estimated Kd of 58 nmol/L. Minimal factor Xa binding was observed on adherent platelets before thrombin stimulation. Factor Xa binding was, however, readily observed on thrombin-stimulated adherent platelets. This factor Xa binding was not saturable, and no Kd value could be estimated. Direct measurement of prothrombinase complex assembly was demonstrated by using an energy transfer phenomenon between fluorescein-labeled factor Va and rhodamine-labeled factor Xa. Prothrombinase complex assembly was observed on both adherent and thrombin-stimulated adherent platelets. The estimated Kd for the factor Va/factor Xa interaction was 4 nmol/L. TIRFS demonstrated that adherent platelets have the ability to support prothrombinase complex assembly, as shown by a direct energy transfer reaction between fluorescently labeled factors Va and Xa.     

Mechanism of the hypercoagulation effect of hyperoxia

It was shown in man and rabbits that a 30-minute inhalation of a 100% O2 under normal atmospheric pressure was accompanied by an elevation of the blood coagulation capacity and a sharp reduction of the count of platelets with the change of their structure. The trigger mechanism of the described hypercoagulation effect is possibly the viscous metamorphosis of platelets developing under the effect of oxygen activation of Hageman's factor.     

Phosphorylation of the thromboxane receptor alpha, the predominant isoform expressed in human platelets

A single gene encodes the human thromboxane receptor (TP), of which there are two identified splice variants, alpha and beta. Both isoforms are rapidly phosphorylated in response to thromboxane agonists when overexpressed in human embryonic kidney 293 cells; this phenomenon is only slightly altered by inhibitors of protein kinase C. Pharmacological studies have defined two classes of TP in human platelets; sites that bind the agonist I-BOP with high affinity support platelet shape change. Low affinity sites, which irreversibly bind the antagonist GR 32191, transduce platelet activation and aggregation. Isoform-specific antibodies permitted detection of TPalpha, but not TPbeta, from human platelets, although mRNA for both isoforms is present. A broad protein band of 50-60 kDa, reflecting the glycosylated receptor, was phosphorylated upon activation of platelets for 2 min with I-BOP. This was a rapid ( approximately 30 s) and transient (maximum, 2-4 min) event and was inhibited by TP antagonists. Both arachidonic acid and low concentrations of collagen stimulated TPalpha phosphorylation, which was blocked by cyclooxygenase inhibition or TP antagonism. Blockade of the low affinity TP sites with GR 32191 prevented I-BOP-induced TPalpha phosphorylation. This coincided with agonist-induced platelet aggregation and activation but not shape change. Also, activation of these sites with the isoprostane iPF2alpha-III induced platelet shape change but not TPalpha phosphorylation. Heterologous TP phosphorylation was observed in aspirin-treated platelets exposed to thrombin, high concentrations of collagen, and the calcium ionophore A 23187. Both homologous and heterologous agonist-induced phosphorylation of endogenous TPalpha was blocked by protein kinase C inhibitors. TPalpha was the only isoform detectably translated in human platelets. This appeared to correspond to the activation of the low affinity site defined by the antagonist GR 32191 and not activated by the high affinity agonist, iPF2alpha-III. Protein kinase C played a more important role in agonist-induced phosphorylation of native TPalpha in human platelets than in human embryonic kidney 293 cells overexpressing recombinant TPalpha.     

Expression of plasminogen activator pla of Yersinia pestis enhances bacterial attachment to the mammalian extracellular matrix

The effect of the plasminogen activator Pla of Yersinia pestis on the adhesiveness of bacteria to the mammalian extracellular matrix was determined. Y. pestis KIM D27 harbors the 9.5-kb plasmid pPCP1, encoding Pla and pesticin; the strain efficiently adhered to the reconstituted basement membrane preparation Matrigel, to the extracellular matrix prepared from human lung NCI-H292 epithelial cells, as well as to immobilized laminin. The isogenic strain Y. pestis KIM D34 lacking pPCP1 exhibited lower adhesiveness to both matrix preparations and to laminin. Both strains showed weak adherence to type I, IV, and V collagens as well as to human plasma and cellular fibronectin. The Pla-expressing recombinant Escherichia coli LE392(pC4006) exhibited specific adhesiveness to both extracellular matrix preparations as well as to laminin. The Pla-expressing strains showed a low-affinity adherence to another basement membrane component, heparan sulfate proteoglycan, but not to chondroitin sulfate proteoglycan. The degradation of radiolabeled laminin, heparan sulfate proteoglycan, or human lung extracellular matrix by the Pla-expressing recombinant E. coli required the presence of plasminogen, and degradation was inhibited by the plasmin inhibitors aprotinin and alpha2-antiplasmin. Our results indicate a function of Pla in enhancing bacterial adhesion to extracellular matrices. Y. pestis also exhibits a low level of Pla-independent adhesiveness to extracellular matrices.     

Mortality mapping in Hong Kong, 1979-83 and 1984-88: the patterns of major non-malignant diseases

In this study, human platelets were used as a cellular model for exploring cytosolic free Ca (Cai) regulation in non-insulin-dependent diabetes mellitus (NIDDM). Cai levels were monitored in resting and thrombin-stimulated platelets from obese females with NIDDM; obese, nondiabetic women, and nonobese, nondiabetic women. All subjects were black. Significant and marked elevation of basal Cai levels was observed in platelets from the diabetic subjects when no aspirin was used during platelet isolation. However, no significant differences were observed in Cai between aspirin-treated platelets from women with NIDDM and platelets from nondiabetic women. The rate of the Cai return to basal level after thrombin stimulation was significantly lower in platelets from the diabetic subjects, suggesting an abnormality in platelet Ca extrusion or sequestration in NIDDM. Platelet Cai levels positively correlated with low-density lipoprotein cholesterol/high-density lipoprotein cholesterol ratio (LDL/HDL) and fasting blood glucose. These findings suggest abnormalities in platelet Cai homeostasis in NIDDM that are influenced by the serum lipid profile and perhaps glucose.     

Identification and partial characterization of factor Va heavy chain kinase from human platelets

Factor Va, the essential cofactor for prothrombinase, is phosphorylated on the acidic COOH terminus of the heavy chain of the cofactor, at Ser692, by a platelet membrane-associated casein kinase II (CKII). Consistent with this observation, phosphorylation of the factor Va heavy chain by the platelet kinase was inhibited by heparin. The membrane-associated platelet CKII kinase was partially purified using heparin-agarose, phosphocellulose, and ion exchange chromatography. CKII antigen was monitored using a polyclonal antibody to the alpha-subunit, and kinase activity in the various fractions was confirmed using human factor Va as a substrate. Immunoblotting experiments using polyclonal antibodies raised against synthetic peptides mimicking a portion of the deduced amino acid sequence of the alpha-, alpha'-, and beta-subunits of human CKII demonstrated the coexistence of both alpha- and alpha'-subunits in platelets and suggested that the platelet CKII kinase may exist in part as an alpha alpha'beta2 complex. It is also possible that there are two distinct populations of CKII in platelets, one that is alphaalpha/betabeta and one that is alpha'alpha'/betabeta. In the presence of the purified platelet-derived CKII, human factor Va incorporates between 0.8 and 1.3 mol of phosphate/mol of factor Va depending on the concentration of the beta-subunit in the kinase preparation. A peptide mimicking the sequence 687-705 of the human factor V molecule incorporates radioactivity in the presence of purified CKII and inhibits factor Va heavy chain phosphorylation by the platelet CKII. In contrast, a peptide with an alanine instead of a serine at position 692 neither incorporates phosphate nor inhibits factor Va phosphorylation by the platelet CKII. Human factor Va is inactivated by activated protein C following three cleavages of the heavy chain at Arg506, Arg306, and Arg679. Cleavage at Arg506 is necessary for efficient exposure of the inactivating cleavage site at Arg306. The phosphorylated cofactor has increased susceptibility to inactivation by activated protein C, since phosphorylated factor Va was found to be inactivated approximately 3-fold faster than its native counterpart. Acceleration of the inactivation process of the phosphorylated cofactor occurs because of acceleration of the rate of cleavage at Arg506. These data suggest a critical role for factor Va phosphorylation on the surface of platelets in regulating cofactor activity.     

The effect of heparin on the interaction of factor VIII and human platelets in vitro

Heparin was found to inhibit the interaction between human factor VIII and platelets. This was noted in two different systems, namely, platelet aggregation induced by neuraminidase-treated human cryoprecipitate and platelet aggregation induced by ristocetin-human factor VIII complex. Heparin appeared to have an inhibitory effect on the above systems similar to that reported on bovine factor VIII-induced platelet aggregation.     

Analysis of the pesticin receptor from Yersinia pestis: role in iron-deficient growth and possible regulation by its siderophore

We have sequenced a region from the pgm locus of Yersinia pestis KIM6+ that confers sensitivity to the bacteriocin pesticin to certain strains of Escherichia coli and Y. pestis. The Y. pestis sequence is 98% identical to the pesticin receptor from Yersinia enterocolitica and is homologous to other TonB-dependent outer membrane proteins. Y. pestis strains with an in-frame deletion in the pesticin receptor gene (psn) were pesticin resistant and no longer expressed a group of iron-regulated outer membrane proteins, IrpB to IrpD. In addition, this strain as well as a Y. pestis strain with a mutation constructed in the gene (irp2) encoding the 190-kDa iron-regulated protein HMWP2 could not grow at 37 degrees C in a defined, iron-deficient medium. However, the irp2 mutant but not the psn mutant could be cross-fed by supernatants from various Yersinia cultures grown under iron-deficient conditions. An analysis of the proteins synthesized by the irp2 mutant suggests that HMWP2 may be indirectly required for maximal expression of the pesticin receptor. HMWP2 likely participates in synthesis of a siderophore which may induce expression of the receptor for pesticin and the siderophore.     

Activation of human platelets by the membrane-expressed A1 domain of von Willebrand factor

Platelet activation and microthrombus formation are invariable features of xenograft rejection and the vascular injury observed when porcine organs are transplanted into primates. This pathological process could be mediated, at least in part, by aberrant interactions of von Willebrand Factor (vWF) associated with the donor vasculature with host platelets. Unlike human vWF, native porcine vWF (pvWF) interacts with human GPIb independently of shear stress or nonphysiological stimuli, eg, ristocetin. We therefore contrasted the potential of isolated human and porcine vWF-A1-domains to interact with human platelets in vitro. Both human and porcine vWF-A1-domains expressed as glycosyl phosphatidylinositol-linked FLAG fusion proteins on COS-7 cells induced GPIb-dependent aggregation and intracellular Ca++ uptake of platelets, independent of both the remainder of the vWF protein and additional modifying factors. Porcine A1-domains were more potent than human homologues, and in addition ristocetin could boost platelet aggregation only with the human A1-domain. Putative conformational changes in the porcine A1-domain could result in the heightened, ristocetin-independent interactions observed with human platelets and may be of importance for xenograft survival.     

The effect of the platelet-derived glycosaminoglycan serglycin on in vitro proplatelet-like process formation

The formation of proplatelet-like processes on megakaryocytes cultured in vitro has been shown to be inhibited by prothrombin, found residually in human serum, which is converted in culture to thrombin. This study reports that another factor found in human serum will counter this inhibition and permit proplatelet-like process formation to occur in vitro even in the presence of inhibitory concentrations of thrombin. The factor was purified from human platelet lysates and identified by amino acid sequence analysis as the proteoglycan serglycin. A similar, if not identical, factor was found at elevated levels in the plasma of thrombocytopenic rabbits. Serglycin probably functions as a proplatelet potentiator by virtue of a tendency to complex with thrombin. Thrombin in complex with serglycin retains its enzymatic properties, but is apparently sterically hindered from interacting with the megakaryocyte cell surface. In preliminary studies, the in vivo administration of serglycin in mice resulted in an increased number of circulating platelets when given in combination with interleukin-6 (IL-6).     

Organization of von Willebrand factor on surface-activated platelets

The distribution and organization of von Willebrand factor (vWF) multimers on platelets after surface activation have not been fully characterized. In the present study, washed human platelets were allowed to interact with Formvar-coated, electron microscope grids for 20 minutes at 37 degrees C and then fixed. After fixation, cells were washed and then incubated with buffer alone, human plasma, human plasma preincubated with ristocetin (1.2 mg/mL), purified human vWF plus ristocetin, or bovine plasma. Macromolecular complexes were revealed by ultrastructural immunocytochemistry employing a polyclonal antibody against vWF and protein A-gold (PAG) as the electron-dense probe. vWF multimers were not present in discoid platelets but appeared on the central zone of dendritic cells and over larger central areas of fully spread platelets. Exposure to human plasma alone did not affect the distribution of electron-dense probes for vWF in central regions of surface-activated cells. Incubation of spread platelets with ristocetin-activated human plasma or bovine plasma resulted in the appearance of randomly dispersed, mottled areas of increased density covering the surface from edge to edge. Exposure to vWF antibody and PAG resulted in specific labeling of the dense areas in a serpentine, linear array. The gold-probe distribution suggested that the vWF multimers were not superimposed and were distributed in a random, irregular manner from edge to edge with label-free, clear areas between them. The results extend previous observations demonstrating that glycoprotein Ib-IX receptors are not spontaneously cleared from the plasma membranes of surface-activated platelets by showing that the receptor function of glycoprotein Ib-IX complex remains unchanged.     

Porcine platelet von Willebrand antigen II (vW AgII): inhibitory effect on collagen-induced aggregation and comparative distribution with human platelets

Von Willebrand factor (vWF) is synthesized by human endothelial cells and megakaryocytes as a large precursor, the pre-provWF, which is finally cleaved into the propolypeptide of vWF (pp-vWF) and the mature vWF. We have purified in parallel the pp-vWF and the GP IIb-IIIa from porcine platelets. The N-terminus comparative analysis of porcine pp-vWF with respect to other species revealed more than a 75% and 65% homology with the bovine and human pp-vWF, respectively. Purified pp-vWF inhibited collagen-induced platelet aggregation in porcine platelet rich plasma (PRP) and specifically binds to collagen. Polyclonal antibody pabBp19 against the purified protein was prepared and characterized by ELISA, Western-blot and immunocytochemistry. The distribution of pp-vWF in soluble and membrane fractions from pig platelets has been performed by immunolabeling detection. pabBp19 did not blot human platelet lysates but human pp-vWF was detectable using the antibody Frieda 013091. Cell distribution and quantification studies of human and porcine platelet pp-vWF showed that the protein exists in the soluble and membrane fractions and its pattern is similar in both species.     

Role of direct revascularization of the internal iliac artery during aortoiliac surgery

BACKGROUND: In the event of hemorrhage and blood loss, platelets play a vital role in the coagulation process. However, there are currently no acceptable protocols for long-term storage of platelets. As a first step toward testing the efficacy of stored platelets or platelet substitutes in vivo, a flow cytometric technique was developed to detect human platelets in rabbit blood. STUDY DESIGN AND METHODS: Human platelets were transfused to rabbits whose reticuloendothelial system was inhibited by the administration of ethyl palmitate. Because human and rabbit platelets display surface molecules with different epitopes, human platelets were selectively labeled with antibodies specific for glycoprotein IX (CD42a). As this antibody does not label rabbit platelets, it allows discrimination of human from rabbit platelets in samples of rabbit blood containing both types of platelets. RESULTS: Survival of human platelets in rabbits was monitored by flow cytometry and fluorescence microscopy in blood drawn at various times after the platelet transfusion. Fresh human platelets transfused to untreated control rabbits (n = 3) were removed from circulation within 10 minutes of the completion of the transfusion. Fresh platelets (1 day old) transfused to rabbits treated with ethyl palmitate (n = 5) survived for 24 hours with an average half-life of 8.6 hours. In contrast, 8-day-old platelets were cleared from the circulation sooner with an average half-life of 2.9 hours (n = 4). CONCLUSION: This report describes a rapid and efficient method of assessing the survival of human platelets in a rabbit model using flow cytometry. This technique will enable the monitoring in rabbits of human platelets prepared by various preservation protocols.     

Platelet agonists enhance the import of phosphatidylethanolamine into human platelets

It is unknown whether the endocytosis-independent transfer of phospholipids from lipoproteins to platelets is regulated by platelet agonists such as thrombin. The movements of the choline phospholipids phosphatidylcholine and sphingomyelin (labeled with either 14C or the fluorescent pyrenedecanoic acid) between low density lipoproteins and platelets were unaffected by thrombin (0.5 unit/ml). In contrast, thrombin accelerated the import of diacyl phosphatidylethanolamine (PE) and alkenylacyl phosphatidylethanolamine into platelets by about 4-fold. Similarly, thrombin receptor-activating peptide (15 microM), collagen (10 microgram/ml), and ADP (10 microM) enhanced PE uptake. High density lipoprotein particles and egg phosphatidylcholine vesicles were also donors for stimulation of platelet PE import. Part of the [14C]arachidonic acid-labeled PE transferred from low density lipoprotein to platelets activated by thrombin and collagen was metabolized to 14C-eicosanoids. Inhibitors of protein kinase C partially prevented thrombin-induced [14C]PE uptake, while direct activators of protein kinase C increased incorporation of [14C]PE into platelets. Proteinaceous factor(s) recovered in the extracellular medium from ADP- and thrombin-activated platelet suspensions were found to accelerate the transfer of pyrenedecanoic acid-labeled PE between donor and acceptor lipid vesicles. The stimulation of import of ethanolamine phospholipids led to a 2-fold enhancement of the prothrombinase activity of thrombin-activated platelets. Our study demonstrates that physiological platelet stimuli increase specifically the transfer of ethanolamine phospholipids from lipoproteins to platelets through a secretion-dependent mechanism. This might contribute to the increase of procoagulant activity of stimulated platelets.     

Platelets inhibit the induction of nitric oxide synthesis by interleukin-1 beta in vascular smooth muscle cells

We have investigated the role of platelets in regulating the hemostatic and vasomotor properties of vascular smooth muscle. Experiments were performed to examine the effect of the releasate from activated platelets on the production of nitric oxide from interleukin-1 beta (IL-1 beta)-treated cultured rat aortic smooth muscle cells. Treatment of vascular smooth muscle cells with IL-1 beta resulted in significant accumulation of nitrite in the culture media and in marked elevation of intracellular cyclic guanosine monophosphate (GMP) levels. The releasate from collagen-aggregated platelets blocked the IL-1 beta-mediated production of nitrite and the accumulation of cyclic GMP in smooth muscle cells in a platelet number-dependent manner. In functional assays, the perfusates from columns containing IL-1 beta-treated smooth muscle cells relaxed detector blood vessels without endothelium and the addition of IL-1 beta-treated smooth muscle cells to suspensions of platelets inhibited their thrombin-induced aggregation. The simultaneous treatment of smooth muscle cells with IL-1 beta and the platelet releasate abolished both the vasorelaxing activities of the perfusates and the inhibition of platelet aggregation. Platelet releasates treated with a neutralizing antibody to platelet-derived growth factor (PDGF) failed to block IL-1 beta-induced nitric oxide production by the smooth muscle cells, as measured by both biochemical and functional assays. The platelet releasate from a patient with gray platelet syndrome likewise failed to block IL-1 beta-induced nitrite release by smooth muscle cells. These results demonstrate that platelets downregulate the production of nitric oxide by IL-1 beta-treated vascular smooth muscle cells through the release of PDGF. This effect may represent a novel mechanism by which platelets regulate vasomotor tone and thrombus formation at sites of vascular injury.     

Increased protein synthesis by human platelets during phagocytosis of latex particles in vitro

A three-fold increase of protein synthesis by human platelets during in vitro phagocytosis of polystyrene latex particles was detected. During the first two hours of incubation, the percentage of phagocytizing platelets and the number of latex particles per platelet increased; by the end of the third hour, the first parameter remained stable, while the number of latex particles per cell had decreased. Vincristine (20 mug/ml of cell suspension) inhibited platelet protein synthesis. This effect was both time- and dose-dependent. The drug also caused a decrease in the number of phagocytizing cells, as well as in their phagocytotic activity.