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 M ADP, similar to normal platelets with blocked P2Y12 receptor. The response to 20 M 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 E1 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 P2Y12 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 forskolininduced 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 P2Y12 required for normal function after ADP binding.ADP ͉ platelet function disorder ͉ G-protein coupled receptors ͉ platelet aggregation P latelets possess at least two P2 receptors whose combined action is required for full activation and aggregation in response to stimulation by ADP (1, 2). One receptor, P2Y 1 , is coupled to the heterotrimeric GTP-binding protein G q and to phospholipase C-␤; it induces mobilization of cytoplasmic calcium and mediates shape change followed by an initial wave of rapidly reversible aggregation (3, 4). The other, P2Y 12 , is negatively coupled to adenylyl cyclase through G i ; it mediates progressive and sustained platelet aggregation in the absence of shape change (2, 3, 5) and plays an important role in the potentiation of secretion induced by several agonists (6, 7). P2Y 12 is the therapeutic target of ticlopidine and clopidogrel (8), two platelet aggregation inhibitors used for the prevention and treatment of arterial thrombosis (9), and its congenital deficiency results in a bleeding disorder (7,10,11). Platelets deficient in P2Y 12 exhibit normal ADP-induced shape change but only slight and rapidly reversible aggregation, as well as a failure of ADP to inhibit the rise of cAMP levels after stimulation with prostaglandin E 1 (PGE 1 ) (1). The P2Y 12 defect is inherited as an autosomal recessive trait (1), and heterozygous patients display a mild abnormality of platelet function similar to that seen in the relatively common primary secretion defects. These are a heterogeneous group of congenital platelet alterations characterized by reduced platelet secretion despite normal thromboxane production and storage granule content (12). In some of these patients, platelets that produce normal amounts of thromboxane A 2 fail to respond to the agonist b...

Abstract-Two unrelated patients with a congenital bleeding diathesis associated with a severe defect of the platelet ADP receptor coupled to adenylate cyclase (P2 CYC ) have been described so far. In one of them, platelet secretion was shown to be abnormal. We recently showed that platelets with the primary secretion defect (PSD; characterized by abnormal secretion but normal granule stores, thromboxane A 2 production, and ADP-induced primary wave of aggregation) have a moderate defect of P2 CYC . Therefore, the interaction of ADP with the full complement of its receptors seems to be essential for normal platelet secretion, and PSD patients may be heterozygotes for the congenital severe defect of P2 CYC .In this study, we describe 2 new related patients with a severe defect of P2 CYC and the son of one of them, who is to be considered an obligate heterozygote for the defect. The 2 patients with the severe defect had lifelong histories of abnormal bleeding, prolonged bleeding times, abnormalities of platelet aggregation and secretion, lack of inhibition of adenylate cyclase by ADP, and a deficiency of platelet-binding sites for

Hyperhomocysteinemia is a frequent risk factor for deep-vein thrombosis. A common mutation (C677T) in the gene encoding for methylenetetrahydrofolate reductase (MTHFR) is responsible, in the homozygous state, for decreased enzyme activity and mild hyperhomocysteinemia and is associated with increased risk for cardiovascular disease. We studied the prevalence of C677T MTHFR in 77 patients with deep-vein thrombosis and in 154 age- and sex-matched healthy control subjects. In the same individuals, we also evaluated the frequency of the coexistence of C677T MTHFR with mutant factor V:Q506, a common risk factor for deep-vein thrombosis. Sixteen patients (20.8%) and 35 control subjects (22.7%) were homozygous for the C677T MTHFR mutation (odds ratio [OR] = 0.8, 95% confidence interval [CI] = 0.4-2.0). Sixteen patients (20.8%) and 4 control subjects (2.6%) had factor V:Q506; of them, 10 patients and 3 control subjects had isolated factor V:Q506 (adjusted OR = 6.3, 95% CI = 1.6-25.3) and 6 patients and 1 control subject also had C677T MTHFR (adjusted OR = 17.3, 95% CI = 2.0-152.9). The OR for the coexistence of the two mutations was 65% to 75% higher than the expected joint effect calculated by either an additive (OR = 6.0) or multiplicative (OR = 4.4) model. The homozygous C677T mutation of MTHFR per se is not a risk factor for deep-vein thrombosis but increases the risk associated with factor V:Q506. Due to the high prevalence of C677T MTHFR, it is likely that previous studies, which did not look for this mutation, overestimated the relative risk of thrombosis associated with factor V:Q506 alone.

Adjusting platelet count (PC) in platelet-rich plasma (PRP) using platelet-poor plasma (PPP) is recommended for platelet aggregation (PA) studies, but it could also affect PA independently of the decrease in PC. Analysis of aggregation tracings from healthy controls showed that PC correlated with PA in 47 diluted-PRPs, but not in 104 undiluted-PRPs. Dilution of 9 PRPs with PPP progressively decreased PA, while dilution of washed platelets with buffer hardly affected PA. Apyrase partially prevented the inhibitory effect of PPP . Therefore, the practice of diluting PRP with PPP to adjust platelet count should be avoided because it artefactually inhibits PA.

Our aim was to determine whether the newly described P2X 1 antagonist NF449 [4,4Ј,4Љ,4ٞ-(carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid octasodium salt] could selectively antagonize the platelet P2X 1 receptor and how it affected platelet function. NF449 inhibited ␣,␤-methyleneadenosine 5Ј-triphosphate-induced shape change (IC 50 ϭ 83 Ϯ 13 nM; n ϭ 3) and calcium influx (pA 2 ϭ 7.2 Ϯ 0.1; n ϭ 3) (pIC 50 ϭ 6.95) in washed human platelets treated with apyrase to prevent desensitization of the P2X 1 receptor. NF449 also antagonized the calcium rise mediated by the P2Y 1 receptor, but with lower potency (IC 50 ϭ 5.8 Ϯ 2.2 M; n ϭ 3). In contrast, it was a very weak antagonist of the P2Y 12 -mediated inhibition of adenylyl cyclase activity. Selective blockade of the P2X 1 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 P2X 1 receptor and decreased intravascular platelet aggregation in a model of systemic thromboembolism (35 Ϯ 4 versus 51 Ϯ 3%) (P ϭ 0.0061; n ϭ 10) but without prolongation of the bleeding time (106 Ϯ 16 versus 78 Ϯ 7 s; n ϭ 10) (N.S.; P ϭ 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 Ϯ 4 versus 42 Ϯ 3%) (P ϭ 0.0002; n ϭ 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 P2X 1 receptor and could be a starting compound in the search for new antithrombotic drugs targeting the platelet P2 receptors.Among the three P2 receptor subtypes present on blood platelets, the G protein-coupled P2Y 1 and P2Y 12 subtypes are ADP receptors, whereas the P2X 1 ion channel is activated by ATP. The P2Y 1 and P2Y 12 receptors are essential for normal aggregation in response to ADP: the Gq-coupled P2Y 1 recepThis work was supported by Institut National de la Santé et de la Recherche Médicale, Association de Recherche et Développement en Médecine et Santé Publique, and Etablissement Français du Sang-Alsace.Article, publication date, and citation information can be found at

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Low vitamin B(6) plasma levels, an independent risk factor for thrombosis, are frequent in patients with IBD, especially those with active disease.

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.