Nitric oxide donors inhibit platelet spreading on surfaces coated with fibrinogen but not with fibronectin

We have studied the effect of nitric oxide (NO) on the interaction of washed human platelets with fibrinogen or fibronectin adsorbed on glass. Platelet contacts were visualized by interference reflection microscopy at 37°C in Tyrode's solution. The areas of spread platelets were measured, using digital video image processing techniques, at times up to 30 minutes after initial surface platelet contact. On fibrinogen spreading was inhibited by NO donors in the order of potency: S-nitroso-acetylpenicillamine > sodium nitroprusside > S-nitroso-glutathione. The inhibitory action of NO donors was prevented by Oxy-haemoglobin, confirming that this inhibition was due to the release of NO. In contrast, NO donors had virtually no effect on platelet spreading on fibronectin. Platelet adhesion to fibrinogen is mediated by GP IIb IIIa and the vitronectin receptor (VNR), while that to fibronectin is via GP IIb IIIa, VNR, Ic IIa and VLA-6. Our results thus indicate that NO inhibits adhesion mediated by GP IIb IIIa and/or VNR but not by the two other receptors. The mechanism of this receptor - specific inhibition of platelet adhesion remains to be elucidated.     

The effect of low dose of 12-0-tetradecanoyl-phorbol-13-acetate on collagen platelet interactions

We and others have reported that phorbol ester doses ranging from 0.25 to 25 μg/ml induce human platelets to aggregate and release serotonin. In this paper, we report the effect of low doses of phorbol (0.5 to 50 ng/ml) on subthreshold amounts of collagen induced platelet aggregation. The platelet aggregation induced by the addition of subthreshold amounts of collagen can be enhanced by low doses of phorbol. The combined low doses of phorbol and subthreshold amounts of collagen induced platelet aggregation can be inhibited by the addition of aspirin and imidazole. The increase in platelet aggregation induce by the combined low doses of phorbol and subthreshold amounts of collagen is probably mediated by the collagen fibril formation.     

Oxidative inactivation of purified human alpha-2-antiplasmin, antithrombin III, and C1-inhibitor

Oxidative inactivation of alpha-1-proteinase inhibitor (A1-PI) and plasminogen activator inhibitor-1 (PAI-1), both members of the serine protease inhibitor (serpin) superfamily, using mild oxidation conditions has been already demonstrated. The oxidation mechanism has been shown to involve conversion of methionine to methionine sulfoxide in the reactive center of the inhibitors.

In this study evidence is presented that alpha-2-antiplasmin (A2-PI) and antithrombin III (AT III) can also be inactivated by means of oxidation. For total inactivation of 50 pM Al-PI about 10 nM chloramine T (CT) and for the same molar concentration of A2-PI and AT III about 250 nM CT were found necessary. C1-inhibitor (C1-INH) showed some resistance to oxidation that could be overcome only by increasing CT to an amount (> 2000 nM) already beginning to inactivate the corresponding C1-esterase. As target enzymes for A2-PI, AT III, and A1-PI plasmin, thrombin and elastase, respectively, were used. Their activity was not impaired by the oxidation conditions applied.

As there is no methionine in the reactive center of AT III an additional mechanism for oxidative inactivation of serpins has to be taken into consideration. Oxidation seems to be a general mechanism for altering the balances between serine proteases and their inhibitors in favour of the protease.     

Species differences in platelet responses to thrombin and SFLLRN. receptor-mediated calcium mobilization and aggregation, and regulation by protein kinases

The thrombin receptor on human platelets is activated by thrombin to stimulate platelet aggregation through the tethered ligand SFLLRN. This study examined the effects of thrombin and SFLLRN on aggregation and calcium mobilization ([Ca²⁺]_i) in rat, guinea pig, rabbit, dog, monkey, and human platelets, and the role of protein kinases in regulating these functions. Thrombin induced platelet aggregation and [Ca²⁺]_i in all species studied; however, only guinea pig, monkey and human platelets were responsive to SFLLRN. Similar species specific effects were obtained with [Ca²⁺]_i studies. The kinetic profile for [Ca²⁺]_i differed among species, suggesting that regulatory mechanisms for calcium differed between agonists and among species. Staurosporine, a non-selective inhibitor of protein kinases, inhibited platelet aggregation induced by thrombin or SFLLRN in all species. Staurosporine inhibited thrombin-induced [Ca²⁺]_i in guinea pigs, had no effect in rat, and increased [Ca²⁺]_i in all other species. Staurosporine inhibited SFLLRN-induced [Ca²⁺]_i in guinea pig, yet had no effect in monkey or human. Tyrphostin 23, a specific inhibitor of tyrosine protein kinases, inhibited thrombin-induced aggregation of rabbit, monkey, dog and human platelets. SFLLRN-induced aggregation was also inhibited by tyrphostin 23. Tyrphostin 23 inhibited [Ca²⁺]_i induced by either thrombin or SFLLRN in all species. Based on the differential response to agonist stimulation, we propose that thrombin can activate platelets via SFLLRN-dependent and independent mechanisms, which could involve yet unrecognized subtypes of the thrombin receptor or distinct cellular activating mechanisms. Furthermore, differential regulation of calcium mobilization and aggregation was observed in those platelets responding to either thrombin or SFLLRN.     

The primary wave of epinephrine-induced platelet aggregation represents alpha 2-adrenoceptor status

We examined relationships between epinephrine-induced slope of primary wave of aggregation and the alpha₂-adrenoceptor status on platelets. A concentration (10^-9 to 10^-6 )-dependent increase in slope of primary wave with EC50 of epinephrine at 4.5 ± 0.4 × 10^-7 was observed. In studies on epinephrine binding to alpha₂adrenoceptors in competition with ³H-yohimbine to platelets, (IC₅₀) of epinephrine was 4.8 ± 3.4 x 10^-7 M. There was a significant (P<0.02) correlation between EC₅₀ of epinephrine to evoke biological response and IC₅₀ of epinephrine to bind to alpha₂-adrenoceptors (r-0.75). There was no relationship between number of receptor sites or dissociation constant of ³H-yohimbine binding and primary wave of platelet aggregation. These data show that the slope of primary wave in response to epinephrine reflects alpha₂-adrenoceptor binding of the agonist.     

Heparin-like glycosaminoglycan is a receptor for Antithrombin III-dependent but not for thrombin-dependent prostacyclin production in human endothelial cells

Antithrombin III (ATIII) induced a marked increase in prostacyclin (PGI₂) release from cultured human umbilical vein endothelial cells (HUVEC) after incubation for more than 2 hr and the induction continued for 8 hr, while thrombin induced the increase within 10 min. ATIII-dependent production of PGI₂ was abolished by addition of heparin, but pretreatment of HUVEC with polyclonal antibody against thrombomodulin could not prevent the PGI₂ productions by ATIII and thrombin. ATIII-dependent PGI₂ production was significantly inhibited by pretreatment of HUVEC with β- -xylosides or heparitinase, though neither pretreatment affected thrombin-induced PGI₂ production. After treatment of HUVEC with 1 μg/ml cycloheximide, ATIII-dependent PGI₂ production was completely abolished. These results indicate that the mechanism of the induction of PGI₂ production by ATIII involves heparin-like glycosaminoglycans on HUVEC and the stimulation of synthesis of a protein related to PGI₂ production. The ATIII-induced PGI₂ production is very different from that induced by thrombin.     

Elevation of platelet cyclic AMP level by thromboxane A2/prostaglandin H2 receptor agonists.

Arachidonic acid (AA)-or thromboxane A₂/prostaglandin H₂ (TXA₂/ PGH₂) analog (STA₂ and U-46619)-induced aggregations yielded a bell-shaped dose-response curve. The inhibitory mechanism by high concentrations of the agonists was examined. STA₂ elevated cAMP level of platelet in a dose-dependent manner. And the aggregation was affected by metabolic inhibitors of cAMP. AA also rised cAMP level, and the rise was suppressed by indomethacin. These results indicate that the reduction of aggregation by high dose of the agonists is through cAMP elevation. The cAMP elevation was not suppressed by ruling out phospholipase C effects by chelation of cytoplasmic Ca²⁺ and inhibition of protein kinase C (PKC). These results suggest that the cAMP elevation is not due to activation of phospholipase C-linked TXA₂/PGH₂ receptor. 13-APA, an antagonist of TXA₂/PGH₂ receptor, suppressed the cAMP elevation, although ONO-3708, another antagonist, had no effect. As to be expected from this result, inhibitory effect of 13-APA on high STA₂ level-induced aggregation was weaker than that of ONO-3708. The antagonists did not inhibit PGE,- or PGD₂-induced cAMP elevation. These findings suggest that platelet has adenylate cyclase-linked TXA₂/PGH₂ receptor.     

Activation of protein kinase C by the action of 9,11-epithio-11,12-methano-thromboxane A2 (STA2), a stable analogue of thromboxane A2, in human platelets

Incubation of human washed platelets with 9,11-epithio-11,12-methano-thromboxane A₂ (STA₂), a stable analogue of thromboxane A₂, caused the activation of protein kinase C and myosin light chain (MLC) kinase to the same extents as those induced by thrombin as judged by measuring the phosphorylation of a 40-kilodalton protein and MLC, respectively. However, STA₂ stimulated much less phosphoinositide turnover than thrombin. Furthermore, the doses of STA₂ necessary for protein kinase C activation and phosphoinositide turnover were higher than those necessary for MLC kinase activation, although the doses of thrombin necessary for these three reactions were nearly the same. These results suggest that protein kinase C may be activated at the Ca²⁺ concentrations higher than those required for MLC kinase activation by the action of STA₂, presumably due to the inability of this agonist to produce diacylglycerol in an amount enough to increase the affinity of the enzyme for Ca²⁺.     

The voltage-gated calcium channel CaV1.2 promotes adult oligodendrocyte progenitor cell survival in the mouse corpus callosum but not motor cortex

Throughout life, oligodendrocyte progenitor cells (OPCs) proliferate and differentiate into myelinating oligodendrocytes. OPCs express cell surface receptors and channels that allow them to detect and respond to neuronal activity, including voltage-gated calcium channel (VGCC)s. The major L-type VGCC expressed by developmental OPCs, CaV1.2, regulates their differentiation. However, it is unclear whether CaV1.2 similarly influences OPC behavior in the healthy adult central nervous system (CNS). To examine the role of CaV1.2 in adulthood, we conditionally deleted this channel from OPCs by administering tamoxifen to P60 Cacna1c ^fl/fl (control) and Pdgfrα-CreER:: Cacna1c ^fl/fl (CaV1.2-deleted) mice. Whole cell patch clamp analysis revealed that CaV1.2 deletion reduced L-type voltage-gated calcium entry into adult OPCs by ~60%, confirming that it remains the major L-type VGCC expressed by OPCs in adulthood. The conditional deletion of CaV1.2 from adult OPCs significantly increased their proliferation but did not affect the number of new oligodendrocytes produced or influence the length or number of internodes they elaborated. Unexpectedly, CaV1.2 deletion resulted in the dramatic loss of OPCs from the corpus callosum, such that 7 days after tamoxifen administration CaV1.2-deleted mice had an OPC density ~42% that of control mice. OPC density recovered within 2 weeks of CaV1.2 deletion, as the lost OPCs were replaced by surviving CaV1.2-deleted OPCs. As OPC density was not affected in the motor cortex or spinal cord, we conclude that calcium entry through CaV1.2 is a critical survival signal for a subpopulation of callosal OPCs but not for all OPCs in the mature CNS.     

Blocking of the receptor-stimulated calcium entry into human platelets by verapamil and nicardipine

Verapamil (ED₅₀=3×10⁻⁶ M) and nicardipine (ED₅₀=10⁻⁶ M) inhibited the platelet activating factor (PAF)-induced increase of free cytosolic calcium concentration ([Ca²⁺]_i) in quin2-loaded human platelets. In a Ca-free medium containing 5 mM BaCl₂, PAF stimulated the inflow of Ba²⁺ ions which is completely abolished by verapamil and nicardipine. Simultaneous determination of quin2 fluorescence and ⁴⁵Ca absorption showed that the action of verapamil is accounted for by blocking of the Ca²⁺ entry. Nicardipine suppresses also Ca²⁺ mobilization from intracellular stores. The effects of verapamil and nicardipine are not competitive with respect to PAF.The blockers reduce the [Ca²⁺]_i increase induced by ADP, vasopressin, and PGH₂ analogue U46619.     

effects of picotamide on human platelet aggregation, the release reaction and thromboxane B2 production

We studied the effects of picotamide (N,N′ bis 3 picolyl-4-methoxy-isophthalamide) on human platelet aggregation, the release reaction and the production of thromboxane B₂ (TxB₂) induced by several platelet agonists. The effects of picotamide were compared to those of acetylsalicylic acid (ASA). Picotamide (0.5 mmol/1) inhibited platelet aggregation, the release of ATP and TxB₂ production induced by ADP, arachidonic acid (AA), collagen or the prostaglandin endoperoxide (PE) analogue U46619. ASA (0.5 mmol/1) did not affect platelet aggregation and the release of ATP induced by U46619. Picotamide and ASA inhibited the AA-induced platelet TxB₂ production both under stirring and non-stirring conditions, whereas the pure thromboxane A₂ receptor antagonist BM13177 (0.5 mmol/1) was inhibitory only under stirring conditions. Since under non-stirring conditions platelet aggregation does not occur, picotamide directly inhibits TxB₂ production, whereas BM13177 inhibits the potentiation of TxB₂ production due to TxA₂/PE-dependent platelet aggregation. Malondialdehyde (MDA) production by unstirred platelets stimulated with AA was not significantly inhibited by picotamide. In conclusion, picotamide inhibits the TxA2/PE-dependent platelet responses to agonists by a double mechanism: (i), TxA₂/PE antagonism; (ii) inhibition of thromboxane synthase.     

A radioimmunoassay for thrombospondin, used in a comparative study of thrombospondin, β-thromboglobulin and platelet factor 4 in healthy volunteers

A radioimmunoassay was developed for the platelet α-granule protein thrombospondin; concentrations of thrombospondin as low as 3 ng ml^?1 could be measured. There was no interference from other components of human biological fluids and no crossreactivity with β-thromboglobulin (β-TG) or platelet factor 4 (PF4). Plasma samples were stable when stored at ?20°C. Normal human plasma contained 105.0 ± 31.0 ng thrombospondin ml^?1 compared with β-TG concentrations of 37.2 ± 10.9 ng ml^?1 and PF4 concentrations of 14.7 ± 10.1 ng ml^?1 when samples were carefully taken into a platelet inhibitor cocktail and processed at 0–4°C. Release of thrombospondin during clotting of blood occurred at the same time as that of β-TG and PF4 and resulted in a serum concentration of 17.5 ± 5.5 μg ml^?1. Assay of whole blood gave a platelet thrombospondin content of 89.1 ± 28.3 ng/ 10⁶ platelets. The concentration in normal urine fluctuated widely from 3 to 22.5 ng ml^?1, and was unrelated to urine flow. The half-life of thrombospondin $\text{in vivo}$ was about 9 h, much longer than that of either β-TG or PF4. Unlike PF4, it was not released into the blood following an intravenous heparin injection. Bovine, ovine, canine and porcine sera contained thrombospondin which crossreacted immunologically with the human molecule; these species would be suitable animal models for the study of thrombospondin and its value as a platelet release marker.     

The influence of time, temperature and packed cell on activated partial thromboplastin time and prothrombin time

Activated partial thromboplastin time (APTT) and prothrombin time (PT) were performed in four groups of studies in order to evaluate the influences of time, temperature, and different forms of plasma storage to the result. Different designs for storage of the plasmas were studied, including the plasmas stored either with or without packed cells, the plasmas stored in the cuvette with exact volume for performing the test or in the test tube. The temperatures for store of the plasma were at room temperature, at 4°C and at −70°C. The time for store of the plasma was from 1 hour up to 7 hours. The plasmas included normal pooled plasmas and diseased plasmas. From this study, it is found that the PT test was not easily affected by the temperature, storage time and the form of storage in comparison with the APTT test which was much easily affected by the above conditions. APTT should be done within 2 hours after sampling and the plasma should be stored with the packed cells at 4°C in order to obtain a reliable result. PT could be done within 7 hours without influence to the result if the plasma was stored with the packed cells at 4°C. No significant cold-induced shortening of PT could be noted when the plasma was incubated at 4°C up to 7 hours. In either PT or APTT, the most suitable condition for storing the plasma should be with the packed cells at 4°C.     

Dorsal striatum and stimulus-response learning: lesions of the dorsolateral, but not dorsomedial, striatum impair acquisition of a simple discrimination task

In the present experiment, the effects of neurotoxic lesions (quinolinic acid) of the dorsolateral or dorsomedial striatum were investigated on a simple instrumental discrimination task (CS+/CS−). Rats with lesions of the dorsolateral striatum were found to be impaired in the acquisition of this task, as compared to rats with either dorsomedial striatal or sham lesions. Furthermore, dorsolateral striatal lesioned animals had significantly lower levels of responding across the course of discrimination training, as assessed both by overall rate of response during CS+ presentations and number of CS+ trials without a response, despite having shown levels of responding during variable interval training that did not differ from that of sham lesioned animals. In contrast, animals with lesions of the dorsomedial striatum did not show an impairment in acquisition of the present task, but had slightly higher rates of responding during CS− presentations. It is argued that the poor acquisition and low response rates observed in animals with dorsolateral striatal lesions reflect a failure in stimulus–response learning, while the performance of animals with dorsomedial striatal lesions may have been the result of an increase in overall activity rate.     

The development, distribution and density of the plasma membrane calcium ATPase 2 calcium pump in rat cochlear hair cells

Calcium is tightly regulated in cochlear outer hair cells (OHCs). It enters mainly via mechanotransducer (MT) channels and is extruded by the plasma membrane calcium ATPase (PMCA)2 isoform of the PMCA, mutations in which cause hearing loss. To assess how pump expression matches the demands of Ca(2+) homeostasis, the distribution of PMCA2 at different cochlear locations during development was quantified using immunofluorescence and post-embedding immunogold labeling. The PMCA2 isoform was confined to stereociliary bundles, first appearing at the base of the cochlea around post-natal day (P)0 followed by the middle and then the apex by P3, and was unchanged after P8. The developmental appearance matched the maturation of the MT channels in rat OHCs. High-resolution immunogold labeling in adult rats showed that PMCA2 was distributed along the membranes of all three rows of OHC stereocilia at similar densities and at about a quarter of the density in inner hair cell stereocilia. The difference between OHCs and inner hair cells was similar to the ratio of their MT channel resting open probabilities. Gold particle counts revealed no difference in PMCA2 density between low- and high-frequency OHC bundles despite larger MT currents in high-frequency OHCs. The PMCA2 density in OHC stereocilia was determined in low- and high-frequency regions from calibration of immunogold particle counts as 2200/μm(2) from which an extrusion rate of ～200 ions/s per pump was inferred. The limited ability of PMCA2 to extrude the Ca(2+) load through MT channels may constitute a major cause of OHC vulnerability and high-frequency hearing loss.     

Possible role of plasminogen activator inhibitor 2 in the prevention of the metastasis of gastric cancer tissues

The concentrations of urinary type plasminogen activator (u-PA), plasminogen activator inhibitor 1 (PAI-1), and PAI-2 were measured in gastric cancer tissues and adjacent healthy mucosal tissues. Levels of u-PA, PAI-1 and PAI-2 were higher in cancer than in control tissues. PAI-1 levels were higher together with the progression of cancer however there were no differences in u-PA or PAI-2 levels. Tumors with higher PAI-1 and lower PAI-2 levels tend to metastasize to remote lymph nodes. When the numbers of involved lymph nodes were analyzed, tumors with the large number of metastatic lymph nodes showed higher PAI-1 and lower PAI-2 level. No difference was shown in u-PA levels among these groups. These tendencies were more significant in patients with progressed gastric cancer. These results suggest that tumor with higher PAI-2 levels tend to localize or have less tendency to metastasize to lymph nodes. On the other hand PAI-1 was generally higher in tumor with invasion into nearby tissue or with nodal metastasis.     

Cannabinoids modulate Olig2 and polysialylated neural cell adhesion molecule expression in the subventricular zone of post-natal rats through cannabinoid receptor 1 and cannabinoid receptor 2

The subventricular zone (SVZ) is a source of post-natal glial precursors that can migrate to the overlying white matter, where they may differentiate into oligodendrocytes. We showed that, in the post-natal SVZ ependymocytes, radial glia and astrocyte-like cells express cannabinoid receptor 1 (CB1), whereas cannabinoid receptor 2 (CB2) is found in cells expressing the polysialylated neural cell adhesion molecule. To study CB1 and CB2 function, post-natal rats were exposed to selective CB1 or CB2 agonists (arachidonyl-2-chloroethylamide and JWH-056, respectively) for 15 days. Accordingly, we found that CB1 activation increases the number of Olig2-positive cells in the dorsolateral SVZ, whereas CB2 activation increases polysialylated neural cell adhesion molecule expression in this region. As intense myelination occurs during the first weeks of post-natal development, we examined how modulating these factors affected the expression of myelin basic protein. Pharmacological administration of agonists and antagonists of CB1 and CB2 showed that the activation of both receptors is needed to augment the expression of myelin basic protein in the subcortical white matter.     

Clorgyline and desipramine alter the sensitivity of [H]noradrenaline release to calcium but not to clonidine

Synaptosomes (P₂) were prepared from cerebral cortices of control rats and from those which had received clorgyline (1 mg/kg/day for 21–28 days) or desipramine (10 mg/kg/day for 21–28 days). Following incubation with [³H]noradrenaline (500 nM/15 min, 37 °C), aliquots of the synaptosomes were gently filtered onto Whatman GF/A filters and superfused with Krebs buffer (pH 7.5, 37 °C) for a maximum period of 2 h. During this time, the basal efflux of tritiated materials (approximately 75% noradrenaline) together with K⁺-evoked release of the amine and metabolites, were measured. Chronic antidepressant drug regimens increased the K⁺-stimulated release, but its attenuation by clonidine was not altered. Thus, chronic antidepressant drug regimens do not apparently alter presynaptic α₂-adrenoceptors. These results suggest that the reported antidepressant drug induced decreases in [³H]clonidine binding, occur on sites which are postsynaptic to noradrenergic neurones. Following the chronic antidepressant drug regimens, the sensitivity of the [³H]noradrenaline release process to Ca²⁺ is significantly increased. This change may explain the enhanced K⁺-evoked release which follows the antidepressant drug regimens. It is proposed that this increased sensitivity of the [³H]noradrenaline release process may be an adaptation to the decreases in neuronal firing which have been reported following antidepressant drug treatments.