Michel Meyer Samama

EFFECTS OF LONG TRAVELS IN SITTING POSITION IN ELDERLY VOLUNTEERS ON BIOLOGICAL MARKERS OF COAGULATION ACTIVATION AND FIBRINOLYSIS

OBJECTIVE To evaluate whether long travel in sitting position is associated with an increase of coagulation activation and/or a decrease of fibrinolytic activity. DESIGN Comparison of blood coagulation and fibrinolysis parameters before and after two pleasure trips by bus organized in winter period (600 km in 8 hours) and in summer period (1200 km in 16 hours). SUBJECTS 31 and 23 healthy elder volunteers for the winter and the summer trip respectively. Nine other elder volunteers were selected as a control group for the winter study. MAIN OUTCOME MEASURES prothrombin fragment 1 + 2 (F1 + 2), thrombin-antithrombin III (TAT), D-dimers (D-D), factor VII activated, plasminogen activator inhibitor (PAI), tissue-type plasminogen activator (t-PA), plasma albumin. RESULTS A significant difference before and after the travel was only observed for TAT in the summer period. However all values of TAT were in the normal range. No volunteer presented with thromboembolic disease during the month following the travel. CONCLUSION In the condition of our study, long travel in sitting position does not lead to an enhanced procoagulant state for elderly with varicose veins. These results suggest that there is no biological support to propose heparin prophylactic therapy for the elderly with varicose veins wishing to travel by bus.

Comparative effects of synthetic pentasaccharide, low-molecular-weight heparin, unfractionated heparin and recombinant hirudin on the generation of factor viia and prothrombin activation after coagulation of human plasma

We studied the effect of synthetic pentasaccharide, a low-molecular-weight heparin (enoxaparin), unfractionated heparin and recombinant hirudin on the generation of factor VIIa (FVIIa) and prothrombin activation after in-vitro clotting of human platelet-poor plasma. FVIIa was measured with a new clotting assay that uses recombinant tissue factor truncated to interact only with FVIIa. Residual prothrombin was measured using the conventional clotting assay. FVIIa and residual FII were measured in the liquid - called pseudo-serum (psi-serum) - obtained 1 h after clotting of normal platelet-poor plasma. A kinetic study of the generation of FVIIa was also performed. Coagulation was initiated by triggering the extrinsic, the intrinsic and both associated clotting pathways. Levels of FVIIa in the psi-sera (55+/-15, 258+/-18, and 164+/-18 ng/ml, in the extrinsic, intrinsic and intrinsic + thromboplastin psi-serum respectively; values are means+/-SEM) were significantly increased compared with those in the platelet-poor plasma (3 ng/ml). Pentasaccharide, low-molecular-weight heparin and unfractionated heparin inhibited the generation of factor VIIa or its activity, or both, in a dose-dependent manner in all the experimental systems (60-90% inhibition). A kinetic study revealed that the inhibition of the generation of FVIIa by pentasaccharide and heparins starts 1 min after triggering either the extrinsic or the intrinsic clotting pathway. The downregulation of FVIIa by heparins was effected mainly by their anti-Xa activity, but also by their inhibitory effect on the generation of prothrombinase. Pentasaccharide, enoxaparin and unfractionated heparin significantly inhibited prothrombin activation in both extrinsic and intrinsic experimental system. Hirudin had no inhibitory effect either on the generation of FVIIa or on prothrombin activation in any experimental system.

Treatment with LMWHs inhibits factor VIIa generation during in vitro coagulation of whole blood

The role of factor VII and activated factor VII (VIIa) is considered to be crucial in the coagulation process. The efficacy of low molecular weight heparins (LMWHs) in the prevention and treatment of thromboembolic episodes has been established in numerous controlled therapeutic trials. However, the mechanisms of their antithrombotic action are still disputed. Heparins exert their anticoagulant effect by enhancing ATIII inhibitory action on factor Xa and thrombin, which results in decreased factor X activation, prothrombinase formation, prothrombin activation and thrombin generation. Moreover, it is clearly established that both kinds of heparins (unfractionated heparin and LMWHs) induce the release of tissue factor pathway inhibitor (TFPI). Therefore, they are involved indirectly in tissue factor (TF)/factor VIIa complex inhibition by the TFPI/factor Xa complex. Factor VII activation is an essential step in the process of blood coagulation and it plays an important role in thrombogenesis. A method for the measurement of factor VIIa has been recently developed. A study on the effects of antithrombotic drugs, as heparins, on factor VIIa generation might allow to better understand the mechanisms that regulate its activation. We investigated ex vivo the effect of treatment with LMWHs on factor VIIa generation during in vitro coagulation of whole blood in order to clarify if LMWHs interfere with factor VIIa generation.

The role of platelet factor 4 in platelet aggregation induced by the antibodies implicated in heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is a severe side effect of heparin treatment. Recent studies using immunological methods demonstrated that antibodies contained in plasma, or in purified total immunoglobulin (Ig)G from patients suffering HIT, recognize as target antigen the complex heparin/platelet factor (PF4). In the present study, the role of PF4 in in-vitro platelet aggregation induced by purified total IgG or platelet-poor plasma from patients suffering HIT was investigated. In order to demonstrate the functional role of PF4, an anti-PF4 antibody that specifically blocked PF4 was used. In an experimental system composed of washed platelet suspension, incubation of F(ab′)2 fragments (0.125 μg/ml) of the polyclonal anti-PF4 antibody resulted in complete inhibition of platelet aggregation triggered by purified total IgG from patients suffering HIT and heparin. In platelet-rich plasma, a significantly higher concentration (4.25 μg/ml) of the anti-PF4 F(ab′)2 was required to inhibit platelet aggregation induced by HIT-PPP and heparin. Intermediate concentrations of the anti-PF4 antibody partially inhibited platelet aggregation. In plasma milieu, the concentration of PF4 was about five-fold higher in comparison with that measured in the purified system. The intensity of platelet aggregation depended on the concentration of HIT-IgG. Platelet aggregation was abolished in the presence of high concentrations of heparin (superior or equal to 10 IU/ml). The present study shows that PF4 is essential for platelet aggregation triggered by the antibodies related to HIT in the presence of heparin. The concentration of PF4 that is available to bind with heparin or with the HIT-related antibodies is critical for platelet aggregation induced by HIT antibodies.

Determination of prothombinase activation after adding human purified prothrombin to human clot: comparison of hirudin, an activated factor II inhibitor, with DX9065a, an activated factor X inhibitor, on clot-associated thrombin and on prothrombin activation.

Clot-associated prothrombinase and thrombin activities may contribute to thrombus extension after thrombolytic and anticoagulant treatment. We studied prothrombin activation after adding human purified prothrombin to human clot. By using two different drugs with an exclusive direct anti-activated factor X activity (DX9065a) or anti-activated factor II activity (r-hirudin), we tried to determine whether clot-bound thrombin and prothrombinase could be inhibited in our experimental system when human purified prothrombin was added. Standard clots were prepared from platelet-poor human plasma after addition of calcium. We measured clot-bound thrombin or free thrombin using a direct simple chromogenic assay. In parallel, prothrombin fragment 1 + 2 measurement was used to monitor prothrombin activation. For this, two protocols were used. We introduced the direct inhibitors before starting the activation process (protocol A) or at the time of the activation process (protocol B). We found a direct correlation between thrombin generation and prothrombin fragment 1 + 2 with an increase of thrombin activity on clots and in the incubation mixtures when clots were incubated in human purified pothrombin alone. Two protocols were used: in the first, clots were pre-incubated in presence of drugs before adding prothrombin; and in the second, clots were incubated in the presence of prothrombin and drugs. Prothrombin activation was not inhibited when clots were incubated with r-hirudin and consequently thrombin generation still occurred. However, added r-hirudin blocks thrombin activity on the clots and in the incubation mixture, but does not prevent prothrombin activation, as shown by the increase of prothrombin fragment 1 + 2. In contrast, DX9065a did not suppress clot-bound thrombin. However, DX9065a blocks prothrombin activation whichever protocol was used. The results show that hirudin is a poor inhibitor of thrombin generation in contrast to DX9065a. On the other hand, DX9065a cannot inhibit thrombin bound to clot in contrast to hirudin.

Pharmacologic modulation of thrombin generation associated with human clots by human purified antithrombin alone or in the presence of low molecular weight heparin or unfractionated heparin.

&NA; Procoagulant activities associated with human clots may contribute to thrombus extension. We investigate the inhibition of clot‐associated factor Xa and thrombin activities by purified human antithrombin either alone or as combination with a low molecular weight heparin (enoxaparin) as compared with unfractionated heparin (UFH). The standard clots were prepared by recalcification of frozen platelet‐poor human plasma. Clot‐associated thrombin was measured on the clot after clot incubation in recalcified buffer or recalcified prothrombin solution. The enzymatic reaction was measured using a specific substrate for thrombin (CBS 3447). The thrombin concentration was determined both on the clots and in the reaction mixtures. In parallel, prothrombin fragment 1.2 and thrombin‐antithrombin complexes (TAT) were measured using enzyme‐linked immunosorbent assay methods. We demonstrated that in the presence of purified human prothrombin and antithrombin (AT), a partial inhibition of clot associated thrombin activity correlated with an increase of TAT complexes. However, antithrombin was unable to inhibit thrombin generation induced by the clot‐associated factor Xa. Enoxaparin (low molecular weight heparin) and UFH did not enhance clot‐bound thrombin inhibition induced by AT. We conclude that clot‐bound thrombin is accessible to human antithrombin alone. AT is also able to inhibit thrombin generated by factor Xa‐associated clot. However, neither a low molecular weight heparin or UFH enhanced the effect of AT alone.

The Heparin Management Test: A New Device for Monitoring Anticoagulation during Coronary Intervention

Whole blood coagulation analysers are widely used during percutaneous coronary interventions. The precise degree of anticoagulation in patients is important in this setting. The aim of this investigation was to compare the results obtained with ACT (Hemochron) and HMT, the Heparin Management Test (TAS) in patients undergoing percutaneous coronary interventions. Patients (n = 100) were enrolled prospectively. Each patient received 10,000 units of heparin. At the end of the procedure, the mean ACT was 284+/-31 seconds and the mean HMT was 292+/-33 seconds. The correlation between the two methods was highly significant (r = 0.64, p<0.001). The HMT correlates well with ACT values in patients undergoing percutaneous coronary interventions. Its use in the management of these patients should be considered.

Comparison of activated clotting times to heparin management test for adequacy of heparin anticoagulation in percutaneous transluminal coronary angioplasty

The aim of this study was to compare the activated clotting time (ACT) obtained with the Hemochron device and the Heparin Management Test (HMT) on a new automated whole-blood coagulometer, the Thrombolytic Assessment System, in patients undergoing angioplasty. Fifty patients undergoing balloon angioplasty were prospectively enrolled. The mean ACT after a 10,000 unit bolus of heparin was 283 +/- 39 sec at the end of the procedure. The mean HMT after 10,000 units of heparin was 286 +/- 31 sec at the end of the procedure in the same patients. The correlation between the two methods was significant (r = 0.6; P < 0.01). The HMT appears to correlate well with standard values obtained with the Hemochron ACT monitor in patients undergoing percutaneous transluminal coronary angioplasty.

Graft thrombosis in small diameter vascular prosthesis: A laboratory model

There are strong demands for innovative anti-thrombogenic materials, such as carbon, because of their necessity in fabricating artificial organs and progressive surgical vascular prostheses. Serial platelet deposition, surface topography, and patency were evaluated in control standard (N 45) and carbon-lined (N 45), small diameter (4 mm inner diameter) polytetrafluoroethylene grafts implanted in the abdominal aortic replacement in rabbits. A pilot study of 80 animals—40 carbon-lined (CL) and 40 standard (ST) grafts were used to develop microsurgical techniques. The 2-hour graft patency (Doppler and angiographic studies) showed better patency rate in the CL group (93% vs 80%). In 10 animals (5 ST and 5 CL grafts) the platelet deposition on each prostheses was quantitated by means of Indium 111 labeled platelets in a dual, isotope-platelet imaging technique. Platelet deposition on ST grafts 2 hours after insertion was significantly higher than on the CL grafts (6.60±1.98×103 platelets/mm2 vs 0.82±0.25×103 platelets/mm2;p<0.05). Light microscopy evaluation of explanted ST midgrafts sections, including both anastomoses, indicated platelet-fibrin deposition (PFD) in nearly all prostheses, whereas only 13% of the CL grafts exhibited PFD (chi-square: 61.117;p<0.001). Gammacamera imaging allowed scintiphotos of the grafts and cinetic acquisition. We conclude that carbon-lining decreases platelet accumulation and improves patency in small-diameter PTFE grafts in the acute phase.

Thrombolysis and adjunctive therapies in acute myocardial infarction.

Thrombolysis and percutaneous transluminal angioplasty represent the cornerstone of the pharmacologic treatment of and the interventional approach to patients with myocardial infarction (MI). They are very effective. However, they are hampered by some critical limitations. Therefore, alternatives to standard thrombolytic therapy have been developed. Platelet glycoprotein (GP) IIb/IIIa blockade is under investigation and seems very attractive. This review will focus on the use of GP IIb/IIIa antagonists and thrombin inhibitors as adjunctive therapies to the thrombolytic treatment of patients with acute MI.