Heparin but not citrate anticoagulation of blood preserves platelet function for prolonged periods.

BACKGROUND: Current guidelines state that platelet aggregation studies should be conducted within 4 h of venepuncture because of the decline in sensitivity to platelet agonists. This constrains studies of platelet activity in clinical situations where samples need to be transported or there are unavoidable delays prior to assessment. OBJECTIVES: The aim of the present study was to compare systematically the responses of platelets stored in the presence of either citrate or heparin, the two most widely used anti-coagulants, using a range of standard techniques. METHODS: Blood was taken from healthy volunteers and either assessed immediately or stored at ambient temperature (18-25 degrees C) for 24 h. Platelet reactivity to a range of agonists was determined by a combination of 96-well plate techniques; light transmission aggregometry, thrombi adhesion, ATP and ADP release, and TxA(2) release; by whole blood aggregometry; and by PFA-100. RESULTS AND CONCLUSIONS: Testing using 96-well plate techniques allowed for the simultaneous measurement of responses to multiple concentrations of multiple agonists. The responses of platelets from blood anti-coagulated with heparin were predominantly preserved in all assays after 24 h storage, whereas, responses of platelets stored in blood anti-coagulated with citrate were greatly diminished. Consequently, anti-coagulation with heparin, but not citrate, preserves platelet responses for up to 24 h as determined by a range of techniques.

Aspirin and the in vitro linear relationship between thromboxane A2-mediated platelet aggregation and platelet production of thromboxane A2.

BACKGROUND: Currently, 'aspirin resistance', the anti-platelet effects of non-steroid anti-inflammatory drugs (NSAIDs) and NSAID-aspirin interactions are hot topics of debate. It is often held in this debate that the relationship between platelet activation and thromboxane (TX) A(2) formation is non-linear and TXA(2) generation must be inhibited by at least 95% to inhibit TXA(2)-dependent aggregation. This relationship, however, has never been rigorously tested. OBJECTIVES: To characterize, in vitro and ex vivo, the concentration-dependent relationships between TXA(2) generation and platelet activity. METHOD: Platelet aggregation, thrombi adhesion and TXA(2) production in response to arachidonic acid (0.03-1 mmol L(-1)), collagen (0.1-30 microg mL(-1)), epinephrine (0.001-100 micromol L(-1)), ADP, TRAP-6 amide and U46619 (all 0.1-30 micromol L(-1)), in the presence of aspirin or vehicle, were determined in 96-well plates using blood taken from naïve individuals or those that had taken aspirin (75 mg, o.d.) for 7 days. RESULTS: Platelet aggregation, adhesion and TXA(2) production induced by either arachidonic acid or collagen were inhibited in concentration-dependent manners by aspirin, with logIC(50) values that did not differ. A linear relationship existed between aggregation and TXA(2) production for all combinations of arachidonic acid or collagen and aspirin (P < 0.01; R(2) 0.92; n = 224). The same relationships were seen in combinations of aspirin-treated and naïve platelets, and in blood from individuals taking an anti-thrombotic dose of aspirin. CONCLUSIONS: These studies demonstrate a linear relationship between inhibition of platelet TXA(2) generation and TXA(2)-mediated aggregation. This finding is important for our understanding of the anti-platelet effects of aspirin and NSAIDs, NSAID-aspirin interactions and 'aspirin resistance'.

The effect of NCX4016 [2-acetoxy-benzoate 2-(2-nitroxymethyl)-phenyl ester] on the consequences of ischemia and reperfusion in the streptozotocin diabetic rat.

The aim of this study was to assess the effect of chronic administration of NCX4016 [2 acetoxy-benzoate 2-(2-nitroxymethyl)-phenyl ester], a nitric oxide-releasing aspirin derivative on the consequences of coronary artery occlusion in streptozotocin-diabetic rats. Rats were made diabetic by injection of streptozotocin (60 mg kg(-1)) and received insulin (2.5 U kg(-1) s.c.) daily for 4 weeks. Animals received vehicle (1 ml kg(-1) polyethylene glycol), aspirin (65.2 mg kg(-1)), NCX4016 (60 mg kg(-1)), or (iv) NCX4016 (120 mg kg(-1)) orally, once daily for the last 5 days before coronary artery occlusion (CAO). One hour after the last dose, pentobarbital-anesthetized rats were subjected to CAO for 30 min followed by 120-min reperfusion. Neither drug significantly modified initial hemodynamics or plasma glucose levels compared with vehicle treatment in either nondiabetic or diabetic rats. Neither drug modified the total ventricular premature beat (VPB) count in normal animals, although NCX4016, but not aspirin, reduced the total VPB count and the incidence of ventricular tachycardia in diabetic rats. In nondiabetic animals, both aspirin and NCX4016 reduced infarct size. However, in diabetic rats, infarct size was reduced only by the larger dose of NCX4016 (120 mg kg(-1)) but not by aspirin or the lower dose of NCX4016. These results demonstrate that the cardioprotective effects of NCX4016 are reduced in the presence of diabetes compared with the effects seen in nondiabetic animals. In summary, the present study confirms the protective effect of NCX4016 against ischemia-reperfusion injury in the normal rat heart and demonstrates for the first time its protective effect in the heart of streptozotocin-diabetic rats.

Effects of non-steroidal anti-inflammatory drugs on cyclo-oxygenase and lipoxygenase activity in whole blood from aspirin-sensitive asthmatics vs healthy donors.

1. Cyclo-oxygenase (COX) and lipoxygenase (LO) share a common substrate, arachidonic acid. Aspirin and related drugs inhibit COX activity. In a subset of patients with asthma aspirin induces clinical symptoms associated with increased levels of certain LO products, a phenomenon known as aspirin-sensitive asthma. The pharmacological pathways regulating such responses are not known. 2. Here COX-1 and LO activity were measured respectively by the formation of thromboxane B(2) (TXB(2)) or leukotrienes (LT) C(4), D(4) and E(4) in whole blood stimulated with A23187. COX-2 activity was measured by the formation of prostaglandin E(2) (PGE(2)) in blood stimulated with lipopolysaccharide (LPS) for 18 h. 3. No differences in the levels of COX-1, COX-2 or LO products or the potency of drugs were found in blood from aspirin sensitive vs aspirin tolerant patients. Aspirin, indomethacin and nimesulide inhibited COX-1 activity, without altering LO activity. Indomethacin, nimesulide and the COX-2 selective inhibitor DFP [5,5-dimethyl-3-(2-isopropoxy)-4-(4-methanesulfonylphenyl)-2(5H)-furanone] inhibited COX-2 activity. NO-aspirin, like aspirin inhibited COX-1 activity in blood from both groups. However, NO-aspirin also reduced LO activity in the blood from both patient groups. Sodium salicylate was an ineffective inhibitor of COX-1, COX-2 or LO activity in blood from both aspirin-sensitive and tolerant patients. 4. Thus, when COX activity in the blood of aspirin-sensitive asthmatics is blocked there is no associated increase in LO products. Moreover, NO-aspirin, unlike other NSAIDs tested, inhibited LO activity in the blood from both aspirin sensitive and aspirin tolerant individuals. This suggests that NO-aspirin may be better tolerated than aspirin by aspirin-sensitive asthmatics.

Placentally derived prostaglandin E2 acts via the EP4 receptor to inhibit IL-2-dependent proliferation of CTLL-2 T cells.

A number of immunomodulatory molecules are present in the placenta, including cytokines, prostaglandins, progesterone and indoleamine 2,3-dioxygenase. An undefined factor capable of down-regulating T-cell activity has recently been reported [1] as being produced by short-term cultures of placental fragments. By careful repetition of these studies we have confirmed that chorionic villi isolated from term placenta produce a low molecular weight, heat stable factor capable of inhibiting the IL-2-dependent proliferation of mouse CTLL-2 cells. This activity was not due, however, to a previously unknown immunosuppressive molecule, but rather to prostaglandin E2 (PGE2). Expression of cyclooxygenase (COX)-2 was detected in the syncytiotrophoblast of chorionic villi explants using immunohistochemistry. Culture of the explants in the presence of the COX-1/COX--2 inhibitors indomethacin and diclofenac, or with the COX-2-selective inhibitor DFP, blocked the production of the immunosuppressive factor. The immunosuppressive activity was restored by adding PGE2 to the supernatants obtained from diclofenac-inhibited explants. A number of different receptors are involved in mediating the biological effects of prostaglandins. By utilizing selective antagonists of individual receptors, we have established that the immunosuppressive effect of PGE2 on CTLL-2 cells is exerted via the EP4 receptor. Thus, addition of an EP4-selective antagonist, but not of EP1 or EP3 antagonists, abolished the immunosuppressive effect of PGE2 on CTLL-2 cells. This may have implications for attempts to selectively manipulate T-cell responses.

A771726, the active metabolite of leflunomide, directly inhibits the activity of cyclo-oxygenase-2 in vitro and in vivo in a substrate-sensitive manner.

1. The immunosuppressive and anti-inflammatory drug leflunomide has several sites of action, although its precise mode of action is unknown. 2. Here we show in vitro and in vivo that leflunomide and/or its active metabolite A771726, inhibit the activity of cyclo-oxygenase (COX) at doses below those that affect protein expression. 3. In J774.2 macrophages treated with endotoxin for 24 h to induce COX-2 and iNOS, leflunomide and A771726 inhibited more potently the accumulation of PGE2 (A771726, IC50 3.5 microg ml-1) than of NO2 (A771726, IC50 380 microg ml-1). At high concentrations (>300 microg ml-1) A771726 also exhibited the expression of COX-2 and iNOS proteins. 4. In A549 cells treated for 24 h with interleukin-1beta, to induce COX-2, A771726 potently inhibited PGE2 synthesis (IC50 0.13 microg ml-1). In the same cells, A771726 was notably less active (IC50, 52 microg ml-1) at inhibiting the formation of PGE2 stimulated by exposure to 30 microM arachidonic acid. 5. In a human whole blood assay, measuring the accumulation of TxB2 in response to calcium ionophore as a measure of COX-1 activity and in response to incubation with bacterial endotoxin as a measure of COX-2 activity, leflunomide inhibited COX-1 and COX-2 with IC50 values of 31 and 185 microg ml-1; for A771726 the corresponding values were 40 and 69 microg ml-1. 6. Pre-treatment of rats with leflunomide or A771726 (10 mg kg-1, i.p.) inhibited the plasma accumulation of 6-keto-PGF1alpha but not NO2/NO3 following infusion of endotoxin. Injection of a bolus of arachidonic acid following 6 h infusion of endotoxin caused a marked acute rise in plasma 6-keto-PGF1alpha which was inhibited only by higher doses of A771726 (50 mg kg-1, i.p.). 7. In conclusion, leflunomide via A771726 can directly inhibit the activity of COX, an effect that appears blunted both by increases in substrate supply and possibly by plasma binding. Only at much higher drug levels does leflunomide and/or A771726 inhibit the induction of COX-2 or iNOS proteins.

Nonsteroid drug selectivities for cyclo-oxygenase-1 rather than cyclo-oxygenase-2 are associated with human gastrointestinal toxicity: a full in vitro analysis.

The beneficial actions of nonsteroid anti-inflammatory drugs (NSAID) can be associated with inhibition of cyclo-oxygenase (COX)-2 whereas their harmful side effects are associated with inhibition of COX-1. Here we report data from two related assay systems, the human whole blood assay and a modified human whole blood assay (using human A549 cells as a source of COX-2). This assay we refer to as the William Harvey Modified Assay. Our aim was to make meaningful comparisons of both classical NSAIDs and newer COX-2-selective compounds. These comparisons of the actions of >40 NSAIDs and novel COX-2-selective agents, including celecoxib, rofecoxib and diisopropyl fluorophosphate, demonstrate a distribution of compound selectivities toward COX-1 that aligns with the risk of serious gastrointestinal complications. In conclusion, this full in vitro analysis of COX-1/2 selectivities in human tissues clearly supports the theory that inhibition of COX-1 underlies the gastrointestinal toxicity of NSAIDs in man.