In view of the tendency toward vascular disease in diabetes mellitus, we studied platelet aggregation in 15 normal, 7 prediabetic, 12 latent, and 20 frankly diabetic subjects. Platelets from latent and frank diabetics showed increased platelet aggregation 4 minutes after adding adenosine 5'-diphosphate (60% verus 29% at 1.0 mu-M), epinephrine (46% versus 14% at 0.25 mu-M), and collagen (72% versus 17% at 0.25 mu-g/ml). Three prediabetics had increased platelet aggregation. Platelet sensitivity to aggregating agents was most marked in frank diabetics, intermediate in latent diabetics, and least in prediabetics. Second-phase platelet aggregation was reversed with acetylsalicylic acid, intravenous tolbutamide, and oral glucose administration. We conclude that platelet aggregation may be increased early in diabetes mellitus and may be involved in the genesis of diabetic microangiopathy. Prospective studies on the effect of therapeutic agents such as acetylsalicylic acid on the natural course of diabetic vascular disease are indicated.

1. An optical method used for measuring platelet aggregation was adapted for measurements of the change in shape of platelets that rapidly follows the addition of the aggregating agent adenosine diphosphate (ADP).2. Measurements were made using dilute suspension of platelets with sufficient EDTA to prevent their aggregation.3. Measurements of the velocity and magnitude of the optical effects of the shape change were highly reproducible.4. Volumetric measurements showed that the shape change is not associated with an increase in mean platelet volume.5. The velocity of the shape change had a temperature coefficient of about 4.5.6. The velocity and magnitude of the shape change were not affected by pH between 5.8 and 9.2.7. The dependence of the velocity of the shape change on the ADP concentration was in accordance with Michaelis-Menton kinetics. The K(m) was about 7.2 x 10(-7)M.8. The velocity of the shape change was inhibited competitively by ATP, adenosine and 2-chloroadenosine but not by AMP. When the inhibitors were added after the maximum of the shape change they caused a concentration-dependent diminution in the record of the change.9. The results suggest that the shape change is initiated by reaction of the agonist ADP with specific receptor sites on the platelet membrane which leads to energy-requiring changes in the structures responsible for maintaining the disk shape of normal platelets.

The endothelium mediates a number of responses (relaxation or contraction) of arteries and veins from animals and humans. The endothelium-dependent relaxations are due to the release, by endothelial cells, of potent non-prostanoid vasodilator substances. Among these, the best characterized is endothelium-derived relaxing factor (EDRF), which is believed to be nitric oxide (NO). Nitric oxide is formed by the metabolism of L-arginine by the constitutive NO synthase of endothelial cells. In arterial smooth muscle, the relaxation evoked by EDRF is explained by the stimulation by NO of soluble guanylate cyclase that leads to the accumulation of cGMP. In a number of animal blood vessels and in human coronary arteries, the endothelial cells release a substance that causes hyperpolarization of the cell membrane (endothelium-derived hyperpolarizing factor, EDHF). The release of EDRF from the endothelium can be mediated by both pertussis toxin-sensitive (alpha 2-adrenoceptor activation, serotonin, aggregating platelets, leukotrienes) and insensitive (adenosine diphosphate (ADP), bradykinin) G proteins. In blood vessels from animals with regenerated and reperfused endothelium, and/or atherosclerosis, there is a selective loss of the pertussin toxin-sensitive mechanism of EDRF release, which favours the occurrence of vasospasm, thrombosis and cellular growth. The available information from isolated human blood vessels or obtained in situ concurs with the conclusions reached from studies with isolated animal tissues. In addition to relaxing factors, the endothelial cells can produce contracting factors (endothelium-derived contracting factors; EDCFs) which include superoxide anions, endoperoxides, thromboxane A2 and endothelin. From animal studies it can be concluded that the propensity to release EDCFs is maintained, or even augmented, in diseased blood vessels. The switch from a normally predominant release of EDRFs to that of EDCFs may play a crucial role in atherosclerosis.

B-cell chronic lymphocytic leukemia (B-CLL) remains an incurable disease that requires innovative new approaches to improve therapeutic outcome. Honokiol is a natural product known to possess potent antineoplastic and antiangiogenic properties. We examined whether honokiol can overcome apoptotic resistance in primary tumor cells derived from B-CLL patients. Honokiol induced caspase-dependent cell death in all of the B-CLL cells examined and was more toxic toward B-CLL cells than to normal mononuclear cells, suggesting greater susceptibility of the malignant cells. Honokiol-induced apoptosis was characterized by the activation of caspase-3, -8, and -9 and cleavage of poly(adenosine diphosphate-ribose) polymerase (PARP). Exposure of B-CLL cells to honokiol resulted in up-regulation of Bcl2-associated protein (Bax) and down-regulation of the expression of the key survival protein myeloid-cell leukemia sequence 1 (Mcl-1), which is associated with response to treatment in B-CLL patients. In addition, B-CLL cells pretreated with interleukin-4 (IL-4), a cytokine known to support B-CLL survival, underwent apoptosis when subsequently incubated with honokiol, indicating that honokiol could also overcome the prosurvival effects of IL-4. Furthermore, honokiol enhanced cytotoxicity induced by fludarabine, cladribine, or chlorambucil. These data indicate that honokiol is a potent inducer of apoptosis in B-CLL cells and should be examined for further clinical application either as a single agent or in combination with other anticancer agents.

PLATELETS IN NATIVE BLOOD ADHERE SPONTANEOUSLY TO GLASS INDEPENDENTLY OF TEMPERATURE: if adenosine diphosphate is added to the blood the adhesiveness of the platelets is increased and this effect is largely independent of temperature. The mono- and triphosphates decrease adhesiveness at 20 degrees C. and 37 degrees C. but have no effect at 0 degrees C.; cocaine inhibits adhesion at 37 degrees C. and at 0 degrees C. Aggregation and viscous metamorphosis of platelets in native plasma is induced at 37 degrees C. by adenosine diphosphate or by thrombin; these reactions do not occur at 0 degrees C. Cocaine and all the other anti-adhesive drugs inhibit thrombin or adenosine diphosphate-induced aggregation. The mono- and tri-phosphates appear to compete with adenosine diphosphate and inhibit aggregation; they also inhibit thrombin-induced aggregation. Aggregation induced by adenosine diphosphate or thrombin is not prevented by any of the usual enzyme inhibitors or uncoupling agents at the appropriate strength. At 37 degrees C. aggregation and viscous metamorphosis induced by adenosine diphosphate or thrombin are reversible, and the addition of more adenosine diphosphate or of thrombin again produces aggregation and viscous metamorphosis. Platelets incubated with adenosine diphosphate but not agitated lose their power to aggregate but when more adenosine diphosphate is added with agitation, then aggregation is again produced. These observations are presumably explained by the finding that intact platelets, but not fragmented platelets, can inactivate adenosine diphosphate. From these results it is tentatively concluded that adhesion may involve intrinsic adenosine diphosphate in the platelet which may be activated by thrombin and inhibited by the added mono- or triphosphate. The anti-adhesive drugs act in a different manner. These phenomena have a remarkable similarity to those concerning mitochondrial swelling.

A short period of ischemia and reperfusion, called ischemic preconditioning, protects various tissues against subsequent sustained ischemic insults. We previously showed that apoptosis of hepatocytes and sinusoidal endothelial cells is a critical mechanism of injury in the ischemic liver. Because caspases, calpains, and Bcl-2 have a pivotal role in the regulation of apoptosis, we hypothesized that ischemic preconditioning protects by inhibition of apoptosis through down-regulation of caspase and calpain activities and up-regulation of Bcl-2. A preconditioning period of 10 minutes of ischemia followed by 15 minutes of reperfusion maximally protected livers subjected to prolonged ischemia. After reperfusion, serum aspartate transaminase (AST) levels were reduced up to 3-fold in preconditioned animals. All animals subjected to 75 minutes of ischemia died, whereas all those who received ischemic preconditioning survived. Apoptosis of hepatocytes and sinusoidal endothelial cells, assessed by in situ TUNEL assay and DNA fragmentation by gel electrophoresis, was dramatically reduced with preconditioning. Caspase activity, measured by poly (adenosine diphosphate ribose) polymerase (PARP) proteolysis and a specific caspase-3 fluorometric assay, was inhibited by ischemic preconditioning. The antiapoptotic mechanism did not involve calpain-like activity or Bcl-2 expression because levels were similar in control and preconditioned livers. In conclusion, ischemic preconditioning confers dramatic protection against prolonged ischemia via inhibition of apoptosis through down-regulation of caspase 3 activity, independent of calpain-like activity or Bcl-2 expression.

The effects of thrombopoietic stimulation on megakaryocytopoiesis, platelet production, and platelet viability and function were examined in normal volunteers randomized to receive single bolus subcutaneous injections of 3 microg/kg pegylated recombinant megakaryocyte growth and development factor (PEG-rHuMGDF) or placebo in a 3:1 ratio. PEG-rHuMGDF transiently doubled circulating platelet counts, from 237 +/- 41 x 10(3)/microL to 522 +/- 90 x 10(3)/microL (P <.0001), peaking on day 12. Baseline and day-12 samples showed no differences in responsiveness of platelets to adenosine diphosphate or thrombin receptor agonist peptide (P>>.4 in all cases); expression of platelet ligand-induced binding sites or annexin V binding sites (P>>.6 in both cases); or density of platelet TPO-receptors (P>>.5). Platelet counts normalized by day 28. The life span of autologous (111)In-labeled platelets increased from 205 +/- 18 hours (baseline) to 226 +/- 22 hours (P <.01) on day 8. Platelet life span decreased from 226 +/- 22 hours (day 8) to 178 +/- 53 hours (P <.05) on day 18. The theoretical basis for senescent changes in mean platelet life span was illustrated by biomathematical modeling. Platelet turnover increased from 43.9 +/- 11.9 x 10(3) platelets/microL/d (baseline) to 101 +/- 27.6 x 10(3) platelets/microL/d (P =.0009), and marrow megakaryocyte mass expanded from 37.4 +/- 18.5 fL/kg to 62 +/- 17 x 10(10) fL/kg (P =. 015). Although PEG-rHuMGDF initially increased megakaryocyte volume and ploidy, subsequently ploidy showed a transient reciprocal decrease when the platelet counts exceeded placebo values. In healthy human volunteers PEG-rHuMGDF transiently increases megakaryocytopoiesis 2-fold. Additionally, peripheral platelets expand correspondingly and exhibit normal function and viability during the ensuing 10 days. The induced perturbation in steady state thrombopoiesis resolves by 4 weeks. (Blood. 2000;95:2514-2522)

Bacterial outer membrane porins have a robust beta-barrel structure and therefore show potential for use as stochastic sensors based on single-molecule detection. The monomeric porin OmpG is especially attractive compared with multisubunit proteins because appropriate modifications of the pore can be easily achieved by mutagenesis. However, the gating of OmpG causes transient current blockades in single-channel recordings that would interfere with analyte detection. To eliminate this spontaneous gating activity, we used molecular dynamics simulations to identify regions of OmpG implicated in the gating. Based on our findings, two approaches were used to enhance the stability of the open conformation by site-directed mutagenesis. First, the mobility of loop 6 was reduced by introducing a disulfide bond between the extracellular ends of strands beta12 and beta13. Second, the interstrand hydrogen bonding between strands beta11 and beta12 was optimized by deletion of residue D215. The OmpG porin with both stabilizing mutations exhibited a 95% reduction in gating activity. We used this mutant for the detection of adenosine diphosphate at the single-molecule level, after equipping the porin with a cyclodextrin molecular adapter, thereby demonstrating its potential for use in stochastic sensing applications.

We used the patch-clamp technique to study the effects of ATP on the small-conductance potassium channel in the apical membrane of rat cortical collecting duct (CCD). This channel has a high open probability (0.96) in the cell-attached mode but activity frequently disappeared progressively within 1-10 min after channel excision (channel "run-down"). Two effects of ATP were observed. Using inside-out patches, low concentrations of ATP (0.05-0.1 mM) restored channel activity in the presence of cAMP-dependent protein kinase A (PKA). In contrast, high concentrations (1 mM) of adenosine triphosphate (ATP) reduced the open probability (Po) of the channel in inside-out patches from 0.96 to 0. 1.2 mM adenosine diphosphate (ADP) also blocked channel activity completely, but 2 mM adenosine 5'-[beta,gamma-imido]triphosphate (AMP-PNP), a nonhydrolyzable ATP analogue, reduced Po only from 0.96 to 0.87. The half-maximal inhibition (Ki) of ATP and ADP was 0.5 and 0.6 mM, respectively, and the Hill coefficient of both ATP and ADP was close to 3. Addition of 0.2 or 0.4 mM ADP shifted the Ki of ATP to 1.0 and 2.0 mM, respectively. ADP did not alter the Hill coefficient. Reduction of the bath pH from 7.4 to 7.2 reduced the Ki of ATP to 0.3 mM. In contrast, a decrease of the free Mg2+ concentration from 1.6 mM to 20 microM increased the Ki of ATP to 1.6 mM without changing the Hill coefficient; ADP was still able to relieve the ATP-induced inhibition of channel activity over this low range of free Mg2+ concentrations. The blocking effect of ATP on channel activity in inside-out patches could be attenuated by adding exogenous PKA catalytic subunit to the bath. The dual effects of ATP on the potassium channel can be explained by assuming that (a) ATP is a substrate for PKA that phosphorylates the potassium channel to maintain normal function. (b) High concentrations of ATP inhibit the channel activity; we propose that the ATP-induced blockade results from inhibition of PKA-induced channel phosphorylation.

Energetic, kinetic and oxygen exchange experiments in the mid-1980s and early 1990s suggested that phosphate (Pi) release from actomyosin-adenosine diphosphate Pi (AM.ADP.Pi) in muscle fibres is linked to force generation and that Pi release is reversible. The transition leading to the force-generating state and subsequent Pi release were hypothesized to be separate, but closely linked steps. Pi shortens single force-generating actomyosin interactions in an isometric optical clamp only if the conditions enable them to last 20-40 ms, enough time for Pi to dissociate. Until 2003, the available crystal forms of myosin suggested a rigid coupling between movement of switch II and tilting of the lever arm to generate force, but they did not explain the reciprocal affinity myosin has for actin and nucleotides. Newer crystal forms and other structural data suggest that closing of the actin-binding cleft opens switch I (presumably decreasing nucleotide affinity). These data are all consistent with the order of events suggested before: myosin.ADP.Pi binds weakly, then strongly to actin, generating force. Then Pi dissociates, possibly further increasing force or sliding.

Experiments were designed to determine the effect of 17 beta-estradiol on endothelium-dependent responses of isolated arteries. Ovariectomized female New Zealand rabbits were treated either with 17 beta-estradiol (100 micrograms i.m.) or with solvent for 4 days. After excision, femoral arteries were cut into rings and suspended for isometric tension recording in organ chambers filled with modified Krebs-Ringer bicarbonate solution. Rings from the 17 beta-estradiol-treated rabbits showed an enhanced endothelium-dependent relaxation to acetylcholine (3 x 10(-9)-3 x 10(-8) M) in the absence or presence of indomethacin. Under the same experimental conditions, the endothelium-dependent responses to the calcium ionophore A23187 were unchanged. In the absence of indomethacin, the response to adenosine diphosphate was depressed in rings with endothelium taken from animals treated with with 17 beta-estradiol; in the presence of the inhibitor of cyclooxygenase, the endothelium-dependent responses were comparable in blood vessels from treated and untreated rabbits. This study suggests that endothelium-dependent responses of arteries can be modulated by steroid hormones.

The particular structural arrangement of chaperonins probably contributes to their ability to assist in the folding of proteins. The interaction of the oligomeric bacterial chaperonin GroEL and its cochaperonin, GroES, in the presence of adenosine diphosphate (ADP) forms an asymmetric complex. However, in the presence of adenosine triphosphate (ATP) or its nonhydrolyzable analogs, symmetric complexes were found by electron microscopy and image analysis. The existence of symmetric chaperonin complexes is not predicted by current models of the functional cycle for GroE-mediated protein folding. Because complete folding of a nonnative substrate protein in the presence of GroEL and GroES only occurs in the presence of ATP, but not with ADP, the symmetric chaperonin complexes formed during the GroE cycle are proposed to be functionally significant.

In this pilot study, membrane phospholipid and high-energy phosphate metabolism were studied in the dorsal prefrontal cortex of 11 drug-naive, first-episode schizophrenic patients and compared with those of 10 healthy control volunteers comparable in age, education, and parental education. The schizophrenic patients had significantly reduced levels of phosphomonoesters and inorganic orthophosphate and significantly increased levels of phosphodiesters and adenosine triphosphate compared with the controls. The levels of phosphocreatine and adenosine diphosphate did not differ in the two subject groups. The adenosine triphosphate and inorganic orthophosphate findings suggest functional hypoactivity of the dorsal prefrontal cortex. The phosphomonoester and phosphodiester findings are compatible with either premature aging or an exaggeration of normal programmed regressive events occurring in the neural systems sampled.

Catalyzed polymerization reactions represent a primary anabolic activity of all cells. It can be assumed that early cells carried out such reactions, in which macromolecular catalysts were encapsulated within some type of boundary membrane. In the experiments described here, we show that a template-independent RNA polymerase (polynucleotide phosphorylase) can be encapsulated in dimyristoyl phosphatidylcholine vesicles without substrate. When the substrate adenosine diphosphate (ADP) was provided externally, long-chain RNA polymers were synthesized within the vesicles. Substrate flux was maximized by maintaining the vesicles at the phase transition temperature of the component lipid. A protease was introduced externally as an additional control. Free enzyme was inactivated under identical conditions. RNA products were visualized in situ by ethidium bromide fluorescence. The products were harvested from the liposomes, radiolabeled, and analyzed by polyacrylamide gel electrophoresis. Encapsulated catalysts represent a model for primitive cellular systems in which an RNA polymerase was entrapped within a protected microenvironment.

Platelet activation and subsequent accumulation at sites of vascular injury are the first steps in hemostasis. Excessive platelet activation after atherosclerotic plaque rupture or endothelial cell erosion may also lead to the formation of occlusive thrombi, which are responsible for acute ischemic events. Multiple pathways are involved in platelet activation, including those activated by adenosine diphosphate (ADP), thromboxane A(2) (TXA(2)), serotonin, collagen, and thrombin. Antiplatelet agents used for prevention of atherothrombosis have focused on blocking the formation of TXA(2) (eg, aspirin) and interfering with ADP stimulation mediated by the P2Y(12) receptor (eg, clopidogrel). These agents, used alone or in combination, significantly decrease the risk for atherothrombotic events, but a significant residual risk for recurrent ischemic events remains. This has been, in part, attributed to persistence of elevated platelet reactivity despite the use of these agents. Several novel antiplatelet agents are currently under clinical development, with the goal of achieving more efficacious platelet inhibition. These include agents that more efficiently block TXA(2)-mediated effects, as well as more potent P2Y(12) receptor antagonists. In addition, inhibition of the protease-activated receptor-1 platelet activation pathway stimulated by thrombin has emerged as a rational target for clinical development. An overview of the basic principles of platelet biology is given and currently available antiplatelet agents, as well as those under clinical development, are reviewed.

BACKGROUND

Epidemiological studies indicate that vitamin E (alpha-tocopherol) exerts a beneficial effect on cardiovascular disease. The effect of vitamin E has generally been attributed to its antioxidant activity and the antioxidant protection of LDL. Distinct from its effect on LDL, vitamin E is also known to inhibit platelet aggregation and adhesion in vitro, but the mechanism(s) responsible for these observations are not known.

RESULTS

Using gel-filtered platelets derived from platelet-rich plasma treated with alpha-tocopherol (500 mumol/L) or vehicle (0.5% ethanol), we found that inhibition of platelet aggregation by alpha-tocopherol was closely linked to its incorporation into platelets (r = -.78; P < .02). Platelet incorporation of alpha-tocopherol was associated with a significant reduction in platelet sensitivity to aggregation by adenosine 5'-diphosphate, arachidonic acid, and phorbol ester (PMA) by approximately, 0.15-, 2-, and 100-fold, respectively. In contrast, platelets treated similarly with butylated hydroxytoluene, another potent lipid-soluble antioxidant, did not demonstrate any change in sensitivity to these agents. Platelet incorporation of alpha-tocopherol inhibited PMA-induced stimulation of platelet protein kinase C (PKC) as determined by phosphorylation of the 47-kD PKC substrate. In 15 normal subjects, oral supplementation with alpha-tocopherol (400 to 1200 IU/d) resulted in an increase in platelet alpha-tocopherol content that correlated with marked inhibition of PMA-mediated platelet aggregation (r = .67; P < .01). Platelets derived from these subjects after supplementation also demonstrated apparent complete inhibition of PKC stimulation by PMA.

CONCLUSIONS

These data indicate that platelet incorporation of alpha-tocopherol at levels attained with oral supplementation is associated with inhibition of platelet aggregation through a PKC-dependent mechanism. These observations may represent one potential mechanism for the observed beneficial effect of alpha-tocopherol in preventing the development of coronary artery disease.

The dynamics of leukocyte-platelet adhesion and platelet-platelet interaction in whole blood are not well understood. Using different platelet agonists, we have studied the whole blood kinetics of these heterotypic and homotypic interactions, the relative abilities of different leukocyte subsets to participate in platelet adhesion, and the ligands responsible for adhesion. When platelet aggregation was inhibited by the Arg-Gly-Asp-Ser (RGDS) peptide, thrombin stimulation of whole blood resulted in platelet expression of granule membrane protein 140 (GMP-140) and, simultaneously, a marked increase in the percentage of monocytes and neutrophils (PMN) binding platelets, as well as an increase in the number of platelets bound per monocyte and PMN. Lymphocytes were unaffected. Monocytes bound more platelets and at an initially faster rate than PMN. This increase in monocyte and PMN adhesion to platelets was completely inhibited by the blocking monoclonal antibody (MoAb), G1, to GMP-140. When the combination of epinephrine and adenosine diphosphate (epi/ADP) was used as a less potent agonist in the presence of RGDS, GMP-140 expression per platelet was less, and while monocyte-platelet conjugates formed, PMN-platelet conjugates did not. With epi/ADP in the absence of RGDS, there was an immediate, marked decrease in the percentage of all leukocytes with bound platelets, simultaneous with an increase in the percentage of unbound platelet aggregates. As these platelet aggregates dissociated, the percentage of monocytes and PMN with adherent platelets increased, with monocytes again binding at a faster initial rate than PMN. This recovery of monocyte and PMN adhesion to platelets was also inhibited by the G1 MoAb. We conclude that: (1) monocytes and PMN bind activated platelets in whole blood through GMP-140; (2) monocytes have a competitive advantage over PMN in binding activated platelets, particularly when less potent platelet agonists are used; and (3) platelet aggregate formation initially competes unactivated platelets off leukocytes; subsequent aggregate dissociation allows the now activated platelets to readhere to monocytes and PMN through GMP-140. These studies further elucidate the dynamic interaction of blood cells and possible links between coagulative and inflammatory processes.

Adenosine diphosphate (ADP) is an important platelet agonist and ADP released from platelet dense granules amplifies responses to other agonists. There are three known subtypes of ADP receptor on platelets: P2X(1), P2Y(1) and P(2T) receptors. Sustained ADP-induced aggregation requires co-activation of P2Y(1) and P(2T) receptors. AR-C69931MX, a selective P(2T) receptor antagonist and novel antithrombotic agent, was studied to characterize further the function of the P(2T) receptor. The roles of the P2Y(1) receptor and thromboxane A(2) were assessed using the selective P2Y(1) antagonist A2P5P and aspirin respectively. Aggregation was measured by whole blood single-platelet counting and platelet-rich plasma turbidimetry, using hirudin anticoagulation. Dense granule release was estimated using ([14)C]-5-hydroxytryptamine (HT)-labelled platelets. Ca(2+) mobilization, P-selectin expression, Annexin V binding and microparticle formation were determined by flow cytometry. P(2T) receptor activation amplified ADP-induced aggregation initiated by the P2Y(1) receptor, as well as amplifying aggregation, secretion and procoagulant responses induced by other agonists, including U46619, thrombin receptor-activating peptide (TRAP) and collagen, independent of thromboxane A(2) synthesis, which played a more peripheral role. P(2T) receptor activation sustained elevated cytosolic Ca(2+) induced by other pathways. These studies indicate that the P(2T) receptor plays a central role in amplifying platelet responses and demonstrate the clinical potential of P(2T) receptor antagonists.

BACKGROUND

Thrombotic events still occur in aspirin-treated patients with coronary artery disease.

RESULTS

To better understand aspirin "resistance," serum thromboxane B2 (TXB2) and flow cytometric measures of arachidonic acid-induced platelet activation (before and after the ex vivo addition of aspirin and indomethacin) were analyzed in 700 consecutive aspirin-treated patients undergoing cardiac catheterization. In 680 of 682 evaluable patients, serum TXB2 concentrations were reduced compared with nonaspirinated healthy donors. Twelve patients had serum TXB2 that was lower than nonaspirinated healthy donors but >10 ng/mL. Arachidonic acid stimulated greater platelet activation in patients with high serum TXB2 (>10 ng/mL) than in patients with low serum TXB2. Addition of ex vivo aspirin reduced arachidonic acid-induced platelet activation to similar levels regardless of serum TXB2 concentrations, which suggests that patients with high residual serum TXB2 concentrations were either noncompliant or underdosed with aspirin. Among the remaining 98% of patients, ex vivo administration of either aspirin or indomethacin failed to prevent platelet activation across all degrees of arachidonic acid-induced platelet activation and aspirin doses. Although the patients were not randomized with respect to clopidogrel treatment, multivariate analysis showed that arachidonic acid-induced platelet activation was less in patients receiving clopidogrel.

CONCLUSIONS

There is a residual arachidonic acid-induced platelet activation in aspirin-treated patients that (1) is caused by underdosing and/or noncompliance in only approximately 2% of patients and (2) in the remaining patients, occurs via a cyclooxygenase-1 and cyclooxygenase-2 independent pathway, in direct proportion to the degree of baseline platelet activation, and is mediated in part by adenosine diphosphate-induced platelet activation.

BACKGROUND

Thiamine (vitamin B1) is an essential molecule for all life forms because thiamine diphosphate (ThDP) is an indispensable cofactor for oxidative energy metabolism. The less abundant thiamine monophosphate (ThMP), thiamine triphosphate (ThTP) and adenosine thiamine triphosphate (AThTP), present in many organisms, may have still unidentified physiological functions. Diseases linked to thiamine deficiency (polyneuritis, Wernicke-Korsakoff syndrome) remain frequent among alcohol abusers and other risk populations. This is the first comprehensive study on the distribution of thiamine derivatives in human biopsies, body fluids and cell lines.

RESULTS

Thiamine derivatives were determined by HPLC. In human tissues, the total thiamine content is lower than in other animal species. ThDP is the major thiamine compound and tissue levels decrease at high age. In semen, ThDP content correlates with the concentration of spermatozoa but not with their motility. The proportion of ThTP is higher in humans than in rodents, probably because of a lower 25-kDa ThTPase activity. The expression and activity of this enzyme seems to correlate with the degree of cell differentiation. ThTP was present in nearly all brain and muscle samples and in ∼60% of other tissue samples, in particular fetal tissue and cultured cells. A low ([ThTP]+[ThMP])/([Thiamine]+[ThMP]) ratio was found in cardiovascular tissues of patients with cardiac insufficiency. AThTP was detected only sporadically in adult tissues but was found more consistently in fetal tissues and cell lines.

CONCLUSIONS

The high sensitivity of humans to thiamine deficiency is probably linked to low circulating thiamine concentrations and low ThDP tissue contents. ThTP levels are relatively high in many human tissues, as a result of low expression of the 25-kDa ThTPase. Another novel finding is the presence of ThTP and AThTP in poorly differentiated fast-growing cells, suggesting a hitherto unsuspected link between these compounds and cell division or differentiation.

BACKGROUND

Introduction of novel agents for the management of advanced prostate cancer provides a range of treatment options with notable benefits for men with metastatic castration-resistant prostate cancer (mCRPC). At the same time, understanding of optimal patient selection, effective sequential use, and development of resistance patterns remains incomplete.

OBJECTIVE

To review current systemic therapies and recent advances in drug development for mCRPC and strategies to aid in patient selection and optimal sequencing.

METHODS

A literature review of PubMed/Medline, Cochrane Library, Current Contents Medicine, Web of Science, Clinical Trial.Gov, WHO-ICTRP (January 2004-November 2017), and the proceedings of major international meetings (2015/2016/2017) was performed in November 2017.

RESULTS

In the last few years, several new options for treatment of mCRPC have shown a survival benefit in phase III trials besides docetaxel:abiraterone, enzalutamide, cabazitaxel, radium-223, and sipuleucel-T. Radium-223 and denosumab have increased options in management of bone metastases. Currently, novel agents such as next-generation androgen receptor (AR) axis-targeting treatments, immunotherapeutics, or therapies targeting other oncogenic and genomic pathways, particularly poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitors and PD-1 inhibitors, are under clinical investigation. With increasing treatment options for mCRPC, information on how to personalize management and how to select and sequence existing therapies is beginning to emerge, as are predictive biomarkers (homologous repair mutations, mismatch repair mutations, AR splice variant 7). Finally, early use of active agents in the castration-sensitive state will likely also change the clinical management of the disease when it becomes castrate resistant.

CONCLUSIONS

The emergence of new drugs for mCRPC has improved treatment options dramatically. Currently, systemic treatment options for mCRPC include hormonal therapy, chemotherapy, immunotherapy, and radionuclide therapy as well as bone-modifying agents and palliative or supportive measures. Further, new genetically targeted agents (PARP inhibitors and PD-1 inhibitors) are on the horizon for certain subsets of biomarker-selected patients. The best strategies for patient selection and optimal sequential use to achieve the longest cumulative survival improvement and to prevent early resistance remain unclear.

UNASSIGNED

The current literature and proceedings from relevant congresses related to available systemic agents for the treatment of metastatic castration-resistant prostate cancer, including novel genetically targeted therapies, including poly(adenosine diphosphate-ribose) polymerase inhibitors and PD-1 inhibitors, were reviewed. Current therapies and ongoing developments are discussed.

Genetic polymorphisms of cytochrome P450 (CYP) isoforms may promote variability in platelet response to clopidogrel. This study was conducted to analyze, in 603 patients with non-ST elevation acute coronary syndromes, the effect of CYP3A4, CYP3A5, and CYP2C19 gene polymorphisms on clopidogrel response and post-treatment platelet reactivity assessed by adenosine diphosphate (ADP)-induced platelet aggregation, vasodilator-stimulated phosphoprotein phosphorylation index, and ADP-induced P-selectin expression. The CYP2C19*2 polymorphism was significantly associated with ADP-induced platelet aggregation, vasodilator-stimulated phosphoprotein phosphorylation index, and ADP-induced P-selectin expression in recessive (p <0.01, p <0.007, and p <0.06, respectively) and codominant (p <0.08, p <0.0001, and p <0.009, respectively) models, but the CYP3A4*1B and CYP3A5*3 polymorphisms were not. The CYP2C19*2 allele carriers exhibited the highest platelet index levels in multivariate analysis (p = 0.03). After covariate adjustment, the CYP2C19*2 allele was more frequent in clopidogrel nonresponders, defined by persistent high post-treatment platelet reactivity (ADP-induced platelet aggregation >70%; p = 0.03). In conclusion, the present data suggest that the CYPC19*2 allele influences post-treatment platelet reactivity and clopidogrel response in patients with non-ST elevation acute coronary syndromes.

1. The functional consequences of P2X receptor activation on peripheral sensory neurones have been investigated in vivo. Behavioural indices of acute nociception were monitored in the conscious rat following subplantar injection of adenosine 5'-triphosphate (ATP), alpha,beta-methylene ATP, adenosine 5'-diphosphate (ADP) and adenosine. 2. Signs of overt nociception, i.e. hindpaw lifting and licking, were apparent in animals injected subplantar with the P2X receptor agonist, alpha,beta-methylene ATP. Nociceptive behaviours continued for 15 min following administration of alpha,beta-methylene ATP (200 nmol) and were dose-related (0-5 min hindpaw lifting times after injection of alpha,beta-methylene ATP 100 nmol and 1000 nmol were 89 +/- 26 s and 232 +/- 11 s, respectively). Subplantar ATP evoked a modest response only at the highest dose tested (1000 nmol; 0-5 min hindpaw lifting time 66 +/- 19 s) whilst ADP or adenosine (both 600 nmol) elicited negligible spontaneous nociceptive activity. 3. Morphine (3 mg kg-1, i.v.) abolished hindpaw licking behaviour induced by subplantar injection of either alpha,beta-methylene ATP (600 nmol) or bradykinin (1 nmol) and substantially reduced (88 +/- 5%) paw licking in formalin (0.5%, 0.1 ml) injected animals. In contrast, hindpaw lifting was only modestly inhibited (34 +/- 11%) in morphine-pretreated animals that had received subplantar bradykinin and was unaffected in rats in which the noxious stimulus was either subplantar alpha,beta-methylene ATP or formalin. Pretreatment of hindpaws with subplantar bupivacaine (1% w/v, 0.1 ml) abolished alpha,beta-methylene ATP-evoked nociceptive behaviours. 4. Hindpaw lifting and licking mediated by alpha,beta-methylene ATP (600 nmol, subplantar) were inhibited (72 +/- 15% and 95 +/- 5%, respectively) by 30 min local pretreatment with 600 nmol alpha,beta-methylene ATP. Subplantar alpha,beta-methylene ATP pretreatment did not inhibit behaviour stimulated by subsequent bradykinin (1 nmol) or formalin (0.5%, 0.1 ml) injection into the hindpaw. 5. Desensitization of small diameter sensory neurones with a single subplantar injection of capsaicin (100 micrograms) abolished all behaviours indicative of spontaneous nociceptive sensation in animals subsequently injected with alpha,beta-methylene ATP (600 nmol), bradykinin (1 nmol) or formalin (0.5%, 0.1 ml). 6. We conclude that activation of P2X receptors present on small diameter (capsaicin-sensitive) primary afferent neurones in the rat hindpaw mediates behaviour indicative of acute nociception.