Pascale Gaussem

Adenosine Diphosphate–Induced Platelet Aggregation Is Associated With P2Y12 Gene Sequence Variations in Healthy Subjects

Background—The adenosine diphosphate (ADP) receptor P2Y12 plays a pivotal role in platelet aggregation, as demonstrated by the benefit conferred by its blockade in patients with cardiovascular disease. Some studies have shown interindividual differences in ADP-induced platelet aggregation responses ex vivo, but the mechanisms underlying this variability are unknown. Methods and Results—We examined ADP-induced platelet aggregation responses in 98 healthy volunteers, and we identified 2 phenotypic groups of subjects with high and low responsiveness to 2 &mgr;mol/L ADP. This prompted us to screen the recently identified Gi-coupled ADP receptor gene P2Y12 for sequence variations. Among the 5 frequent polymorphisms thus identified, 4 were in total linkage disequilibrium, determining haplotypes H1 and H2, with respective allelic frequencies of 0.86 and 0.14. The number of H2 alleles was associated with the maximal aggregation response to ADP in the overall study population (P =0.007). Downregulation of the platelet cAMP concentration by ADP was more marked in 10 selected H2 carriers than in 10 noncarriers. Conclusions—In healthy subjects, ADP-induced platelet aggregation is associated with a haplotype of the P2Y12 receptor gene. Given the crucial role of the P2Y12 receptor in platelet functions, carriers of the H2 haplotype may have an increased risk of atherothrombosis and/or a lesser clinical response to drugs inhibiting platelet function.

P2Y12 H2 Haplotype Is Associated With Peripheral Arterial Disease A Case-Control Study

Background—We recently described a gain-of-function haplotype, called H2, of the adenosine diphosphate (ADP) receptor P2Y12 gene associated with increased ADP-induced platelet aggregation ex vivo in healthy volunteers. Because platelets play a key role in atherosclerosis and arterial thrombosis, we tested the possible link between the H2 haplotype and the risk of peripheral arterial disease (PAD) in a case-control study. Methods and Results—We studied 184 consecutive male patients under 70 years of age with PAD and 330 age-matched control subjects free of symptomatic PAD and with no cardiovascular history. Mean age was 57.1±7.2 years (cases) and 56.7±7.6 years (control subjects). The H2 haplotype was more frequent in patients with PAD than in control subjects (30% and 21%, respectively; OR, 1.6; CI, 1.1 to 2.5; P =0.02 in univariate analysis). This association with PAD remained significant in multivariate regression analysis (OR, 2.3; CI, 1.4 to 3.9; P =0.002) after adjustment for diabetes, smoking, hypertension, hypercholesterolemia, and other selected platelet receptor gene polymorphisms. Conclusions—These data point to a role of the H2 haplotype in atherosclerosis and raise the possibility of relative thienopyridine resistance in carriers of the P2Y12 H2 haplotype.

Haemorrhagic complications of vitamin k antagonists in the elderly: risk factors and management.

In patients >75 years of age, the two main indications for oral anticoagulant therapy with vitamin K antagonists (VKAs) are treatment of venous thromboembolic disease and prevention of systemic embolism in patients with nonvalvular atrial fibrillation. In both indications, a target International Normalized Ratio of 2.5 (range 2.0–3.0) is recommended. Bleeding is the adverse effect feared by physicians that most limits the use of VKAs in older frail patients. In this paper, we discuss (i) the risk of VKA-related bleeding with advancing age; (ii) the severity of bleeding complications and particularly the risk of intracranial haemorrhage in older patients; (iii) the risk factors for bleeding related to patient characteristics; and (iv) the risk factors or determinants for bleeding related to treatment variables (warfarin induction and maintenance administration, instability of anticoagulation, poor compliance and patient’s education level, and concomitant use of drugs). Avoiding over-anticoagulation and/or reducing periods of overdosing in the course of oral anticoagulant treatment with tailored monitoring may help to minimise the risk of bleeding in older patients.

Expression and Characterization of Recombinant Protein S with the Ser 460 Pro Mutation

To characterize the putative biochemical modifications induced by the Ser 460 to Pro (Heerlen) mutation in protein S (PS), we expressed both wild-type (wt) and mutated recombinant PS in HEK cells. In SDS-polyacrylamide gels, r-PS Heerlen migrated at 71 kDa whereas r-wt PS migrated at 73 kDa, a difference abolished after deglycosylation by N-glycosidase, suggesting that the Ser 460 Pro mutation abolishes N-glycosylation of Asn 458. The affinity of r-wt PS and r-PS Heerlen for C4b-binding protein (C4b-BP) and for phospholipid vesicles was similar. Neither the enhancement of APC-dependent prolongation of the APTT, nor the specific enhancement of FVa and FVIIIa proteolysis by APC in purified systems was affected by the mutation. However, the Ser 460 Pro mutation induced a slight conformational change in the SHBG domain of the PS molecule, as shown by reduced binding affinity for monoclonal antibodies. The type III phenotype associated with the Heerlen mutation might thus result from a slightly modified rate of synthesis or catabolism. The resulting moderate decrease in the circulating PS concentration may modify the equilibrium between free PS and C4b-BP/PS complexes.

Amino acids 225–235 of the protein C serine-protease domain are important for the interaction with the thrombin-thrombomodulin complex

Protein C (PC) is a vitamin K‐dependent zymogen that inactivates factors Va and VIIIa after its activation by thrombin complexed to thrombomodulin. We characterized a monoclonal antibody (mAb) against PC, whose only influence on PC functions was to inhibit PC activation by the thrombin‐thrombomodulin complex. It recognized an epitope in the PC heavy chain, the conformation of which is calcium‐dependent. The mAb did not recognize a natural PC variant that was not activated by the thrombin‐thrombomodulin complex (mutation R229Q) and did recognize a synthetic peptide corresponding to PC amino acids 225–235 in an Elisa assay. The peptide inhibited PC activation by the thrombin‐thrombomodulin complex. These data confirm that the calcium‐binding loop of the serine‐protease domain is involved in the interaction of PC with the thrombin‐thrombomodulin complex.

P2RY1 and P2RY12 polymorphisms and on‐aspirin platelet reactivity in patients with coronary artery disease

Introduction:u2002 Association of P2RY1 and P2RY12 polymorphisms with on‐aspirin platelet reactivity was investigated.

P2Y1 gene polymorphism and ADP-induced platelet response.

1 Mannucci PM, Duga S, Peyvandi F. Recessively inherited coagulation disorders. Blood 2004; 104: 1243–52. 2 Maghzal GJ, Brennan SO, Homer VM, George PM. The molecular mechanisms of congenital hypofibrinogenaemia. Cell Mol Life Sci 2004; 61: 1427–38. 3 Neerman-Arbez M. The molecular basis of inherited afibrinogenaemia. Thromb Haemost 2001; 86: 154–63. 4 Hanss MM, Ffrench PO, Mornex JF, Chabuet M, Biot F, De Mazancourt P, Dechavanne M. Two novel fibrinogen variants found in patients with pulmonary embolism and their families. J Thromb Haemost 2003; 1: 1251–7. 5 Homer VM, Brennan SO, Ockelford P, George PM. Novel fibrinogen truncation with deletion of Bbeta chain residues 440–461 causes hypofibrinogenaemia. Thromb Haemost 2002; 88: 427–31. 6 Zhang JZ, Redman CM. Identification of Bbeta chain domains involved in human fibrinogen assembly. J Biol Chem 1992; 267: 21727– 32. 7 Brennan SO, Maghzal G, Shneider BL, Gordon R, Magid MS, George PM. Novel fibrinogen gamma 375 Arg fi Trp mutation (fibrinogen Aguadilla) causes hepatic endoplasmic reticulum storage and hypofibrinogenemia. Hepatology 2002; 36: 652–8. 8 Huang S, Mulvihill ER, Farrell DH, Chung DW, Davie EW. Biosynthesis of human fibrinogen. Subunit interactions and potential intermediates in the assembly. J Biol Chem 1993; 268: 8919–26. 9 Okumura N, Terasawa F, Yonekawa O, Hamada E, Kaneko H. Hypofibrinogenemia associated with a heterozygous C>T nucleotide substitution at position )1138 bp of the 5¢flanking region of the fibrinogen Aa-chain gene. Ann NY Acad Sci 2001; 936: 526–30. 10 Brennan SO, Hammonds B, George PM. Aberrant hepatic processing causes removal of activation peptide and primary polymerisation site from fibrinogen Canterbury (A alpha 20 Val fi Asp). J Clin Invest 1995; 96: 2854–8. 11 Farrell DH, Huang S, Davie EW. Processing of the carboxyl 15-aminoacid extension in the alpha-chain of fibrinogen. J Biol Chem 1993; 268: 10351–5.

LETTER TO THE EDITOR: P2Y1 gene polymorphism and ADP-induced platelet response

1 Mannucci PM, Duga S, Peyvandi F. Recessively inherited coagulation disorders. Blood 2004; 104: 1243–52. 2 Maghzal GJ, Brennan SO, Homer VM, George PM. The molecular mechanisms of congenital hypofibrinogenaemia. Cell Mol Life Sci 2004; 61: 1427–38. 3 Neerman-Arbez M. The molecular basis of inherited afibrinogenaemia. Thromb Haemost 2001; 86: 154–63. 4 Hanss MM, Ffrench PO, Mornex JF, Chabuet M, Biot F, De Mazancourt P, Dechavanne M. Two novel fibrinogen variants found in patients with pulmonary embolism and their families. J Thromb Haemost 2003; 1: 1251–7. 5 Homer VM, Brennan SO, Ockelford P, George PM. Novel fibrinogen truncation with deletion of Bbeta chain residues 440–461 causes hypofibrinogenaemia. Thromb Haemost 2002; 88: 427–31. 6 Zhang JZ, Redman CM. Identification of Bbeta chain domains involved in human fibrinogen assembly. J Biol Chem 1992; 267: 21727– 32. 7 Brennan SO, Maghzal G, Shneider BL, Gordon R, Magid MS, George PM. Novel fibrinogen gamma 375 Arg fi Trp mutation (fibrinogen Aguadilla) causes hepatic endoplasmic reticulum storage and hypofibrinogenemia. Hepatology 2002; 36: 652–8. 8 Huang S, Mulvihill ER, Farrell DH, Chung DW, Davie EW. Biosynthesis of human fibrinogen. Subunit interactions and potential intermediates in the assembly. J Biol Chem 1993; 268: 8919–26. 9 Okumura N, Terasawa F, Yonekawa O, Hamada E, Kaneko H. Hypofibrinogenemia associated with a heterozygous C>T nucleotide substitution at position )1138 bp of the 5¢flanking region of the fibrinogen Aa-chain gene. Ann NY Acad Sci 2001; 936: 526–30. 10 Brennan SO, Hammonds B, George PM. Aberrant hepatic processing causes removal of activation peptide and primary polymerisation site from fibrinogen Canterbury (A alpha 20 Val fi Asp). J Clin Invest 1995; 96: 2854–8. 11 Farrell DH, Huang S, Davie EW. Processing of the carboxyl 15-aminoacid extension in the alpha-chain of fibrinogen. J Biol Chem 1993; 268: 10351–5.

P2Y1 gene polymorphism and ADP-induced platelet response: Letter to the Editor

1 Mannucci PM, Duga S, Peyvandi F. Recessively inherited coagulation disorders. Blood 2004; 104: 1243–52. 2 Maghzal GJ, Brennan SO, Homer VM, George PM. The molecular mechanisms of congenital hypofibrinogenaemia. Cell Mol Life Sci 2004; 61: 1427–38. 3 Neerman-Arbez M. The molecular basis of inherited afibrinogenaemia. Thromb Haemost 2001; 86: 154–63. 4 Hanss MM, Ffrench PO, Mornex JF, Chabuet M, Biot F, De Mazancourt P, Dechavanne M. Two novel fibrinogen variants found in patients with pulmonary embolism and their families. J Thromb Haemost 2003; 1: 1251–7. 5 Homer VM, Brennan SO, Ockelford P, George PM. Novel fibrinogen truncation with deletion of Bbeta chain residues 440–461 causes hypofibrinogenaemia. Thromb Haemost 2002; 88: 427–31. 6 Zhang JZ, Redman CM. Identification of Bbeta chain domains involved in human fibrinogen assembly. J Biol Chem 1992; 267: 21727– 32. 7 Brennan SO, Maghzal G, Shneider BL, Gordon R, Magid MS, George PM. Novel fibrinogen gamma 375 Arg fi Trp mutation (fibrinogen Aguadilla) causes hepatic endoplasmic reticulum storage and hypofibrinogenemia. Hepatology 2002; 36: 652–8. 8 Huang S, Mulvihill ER, Farrell DH, Chung DW, Davie EW. Biosynthesis of human fibrinogen. Subunit interactions and potential intermediates in the assembly. J Biol Chem 1993; 268: 8919–26. 9 Okumura N, Terasawa F, Yonekawa O, Hamada E, Kaneko H. Hypofibrinogenemia associated with a heterozygous C>T nucleotide substitution at position )1138 bp of the 5¢flanking region of the fibrinogen Aa-chain gene. Ann NY Acad Sci 2001; 936: 526–30. 10 Brennan SO, Hammonds B, George PM. Aberrant hepatic processing causes removal of activation peptide and primary polymerisation site from fibrinogen Canterbury (A alpha 20 Val fi Asp). J Clin Invest 1995; 96: 2854–8. 11 Farrell DH, Huang S, Davie EW. Processing of the carboxyl 15-aminoacid extension in the alpha-chain of fibrinogen. J Biol Chem 1993; 268: 10351–5.

Human thrombin variable region 1, including E39, is involved in interactions with α1‐antitrypsin M358R and protein C

We used an antithrombin autoantibody (IgG D), the epitope of which encompasses ABE1 and amino acids located within variable region 1, to study thrombin interactions with R358 α1‐AT and protein C. IgG D inhibited the thrombin interaction with R358 α1‐AT, while hirugen had no effect, indicating that the interaction of R358 α1‐AT with thrombin may involve the VR1 subsite. We also obtained evidence that VR1 may be involved in the activation of protein C by thrombin in the absence of thrombomodulin. Moreover, IgG D attenuated the inhibitory effect of calcium ions during protein C activation by thrombin, probably by masking E 39 within the VR1 site.