Effects of chronic administration of valproic acid to epileptic patients on coagulation tests and primary hemostasis.

Valproic acid (VPA) is an antiepileptic drug that has been associated with impaired hemostasis and increased risk for postsurgical bleeding. However, the published reports provide controversial results. We measured parameters of primary hemostasis in VPA-treated patients with epilepsy, focusing on adenosine nucleotide-dependent platelet responses, which play a central role in primary hemostasis. We enrolled 20 cases (epileptic patients receiving treatment with VPA) and 20 controls (12 epileptic patients receiving treatment with drugs different from VPA and 8 healthy subjects). Measurements included prothrombin time (PT), activated partial thromboplastin time (APTT), platelet count, platelet function analyzer (PFA)-100 closure times, plasma von Willebrand factor levels, platelet content of ADP, ATP, and serotonin (all stored in platelet dense granules), and platelet shape change and aggregation induced by ADP and other platelet agonists, including the ATP analog α,ß-methylene-ATP. The plasma concentration of VPA was in the therapeutic range in 17 patients and slightly above the upper limit in 3 patients. There were no statistically significant differences in any of the studied parameters in cases versus controls. Our thorough controlled study failed to show that chronic treatment with VPA induces significant abnormalities of coagulation and primary hemostasis. Therefore, VPA, when present in the circulation in the therapeutic range, does not impair hemostasis.

Sex, gender and venous thromboembolism: do we care enough?

: The role of sex and gender in determining clinical presentation, diagnostic approach and outcomes of venous thromboembolism is not fully and systematically addressed, except for hormone-related events in women. A lack of knowledge is also apparent regarding drug prescription patterns, physician bias, enrolment in clinical studies and analysis of sex-related confounders in preclinical and clinical studies. As was shown for cardiovascular disease, ignoring sex and gender in medicine can have important impact on outcomes, including mortality. In this review, we seek to address some aspects of venous thromboembolism such as epidemiology and clinical presentation, recurrence, risk factors, animal studies, safety and efficacy of antithrombotic drugs, highlighting what is known and what is not regarding the role of sex and gender, and hoping to focus some interest and to promote the inclusion of these variables in all future studies on venous thromboembolism.

Effects of 5'-phosphate derivatives of 2-hexynyl adenosine and 2-phenylethynyl adenosine on responses of human platelets mediated by P2Y receptors.

Newly synthesized mono-, di-, and triphosphate of 2-alkynyl adenosines showed very different behavior in human platelet P2Y receptor models, according to the different alkynyl chains. In fact, 2-hexynyladenosine di- (5) and triphosphate (7) induced platelet shape change and aggregation and inhibited PGE(1)-induced increase in platelet cyclic AMP. On the contrary, the corresponding 2-phenylethynyladenosine di- (6) and triphosphate (8) did not induce platelet shape change or aggregation, but inhibited platelet aggregation induced by ADP.

Acyclic analogues of adenosine bisphosphates as P2Y receptor antagonists: phosphate substitution leads to multiple pathways of inhibition of platelet aggregation.

Activation by ADP of both P2Y(1) and P2Y(12) receptors in platelets contributes to platelet aggregation, and antagonists at these receptor subtypes have antithrombotic properties. In an earlier publication, we have characterized the SAR as P2Y(1) receptor antagonists of acyclic analogues of adenine nucleotides, containing two phosphate groups on a symmetrically branched aliphatic chain, attached at the 9-position of adenine. In this study, we have focused on antiaggregatory effects of P2Y antagonists related to a 2-chloro-N(6)-methyladenine-9-(2-methylpropyl) scaffold, containing uncharged substitutions of the phosphate groups. For the known nucleotide (cyclic and acyclic) bisphosphate antagonists of P2Y(1) receptors, there was a significant correlation between inhibition of aggregation induced by 3.3 microM ADP in rat platelets and inhibition of P2Y(1) receptor-induced phospholipase C (PLC) activity previously determined in turkey erythrocytes. Substitution of the phosphate groups with nonhydrolyzable phosphonate groups preserved platelet antiaggregatory activity. Substitution of one of the phosphate groups with O-acyl greatly reduced the inhibitory potency, which tended to increase upon replacement of both phosphate moieties of the acyclic derivatives with uncharged (e.g., ester) groups. In the series of nonsymmetrically substituted monophosphates, the optimal antagonist potency occurred with the phenylcarbamate group. Among symmetrical diester derivatives, the optimal antagonist potency occurred with the di(phenylacetyl) group. A dipivaloyl derivative, a representative uncharged diester, inhibited ADP-induced aggregation in both rat (K(I) 3.6 microM) and human platelets. It antagonized the ADP-induced inhibition of the cyclic AMP pathway in rat platelets (IC(50) 7 microM) but did not affect hP2Y(1) receptor-induced PLC activity measured in transfected astrocytoma cells. We propose that the uncharged derivatives are acting as antagonists of a parallel pro-aggregatory receptor present on platelets, that is, the P2Y(12) receptor. Thus, different substitution of the same nucleoside scaffold can target either of two P2Y receptors in platelets.

Blood levels of homocysteine, folate, vitamin B6 and B12 in women using oral contraceptives compared to non-users.

BACKGROUND AND OBJECTIVES: To compare the levels of total homocysteine (tHcy), folate, vitamin B6 and B12, in women not using oral contraceptives (OC) vs. those using OC. MATERIALS AND METHODS: 219 healthy women were enrolled in the study; 159 of them had not been using OC for at least 12 months prior to their enrollment, while 60 were on regular OC treatment. RESULTS: The median levels of vitamin B6 and B12 were significantly lower in OC users than in non-users (24.2 vs. 32.9 nmol/l, p=0.029; 278 vs. 429 ng/ml, p<0.001). There were no statistically significant differences in the levels of tHcy (fasting and post-methionine loading) and folate. CONCLUSIONS: In our cross-sectional study, OC use was associated with low vitamin B6 and B12 levels. Since low vitamin B6 levels are independently associated with heightened risks for arterial and venous thromboembolism (TE), they could partly account for the increased TE risk of OC users.

Inhibition of platelet functions and thrombosis through selective or nonselective inhibition of the platelet P2 receptors with increasing doses of NF449 [4,4',4'',4'''-(carbonylbis(imino-5,1,3-benzenetriylbis-(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid octasodium salt].

Our aim was to determine whether the newly described P2X1 antagonist NF449 [4,4',4'',4'''-(carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid octasodium salt] could selectively antagonize the platelet P2X1 receptor and how it affected platelet function. NF449 inhibited alpha,beta-methyleneadenosine 5'-triphosphate-induced shape change (IC50 = 83 +/- 13 nM; n = 3) and calcium influx (pA2 = 7.2 +/- 0.1; n = 3) (pIC50 = 6.95) in washed human platelets treated with apyrase to prevent desensitization of the P2X1 receptor. NF449 also antagonized the calcium rise mediated by the P2Y1 receptor, but with lower potency (IC50 = 5.8 +/- 2.2 microM; n = 3). In contrast, it was a very weak antagonist of the P2Y12-mediated inhibition of adenylyl cyclase activity. Selective blockade of the P2X1 receptor with NF449 led to reduced collagen-induced aggregation, confirming a role of this receptor in platelet activation induced by collagen. Intravenous injection of 10 mg/kg NF449 into mice resulted in selective inhibition of the P2X1 receptor and decreased intravascular platelet aggregation in a model of systemic thromboembolism (35 +/- 4 versus 51 +/- 3%) (P = 0.0061; n = 10) but without prolongation of the bleeding time (106 +/- 16 versus 78 +/- 7 s; n = 10) (N.S.; P = 0.1209). At a higher dose (50 mg/kg), NF449 inhibited the three platelet P2 receptors. This led to a further reduction in platelet consumption compared with mice injected with saline (13 +/- 4 versus 42 +/- 3%) (P = 0.0002; n = 5). NF449 also reduced dose-dependently the size of thrombi formed after laser-induced injury of mesenteric arterioles. Overall, our results indicate that NF449 constitutes a new tool to investigate the functions of the P2X1 receptor and could be a starting compound in the search for new antithrombotic drugs targeting the platelet P2 receptors.

Effects of some pre-analytical conditions on the measurement of homocysteine and cysteine in plasma.

The association of hyperhomocysteinemia and hypercysteinemia with the risk of arterial and venous thrombosis is well documented. While it is known that standardized pre-analytical conditions are necessary for reliable measurement of plasma total homocysteine, the effects of pre-analytical conditions on cysteine measurement are less well known. The aim of this study was to evaluate the effects of pre-analytical conditions on the measurement of homocysteine and cysteine. We observed that the concentration of total homocysteine in plasma increased significantly with time (38% after 6 h), whereas total cysteine decreased (5% after 2 h) when blood anticoagulated with ethylenediaminetetraacetic tripotassium salt was kept at room temperature. These changes were minimized when acidic citrate dextrose was used as an anticoagulant and were abolished when blood samples were immediately placed on crushed ice, independently of the anticoagulant. Storage of plasma for 72 h at room temperature induced a small (approximately equal to 6%), but significant, decrease in cysteine when blood was collected in ethylenediaminetetraacetic tripotassium salt. In contrast, homocysteine was stable in plasma for 72 h, independently of the anticoagulant used. In conclusion, if blood samples for plasma total homocysteine and cysteine measurement cannot be kept on ice, they should be collected in acidic citrate dextrose to minimize the artifactual changes.

Low risk of thrombosis in family members of patients with hyperhomocysteinaemia.

Mild to moderate hyperhomocysteinaemia, a metabolic disorder due to genetic and/or acquired factors, is associated with an increased risk of venous and arterial thrombosis. To establish whether measuring homocysteine in members of families of hyperhomocysteinaemic patients is warranted, we investigated 169 relatives of patients diagnosed with hyperhomocysteinaemia after they developed arterial or venous thrombosis. The prevalence of hyperhomocysteinaemia was 16.6%; the relative risk of thrombosis in relatives with hyperhomocysteinaemia compared to those without was 1.2 (odds ratio; 95% CI 0.24-4.2), with similarly low absolute annual incidences of thrombosis (0.28% and 0.24%). The low prevalence of hyperhomocysteinaemia among relatives of patients with this metabolic disorder, and their low risk of thrombosis, do not justify family screening.

Molecular bases of defective signal transduction in the platelet P2Y12 receptor of a patient with congenital bleeding.

We have identified structural attributes required for signal transduction through a seven-transmembrane-domain receptor. Platelets from a patient (AC) with a congenital bleeding disorder had normal shape change but reduced and reversible aggregation in response to 4 microM ADP, similar to normal platelets with blocked P2Y(12) receptor. The response to 20 microM ADP, albeit still decreased, was more pronounced and was reduced by a P2Y(12) antagonist, indicating some residual receptor function. ADP failed to lower the adenylyl cyclase activity stimulated by prostaglandin E(1) in the patient's platelets, even though the number and affinity of 2-methylthioadenosine 5'-[(33)P]diphosphate-binding sites was normal. Analysis of the patient's P2Y(12) gene revealed a G-to-A transition in one allele, changing the codon for Arg-256 in the sixth transmembrane domain to Gln, and a C-to-T transition in the other allele, changing the codon for Arg-265 in the third extracellular loop to Trp. Neither mutation interfered with receptor surface expression but both altered function, since ADP inhibited the forskolin-induced increase of cAMP markedly less in cells transfected with either mutant P2Y(12) as compared with wild-type receptor. These studies delineate a region of P2Y(12) required for normal function after ADP binding.