Per A Whiss

IN VITRO EFFECTS OF ANTIPSYCHOTICS ON HUMAN PLATELET ADHESION AND AGGREGATION AND PLASMA COAGULATION

1 Several studies suggest an association between venous thromboembolism and the use of antipsychotic drugs, especially clozapine, but the biological mechanisms are unknown. It has been suggested that antipsychotic drugs enhance aggregation of platelets and thereby increase the risk of venous thrombosis. The purpose of the present study was to examine the effects of clozapine and its main metabolite, N‐desmethyl clozapine, as well as olanzapine, risperidone and haloperidol, on platelet adhesion and aggregation and on plasma coagulation in vitro. 2 Blood was collected from healthy subjects free of medication. Platelet adhesion to different protein surfaces and aggregation were measured in microplates. The coagulation methods of activated partial thromboplastin time (APTT) and prothrombin time were performed in platelet‐poor plasma. 3 Clozapine was the only compound that increased platelet adhesion and aggregation and shortened APTT. The effect appeared at therapeutic concentrations and was significant but weak. 4 This weak effect of clozapine on haemostasis may explain, in part, the association of this compound and venous thromboembolism.

Expression of platelet P-selectin and detection of soluble P-selectin, NPY and RANTES in patients with inflammatory bowel disease

Abstract:Objective and Design: P-selectin, a membrane glycoprotein which is expressed on activated platelets and endothelial cells, plays a crucial role in the inflammatory response. The main action is adhesion of leukocytes, facilitation of diapedesis and induction of cytokine production from monocytes (MCP-1 and IL-8), mediated via RANTES released from activated platelets. An abnormal platelet activity has been reported in patients with ulcerative colitis (UC) and Crohns disease (CD), jointly referred to as inflammatory bowel disease (IBD), which could have an aggravating influence on the inflammatory response. In addition, an up-regulation of platelet IL-8 receptors among patients with IBD has been reported. To reveal a presumptuous platelet dysfunction we analysed the expression of platelet surface P-selectin at resting state and after stimulation with thrombin, collagen, epinephrine and interleukin 8 (IL-8), and plasma levels of soluble P-selectin, neuropeptide Y (NPY) and RANTES in patients with IBD.¶Subjects: Blood from twelve healthy subjects (control group) and twenty-one patients with IBD who had not taken any anti-platelet drugs or steroids were analysed.¶Methods: Patients were sub-grouped according to disease entity, disease activity and 5ASA medication. Surface P-selectin expression on isolated human platelets and plasma P-selectin, NPY and RANTES were analysed with ELISA. All values are presented as mean ± standard error of the mean (SEM). Mann-Whitney U test and Wilcoxon matched rank test were used for statistical analyses.¶Results: Patients with IBD in remission (n = 9) had higher basal P-selectin expression, 0.38 ± 0.04, compared to the control group (n = 12), 0.22 ± 0.03, p < 0.01. UC patients (n = 16) showed down-regulation of P-selectin expression after stimulation with IL-8, 0.26 ± 0.03 to 0.22 ± 0.02, p < 0.05. No significant differences could be observed concerning soluble P-selectin and NPY in plasma. Patients with 5ASA (n = 12) had lower levels of plasma RANTES, 2.39 ± 0.06 μg/l, compared to the control group (n = 12), 3.29 ± 0.19 μg/l, p < 0.01, and patients without 5ASA (n = 9), 2.90 ± 0.17 μg/l, p < 0.05.¶Conclusions: Patients with IBD in remission have higher basal platelet surface P-selectin expression. An exaggerated platelet activity with increased expression of platelet P-selectin and release of inflammatory mediators such as RANTES, which is chemotactic and induce chemokine production, could have a reinforcing and aggravating influence on the inflammatory response and increase the susceptibility to IBD. In addition IL-8 has a down-regulating effect on platelet surface P-selectin expression and 5ASA medication seems to lower plasma RANTES. If 5ASA is responsible for lowering the concentration of RANTES this could be one of the beneficial outcomes of 5ASA medication.¶

Platelets enhance neutrophil locomotion: evidence for a role of P-selectin

It has been suggested that the accumulation of platelets at sites of vascular damage and inflammation regulates the function of leukocytes. In this study, we investigated the effects of platelets on the transmigration of neutrophil granulocytes through microporous membranes. We demonstrate that platelets markedly enhance both the random and the chemotactic migration of neutrophils. Stimulatory effects were acquired by adding paraformaldehyde-fixed platelets or the supernatants of platelets; however, the effects were lower or significantly higher, respectively, compared with viable platelets. The increased neutrophil migration was associated with an amplified polymerization of actin filaments and expression of CD11b/CD18. Previous investigations indicate that the initial adhesion between platelets and neutrophils is mediated by P-selectin exposed on the surface of platelets. In this study, the following observations suggest a role for P-selectin in the platelet-induced enhancement of neutrophil motility: (i) platelet supernatants contained substantial amounts of P-selectin, (ii) filtration of platelet supernatants markedly reduced the content of P-selectin and simultaneously decreased the potentiating effects on neutrophil motility, (iii) inhibition of P-selectin-mediated cell cell adhesion with sialyl Lewis X or by incubation in calcium-free medium reduced the enhancing effects of platelets on neutrophil responses, and (iv) purified and recombinant P-selectin mimicked the effects of platelets on neutrophil locomotion. In conclusion, we propose that platelets through P-selectin promote accumulation and emigration of neutrophils during inflammatory and thrombotic processes.

Modulation of P-selectin expression on isolated human platelets y an NO donor assessed by a novel ELISA application

Adhesion molecules such as P-selectin are potential markers for evaluating platelet activation and studying the role of cell-cell interactions in numerous biological processes related to hemostasis and inflammation. The expression of P-selectin and related molecules has previously been quantified with different techniques. As an alternative to the most common method. flow cytometry, we have developed a useful ELISA method to simultaneously analyse 96 samples for platelet expression of P-selectin. Samples may be stored for at least 7 days at 4 degrees C prior to analysis. The method is simple, reproducible, flexible and requires only standard equipment. Washed platelets (WP) from healthy male volunteers, at a concentration of 1 x 10(7)/microtiter plate well, were stimulated with various known platelet activators and fixed with 0.1% formaldehyde for 10 min. The fixed WP were centrifuged to form a confluent layer in the wells and then incubated with optimal dilutions of primary antibodies (1/2000) directed against P-selectin, CD41, CD9 and secondary antibodies conjugated with alkaline phosphatase. Our results show that P-selectin expression on WP increases significantly upon stimulation with thrombin (0.1-1.0 U/ml), ADP (10 microM) and epinephrine (100 microM). The induction of P-selectin expression by thrombin is fast and has different kinetics depending on the concentration of the agonist. Prior incubation with the nitric oxide donor SNAP (10 microM) inhibits the up-regulation of P-selectin induced by sub-maximal concentrations of thrombin (p < 0.05). This ELISA is suitable for studying the expression and regulation of P-selectin and other surface molecules on human platelets in various pathological states.

Weak relationship between ionized and total magnesium in serum of patients requiring magnesium status

Measurement and monitoring of magnesium (Mg) are important to prevent the development of serious and potentially fatal complications in critically ill patients. Although ion-selective electrodes are available and earlier reports suggest that free ionized magnesium (iMg2+) is the most useful test to estimate Mg status, most clinical laboratories still only measure total Mg. To compare the relationship among iMg2+, total Mg, and albumin in serum, samples were collected from 48 consecutive patients admitted to an intensive care unit or a primary health center. The mean serum level of iMg2+ in 44 patients was 0.53 mmol/L, the total Mg was 0.06 mmol/L, and the albumin was 34.93 g/L. The correlation between iMg2+ and total Mg in serum was r=0.585; the correlation between iMg2+ and albumin in serum was r=378; and the correlation between total Mg and albumin in serum was r=0.340. The mean percent iMg2+ in relation to total Mg in serum was calculated to be 55% in the patient samples. The important level of biologically active iMg2+ was not reflected upon analysis of total Mg in 25% of consecutive patients. This report shows that the correlation of iMg2+ and total Mg is weak, not only in critically ill patients but also in patients in whom Mg status is inquired as a whole.

Divalent cations and the protein surface co-ordinate the intensity of human platelet adhesion and P-selectin surface expression.

At sites of blood vessel injury, platelets adhere to exposed vessel components, such as collagen, or immobilized fibrinogen derived from plasma or activated platelets. The divalent cations Mg2+ and Ca2+ are essential for platelet adhesion and activation, but Mg2+ can also inhibit platelet activation. The present study evaluates, by an enzymatic method, the effects of various divalent cations on the adhesion of isolated human platelets to collagen, fibrinogen, albumin or plastic in vitro. By enzyme-linked immunosorbent assay, platelet surface expression of P-selectin was measured to estimate the state of activation on adherence. Mg2+ increased platelet adhesion exclusively to collagen and fibrinogen at physiologically relevant concentrations. At higher concentrations, the adhesion declined. Ca2+ induced a weak adhesion only to fibrinogen at physiological doses and a peak of increased adhesion to all protein-coated surfaces at 10 mmol/l. Mn2+ elicited dose-dependent adhesion only to collagen and fibrinogen. Zn2+, Ni2+ and Cu2+ increased the adhesion of platelets independently of the surface. Ca2+ dose-dependently inhibited adhesion elicited by Mg2+ to collagen and fibrinogen. No other combination of divalent cations elicited such an effect. Mg2+-dependent platelet adhesion to collagen and Ca2+-dependent adhesion to fibrinogen increased P-selectin expression. Thus, the present study shows that the outcome of the platelet adhesion depends on the surface and the access of divalent cations, which co-ordinate the intensity of platelet adhesion and P-selectin surface expression.

Kinetics of platelet P-selectin mobilization : Concurrent surface expression and release induced by thrombin or PMA, and inhibition by the NO Donor SNAP

Activated platelets and endothelium surface express the cell adhesion molecule P-selectin (CD62P), which plays an important role in mediating interactions with leukocytes. Increased levels of a functional soluble form of P-selectin (sP-selectin) have been reported in several pathological states but it is not clear whether this circulating sP-selectin originates from platelets and/or endothelial cells. Here we describe the concurrent kinetics of intracellular storage, surface expression and release of platelet P-selectin induced by thrombin or the protein kinase C activator PMA. Platelet activation with submaximal concentrations of thrombin (0.1 U/ml) resulted in a rapid decrease of intracellular P-selectin. This decrease of intracellular P-selectin concurred with a gradual increase of surface expression and an initial increase of sP-selectin. Our results indicate that intracellular stores of P-selectin were only partly mobilized upon activation with submaximal concentrations of thrombin. A high concentration of thrombin (1.0 U/ml) induced a rapid and nearly total decrease of intracellular stores and a more pronounced, but transient, increase of surface expression. The release of P-selectin was fast and occurred during the initial activation phase. The NO donor SNAP inhibited both surface expression and release of platelet P-selectin in a similar manner. PMA (0.1-1.0 microM) mediated a more slow, gradual and sustained surface expression and release of P-selectin than thrombin. Thus, surface expression and release of platelet P-selectin show different kinetics depending on the mode of activation.

Adhesion of human platelets to albumin is synergistically increased by lysophosphatidic acid and adrenaline in a donor-dependent fashion

Lysophosphatidic acid (LPA) and adrenaline are weak platelet activators considered important for thrombus formation, and were previously shown to synergistically increase platelet aggregation. Here we investigate synergistic activation by LPA and adrenaline when measuring platelet adhesion. Platelet-rich plasma from healthy blood donors together with adrenaline and/or LPA were added to protein-coated microplates. Platelets were allowed to adhere and the amount of adhesion detected enzymatically. The LPA and adrenaline combination induced a synergistic increase of platelet adhesion to a normally non-adhesive albumin surface. The degree of synergy varied markedly between individuals; these variations could not be explained by age, gender, blood type or different amounts of platelets, oxidized low-density lipoprotein, insulin or glucose in plasma. There was a trend indicating increased synergistic effect for platelets sensitive to adrenaline stimulation. The synergistic effect was blocked by the α2-adrenoceptor antagonist yohimbine and inhibited by the ADP scavenger system creatine phosphate/creatine phosphokinase and antibodies against αIIbβ3. Furthermore, platelets adhering to albumin after adrenaline and LPA treatment expressed P-selectin. In conclusion, LPA and adrenaline act synergistically to increase αIIbβ3-mediated platelet adhesion to albumin, dependent on α2-adrenoceptor signalling and platelet secretion. We also confirm that synergistic platelet activation achieved with LPA and adrenaline is highly donor dependent.

Characterization of static adhesion of human platelets in plasma to protein surfaces in microplates.

Platelet adhesion is a complex and important event for prevention of blood loss after vessel injury. This study investigated fundamental adhesive mechanisms occurring in an in-vitro assay developed for the measurement of static adhesion of human platelets in plasma. The aim was to gain methodological knowledge that could be used for interpretations of results from other studies using this specific assay. Involvement of adhesive receptors was investigated by the use of various antibodies as well as therapeutic drugs (abciximab, eptifibatide and tirofiban). Inhibitors of adenosine 5′-diphosphate receptors (cangrelor, MRS2179) and of thromboxane A2 signalling (BM-531) were used to estimate the role of autocrine activation. Adhesion to collagen was found to be mainly mediated by α2β1 and to some extent by αIIbβ3. Adhesion to fibrinogen was mediated by αIIbβ3. In addition, adenosine 5′-diphosphate-induced adhesion to albumin was dependent on αIIbβ3. Furthermore, experiments with cangrelor and BM-531 showed that the majority of the adhesive interactions tested were dependent on adenosine 5′-diphosphate or thromboxane A2. We conclude that the mechanisms of adhesion measured by the static platelet adhesion assay are in accordance with the current knowledge regarding platelet activation and adhesion. Despite its simplicity, we suggest that this adhesion assay could be used as a screening device for the study of the influence of various surfaces and soluble substances on platelet adhesion.

Adenosine 5’-Diphosphate-Induced Platelet Aggregation in Uremia Shows Resistance to Inhibition by the Novel Nitric Oxide Donor GEA 3175 but Not by S-Nitroso-N-Acetylpenicillamine

Both bleeding and thrombosis are complications of uremia in patients on regular hemodialysis. An excessive endogenous formation of the vasodilator and platelet inhibitor nitric oxide (NO) has been proposed to contribute to the bleeding defect. Since exposure to pharmacological donors of NO, nitrovasodilators, can cause tolerance to NO, we investigated whether platelets from uremic patients on regular hemodialysis are influenced differently by NO donors than platelets from healthy subjects. A frequently used S-nitrosothiol, S-nitroso-N-acetylpenicillamine (SNAP), was compared to a recently synthezised mesoionic oxatriazole derivate, GEA 3175, regarding its capacity to inhibit adenosine 5′-diphosphate (ADP)-induced platelet aggregation in vitro. The final products of NO production, nitrite + nitrate, were found to be significantly increased in uremic patients. The capacity to inhibit platelet aggregation by SNAP was only slightly different between the groups. However, GEA 3175 showed a significantly marked and reduced capacity to inhibit aggregation of uremic platelets compared to controls. Interactions of erythropoietin (EPO) with NO have earlier been reported. Addition of EPO to platelets from healthy donors in vitro did not significantly influence the NO donor capacity to inhibit platelet aggregation, but showed a tendency to enhance the effect of SNAP while the effect of GEA 3175 was inhibited. These results suggest compound-specific resistance to NO donors in uremic platelet activation.