Increased platelet responsiveness following coronary stenting. Heparin as a possible aetiological factor in stent thrombosis

AIMS: Platelet activation may be a determinant of thrombotic and restenotic complications following intracoronary stenting. In order to measure the effect of stenting on platelet activation antigen expression we used whole blood flow cytometry in 18 patients undergoing Palmaz-Schatz stenting (treated with full anticoagulation) and compared these with a group of 18 patients undergoing elective angioplasty. The effects of low molecular weight heparin and unfractionated heparin on platelet behaviour were also studied, both in vitro and in vivo to determine the contribution of prolonged heparin therapy to platelet activation following stenting. METHODS AND RESULTS: Fibrinogen binding to activated GPIIb-IIIa, and surface expression of P-selectin, GPIb and GPIIb-IIIa antigens were measured in unstimulated peripheral blood samples (rest) and on stimulation with adenosine diphosphate (0.1-10 micromol x 1(-1)) and thrombin (0.02-0.16 U x ml(-1)). No changes were seen in resting samples following angioplasty or stenting. Agonist responsiveness was unaltered after angioplasty, but in stented patients antigen expression in response to thrombin was significantly reduced (P< or =0.04), whilst the adenosine diphosphate response was significantly increased (P=0.01). Similar effects were observed in patients with unstable angina treated with either low molecular weight heparin or unfractionated heparin in vivo. In vitro, both unfractionated and low molecular weight heparin inhibited thrombin-induced platelet activation, but stimulation of adenosine diphosphate responses was more marked with unfractionated than low molecular weight heparin. CONCLUSIONS: There was a significant increase in platelet responsiveness to adenosine diphosphate following intracoronary stenting in patients treated with conventional anticoagulants. This was probably a consequence of treatment with heparin. Activation of platelets by heparin may explain the increased rate of stent thrombosis in patients treated with anticoagulant therapy. Low molecular weight heparins stimulate platelets less than unfractionated heparin.     

XV454, a novel nonpeptide small-molecule platelet GIIb/IIIa antagonist with comparable platelet alpha(IIb)beta3-binding kinetics to c7E3

XV454 demonstrated high potency (IC50 = 14-25 nM) in inhibiting human platelet aggregation induced by adenosine diphosphate (ADP, 10 microM), thrombin receptor agonist peptide (TRAP) (10 microM), or collagen (20 microg/ml). XV454 exhibited a high degree of selectivity for platelet alpha(IIb)beta3 in comparison with c7E3, which is a nonspecific antagonist for both alpha(IIb)beta3 and alpha(v)beta3. Both XV454 and c7E3 bind with high affinity to either activated (A) or unactivated (U) human, baboon, or canine platelets. XV454 binds with a relatively higher affinity [Kd = 0.5 nM (A), 0.6 nM (U)] as compared with c7E3 [Kd = 9.1 nM (A), 9.2 (U) nM]. XV454 demonstrated a tight association with human, baboon, and, to a lesser extent, with canine platelets (t(1/2) of dissociation = 110 +/- 6, 80 +/- 10, and 23 +/- 2 min, respectively). Both c7E3 and XV454 associate tightly with a slower dissociation rate with unactivated human platelets: t(1/2) of 42 and 116 min, respectively. In non-human primates, oral (0.1 mg/kg, p.o.) and intravenous (0.05 mg/kg, i.v. bolus administration of XV454 methyl ester pro-drug resulted a long-lasting maximal antiplatelet efficacy for < or = 72 h with significant but reversible prolongation of bleeding time and without effects on platelet count, clinical chemistry, or hemodynamic profile. In conclusion, XV454 represents a potent antiplatelet agent in inhibiting platelet aggregation along with a high affinity and relatively slow dissociation rate from human platelet GPIIb/IIIa receptors that allow a long-lasting antiplatelet efficacy after single i.v. or oral administration.     

Regulation of the paracellular permeability of cultured human cervical epithelium by a nucleotide receptor

OBJECTIVE: We sought to determine what type of regulation of transepithelial transport in leaky epithelium can occur at the level of paracellular permeability. METHODS: The epithelial permeability to the polar acid pyranine (Ppyr) and the transepithelial electrical resistance (R) were determined in cultured human cervical epithelium. RESULTS: Extracellular adenosine triphosphate (ATP) acutely and reversibly decreased the paracellular permeability, as evidenced by an increase of R from 10 +/- 3 to 16 +/- 2 omega/cm2 and a decrease in Ppyr from 13 +/- 3 to 9 +/- 3 x 10(-6) cm/sec and (P < .01 for both). The ATP effect was dose related (average median effective concentration 2 +/- 1 microM), saturable at 50 microM, and desensitized with repeated administration; it was mimicked by uridine triphosphate and ATP-gamma-S but not by adenosine monophosphate, adenine, adenosine, or adenosine diphosphate. The ATP effect on resistance remained intact even when the intercellular resistance was decreased with a basal-to-apical pressure gradient, but was abolished by lowering extracellular calcium. CONCLUSIONS: These results indicate the following: 1) Paracellular permeability in the cervical epithelial cell line is regulated by a nucleotide receptor; and 2) the tight junctions are effectors of nucleotide-receptor stimulation. We suggest that extracellular ATP may regulate mucus production in the cervix in vivo by acting on a surface receptor and by increasing the resistance of the tight junctions.     

Labeling of A2A adenosine receptors in human platelets by use of the new nonxanthine antagonist radioligand [3H]SCH 58261

The present study describes the binding to human platelet A2A adenosine receptors of the new potent and selective antagonist radioligand [3H]5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo [1,5-c] pyrimidine ([3H]SCH 58261). Saturation experiments revealed that [3H]SCH 58261 labels a single class of recognition sites with high affinity (Kd = 0.85 nM), limited capacity (apparent Bmax = 85 fmol/mg of protein) and good specific binding (about 60%). [3H]SCH 58261 binding was not modulated by either the divalent cation Mg(+2) or guanine nucleotides. In competition experiments, a series of both adenosine agonists and antagonists inhibited [3H]SCH 58261 binding to A2A platelet receptors with rank order of potency and affinity similar to those observed in rat striatal membranes with the same radioligand. This confirms that the platelet A2A receptor is similar to that labeled in the brain striatum. Binding data were also found to be in good agreement with the results from functional studies such as A2A agonist-induced stimulation of adenylate cyclase or platelet aggregation inhibition. The present findings indicate that [3H]SCH 58261 is the first radioligand available for the characterization of the A2A receptor subtype in platelets.     

Inhibitory mechanisms of naloxone on human platelets

1. In the present study, naloxone was tested for its antiplatelet activities in human platelet-rich plasma (PRP). In human PRP, naloxone (0.1-0.5 mmol/L) inhibited aggregation stimulated by a variety of agonists (i.e. collagen, adenosine diphosphate (ADP), U46619 and adrenaline). 2. Naloxone (0.1-0.5 mmol/L) did not significantly affect cyclic adenosine monophosphate and cGMP levels in human washed platelets, whereas naloxone (0.5 mmol/L) significantly inhibited thromboxane B2 formation stimulated by collagen (5 micrograms/mL) in human washed platelets. 3. Naloxone (0.5 mmol/L) significantly inhibited [3H]-inositol monophosphate formation of [3H]-myoinositol-loaded platelets stimulated by collagen and U46619. Moreover, naloxone did not influence the binding of 125I-triflavin to platelet membranes. Triflavin is an Arg-Gly-Asp-containing specific fibrinogen receptor antagonist. 4. Addition of naloxone (0.5 mmol/L) to platelet preparations tagged with diphenylhexatriene (DPH) resulted in a considerable decrease in relative fluorescence intensity. 5. It is suggested that the anti-platelet effects of naloxone may be caused, at least partly, by the induction of conformational changes in the platelet membrane initially, followed by the inhibition of thromboxane A2 formation and phosphoinositide breakdown of platelets stimulated by agonists.     

Rat platelet aggregation by ATP. Aggregometrical and ultrastructural comparison with aggregations induced by ADP and collagen

This paper describes the aggregation of rat platelets by adenosine triphosphate (ATP). The aggregometry of ATP-induced aggregation and the ultrastructure of ATP-aggregated platelets were compared and contrasted with those of adenosine diphosphate (ADP)-treated and collagen-treated samples. Human platelets were also studied alongside with rat specimens. Several lines of evidence indicate that the ATP-induced aggregation of rat platelet-rich plasma (PRP) is not a result of contaminating ADP in the ATP preparation. ATP did not cause aggregation of human platelets; it inhibited ADP- and collagen-induced human platelet aggregation. ATP pretreated with a creatine phosphate/creatine phosphokinase system caused similar rat platelet aggregation as did ATP not treated with this system. The aggregometry of ATP-induced aggregation of rat PRP was similar to that of collagen-induced aggregation but markedly different from that of ADP-induced aggregation. However, the nature of ATP-induced aggregation was similar to that induced by ADP. Both ATP- and ADP-induced rat platelet aggregations were not affected by adenosine, adenosine monophosphate, or acetylsalicylic acid. The ultrastructure of ATP-aggregated platelets was similar to that of ADP-aggregated ones. It appears that either platelets of rats possess specific ATP receptors or the rat plasma contains a material, lacking or insufficiently present in human plasma, that converts ATP to ADP in a fashion similar to the release of ADP from platelet storage granules.     

Intravenous cocaine induces platelet activation in the conscious dog

BACKGROUND: Cocaine consumption has been associated with thrombosis of coronary and peripheral arteries. Since cocaine has been found to induce platelet activation in vitro, we sought to establish whether cocaine induced platelet activation in vivo. METHODS AND RESULTS: Chronically instrumented, conscious dogs were infused with cocaine (1 mg/kg), norepinephrine (0.2 to 0.4 mg/kg), or saline intravenously over 1 minute. Activated canine platelets were identified in whole blood collected from an indwelling aortic catheter by flow cytometric detection of the binding of a monoclonal antibody directed against the activation-dependent antigen P-selectin. Infusion of cocaine resulted in an elevation of mean arterial pressure (91 +/- 3 to 128 +/- 7 mm Hg [P < .01]) and heart rate (87 +/- 9 to 125 +/- 11 beats per minute [P < .01]). A similar change (P = NS) in mean arterial pressure followed norepinephrine infusion (100 +/- 5 to 137 +/- 13 mm Hg [P < .04]), whereas saline infusion had no effect. Cocaine resulted in a substantial but delayed increase in platelet P-selectin expression (14 +/- 7% [P < .08], 31 +/- 12% [P < .04], and 55 +/- 22% [P < .04] at 17, 22, and 27 minutes after drug infusion, respectively). The magnitude of this increase was similar to that found in blood treated ex vivo with the agonists ADP or PAF (23 +/- 7% and 53 +/- 15%, respectively). No significant increase in P-selectin expression was detected in the blood of animals that received norepinephrine or saline. Serum cocaine concentrations were highest immediately after infusion (538 +/- 55 ng/mL at 2 minutes) but declined rapidly (185 +/- 22 and 110 +/- 25 ng/mL at 17 and 32 minutes after infusion); in contrast, the increase in benzoylecgonine concentrations was delayed (from < 25 ng/mL in all but one animal [34 ng/mL] at 2 minutes to 46 +/- 4 and 71 +/- 11 ng/mL at 17 and 32 minutes, respectively, after infusion). CONCLUSIONS: Intravenous cocaine induces activation of individual circulating platelets; this effect is not reproduced by infusion of norepinephrine at doses sufficient to exert similar hemodynamic effects. The delay in detection of activated platelets after treatment with cocaine may result from the adhesion and subsequent detachment of activated platelets; alternatively, cocaine metabolites, rather than the drug itself, may induce platelet activation.     

Toxicity assessment of 16 inorganic environmental pollutants by six bioassays

ATP and ADP are simultaneously released from activated platelets in equimolar concentrations. Micromolar concentrations of ATP inhibit platelet aggregation by both competitive and non-competitive mechanisms. The current studies addressed the question of how platelets respond to agonists in the presence of nanomolar and micromolar concentrations of ATP and ADP alone or in combination. This is a significant issue since the concentration of ATP +/- ADP may vary widely within a microenvironment depending upon the source and cause for the release of the nucleotides. ATP (1-10 nM) was found to significantly enhance the thromboxane A2 analog, U44619-, collagen- and thrombin-induced platelet aggregations. Conversely, ATP at 1-100 microM inhibited these same reactions. ADP, in general, behaved exactly opposite to ATP. When equal amounts of ATP and ADP were added together the ADP response appeared to predominate. The observed ATP-induced response was not due to a hydrolytic product as evidenced by an unaltered response to ATP in the presence of adenosine deaminase or the ATP generating system, creatine phosphate plus creatine phosphokinase. Adenosine (1-10 nM), like ADP, inhibited agonist-induced platelet aggregation. The stimulation of agonist-induced platelet aggregation by 1-10 nM extracellular ATP appears to depend upon the phosphorylation of platelet membrane ecto proteins. The ATP analog, beta gamma-methylene ATP, that is incapable of serving as a phosphate donor for protein kinases, inhibited rather than stimulated agonist-induced platelet aggregation. The dual response of platelets to low and high concentrations of extracellular ATP +/- ADP may play a physiological role in hemostasis and thrombosis under normal and pathological conditions.     

Increased fraction of circulating activated platelets in acute and previous cerebrovascular ischemia

Determination of circulating activated platelets may be helpful to estimate the prognosis and to stratify therapies in arterial vascular disorders including stroke. We used flow cytometry and phase contrast microscopy to study whether the fraction of platelets expressing p-selectin and CD63 and the fraction of platelets with shape change are increased in patients with acute and previous cerebrovascular ischemia. The proportion of platelets expressing activation dependent antigens was higher in patients with acute (n = 24; p-selectin: 8.23 +/- 4.21%; CD63: 3.53 +/- 2.53%) and with previous cerebrovascular ischemia (n = 46; 3.86 +/- 1.98%; 2.80 +/- 1.79%) as compared to age- and sex-matched control subjects (n = 35; 2.17 +/- 0.96%; 1.79 +/- 0.75%; p < or = 0.005, respectively). In patients with previous ischemia, there was no difference between treatment with aspirin (n = 25) or phenprocoumon (n = 21). Hypertension, diabetes mellitus and smoking were not associated with increased antigen expression (analysis of variance). The fraction of discoid platelets and platelet counts were not significantly different between groups. Our results indicate increased expression of platelet neoantigens in acute and to a less degree in previous cerebrovascular ischemia. Ongoing platelet activation after cerebrovascular ischemia despite therapy with aspirin or phenprocoumon indicates that new anti-platelet drugs may be of benefit for these patients. Flow cytometry appears to be a useful tool to assess platelet function in cerebrovascular ischemia.     

Comparison of inotropic and chronotropic effects of vasoactive intestinal peptide in isolated dog atria

In an in vitro study the effect of various thrombin inhibitors (argatroban, efegatran, DuP 714, recombinant hirudin and PEG-hirudin) on platelet activation in whole blood was investigated. Blood was drawn from normal human volunteers using the double syringe technique without use of a tourniquet to avoid autoaggregation of platelets. Blood was anticoagulated with either argatroban, efegatran, DuP 714, hirudin or PEG-hirudin at final concentrations of 10 micrograms/ml. Blood samples were then incubated at 37 degrees C either with saline, r-tissue factor, arachidonic acid, adenosine diphosphate or collagen. At definite times (1, 2.5, 5, 10 min) aliquots were taken and after various steps of fixative procedure the percentage of platelet activation was measured using fluorescent monoclonal antibodies to platelet surface receptors GPIIIa (CD-61) and P-selectin (CD-62). Flow cytometric analysis showed a platelet activation after all agonists used. All thrombin inhibitors studied caused a nearly complete inhibition of r-tissue factor-mediated platelet activation. In contrast, after activation with the other agonists an increased percent CD-62 expression was found with a maximum after 2.5 to 5 min. The results show that in whole blood thrombin inhibitors are effective in preventing platelet activation induced by r-tissue factor. The formation of active serine proteases including thrombin may be effectively inhibited by these agents. The observations further suggest that while thrombin inhibitors may control serine proteases, these agents do not inhibit the activation of platelets mediated by other agonists.     

Comparison of three different methods for radiolabelling human activated T lymphocytes

The purpose of this study was to determine both the selenium (Se) level and glutathione peroxidase (GSH-Px) activity in plasma, erythrocytes and platelets from 51 healthy Japanese individuals. The Se levels (mean +/- SD) of plasma, erythrocytes and platelets were 117.4 +/- 15.7 micrograms/L, 0.954 +/- 0.159 microgram/g hemoglobin, and 4.93 +/- 1.52 ng/mg protein, respectively, and GSH-Px activity was 318 +/- 50 U/L, 18.0 +/- 5.0 U/g hemoglobin, and 0.142 +/- 0.035 U/mg protein, respectively. There was a negative correlation between age and the platelet Se level in men (r = -0.761, p < 0.001), and a positive correlation between the plasma and platelet GSH-Px activities in women (r = 0.663, p < 0.001).     

Morphologic comparison of atherosclerotic lesions in native coronary arteries and saphenous vein graphs with intracoronary angioscopy in patients with unstable angina

The purpose of this study was to investigate platelet effects on postischemic heart function in conjunction with adenosine effects on intracoronary platelet adhesion. Homologous platelets were infused into the coronaries of isolated guinea pig hearts, either during low-flow ischemia or during reperfusion, and external heart work (EHW) and intracoronary platelet adhesion were determined. In most experiments, thrombin was added to the perfusate. The influence of endogenous adenosine was studied by use of the uptake blocker dipyridamole and the unspecific adenosine-receptor blocker theophylline, the A1-receptor blocker 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), and the A2-receptor blocker 3,7-dimethyl-1-propargylxanthine (DMPX). The importance of nitric oxide and prostaglandin I2 (PGI2) was tested by using nitro-L-arginine (NOLAG) and indomethacin, respectively. When platelets were applied with thrombin during low-flow ischemia, EHW recovered to only 63 +/- 4% of the preischemic value, as compared with 89 +/- 3% without platelets (p < 0.05). Despite thrombin, platelets incurred no significant functional loss when applied in the first minute of reperfusion (but again in the fifth minute); however, when theophylline was also present, recovery of EHW amounted to only 42 +/- 12%. Intracoronary adhesion of platelets was negligible without thrombin, and highest during low-flow ischemia with thrombin (35 +/- 3% of the applied number). No adhesion occurred during the first minute of reperfusion, whereas in the fifth minute, adhesion was again 20.8 +/- 4%. Dipyridamole increased adenosine release and attenuated adhesion at this time. Theophylline increased adhesion in the first minute of reperfusion (33 +/- 6.4%), whereas NOLAG and indomethacin proved to be ineffective. DPCPX and DMPX each increased platelet retention during the first minute of reperfusion, their effects being additive. Intracoronary adhesion of platelets induced by thrombin in isolated hearts can reduce postischemic recovery of heart function. During reperfusion, but not during low-flow, endogenous adenosine can prevent platelet adhesion and loss of myocardial function, an action mediated both by A1- and A2-receptor-dependent mechanisms.     

Morphine reverses the inhibitory effects of repeated saline injections on peritoneal inflammation in mice

AIMS: The lipid composition of platelets and the function of these cells in patients with uremia were studied. METHODS: Fourteen patients and 14 normal volunteers were studied. Platelet lipids including phospholipids and cholesterol, as well as the platelet aggregation response to agonists, were studied. RESULTS: The amount of platelet phospholipids was decreased in patients compared to controls (338.0 +/- 79 vs. 511.6 +/- 125 nmol/10(9) cells; p < 0.001), while the percentage of the five main specimens of these compounds was normal. The content of platelet cholesterol in patients (97.8 +/- 17.0 microg/10(9) cells) was similar to that in controls (91.7 +/- 26.0 microg/10(9) cells). Consequently, the cholesterol:phospholipid ratio in uremic platelets was increased (0.75 +/- 0.1 vs. 0.46 +/- 0.1; p < 0.01). Although this feature is associated with hyperreactive platelets, the aggregation tests were defective for adenosin diphosphate (p < 0.01), arachidonic acid (p < 0.01), epinephrine (p < 0.01) and collagen (p < 0.001). This behavior is probably due to the multifactorial platelet defect described in uremia.     

The F2-isoprostane 8-epiprostaglandin F2alpha increases platelet adhesion and reduces the antiadhesive and antiaggregatory effects of NO

F2-isoprostanes are prostaglandin (PG) isomers produced in vivo through free radical-catalyzed peroxidation of arachidonic acid, which may affect platelet function. The current study investigated the effects of 8-epiprostaglandin F2alpha (8-epi-PGF2alpha) on critical events of platelet activation. A dose-dependent increase in platelet adhesion to fibrinogen- and plasma-coated microwells by 8-epi-PGF2alpha (1 to 1000 nmol/L) was observed when resting platelets (plasma from 1.3+/-0.2% to 5.5+/-0.2%, EC50 of 48 nmol/L; fibrinogen from 3.3+/-0.3% to 6.4+/-0.2%, EC50 of 35 nmol/L; mean+/-SEM, n=8, P<0.001) and thrombin-stimulated human platelets were used. The expression of the adhesion molecule glycoprotein IIb/IIIa was increased by 10 to 1000 nmol/L 8-epi-PGF2alpha in resting platelets (from 64.8+/-2.1% to 83.9+/-1.3%; n=5, P<0.01) and in stimulated platelets. The secretion of the glycoprotein GMP-140 increased only in the presence of both thrombin and 10 to 1000 nmol/L 8-epi-PGF2alpha (from 48.5+/-3.1% to 63.1+/-2.0%, P<0.05). The antiaggregatory effects of both the NO donor NOR-3 (basal, 21.4+/-4.6%; with 8-epi-PGF2alpha, 30.8+/-6.9%; n=14, P<0.05) and endothelial cells that release NO (basal, 18.5+/-4.6%; with 8-epi-PGF2alpha, 30.7+/-5.3%; n=15, P<0.001) were also reduced. All of these effects were prevented by the thromboxane receptor antagonist GR32191 but not affected by acetylsalicylic acid. An increase in free intracellular calcium concentration, measured with the use of fura 2, was observed with 8-epi-PGF2alpha. In conclusion, F2-isoprostanes may participate in oxidative injury by inducing platelet activation and by reducing the antiplatelet activity of NO: increased platelet adhesiveness and expression of the fibrinogen receptor are induced by nanomolar amounts of 8-epi-PG-F2alpha. Platelet secretion and aggregation can also be induced in the presence of platelet agonists.     

Ultrastructural observation of platelets from patients with progressive systemic sclerosis (PSS)

We observed the ultrastructure of platelets from patients with PSS (7 cases; 48.2 +/- 12.3 y-old; M:F = 1:6_ and healthy controls (HC) (5 cases; 44.8 +/- 8.0 y-old; M:F = 1:4) by using transmission (TEM) and freeze-fracture electron microscopy (FEM). The open canalicular system (OCS) connected with the plasma membrane (PM) formed pinhole-like invaginations (50 nm in diameter) in the cleaved face (P-face) of the plasma membrane seen from the outside of the platelets and sharply elevated structures in the cleaved face (E-face) of PM seen from the inside of the platelets by FEM. The density of OCS on the surface of the platelets from PSS patients was 3 +/- 1/micron 2, which was higher than that from HC (1 +/- 0.5/micron 2) (p < 0.02). Dome-shaped structures, which clearly differ from OCS and were 80-150 nm in diameter without intramembranous particles, were seen in the P-face, and the complementary depressed structures were seen in the E-face. These structures were thought to be vesicles fused onto the PM of the platelets. The total volume of platelets (7.62 +/- 0.11 micron 3), total volume of granules (0.79 +/- 0.01 micron 3) and vacuoles including OCS (0.78 +/- 0.05 micron 3), and the total surface area of platelets (17.25 +/- 1.30 micron 2) from four PSS patients calculated by the morphometrical method were similar to those from four HC (7.32 +/- 0.25 micron 3, 0.76 +/- 0.03 micron 3, 0.80 +/- 0.05 micron 3, 18.75 +/- 0.35 micron 2, respectively); there were no statistical significances between the data from PSS patients and HC. The total volumes of vacuoles in platelets from both PSS patients and HC significantly decreased after a 2 min-vibration stress of the hands (p < 0.02) and the total volume of granules in platelets from PSS patients decreased significantly after the same stress (p < 0.002), although that from HC showed no similar significant change. However, there were no statistically significant differences in total volume or total surface of platelets from PSS patients and HC after the stress. These data may suggest that depletion of granules occurred due to activation of platelets from PSS patients following a secretion of their proteins, because their plasma protein levels were elevated after the stress (Jpn J Dermatol, 98; 1205, 1988). Higher density of OCS on the surface of the platelets from PSS patients may play an important role in secretion of their proteins, although the detailed mechanism of secretion of specific proteins derived from platelet granules is still unknown. These ultrastructural abnormalities of platelets may correlate with some involvement of a platelet disorder and with a possible role for the activation of platelets from PSS patients.     

Effects of a new platelet glycoprotein IIb/IIIa antagonist, SR121566, on platelet activation, platelet-leukocyte interaction and thrombin generation

The effects of SR121566, a new inhibitor of the glycoprotein (GP) IIb/IIIa complex on platelet activation and platelet-leukocyte interactions, as well as on thrombin generation were investigated. SR121566 dose-dependently inhibited adenosine diphosphate (ADP)-induced platelet fibrinogen binding determined either by flow cytometry analysis (IC50=50 nmol/l) or by measuring the binding of 125I-fibrinogen to activated human gel-filtered platelets (IC50=20 nmol/l). Consistent with its inhibitory effects on platelet fibrinogen binding, SR121566 demonstrated a dose-dependent inhibition of collagen-, ADP- or thrombin-induced platelet aggregation with IC50 values ranging between 20 and 60 nmol/l. SR121566, even tested at high concentrations, did not significantly affect ADP-induced platelet-leukocyte aggregate formation. The GPIIb/IIIa antagonist strongly inhibited thrombin generation in both native clotting blood and recalcified whole blood, suggesting that SR121566, by interfering with the platelet-activation events involved in facilitating thrombin generation, may also function as an anticoagulant, an effect which may contribute to its antithrombotic properties in humans.     

Role of chondroitin 4-sulphate as a receptor for polycation induced human platelet aggregation

1. Proteoglycans provide negatively charged sites on the surface of platelets, leukocytes and endothelial cells. Since chondroitin 4-sulphate is the main proteoglycan present on the platelet surface, the role of this molecule in mediating the activation of human platelets by polylysine was studied. 2. Platelets were desensitized with phorbol 12-myristate 13-acetate (PMA, 10 nM) 5 min before the addition of polylysine to platelet-rich plasma (PRP). Changes in the intracellular Ca2+ concentration were measured in fura2-am (2 microM) loaded platelets and protein phosphorylation was assessed by autoradiography of the electrophoretic profile obtained from [32P]-phosphate labelled platelets. The release of dense granule contents was measured in [14C]-5-hydroxytryptamine loaded platelets and the synthesis of thromboxane (TXA2) was assessed by radioimmunoassay. Surface chondroitin 4-sulphate proteoglycan was degraded by incubating platelets with different concentrations of chondroitinase AC (3 min, 37 degrees C). The amount of chondroitin 4-sulphate remaining in the platelets was then quantified after proteolysis and agarose gel electrophoresis. 3. The addition of PMA to PRP before polylysine inhibited the aggregation by 88 +/- 18% (n = 3). Staurosporine (1 microM, 5 min) prevented the PMA-induced inhibition. Chondroitinase AC (4 pu ml-1 to 400 muu ml-1, 3 min) abolished the polylysine-induced aggregation in PRP but caused only a discrete inhibition of ADP-induced aggregation. The concentration of chrondroitin 4-sulphate in PRP (0.96 +/- 0.2 microgram/10(8) platelets, n = 3) and in washed platelets (WP; 0.35 +/- 0.1 microgram/10(8) platelets, n = 3) was significantly reduced following incubation with chondroitinase AC (PRP = 0.63 +/- 0.1 microgram/10(8) platelets and WP = 0.08 +/- 0.06 microgram/10(8) platelets). 4. Washed platelets had a significantly lower concentration of chondroitin 4-sulphate than platelets in PRP. The addition of polylysine to WP induced a rapid increase in light transmission which was not accompanied by TXA2 synthesis or the release of dense granule contents. This effect was not inhibited by sodium nitroprusside (SNP), iloprost, EDTA or the peptide RGDS. This event was accompanied by the discrete phosphorylation of plekstrin and myosin light chain, which were inhibited by staurosporine (10 microM, 10 min). The hydrolysis of platelet surface chondroitin 4-sulphate strongly reduced the polylysine-induced phosphorylation. 5. Our results indicate that polylysine activates platelets through a specific receptor which could be the proteoglycan chondroitin 4-sulphate present on the platelet membrane.     

Ristocetin- and thrombin-induced platelet aggregation at physiological shear rates: differential roles for GPIb and GPIIb-IIIa receptor

We recently reported that washed platelets (WP) activated with ADP and expressing surface-bound vWF aggregated in flow through small tubes or in a cylindrical couette device at physiological shear rates of G = 300 s(-1)-1000 s(-1) in the absence of exogenous ligands, with GPIb-vWF partially, and activated GPIIb-IIIa totally required for the aggregation. We have now extended these studies to aggregation of platelets "activated" with ristocetin or thrombin. Washed platelet suspensions with added soluble vWF and ristocetin (0.3-0.75 mg/ml), or activated with thrombin (0.01-0.5 U/ml) but no added ligand, were sheared in a coaxial cylinder device at uniform shear rate, G = 1000 s(-1). The collision capture efficiency (alphaG) with which small aggregates form (= experimental/calculated initial rates of aggregation) was correlated with vWF platelet binding assessed by flow cytometry. The vWF-GPIb interaction was exclusively able to support ristocetin-mediated shear aggregation of metabolically active platelets, with very few vWF monomer equivalents bound per platelet (representing < or = 10 molecules of 10 million Da) required to yield high capture efficiencies (alphaG = 0.38+/-.02; n = 11), suggesting rapid and stable bond formations between vWF and GPIb. However, platelet surface-expressed vWF, generated by addition of thrombin to washed platelets, was found to mediate platelet aggregation with alphaG = 0.08+/-.01 (n = 6), surprisingly comparable to that previously reported for WP and ADP activation. Blocking the GPIIb-Illa receptor decreased alphaG by 95+/-3% (n =3), while a monoclonal antibody to the vWF site on GPIb caused a 49+/-7% (n = 8) decrease in alphaG. The partial role for GPIb thus appears to reflect a facilitative function for increasing contact time between flowing platelets, and allowing engagement of the GPIIb-IIa receptor to yield stable attachment.     

Polyps of the colon and rectum

The uptake and metabolism of [14C]- or E[3H] adenosine have been studied in suspensions of washed platelets and in platelet rich plasma. The appearance of radioactivity in the platelets and the formation of radioactive adenosine metabolites have been used to determine the uptake. Adenosine is transported into human blood platelets by two different systems: a low Km system (9.8 muM) which is competitively inhibited by papaverine, and a high Km system (9.4 mM) which is competitively inhibited by adenine. Adenosine transported via the low Km system is probably directly incorporated into adenine nucleotides, while adenosine transported through the high Km system arrives unchanged inside the platelet and is then converted into inosine and hypoxanthine or incorporated into adenine nucleotides.     

Hormonal therapy increases arterial compliance in postmenopausal women

Vessel injury and thrombus formation are the cause of most ischemic coronary syndromes and, in this setting, activated platelets stimulate platelet recruitment to the growing thrombus. Recently, a constitutive nitric oxide synthase (NOS) has been identified in human platelets. To further define the capacity of platelets to produce nitric oxide (NO), as well as to study the role of this NO in platelet recruitment, we adapted a NO-selective microelectrode for use in a standard platelet aggregometer, thereby permitting simultaneous measurement of platelet aggregation and NO production. Treatment of platelets with the NO synthase inhibitor -NG-nitroarginine methyl ester (L-NAME), reduced NO production by 92+/-8% in response to 5 microM ADP compared to control but increased aggregation by only 15+/-2%. In contrast, L-NAME had a more pronounced effect on platelet recruitment as evidenced by a 35+/-5% increase in the extent of aggregation, a 33+/-3% decrease in cyclic GMP content, and a 31+/-5% increase in serotonin release from a second recruitable population of platelets added to stimulated platelets at the peak of NO production. To study platelet recruitment accurately, we developed an assay that monitors two platelet populations simultaneously. Nonbiotinylated platelets were incubated with L-NAME or vehicle and activated with ADP. At peak NO production, biotinylated platelets were added. As measured by three-color flow cytometry, there was a 56+/-11% increase in the number of P selectin- positive platelets in the nonbiotinylated population treated with L-NAME as compared to control. When biotinylated platelets were added to the L-NAME-treated nonbiotinylated population, the number of P selectin positive biotinylated plate-lets increased by 180+/-32% as compared to biotinylated platelets added to the control. In summary, stimulated platelets produce NO that modestly inhibits platelet activation but markedly inhibits additional platelet recruitment. These data suggest that platelet-derived NO may regulate platelet recruitment to a growing thrombus.