Reversibility of platelet P2Y12 inhibition by platelet supplementation: ex vivo and in vitro comparisons of prasugrel, clopidogrel and ticagrelor.

Essentials Successful outcome of platelet transfusion depends on specific antiplatelet therapy in use. We assessed if ticagrelor, clopidogrel or prasugrel impacts on donor platelet activity ex vivo. Ticagrelor and/or its active metabolite in plasma or bound to platelets can inhibit donor platelets. This might compromise the effectiveness of platelet transfusion therapy. SUMMARY: Background Platelet transfusion is the conventional approach to restore platelet function during acute bleeds or surgery, but successful outcome depends on the specific antiplatelet therapy. Notably ticagrelor is associated with inadequate recovery of platelet function after platelet transfusion. We examined whether plasma and/or platelets from ticagrelor-treated patients influence donor platelet function, in comparison with clopidogrel and prasugrel. Methods Platelet transfusion was mimicked ex vivo by mixing naïve donor platelet-rich plasma (PRP) or gel-filtered platelets (GFP) in defined proportions with PRP, plasma or GFP from cardiovascular patients receiving standard care including medication with prasugrel, clopidogrel or ticagrelor (n = 20 each). Blood was taken 4 h after the previous dose. HLA2/HLA28 haplotyping let us distinguish net (all platelet) and individual patient/donor platelet reactivity in mixtures of patient/donor platelets, measured by flow cytometry analysis of ADP-induced fibrinogen binding and CD62P expression. Results ADP responsiveness of donor platelets was dramatically reduced by even low (10%) concentrations of PRP or plasma from ticagrelor-treated patients. Clopidogrel and prasugrel were associated with more modest donor platelet inhibition. GFP from ticagrelor-treated patients but not patients receiving clopidogrel or prasugrel also suppressed donor GFP function upon mixing, suggesting the transfer of ticagrelor from patient platelets to donor platelets. This transfer did not lead to recovery of ADP responsiveness of patient's platelets. Conclusion Collectively, these observations support the concept that ticagrelor and/or its active metabolite in plasma or bound to platelets can inhibit donor platelets, which might compromise the effectiveness of platelet transfusion therapy.

Cytochrome P450 3A inhibition by ketoconazole affects prasugrel and clopidogrel pharmacokinetics and pharmacodynamics differently.

Prasugrel and clopidogrel inhibit platelet aggregation through active metabolite formation. Prasugrel's active metabolite (R-138727) is formed primarily by cytochrome P450 (CYP) 3A and CYP2B6, with roles for CYP2C9 and CYP2C19. Clopidogrel's activation involves two sequential steps by CYP3A, CYP1A2, CYP2C9, CYP2C19, and/or CYP2B6. In a randomized crossover study, healthy subjects received a loading dose (LD) of prasugrel (60 mg) or clopidogrel (300 mg), followed by five daily maintenance doses (MDs) (15 and 75 mg, respectively) with or without the potent CYP3A inhibitor ketoconazole (400 mg/day). Subjects had a 2-week washout between periods. Ketoconazole decreased R-138727 and clopidogrel active metabolite Cmax (maximum plasma concentration) 34-61% after prasugrel and clopidogrel dosing. Ketoconazole did not affect R-138727 exposure or prasugrel's inhibition of platelet aggregation (IPA). Ketoconazole decreased clopidogrel's active metabolite AUC0-24 (area under the concentration-time curve to 24 h postdose) 22% (LD) to 29% (MD) and reduced IPA 28% (LD) to 33% (MD). We conclude that CYP3A4 and CYP3A5 inhibition by ketoconazole affects formation of clopidogrel's but not prasugrel's active metabolite. The decreased formation of clopidogrel's active metabolite is associated with reduced IPA.

Elevated glucose alters eicosanoid release from porcine aortic endothelial cells.

Cultured porcine aortic endothelial cells were conditioned through two passages to mimic euglycemic and hyperglycemic conditions (5.2 mM, normal glucose; 15.6 mM, elevated glucose). After incubation with 1 microM [14C]arachidonic acid for 24 h, the cells were stimulated with 1 microM A23187 for times up to 30 min. Uptake of [14C]arachidonic acid and its distribution among cell lipids were unaffected by the increased glucose concentration. The release of eicosanoids from labeled cells and unlabeled cells was measured by reverse-phase HPLC and by RIA, respectively. Compared with cells stimulated in the presence of normal glucose concentrations, cells stimulated in the presence of elevated glucose released 62.6% less free [14C]arachidonic acid, but released 129% more 14C-labeled 15-hydroxyeicosatetraenoic acid (HETE). Increased release of 15-HETE in the presence of elevated glucose in response to A23187, bradykinin, and thrombin was confirmed by RIA. A similar increase in 5-HETE release was observed by RIA after A23187 treatment. The release of both radiolabeled and unlabeled prostanoids was equal at both glucose concentrations. The data indicate that glucose may play an important role in the regulation of release and metabolism of arachidonic acid after agonist stimulation. In the presence of elevated glucose concentrations, such as those associated with diabetes mellitus, the extent and pattern of eicosanoid release from endothelial cells is markedly altered.

The greater in vivo antiplatelet effects of prasugrel as compared to clopidogrel reflect more efficient generation of its active metabolite with similar antiplatelet activity to that of clopidogrel's active metabolite.

BACKGROUND AND METHODS: Prasugrel is a novel orally active thienopyridine prodrug with potent and long-lasting antiplatelet effects. Platelet inhibition reflects inhibition of P2Y(12) receptors by its active metabolite (AM). Previous studies have shown that the antiplatelet potency of prasugrel is at least 10 times higher than that of clopidogrel in rats and humans, but the mechanism of its higher potency has not yet been fully elucidated. RESULTS: Oral administration of prasugrel to rats resulted in dose-related and time-related inhibition of ex vivo platelet aggregation, and its effect was about 10 times more potent than that of clopidogrel. The plasma concentration of prasugrel AM was higher than that of clopidogrel AM despite tenfold higher doses of clopidogrel, indicating more efficient in vivo production of prasugrel AM than of clopidogrel AM. In rat platelets, prasugrel AM inhibited in vitro platelet aggregation induced by adenosine 5'-diphosphate (ADP) (10 microm) with an IC(50) value of 1.8 microm. Clopidogrel AM similarly inhibited platelet aggregation with an IC(50) value of 2.4 microm. Similar results were also observed for ADP-induced (10 microm) decreases in prostaglandin E(1)-stimulated rat platelet cAMP levels. These results indicate that both AMs have similar in vitro antiplatelet activities. CONCLUSIONS: The greater in vivo antiplatelet potency of prasugrel as compared to clopidogrel reflects more efficient in vivo generation of its AM, which demonstrates similar in vitro activity to clopidogrel AM.

Induction of Diabetes Abolishes the Antithrombotic Effect of Clopidogrel in Apolipoprotein E-Deficient Mice.

Patients with acute coronary syndrome with diabetes mellitus (DM) exhibit an impaired platelet inhibitory response to clopidogrel which is only partially understood. DM was induced by the administration of streptozotocin (STZ) to 9-week-old mice. The antithrombotic effects of clopidogrel (10 mg/kg/d, orally × 5 days) were determined using a FeCl 3 -induced thrombosis model employing wild-type (WT), apolipoprotein E (apoE)-deficient, and diabetic apoE-deficient mice at 21 weeks. Antiplatelet effects were determined using flow cytometry. The antithrombotic effects of clopidogrel were similar in WT and apoE-deficient mice but were attenuated in diabetic apoE-deficient mice with the percent inhibition of thrombus area (µm 2 ) by clopidogrel being 85.5% (WT mice), 75.0% (apoE-deficient mice), and 1.9% (diabetic apoE-deficient mice). The time to first occlusion and lumen stenosis also reflected a significant loss of the antithrombotic effects of clopidogrel in diabetic apoE-deficient mice. Ex vivo platelet activation, which was assessed using ADP-induced expression of activated glycoprotein IIb/IIIa, was completely inhibited by clopidogrel in these three groups of mice. In contrast, the effect of clopidogrel on the ex vivo expression of platelet P-selectin induced by protease-activated receptor 4-activating peptide was diminished in diabetic apoE-deficient mice compared with that in WT and apoE-deficient mice. These data suggest that diabetic apoE-deficient mice may serve as a useful model to better understand the impaired responses to clopidogrel in patients with DM, which may partially reflect a reduction of the effect of clopidogrel on thrombin-induced platelet activation.

Incomplete reversibility of platelet inhibition following prolonged exposure to ticagrelor.

Essentials Irreversible platelet inhibition persists after reversibly-binding ticagrelor is discontinued. Reversibility of platelet inhibition by ticagrelor and its active metabolite was assessed. Incomplete recovery was observed after prolonged exposure to ticagrelor. Activated GPIIb-IIIa and P-selectin, not platelet reactivity index, showed irreversibility. SUMMARY: Introduction Ticagrelor is described as a reversible P2Y12 antagonist. However, residual platelet inhibition persists after discontinuation of ticagrelor when plasma levels are undetectable. We assessed the reversibility of platelet inhibition by ticagrelor and its active metabolite (T-AM) in comparison with cangrelor and prasugrel's active metabolite (P-AM). Methods Whole blood was treated in vitro with ~ 50% inhibitory concentrations of ticagrelor, T-AM, cangrelor, P-AM and assessed for ADP-stimulated activated GPIIb-IIIa and P-selectin and vasodilator-stimulated phosphoprotein (VASP) platelet reactivity index (PRI) before and after 100-fold dilution. Results Platelets exposed for 30 min to ticagrelor, T-AM or cangrelor showed full recovery of activated GPIIb-IIIa but only partial recovery of P-selectin. Longer exposure (24 h) to the drug decreased reversibility of activated GPIIb-IIIa by ticagrelor (65.1% [49.5-80.6], % of vehicle with 95% confidence interval [CI]) and T-AM (88.8% [79.2-98.3]), but not by cangrelor (101.4% [96.4-106.4]). Compared with 30 min exposure, the reversibility of P-selectin further decreased after 24 h exposure to ticagrelor (from 91.8% [82.1-101.5] to 51.8% [45.5-85.0]), but not T-AM (from 79.0% [67.8-90.3] to 77.4% [61.8-93.1]) or cangrelor (from 76.0% [67.6-84.4] to 76.2% [70.6-81.8]). In contrast, 24 h exposure to ticagrelor, T-AM and cangrelor resulted in full recovery of platelet reactivity as measured by PRI. Platelets exposed to P-AM showed no recovery of ADP reactivity. Conclusions Incomplete recovery after prolonged exposure to ticagrelor, observed by activated GPIIb-IIIa and P-selectin but not upstream VASP signaling, suggests that P2Y12 regains functionality and irreversible changes occur independent of VASP signaling.

Hydrolysis of 1-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine, a common precursor of platelet-activating factor and eicosanoids, by human platelet phospholipase A2.

The metabolism of platelet-activating factor (PAF) and arachidonic acid is linked through the common intermediate 1-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine (alkylarachidonoyl-GPC). Hydrolysis of alkylarachidonoyl-GPC by phospholipase A2 may initiate the biosynthesis of both PAF and eicosanoids, since alkyllyso-GPC is formed for acetylation to PAF and arachidonic acid is liberated for conversion to biologically active metabolites. In order to elucidate the regulation and functional role of human platelet phospholipase A2 in the pathway leading to the formation of both classes of lipid mediators, we have characterized its action upon alkylarachidonoyl-GPC. Human platelet phospholipase A2 was solubilized and then partially purified in the presence of n-octyl-beta-D-glucopyranoside (octyl glucoside). Hexadecylarachidonoyl-GPC was prepared biosynthetically using platelet sonicates, purified by two-step high-performance liquid chromatography (HPLC) and suspended in buffer by sonication. Our results indicate that deacylation of alkylarachidonoyl-GPC by platelet phospholipase A2 has an absolute requirement for Ca2+. It occurs at submicromolar concentrations of free Ca2+ and exhibits a biphasic Ca2+-dependence with activity plateaus at 10 microM and 2 mM. Phospholipase A2-mediated hydrolysis of alkylarachidonoyl-GPC is increased 2-fold by albumin and is enhanced 5-fold if 1,2-dioleoylglycerol is incorporated into the substrate dispersion. The substrate dependence and specificity of platelet phospholipase A2 for 1-alkyl- vs. 1-acyl-linked subclasses of arachidonic acid containing phosphatidylcholine was examined with 1-O-hexadecyl-2-arachidonoyl-sn-glycero-3-phosphocholine (hexadecylarachidonoyl-GPC) and 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (palmitoylarachidonoyl-GPC). We found that the substrates were deacylated equivalently. We conclude that, in stimulated platelets, in the presence of increased levels of cytoplasmic free Ca2+ and newly generated diacylglycerol, alkylarachidonoyl-GPC may be rapidly hydrolyzed by phospholipase A2 and may serve as a precursor of both PAF and eicosanoids.

Incorporation and redistribution of arachidonic acid in diacyl and ether phospholipids of bovine aortic endothelial cells.

The present experiments characterized the incorporation and redistribution of arachidonic acid in diacyl and ether phospholipids of bovine aortic endothelial cells. Confluent cultures were either continuously labeled or pulse labeled with [14C]arachidonic acid. Major lipid classes and ether-linked subclasses of phosphatidyl-ethanolamine (PE) and phosphatidylcholine (PC) were separated by high-performance liquid chromatography and thin-layer chromatography. During continuous labeling, total incorporation of arachidonic acid reached a peak at 8 h and was essentially constant up to 24 h. After 8 h, net label in total PC declined, whereas that in total PE continued to rise. In pulse labeling experiments radioactivity in diacyl PC continuously declined with concomitant increases in both diacyl- and alkenylacyl PE. The data demonstrate that transfer of arachidonic acid from diacyl PC to both diacyl- and alkenylacyl PE occurs in endothelial cells. In contrast to previous observations in platelets, transfer of arachidonic acid to alkenylacyl PE did not require agonist stimulation. This pathway may contribute to the enrichment of endothelial cell PE with arachidonic acid with the potential for subsequent metabolism to prostacyclin.

Ionophore-induced metabolism of phospholipids and eicosanoid production in porcine aortic endothelial cells: selective release of arachidonic acid from diacyl and ether phospholipids.

Confluent cultures of porcine aortic endothelial cells were prelabeled with 1 microM [14C]arachidonic acid complexed to 1 microM bovine serum albumin. After washing, the cells were stimulated with 1 microM A23187 for time intervals between 30 s and 30 min. Cellular lipids were extracted and separated into major lipid classes and phospholipid subclasses. The external medium was analyzed for released radioactive eicosanoids. The time-course of total release of 14C radioactivity demonstrated a biphasic nature of A23187-induced changes in endothelial cell lipids. Early, from 30 s to 5 min, substantial losses of [14C]arachidonic acid from diacylphosphatidylethanolamine and phosphatidylinositol, as well as an abrupt increase in diacylphosphatidylcholine-associated radioactivity were observed. These initial changes coincided with the release of 14C-labeled cyclooxygenase products. Later changes (5-30 min) included a sustained progressive loss of 14C radioactivity from alkenyl (alk-1-enyl) acylphosphatidylethanolamine and diacylphosphatidylcholine. These later changes coincided with the elaboration of 14C-labeled lipoxygenase products. Although unequivocal assignments cannot be made, the data suggest that specific pools of arachidonic acid provide precursors for individual classes of eicosanoids.

Vitamin E supplementation effect on human platelet function, arachidonic acid metabolism, and plasma prostacyclin levels.

A randomized, placebo-controlled double-blind trial was conducted on 20 adults to assess the effect of vitamin E (800 IU/d 727 mg/d for 5 wk) on platelet function, arachidonic acid metabolism, and prostacyclin generation. Platelet aggregation was measured in response to collagen, arachidonic acid, and adenosine diphosphate. Thromboxane B2 was assayed in serum and in the supernatant plasma after platelet aggregation. Platelets were labeled with [3H]arachidonic acid to assess production and release of cyclooxygenase products (MDA, TXB2, and HHT), a lipoxygenase product (12-HETE), and arachidonic acid in response to stimulation by thrombin or collagen. Prostacyclin was measured in plasma and in blood collected from bleeding-time incisions by a sensitive HPLC-RIA procedure. Despite marked increases in plasma and erythrocyte vitamin E levels in the vitamin E group, there were no significant differences between the vitamin E and placebo groups in any of the variables measured.

Modification of human platelet function by a diet enriched in saturated or polyunsaturated fat.

Twelve healthy male subjects were maintained on a saturated fat (SF) dietary regimen followed by a polyunsaturated fat (PUF) regimen. At selected intervals a number of tests were carried out to assess the effect of SF or PUF on platelet composition and activation. Concomitant with the fall in serum cholesterol, associated with the PUF diet, there was a decrease in plasma heparin neutralizing activity (as measured by the heparin--thrombin clotting time), and a fall in the number of circulating platelet aggregates was also observed. These two parameters suggest diminished platelet activation. Malondialdehyde production (an index of prostaglandin synthesis) was unchanged throughout the two dietary periods. Changes in the quality of the dietary fat were manifested in the phospholipid fraction of platelet lipids, particularly phosphatidyl choline and sphingomyelin. Platelet counts of whole blood were significantly decreased when subjects were consuming PUF, but not all of these alterations were reflected in platelet-rich plasma. These results indicate that platelets may be activated in apparently normal people consuming a SF diet (the standard diet of developed countries) and that this activation may be decreased by replacement of dietary SF with PUF.

Further observations on the effects of dietary fatty acid composition on platelet reactivity and blood coagulation in man and the influence of methodology on findings.

Eight human subjects were fed diets enriched in saturated fat (SF), or polyunsaturated fat (PUF) and after each dietary regimen the plasma heparinthrombin clotting time (HTCT) was determined. The HTCT of citrated plasma indicated reduced heparin-neutralizing activity (HNA) after PUF feeding compared with SF feeding. Platelet factor 4 (PF4) levels in the citrated plasma samples demonstrated an inverse correlation with the HTCT (r = 0.62). Experiments with purified PF4 indicated that the PF4 present in citrated plasma could only account for approximately 10% of the HNA. Plasma prepared in a manner which minimized in vitro release of platelet constituents contained significantly less PF4 after PUF feeding and indicated that most of the PF4 found in citrated plasma resulted from in vitro release. The factor Xa inhibitory activity of citrated plasma was not significantly altered by either of the dietary regimens.

Effect of alternate-day regular and enteric-coated aspirin on platelet aggregation, bleeding time, and thromboxane A2 levels in bleeding-time blood.

The effectiveness of low-dose aspirin for primary prevention of cardiovascular mortality is being assessed among the nearly 22,000 United States physicians currently participating in the Physicians' Health Study. Because of occasional reports of gastric irritation among study participants, two enteric-coated aspirin preparations were tested as possible alternatives to regular compressed aspirin for platelet inhibition. Thirty-three volunteers were assigned randomly to one of four treatment groups: regular aspirin (325 mg), placebo, and two enteric-coated aspirin preparations (325 mg). Pills were administered every other day, duplicating the regimen used in the Physicians' Health Study. Bleeding times, platelet aggregation, and thromboxane A2 levels produced by aggregating platelets in vitro, as well as in collected bleeding-time blood, were determined. Measurements were taken before and after a single dose as well as after seven alternate-day doses. Regular and enteric-coated aspirin preparations were equally efficacious in prolonging the bleeding time, inhibiting platelet aggregation, and suppressing thromboxane A2 production. There was virtually complete suppression of thromboxane A2 production (over 99 percent), by platelets in vitro and in collected bleeding-time blood. The levels were still profoundly reduced (89 percent) 48 hours after the last dose. Enteric-coated aspirin may provide an alternative to regular aspirin in a low-dose regimen designed to inhibit platelet activity.

Development of dual-acting agents for thromboxane receptor antagonism and thromboxane synthase inhibition. 3. Synthesis and biological activities of oxazolecarboxamide-substituted omega-phenyl-omega-(3-pyridyl)alkenoic acid derivatives and related compounds.

A novel series of oxazolecarboxamide-substituted omega-phenyl-omega-(3-pyridyl)alkenoic acid derivatives was discovered as potent dual-acting agents to block the TXA2 receptor and to inhibit the thromboxane synthase (TRA/TSI). Synthesis, structure-activity relationship (SAR), and in vitro and in vivo pharmacology of this series of compounds are described. Modification of the series revolved around the oxazole moiety to increase the hydrophilicity of the compounds and to correlate the biological activity with lipophilicity of the compounds. The most potent in the series was (E)-7-[4-[4-[[(4-cyclohexylbutyl)amino]carbonyl]-2-oxazolyl] phenyl]-7 -(3-pyridyl)hept-6-enoic acid (14) with Kd = 9.9 +/- 0.4 nM for the thromboxane receptor antagonism and IC50 = 55.0 +/- 17.9 nM for thromboxane synthase inhibition. The compound 14 was a selective TRA/TSI which exhibited desirable characteristics for oral activity, "shunt" effect to elevate PGI2 level, and absence of agonist activity.

Fibrinogen receptor (GPIIb-IIIa) antagonists derived from 5,6-bicyclic templates. Amidinoindoles, amidinoindazoles, and amidinobenzofurans containing the N-alpha-sulfonamide carboxylic acid function as potent platelet aggregation inhibitors.

A series of highly potent and specific fibrinogen receptor antagonists have been discovered and optimized through structural modification of the novel amidinoindole and benzofuran compounds, I and II. Systematic linker optimization afforded the amidinobenzofuran-containing inhibitor 29, which displayed an IC50 value of 250 nM in platelet aggregation assays. Attempts to enhance activity by modification of the beta-position of the beta-alanyl carboxylate group of 29 had only a modest effect on inhibitory activity in aggregation assays. Analogues prepared to enhance the activity by conformational restriction were also found to be equally or less potent. In contrast, modification at the alpha-position of the beta-alanyl carboxylate group resulted in the identification of extremely potent and novel amidinobenzofuran-containing derivatives 46-49. Reexamination of 5,6-bicyclic aromatic nucleus led to the further identification of amidinoindole- and amidinoindazole-containing derivatives 53-55. These analogues, 46-49 and 53-55, exhibited potent in vitro activity with IC50 values of 25-65 nM in platelet aggregation assays and an IC50 value of 2 nM in fibrinogen binding assays and demonstrated a selectivity of > 50,000-fold for GPIIb-IIIa versus the most closely related integrin, the vitronectin receptor, alpha v beta 3.

Non-peptide RGD surrogates which mimic a Gly-Asp beta-turn: potent antagonists of platelet glycoprotein IIb-IIIa.

Cyclic heptapeptide 1, which contains an Arg-Gly-Asp sequence, has good affinity for the platelet receptor GPIIb-IIIa and was chosen for study by 1H NMR techniques. The key RGD sequence of this molecule was found to reside in a conformationally defined type II' Gly-Asp beta-turn, and this information was used in the design of simple non-peptide RGD mimics. Disubstituted isoquinolones, bearing an acidic side chain at position 2 and a basic side chain at position 6, were prepared and were found to have modest affinity for GPIIb-IIIa. Systematic modification of the basic residue contained in these molecules yielded compounds with high affinity for GPIIb-IIIa.

Use of conformationally restricted benzamidines as arginine surrogates in the design of platelet GPIIb-IIIa receptor antagonists.

The use of 5,6-bicyclic amidines as arginine surrogates in the design of a novel class of potent platelet glycoprotein IIb-IIIa receptor (GPIIb-IIIa) antagonists is described. The additional conformational restriction offered by the bicyclic nucleus results in 20-400-fold increases in potency compared to the freely flexible, acyclic benzamidine counterpart. The design, synthesis, structure-activity relationships (SAR), and in vitro activity of this novel class of GPIIb-IIIa antagonists are presented.

Efficacy of tissue plasminogen activator and urokinase in a canine model of prosthetic graft thrombosis.

Tissue plasminogen activator and urokinase were evaluated in a model of prosthetic graft thrombosis. In addition, the effects of thrombus age on lysability and the effect of thrombolytic agents on endothelium were examined. Polytef (polytetrafluoroethylene [PTFE]) grafts (3 mm X 3.5 cm) were placed in femoral arteries of dogs and graft thrombosis was induced. Grafts were treated with a local infusion of either urokinase or tissue plasminogen activator (4000 units/min) and the times for initial flow, complete thrombolysis, and anastomotic bleeding were noted. The luminal surfaces of the grafts and the proximal arterial segments were assayed for the production of thromboxane A2 and prostacyclin and examined with scanning electron microscopy. No difference in the ease of graft lysis was observed, but 50% of tissue plasminogen activator-treated vs 0% of urokinase treated grafts had extravasation of blood through the wall. Grafts treated with tissue plasminogen activator produced less thromboxane A2 and had less thrombus than those treated with urokinase. No differences between arteries exposed to either agent and control arteries were seen. Grafts treated 1,3,5, and 7 days after thrombosis were progressively more difficult to lyse. We conclude that tissue plasminogen activator is an effective thrombolytic agent, but has a potential for local bleeding complications. Grafts of PTFE are thrombogenic after lysis, but may be less so with tissue plasminogen activator than with urokinase. No effect on arterial endothelium was seen, and our studies confirm the clinical impression that older thrombi are more difficult to lyse.

Effect of streptozotocin-induced diabetes on prostaglandin production by rat cerebral microvessels.

Diabetes has been shown to result in reduced prostacyclin synthesis by macrovascular tissue in rats and humans. Other studies have shown that plasma levels of 6-keto-prostaglandin-F1 alpha (6-keto-PGF1 alpha) and synthesis of 6-keto-PGF1 alpha by isolated glomeruli are either unchanged or increased in diabetes. Thus, microvascular tissue may respond differently to diabetes than macrovascular tissue. Accordingly, we have studied the effects of streptozotocin-induced diabetes mellitus on prostaglandin synthesis by rat cerebral microvessel (RCMV). Prostaglandin synthesis from both endogenous and exogenous arachidonic acid was determined. The yield of RCMV from diabetic and control animals was similar. Under basal conditions and following stimulation of prostaglandin synthesis by melittin (5 micrograms/ml), RCMV production of prostacyclin (measured as immunoreactive 6-keto-PGF1 alpha) was greater than production of PGE2. Both 6-keto-PGF1 alpha and PGE2 production under basal and stimulated conditions were found to be similar for control and diabetic RCMV. The RCMV converted exogenous [3H]arachidonic acid predominately to PGD2 and to a lesser extent to 6-keto-PGF1 alpha. No significant differences in the conversion of exogenous [3H]arachidonic acid to PGD2 and 6-keto-PGF1 alpha was observed between control and diabetic RCMV. This study suggests that the effect of streptozotocin-induced diabetes mellitus on prostaglandin formation by microvascular endothelium is different from its effect on macrovascular tissue.

Acute postprandial lipaemia does not influence the in vivo activity of human platelets.

Platelet function and serum lipid studies were conducted on nine healthy male subjects before and two hours after three separate meals, two of which were rich in fat: saturated fat (SF) or polyunsaturated fat (PUF), and a third control meal which contained no fat. Platelet activity was assessed by determination of in vivo platelet aggregate formation, and plasma levels of the platelet specific proteins platelet factor 4 (PF4) and beta-thromboglobulin (beta-TG). Serum triglyceride levels increased significantly after both fat containing meals but were unaffected by the fat free meal. Serum cholesterol levels were not affected by any of the three meals. Platelet function tests could not detect any alteration of in vivo platelet activity in terms of platelet aggregate formation or plasma concentrations of PF4 and beta-TG. These results are at variance with previously published studies that used less specific assays of platelet activity and which suggested platelet activation shortly after meals rich in SF.