Bernard Boneu

Early Potent Antithrombotic Effect With Combined Aspirin and a Loading Dose of Clopidogrel on Experimental Arterial Thrombogenesis in Humans

BACKGROUND We conducted a double-blind, randomized, crossover study to assess the antithrombotic effects of the combination of aspirin (acetylsalicylic acid, ASA) and clopidogrel, with or without a loading dose, versus ASA alone in a model of arterial thrombosis in humans. METHODS AND RESULTS Eighteen male volunteers received the following 3 regimens for 10 days separated by a 1-month period: (1) 325 mg ASA daily, (2) 325 mg ASA+75 mg clopidogrel daily, (3) 325 mg ASA daily+300-mg clopidogrel loading dose on day 1 and +75 mg clopidogrel per day on days 2 to 10. The antithrombotic effect was measured 1.5, 6, and 24 hours after drug intake on day 1 and 6 hours after drug intake on day 10. Arterial thrombus formation was induced ex vivo by exposing a collagen-coated coverslip in a parallel-plate perfusion chamber to native blood for 3 minutes at an arterial wall shear rate. Without a loading dose, clopidogrel+ASA developed an antithrombotic effect within 6 hours after the first intake. It was superior to that produced by ASA, but it was moderate (P</=0.03). However, with the loading dose, the antithrombotic effect of clopidogrel+ASA appeared within 90 minutes, and after 6 hours it was comparable to that on day 10. On day 10, clopidogrel+ASA decreased platelet thrombus formation by approximately 70%, and the effect was significantly more potent than that produced by ASA alone (P<0.001). CONCLUSIONS This study confirms the synergistic antithrombotic effects of a combined ASA and clopidogrel therapy and shows the early benefit obtained with a loading dose of clopidogrel.

Evaluation of six markers of haemostatic system in normal pregnancy and pregnancy complicated by hypertension or pre-eclampsia

Objective To establish the plasma evolution of prothrombin fragments 1+2 (F1+2), thrombin–antithrombin III complexes (TAT), fibrin fragment D‐Dimers (DD), von Willebrand factor antigen (vWf), Type 1 plasminogen activator inhibitor antigen (PAI) and blood platelet count during normal pregnancy and to compare these values with those obtained in hypertensive or pre‐eclamptic pregnancies.

CONSTITUTIONAL HEPARIN CO-FACTOR II DEFICIENCY ASSOCIATED WITH RECURRENT THROMBOSIS

The family of a 36-year-old man with recurrent deep venous thrombosis and heparin co-factor II (HC II) deficiency was investigated for this deficiency. The deficiency was inherited as an autosomal dominant trait. 4 members of the family had low HC II levels but only the proband had a history of thromboses; however, 2 of 4 affected were only 14 and 23 years old. The deficiency did not affect the anticoagulant action of heparin, with which the patient was treated.

Prothrombin fragment 1 + 2, thrombin–antithrombin III complexes and D‐dimers in acute deep vein thrombosis: effects of heparin treatment

Summary. Plasma levels of prothrombin fragment 1 + 2 (F 1 + 2), of thrombin–antithrombin III complexes (TAT) and of D‐dimers were evaluated at several time intervals in 15 patients affected by acute proximal deep vein thrombosis, complicated or not by pulmonary embolism, and treated by conventional heparin therapy for 9 d. The mean levels of the three markers remained significantly increased throughout the period of observation, except for F 1 + 2 on day 9, when compared to normal values established in a population of normal healthy blood donors. However, whereas heparin significantly decreased the plasma levels of F 1 + 2 and of TAT complexes in less than 3 d, D‐dimer levels were not significantly altered. Significant correlations were observed between the plasma levels of the three markers but they were not correlated to the actual intensity of heparin treatment evaluated as the activated partial thromboplastin time prolongation. These results indicate that heparin improves the hypercoagulable state associated with a deep vein thrombosis within the first days of treatment as indicated by TAT and F 1 + 2. They also account for the performances of D‐dimer assay for the diagnosis of deep vein thrombosis in patients already receiving heparin, a common situation in routine hospital practice.

3 Pharmacokinetics of heparin and low molecular weight heparin

After parenteral injection, heparin is removed from the blood via two mechanisms, saturable and non-saturable. The saturable mechanism represents clearance by the reticuloendothelial system and endothelial cells, to which heparin binds with a high affinity. The non-saturable mechanism is represented by renal excretion. The contribution of the two mechanisms to the clearance of heparin varies according to the dose delivered and the molecular weight of the heparin preparation. At low doses, unfractionated heparin (UH) is removed mainly via the saturable mechanism, while at higher doses the contribution of the non-saturable mechanism to its clearance becomes pre-eminent. This model accounts for the major pharmacokinetic properties of UH. After bolus intravenous injection of low doses, UH disappears from the blood exponentially with a dose-dependent half-life; at higher doses, UH disappears with a concave-convex pattern. Under continuous intravenous infusion there is a non-linear relationship between the dose of UH injected and the steady-state plasma concentration. After subcutaneous injection, the bioavailability of the anti-factor Xa activity increases with the dose delivered and tends toward 100% at high doses. In contrast, low molecular weight heparins (LMWH) are mainly removed by non-saturable renal excretion. This explains the dose independence of the pharmacokinetic parameters of LMWH, the excellent bioavailability of the subcutaneous route at any dose, and the prolongation of LMWH half-life in cases of chronic renal insufficiency. However, the model does not explain the large interindividual variability of the pharmacokinetic parameters of both UH and LMWH.

Respective role of antithrombin III and heparin cofactor II in the in vitro anticoagulant effect of heparin and of various sulphated polysaccharides

Summary. The in vitro anticoagulant effects of standard heparin (SH) and of seven other sulphated polysaccharides (SPS) were investigated by measuring activated partial thromboplastin time (APTT) prolongation of normal plasma and of plasmas selectively depleted of antithrombin III (AT III), of heparin cofactor II (HCII) and of both heparin cofactors. This allowed the determination of the relative contribution of each of the two heparin cofactors to the SPS anticoagulant effect. The SPS varied in their relative activities as catalysts of thrombin inhibition by purified AT III or HC II. The anticoagulant activities of heparin and dermatan sulphate were primarily attributable to their ability to enhance thrombin inhibition by AT III and HC II respectively. Heparin had an additional minor anticoagulant activity which was independent of both AT III and HC II. Pentosan polysulphate, high molecular weight dextran sulphate, heparin with low affinity for AT III and a sulphated heparin derivative had weaker anticoagulant activities in normal plasma than standard heparin. The anticoagulant activities of these last four SPS in plasma depleted of both AT III and HC II were similar to their respective activities in normal plasma. This suggests that these SPS act by directly preventing thrombin generation rather than by enhancing thrombin inhibition.

Low Molecular Weight Heparins: Are They Superior to Unfractionated Heparins to Prevent and to Treat Deep Vein Thrombosis?

In many countries, low molecular weight heparins (LMWHs) have replaced unfractionated heparin (UH) for prevention and treatment of venous thromboembolism. The present paper reviews the possible advantages of LMWHs over UH. In spite of their lower molecular weight distribution, LMWHs are functionally more heterogeneous than UH. Their anti-Xa/anti-IIa ratio varies significantly, and the injection of the same dose generates different anti-Xa activities and activated partial thromboplastin time (APTT) prolongations. Their pharmacodynamic properties account for their more convenient use in comparison with UH; however, there is a risk of accumulation in case of renal insufficiency. Even if they are less anticoagulant on the basis of the APTT prolongation, they are not less prohemorrhagic than UH. LMWHs are probably less immunogenic and probably induce less osteoporosis. Several meta-analyses published between 1992 and 1999 indicate that LMWHs are as efficient as UH in preventing postoperative deep vein thrombosis (DVT) in general surgery and more efficient than UH in preventing DVT in orthopedic surgery and treating established DVT.

Effects of lysophosphatidic acid on proliferation and cytosolic Ca++ of human adult vascular smooth muscle cells in culture.

Lysophosphatidic acid (LPA) is a lipid mediator generated by activated platelets and having various effects on numerous cell types. We investigated some effects of 1-oleyl LPA on vascular smooth muscle cells cultured from adult human normal arteries. At micromolar concentrations, LPA induced a mitogenic effect ([3H]-thymidine incorporation and cell proliferation) on quiescent cells, without an additional growth factor being required. This effect was equipotent to that of 10% fetal calf serum, and it was accompanied by early (5 minutes) and late (1-3 hours) phosphorylation of mitogenactivated protein kinase. LPA inhibited cell migration through collagen coated membranes, with or without platelet-derived growth factor BB as chemoattractant. LPA induced a typical biphasic Ca2+ signal response made up of a rapid first phase due to Ca2+ release from intracellular stores followed by a second wave due to external Ca2+ influx. These findings support the proposal that LPA released from activated platelets is a mediator for smooth muscle cell response at the site of vessel injury in humans.

Polymorphonuclear Leukocytes Modulate Tissue Factor Production by Mononuclear Cells: Role of Reactive Oxygen Species

To determine whether polymorphonuclear leukocytes (PMN) modulate the production of tissue factor (TF) by monocytes, PBMC were incubated with increasing concentrations of PMN. PMN did not express any procoagulant activity. After 20-h cocultures, PMN enhanced or inhibited the TF production of PBMC, and this effect depended on the PMN/PBMC ratio. When the ratio increased from 1/1000 to 1/5, without or with LPS, the TF activity of PBMC increased to peak at 2.5-fold the baseline value (p < 0.01). The TF Ag and TF mRNA also increased. This potentiating effect was mediated by reactive oxygen species (ROS) released by PMN during the coculture; it did not require direct cell contact between PMN and PBMC, it was enhanced when PMN were stimulated by fMLP (a chemotactic peptide), and it was inhibited by two antioxidants, N-acetyl cysteine and pyrrolidine dithiocarbamate. In contrast, when the PMN/PBMC ratio was further increased from 1/2 to 2/1, the PBMC TF activity, Ag, and mRNA decreased and were inhibited compared with those of PBMC cultured alone (p < 0.01). This inhibitory effect required direct cell contact between PMN and PBMC, and it was not due to a PMN-mediated cytotoxicity. To confirm the role of ROS, H2O2 enhanced then inhibited the TF activity of PBMC in a dose-dependent manner, similarly to PMN. Thus, PMN may play an important role in the pathogenesis of thrombosis and atherosclerosis by exerting concentration-dependent regulatory effects on the TF production by PBMC via the release of ROS.

Evidence for a saturable mechanism of disappearance of standard heparin in rabbits

This work demonstrates that after bolus intravenous injection standard heparin (SH) disappearance results from the combination of a saturable and a non saturable mechanism. Pharmacokinetics and pharmacodynamics of SH were studied by measuring the disappearance of increasing doses (5 - 500 anti-factor Xa U/kg) of 125I-heparin and of its biological effects. CPM curves allowed to determine the half lives of heparin according to the dose injected. The half lives were clearly dose dependent and reached a plateau over 100 anti-factor Xa U/kg. The complex curve which describes the amount of heparin cleared per time unit after any given dose has been resolved into its two components reflecting a saturable and a non saturable mechanism of disappearance. For the doses less than 100 anti-factor Xa U/kg the saturable mechanism was preeminent and the anti-factor Xa activity disappearance followed an exponential pattern; for the doses less than 100 anti-factor Xa U/kg the contribution of the non saturable mechanism becomes more important and the anti-factor Xa activity disappearance followed a concave-convex pattern. Further experiments showed that the heparin half life shortened as the circulating anti-factor Xa activity decreased; this phenomenon may explain the concave-convex pattern of the curve of the anticoagulant effect observed after injection of large doses of SH.