Jozef Arnout

Dimers of beta 2-glycoprotein I increase platelet deposition to collagen via interaction with phospholipids and the apolipoprotein E receptor 2'.

Patients with prolonged clotting times caused by lupus anticoagulant (LAC) are at risk for thrombosis. This paradoxal association is not understood. LAC is frequently caused by anti-β2-glycoprotein I (β2GPI) antibodies. Antibody-induced dimerization of β2GPI increases the affinity of β2GPI for phospholipids, explaining the observed prolonged clotting times. We constructed dimers of β2GPI that mimic effects of β2GPI-anti-β2GPI antibody complexes, and we studied their effects on platelet adhesion and thrombus formation in a flow system. Dimeric β2GPI increased platelet adhesion to collagen by 150% and increased the number of large aggregates. We also observed increased platelet adhesion to collagen when whole blood was spiked with patient-derived polyclonal anti-β2GPI or some, but not all, monoclonal anti-β2GPI antibodies with LAC activity. These effects could be abrogated by inhibition of thromboxane synthesis. A LAC-positive monoclonal anti-β2GPI antibody, which did not affect platelet adhesion, prevented the induced increase in platelet adhesion by β2GPI dimers. Furthermore, increased platelet adhesion disappeared after preincubation with receptor-associated protein, a universal inhibitor of interaction of ligands with members of the low density lipoprotein receptor family. Using co-immunoprecipitation, it was shown that dimeric β2GPI can interact with apolipoprotein E receptor 2 (apoER2′), a member of the low density lipoprotein receptor family present on platelets. These results demonstrate that dimeric β2GPI induces increased platelet adhesion and thrombus formation, which depends on activation via apoER2′.

MK-383 (L-700,462), a selective nonpeptide platelet glycoprotein IIb/IIIa antagonist, is active in man

BackgroundFibrinogen-dependent cross-linking of glycoprotein (GP) lIb/IIIa on activated platelets is the final mechanism leading to platelet aggregation. Inhibition of this mechanism may result in a novel antithrombotic agent. We studied the activity of MK-383 (L-700,462), a new, nonpeptide GPIIb/IIIa antagonist, in vitro and in vivo, in man. Methods and ResultsMK-383, a nonpeptide trosine derivative, dose-dependently inhibited fibrinogendependent platelet aggregation, in vitro. Binding of 125I-labeled fibrinogen to activated platelets was prevented in a competitive manner with an IC50 of 10±4.2 nmol/L. The activity and tolerability of MK-383 were evaluated in a two-part double-blind, placebo-controlled, dose-escalation study in healthy male subjects using 1- and 4-hour intravenous infusions. Effects on ADP- and collagen-induced ex vivo platelet aggregation (APA or CPA) and template bleeding time (TBT) were evaluated. Twenty-four subjects participated in the 1-hour part. Six received placebo and 18 MK-383 in doses ranging from 0.05 to 0.40 μg · kg-1 · min-1. MK-383 inhibited platelet aggregation and prolonged bleeding time in a dose-dependent manner. APA and CPA were totally inhibited at the end of infusion of 0.4 μg · kg-1 · min-1 and returned to 55% and 89% of baseline, respectively, at 3 hours after infusion. TBT was prolonged at this dose from 5.0±1.3 minutes predose to 22.7±6 minutes at the end of the infusion (P<.01) and was normalized by 3 hours after infusion. In the 4-hour infusion part, 15 subjects received MK-383 (0.1 to 0.2 μg · kg-1 · min-1), and five received placebo. Complete inhibition of ex vivo platelet aggregation was seen at 0.15 and 0.2 μg · kg-1 · min-1. At 0.2 μg · kg-1 · min-1, TBT was prolonged from 4.4±1.2 to 23.9±4.3 minutes at the end of infusion (P<.01) and remained slightly prolonged 3 hours after infusion (7.2±1.8 minutes). No adverse effects were observed in any of the 33 subjects receiving MK-383. ConclusionsThe results from this study indicate that MK-383 appears to be well tolerated and active in man. It is the first nonpeptide GPIIb/IIMa antagonist that can be used to investigate the antithrombotic potential of this new class of antiplatelet agents.

Current status and implications of autoimmune antiphospholipid antibodies in relation to thrombotic disease

Summary.  This review briefly describes the development of the concepts of antiphospholipid antibody and of antiphospholipid syndrome. It focuses on the two main antigenic targets, β2 glycoprotein I and prothrombin. An excessive production of natural antibodies rather than an immune response to exogenous antigen is proposed as pathogenetic for the development of these antibodies. The review attempts to explain how some of these antibodies are anticoagulant in vitro yet prothrombotic in vivo. The final section discusses when to test for such antibodies, how to test and how to consider treatment of patients with the antiphospholipid syndrome.

Role of proaggregatory and antiaggregatory prostaglandins in hemostasis. Studies with combined thromboxane synthase inhibition and thromboxane receptor antagonism.

Thromboxane synthase inhibition can lead to two opposing effects: accumulation of proaggregatory cyclic endoperoxides and increased formation of antiaggregatory PGI2 and PGD2. The elimination of the effects of the cyclic endoperoxides by an endoperoxide-thromboxane A2 receptor antagonist should enhance the inhibition of hemostasis by thromboxane synthase blockers. We have carried out a series of double-blind, placebo-controlled, crossover studies in healthy volunteers to check if this hypothesis may be operative in vivo in man. In a first study, in 10 healthy male volunteers, the combined administration of the thromboxane receptor antagonist BM 13.177 and the thromboxane synthase inhibitor dazoxiben gave stronger inhibition of platelet aggregation and prolonged the bleeding time more than either drug alone. In a second study, in 10 different healthy male volunteers, complete inhibition of cyclooxygenase with indomethacin reduced the prolongation of the bleeding time by the combination BM 13.177 plus dazoxiben. In a third study, in five volunteers, selective cumulative inhibition of platelet TXA2 synthesis by low-dose aspirin inhibited platelet aggregation and prolonged the bleeding time less than the combination BM 13.177 plus dazoxiben. In vitro, in human platelet-rich plasma stimulated with arachidonic acid, the combination of BM 13.177 and dazoxiben increased intraplatelet cAMP while the single drugs did not affect it. Our results indicate that prostaglandin endoperoxides can partly substitute for the activity of TXA2 in vivo in man and that an increased formation of endogenous antiaggregatory and vasodilatory prostaglandins, as obtained with selective thromboxane synthase inhibitors, may contribute to the impairment of hemostasis.

A simple method to discriminate between beta2-glycoprotein I- and prothrombin-dependent lupus anticoagulants.

Summary.  Lupus anticoagulants (LAC) are a heterogeneous group of autoantibodies that prolong phospholipid‐dependent clotting assays. The autoantibodies that cause LAC activity are predominantly directed against β2‐glycoprotein I (β2GPI) or prothrombin. In the present study, we describe a method to differentiate between LAC caused by antibodies directed against β2GPI or prothrombin. Monoclonal antibodies, affinity purified patient antibodies, and selected patient samples were used to show that in an aPTT‐based clotting assay (PTT‐LA; Diagnostica Stago), the use of cardiolipin vesicles in the neutralization procedure discriminates between β2GPI‐ or prothrombin‐dependent LAC activities. Addition of cardiolipin vesicles shortened the prolonged clotting time caused by anti‐β2GPI antibodies with LAC activity, whereas this procedure further prolonged clotting times caused by antiprothrombin antibodies with LAC activity. In contrast, addition of phosphatidylcholine/phosphatidylserine vesicles corrected prolonged clotting times caused by either anti‐β2GPI or antiprothrombin antibodies with LAC activity. The effects of cardiolipin (CL) on β2GPI‐induced LAC activity were specific for contact activation mediated clotting assays. Possible explanations for these findings are the relatively high affinity of β2GPI for cardiolipin, as determined by surface plasmon resonance analysis, and inhibition by anti‐β2GPI antibodies of the CL‐induced prolongation of the PTT‐LA.

Effects of 6% hydroxyethyl starch and 3% modified fluid gelatin on intravascular volume and coagulation during intraoperative hemodilution.

In the perioperative period, artificial colloids are most often infused in doses of 500-1000 mL intravenously. This randomized study compared the effects on intravascular volume and coagulation of approximate 2000 mL of two isooncotic artificial colloids: 6% hydroxyethyl starch (HES; MW 200,000; substitution ratio 0.40-0.55) and 3% modified fluid gelatin (GEL). We hypothesized more pronounced hypocoagulation with HES and a weaker intravascular volume effect of GEL. Forty-two patients, scheduled for primary total hip replacement, were allocated randomly to receive HES or GEL during acute normovolemic hemodilution and subsequent further intraoperative hemodilution. Blood samples were taken before and after 500 mL and 1000 mL of acute normovolemic hemodilution; intraoperatively after 20 mL/kg of artificial colloid and at the end of colloid infusion; on arrival in the recovery room; and 3 h later. We quantified: 1) coagulation variables; 2) blood loss; 3) hemodynamic stability; 4) necessary infusion volume; 5) interstitial extravasation, calculated from plasma volumes measured using albumin marked with technetium-99m and iodine-125, respectively; 6) percentage volume effect at the end of the study as well as hematocrit, total serum protein, and colloid osmotic pressure. Intraoperative volume therapy was guided by radial systolic pressure and systolic pressure variation, mixed venous hemoglobin saturation in the pulmonary artery, and pulmonary capillary occlusion pressure. The following differences (HES vs GEL, P < 0.05) were found: 382 vs 725 mL extravasation; 76% vs 56% intravascular volume expansion 7 h after the median point of artificial colloid infusion; 27% vs 29% hematocrit and 35 vs 45 g/L total serum protein on arrival in recovery; 4 vs 0 abnormal bleeding times (>900 s); 3437 vs 2778 mL blood loss. This study quantifies a poorer volume effect of GEL and a higher blood loss with HES. The higher blood loss was significant with one-tailed testing only. These observations warrant extra GEL infusion to avoid hemoreconcentration and caution with large dose HES. (Anesth Analg 1995;81:1235-42)

Influence of exercise at lower and higher intensity on blood pressure and cardiovascular risk factors at older age

Objective It is not well known which exercise intensity is needed to obtain blood pressure reductions in response to endurance training. We therefore compared the effect of training at lower and higher intensity on blood pressure, and, in addition on other cardiovascular risk factors, in at least 55-year-old sedentary men and women. Methods We used a randomized crossover design comprising three 10-week periods. In the first and third periods, participants exercised at, respectively, lower and higher intensity (33 and 66% of heart rate reserve) in random order, with a sedentary period in between. Training programmes were identical except for intensity and were performed three times, 1 h per week. Thirty-nine (18 men) out of 48 randomized participants completed the study; age averaged 59 years. Results The change of aerobic power from baseline to the end of each period was more pronounced (P < 0.05) with higher intensity (+3.70 ml/kg min; P < 0.001) than with lower intensity training (+2.31 ml/kg min; P < 0.001). Systolic blood pressures at rest and during submaximal exercise were reduced with both intensities (P < 0.01), whereas diastolic office blood pressure was significantly reduced after higher intensity only (P < 0.01). There were no significant differences in blood pressure reduction between intensities. Ambulatory blood pressure remained unchanged after training. Only higher intensity training reduced weight (−1.09 kg; P < 0.001), body fat (−0.85%; P < 0.001), plasma triglycerides (−0.17 mmol/l; P < 0.05) and oxidized low-density lipoprotein (−5.92 U/l; P < 0.01). Conclusion Higher and lower intensity training reduces systolic office and exercise blood pressure to a similar extent, but does not alter ambulatory blood pressure; only higher intensity training favourably affects anthropometric characteristics and blood lipids.

Plasma fibrinopeptide A levels in patients with acute myocardial infarction treated with alteplase. Correlation with concomitant heparin, coronary artery patency, and recurrent ischemia. The European Cooperative Study Group.

BackgroundFibrin generation during and after therapy with alteplase may depend on the level of concomitant anticoagulation. The hypothesis that fibrinopeptide A (FPA) levels, as markers of ongoing in vivo fibrin formation, correlate with the angiographic and clinical outcome of thrombolysis is tested. Methds and ResultsSerial plasma FPA levels were determined in 334 patients of the randomized European Cooperative Study Group trial comparing heparin versus placebo plus alteplase and aspirin in patients with acute myocardial infarction. Median FPA levels (with the 10th to 90th percentiles) were 21 ng/ml (2–390 ng/ml) before treatment in placebo-allocated patients (n = 166) and increased to 49 (15–580), 34 (4–320), 27 (2–240), 29 (2–430), and 30 (3–390) ng/ml after 0.75, 3, 12, 24, and 36 hours, respectively. In heparin-allocated patients (n = 168), median baseline FPA values were 18 ng/ml (2–210 ng/ml) and decreased to 6 (1–110), 5 (1–75), 5 (1–60), 7 (1–100), and 10 (1–170) ng/ml at corresponding time points (p<0.0001 for the difference at each time point). Adequate anticoagulation, defined as no activated partial thromboplastin time value below twice the pretreatment value at 3, 12, 24, and 36 hours after initiation of treatment, was obtained in 48 patients assigned to heparin. It was associated with normal median FPA levels (≤4 ng/ml) at all time points compared with 12 (2–80), 16 (2–240), and 15 (2–240) nglml at 12, 24, and 36 hours, respectively, in heparin-assigned but inadequately anticoagulated patients (n = 102, p<0.001 for each time point). In the heparin-treated group, median FPA values tended to be lower at all time points in patients with patent vessels than in patients with occluded arteries, but the difference was significant only at 24 hours (p = 0.04). FPA levels did not correlate with clinically apparent recurrent ischemia or with left ventricular thrombosis on two-dimensional echocardiography. ConclusionsDuring and after thrombolytic therapy with alteplase, the enhanced fibrin generation is suppressed by sustained concomitant anticoagulation with intravenous heparin. Adequate anticoagula-tion warrants individual titration of the heparin dose. High plasma FPA levels 24 hours after alteplase therapy are specific but insensitive markers of vessel occlusion in anticoagulated patients. They do not correlate with clinical outcome.

Influence of hydroxyethyl starch on coagulation in patients during the perioperative period

The perioperative use of hydroxyethyl starch (HES) has been implicated as a possible cause of intracranial bleeding. The purpose of this study was to compare the influence on blood coagulation of the isovolemic replacement of 1-L blood loss with either 6% HES (molecular weight [MW] average: 450,000) or 5% human albumin during neurosurgery or lower abdominal surgery. Twenty patients scheduled for brain tumor surgery and 20 patients undergoing transabdominal hysterectomy were studied. The activated partial thromboplastin time, prothrombin time, fibrinogen concentration, factor VIII coagulant, von Willebrand factor antigen, platelet count, and the activated clotting time were compared after induction of anesthesia, after administration of 500 and 1000 mL of colloid solution, and 24 and 48 h postoperatively. All measured coagulation variables remained within physiologic range. Changes in coagulation indices were identical in neurosurgical and hysterectomy patients, except for a larger increase in fibrinogen concentration 24 and 48 h after hysterectomy. The acute phase reaction of factor VIII coagulant and von Willebrand factor, which plays a role in postoperative hypercoagulability, was attenuated by the use of HES. We conclude that isovolemic replacement of 1-L blood loss with either 6% HES (MW average: 450,000) or 5% human albumin does not interfere with normal hemostasis during and after neurosurgery or lower abdominal surgery.

Leukotriene B4 production by stimulated whole blood: comparative studies with isolated polymorphonuclear cells.

A new method was developed to study leukotriene B4 (LTB4) production by stimulated whole blood. The calcium ionophore A23187 and serum-treated zymosan induced LTB4 production, measured by radioimmunoassay, in a dose- and time-dependent manner. The pattern of LTB4 production by whole blood differed markedly from that observed with isolated, purified polymorphonuclear leukocytes. Higher levels of LTB4 were reached and maintained in whole blood. The system allowed to detect drug effects on LTB4 synthesis in vitro. This new method to study the synthesis of LTB4 takes into account the complex interactions between different cell types which can modulate LTB4 metabolism.