Comparison of the effect of fondaparinux and enoxaparin on thrombin generation during in-vitro clotting of whole blood and platelet-rich plasma.

Fondaparinux, a selective antithrombin-dependent inhibitor of activated factor X (FXa), is effective in the prevention and treatment of deep vein thrombosis and seems to be superior to enoxaparin. However, the exact mechanism of fondaparinux antithrombotic action is still unclear. We compared the effect of clinically relevant concentrations of fondaparinux and enoxaparin on the initiation and propagation phase of prothrombin activation and on the endogenous thrombin potential (ETP). Coagulation was triggered either in whole blood or in platelet-rich plasma (PRP) by recalcification in the presence of diluted thromboplastin. Prothrombin activation in whole blood was assessed with an original method by measuring the kinetics of prothrombin F1+2 formation using an enzyme-linked immunosorbent assay. We also assessed the maximum concentration of thrombin (Cmax) and the ETP in PRP using the Thrombogram-Thrombinoscope assay. Concentrations of fondaparinux achieved in prophylaxis (0.11-0.28 anti-FXa IU/ml) prolonged the initiation phase and reduced the velocity of the propagation phase of F1+2 formation. Concentrations of enoxaparin achieved in prophylaxis (0.1-0.25 anti-FXa IU/ml) did not significantly modify these parameters. Concentrations of fondaparinux equal to or higher than 0.57 anti-FXa IU/ml significantly reduced the Cmax of F1+2 or thrombin as well as the ETP. At fondaparinux concentrations equal to or higher than 0.91 anti-FXa IU/ml, a maximum 60% inhibition of thrombin generation was observed. In the presence of enoxaparin concentrations equal to or higher than 0.8 anti-FXa IU/ml, the inhibition of thrombin generation was higher than 80%. Fondaparinux prolonged the initiation phase, decreased the velocity of the propagation phase of thrombin generation and partially reduced the total amount of generated thrombin. The inhibitory effect of fondaparinux on the initiation and propagation phase of thrombin generation seems to be responsible for its antithrombotic action. The more profound inhibition of thrombin generation induced by enoxaparin is due to its supplementary anti-activated factor II activity.     

Platelet-dependent thrombin generation assay for monitoring the efficacy of recombinant Factor VIIa

Although the administration of recombinant coagulation factor VIIa (rFVIIa) is a well established treatment in haemophilia with inhibitory antibodies, monitoring the therapeutic efficacy is still a problem. This is because the complete haemostatic effect in vivo depends on negatively charged surfaces provided by exposed lipids from activated platelets which are not present in standard clinical assays using plasma. The thrombin generation assay, however, measures the endogenous thrombin potential (ETP) in platelet-rich plasma (PRP) and this assay might be useful for monitoring the haemostatic response to rFVIIa. We characterized the in vitro concentration-response relationship of rFVIIa and the thrombin generation parameters ETP, PEAK and TIME TO PEAK using platelet-rich plasma (PRP) and rFVIIa at concentrations between one and five times the therapeutic dose. We also studied the effect of inhibiting tissue-factor and the intrinsic coagulation pathway using excess TF-neutralizing antibodies and corn trypsin inhibitor (CTI), respectively. There was a sigmoid relationship between ETP and PEAK and the dose of rFVIIa. Increasing rFVIIa concentrations between 100 and 500 U/ml resulted in a progressive increase in ETP, whereas supratherapeutical concentrations led to a plateau phase. The plateau phase differed between patients, suggesting a biological variation in the maximum ETP. Neutralization of plasma TF with TF-Ab partially decreased FVIIa efficacy. The inhibitory effect of CTI on rFVIIa-induced thrombin generation via the intrinsic pathway was negligible. The thrombin generation assay using PRP is a useful test for determining the sufficient efficacy of rFVIIa in blood. Once a plateau level is reached, higher doses of rFVIIa have no additional effect on haemostatic efficacy. The variation in plateau levels between subjects indicates that there are inter-individual differences in the level of thrombin activity that can be generated. High doses of rFVIIa are effective even in the absence of TF.     

Effects of ximelagatran,an oral direct thrombin inhibitor,r-hirudin and enoxaparin on thrombin generation and platelet activation in healthy male subjects

OBJECTIVES: The effects of ximelagatran, an oral direct thrombin inhibitor (DTI), recombinant hirudin (r-hirudin) and enoxaparin on thrombin generation and platelet activation were studied in humans. BACKGROUND: Recombinant hirudin (parenteral DTI) and enoxaparin (low molecular weight heparin) have been demonstrated to be clinically effective in acute coronary syndromes. Ximelagatran is currently under investigation for the prevention and treatment of thromboembolism. The shed blood model allows for the study of thrombin generation and platelet activation in humans in vivo. METHODS: This was an open-label, parallel-group study involving 120 healthy male volunteers randomized to receive one of three oral doses of ximelagatran (15, 30 or 60 mg), r-hirudin (intravenous) or enoxaparin (subcutaneous) at doses demonstrated to be clinically effective in acute coronary syndromes, or to serve as a control. Thrombin generation (prothrombin fragment 1+2 [F1+2] and thrombin-antithrombin complex [TAT]) and platelet activation (beta-thromboglobulin [beta-TG]) biomarkers were studied using a shed blood model involving blood collection from skin incisions made using standardized bleeding time devices. RESULTS: Oral ximelagatran, intravenous r-hirudin and subcutaneous enoxaparin rapidly and significantly (p < 0.05) decreased F1+2, TAT and beta-TG levels in shed blood, indicating inhibition of thrombin generation and platelet activation. Statistically significant concentration (melagatran, the active form of ximelagatran)-response relationships for F1+2 (p = 0.005), TAT (p = 0.005) and beta-TG (p < 0.001) levels, with IC(50)s of 0.376 (F1+2), 0.163 (TAT) and 0.115 (beta-TG) micromol/l, were detected. Melagatran showed dose-proportional pharmacokinetics with low variability. All drugs were well tolerated. CONCLUSIONS: Oral administration of the DTI ximelagatran resulted in a rapid inhibition of both thrombin generation and platelet activation in a concentration-dependent manner using a human shed blood model. The inhibition of thrombin generation by 60 mg ximelagatran was comparable to that observed with doses of r-hirudin and enoxaparin demonstrated to be effective for the treatment of acute coronary syndromes.     

Platelet aggregation impacts thrombin generation assessed by calibrated automated thrombography

A calibrated automated thrombogram (CAT) is performed usually with human platelet-free plasma (PFP) but may be more relevant with platelet-rich plasma (PRP). In this case, platelets are not stimulated by subendothelial molecules like collagen. Our aim was to assess the consequence of strong (collagen) or weak (ADP) induction of platelet release and aggregation on thrombin generation. Platelet aggregation in PRP was triggered with 10 µg/mL collagen or 10 µM ADP using a lumi-aggregometer. Thrombin generation curves were monitored by CAT in different conditions: PRP, PRP with activated platelets (actPRP), aggregated PRP (agPRP), aggregated platelets resuspended in autologous PFP (resPRP), PFP and PFP obtained after aggregation (agPFP). We found a 3-fold shortening of the lag time and time to peak and a marked increase in velocity and thrombin peak without changes in endogenous thrombin potential (ETP) in agPRP with both agonists compared with PRP. The same holds true in agPFP but with a marked increase in ETP compared with PFP. Similar changes in the kinetics of thrombin generation were observed with actPRP-collagen and to a lesser extent in resPRP-collagen compared with PRP. By contrast, there were no modifications of the thrombin generation curves in actPRP-ADP. Alpha-2-macroglobin-thrombin complexes were unchanged in the different PRP conditions but were increased in PFP prepared from agPFP compared to control PFP. Platelet aggregation during activation by agonists other than thrombin did not increase thrombin generation but accelerated its kinetics mainly via platelet content release and platelet-derived extracellular vesicules formation. In diseases characterized by altered platelet granule content or release as well as altered platelet activation, a platelet aggregation step prior to CAT analysis may be clinically relevant to improve laboratory estimation of the bleeding/thrombotic balance.     

Measuring thrombin generation based sensitivity to activated protein C using an automated coagulometer (ACL 9000)

Increased thrombin generation in the presence of activated protein C (APC) is known to be an independent risk factor for thrombosis. Although commercially available methods for measuring thrombin generation are available, none measure sensitivity to APC. We have developed an automated thrombin generation based APC sensitivity test, for use with defibrinated plasma using the automated coagulometer (ACL9000). A chromogenic substrate that is cleaved by thrombin at a slow rate, 7 pm tissue factor and 20 mum phospholipid (DOPC : DOPE : DOPS, 60 : 20 : 20) with and without 5 nm APC. Thrombin generation with [endogenous thrombin potential (ETP)(+APC)] and without APC (ETP), was expressed as a ratio relative to pooled normal plasma. Normal reference ranges were established in 30 normal subjects: ETP 0.73-1.06; ETP(+APC) 0.42-1.07. ETP(+APC) was found to be sensitive to factor V Leiden, oral contraceptive use, low levels of protein S and tissue factor pathway inhibitor and increased plasma factor VIII.     

A cell-based model of thrombin generation

We have developed a cell-based model of thrombin generation using activated monocytes as a source of tissue factor (TF) and platelets serving as a surface for thrombin generation. Monocytes are activated by lipopolysaccharide and express cell-bound TF. To these are added physiologic (plasma) concentrations of all the plasma procoagulants as well as TF pathway inhibitor, antithrombin, and C1-esterase inhibitor. Coagulation takes place in microtiter wells and is initiated by factor VIIa (FVIIa) and calcium. At time intervals, aliquots are removed, platelet activation is measured by the expression of P-selectin, and thrombin generation is measured by chromogenic assay. In addition, one can measure the activation of FIX, FX, FVIII, FV, and FXI. Initial results reveal that the FVIIa-TF interaction results in the activation of FX to FXa and FIX to FIXa. FXa stays in the vicinity of the TF-bearing cell and, in the presence of FVa, converts a small amount of prothrombin to thrombin on the surface of the TF cell. This small amount of thrombin is not sufficient to clot fibrinogen, but is sufficient to activate platelets and FVIII, FV, and FXI. Following platelet activation, FVIIIa, FVa, and FXa occupy sites on the activated platelet surface. FIXa, activated by TF-FVIIa, does not remain on the TF cell, but converts FX to FXa on the platelet surface. FXIa acts to boost FIXa generation on the activated platelet, increasing FXa and subsequent thrombin generation. We have also shown that activated protein C does not inactivate Va on the platelet surface but rather on endothelial cell surfaces.     

Effects of melagatran, the active form of the oral direct thrombin inhibitor ximelagatran, and dalteparin on the endogenous thrombin potential in venous blood from healthy male subjects.

The effect of the oral direct thrombin inhibitor ximelagatran and its active form, melagatran, on thrombin generation was investigated in vitro and ex vivo using a thrombin generation assay. In-vitro thrombin generation was triggered in human platelet-poor plasma by the addition of tissue factor, and the endogenous thrombin potential (ETP) was measured. The ETP IC(50) values for melagatran and the low-molecular-weight heparin dalteparin were 0.44 micromol/l and 0.06 IU/ml, respectively. In contrast to dalteparin, melagatran increased the time-to-thrombin peak in a concentration-dependent manner. ETP was also studied ex vivo in platelet-poor plasma collected from healthy male subjects (n = 54) at pre-dose and 2 h post-dose, with ximelagatran (60 mg) orally, dalteparin (120 IU/kg) subcutaneously, or control (water) orally. After ximelagatran or dalteparin administration, the time-to-thrombin peak was prolonged by 41 and 95%, and the ETP was decreased by 61 and 77%, respectively. Thus, melagatran, the active form of the oral direct thrombin inhibitor ximelagatran, efficiently delays and inhibits the generation of thrombin in plasma both in vitro and ex vivo.     

Influence of the surface on thrombin generation

Thrombin generation depends on the surface of the blood vessel or container. With a new ultra-sensitive and -specific thrombin assay the surface-dependent thrombin generation was quantified. Citrated blood or plasma was preincubated for 1 h (37 degrees C). Citrated blood, plasma, or plasma with 0-10 g/l hemoglobin-erythrocyte microparticles (Hb-MP) were preincubated at 23 degrees C or at 37 degrees C. Plasma samples (50 microl) were recalcified in polystyrol (PS) wells and incubated for different coagulation reaction times (CRT). Final supramolar arginine concentrations, 0.1% Triton X 100, and chromogenic thrombin substrate concentrations in the onefold km-range were added and the linear DeltaA/t was measured in the recalcified coagulation activity assay (RECA). Aprotinin or corn trypsin inhibitor were added. (i) Recalcification of plasma (in different monovettes) pre-incubated for 1 h (37 degrees C) generated the following thrombin activities after 7 min (37 degrees C): 0.74 IU/ml (polypropylene (PP)-citrate), 0.39 IU/ml (PP-EDTA), 0.06 IU/ml (PP-heparin), 1.38 IU/ml (PS), 0.63 IU/ml (1 ml volume PP), 0.13 IU/ml (15 ml volume PP), and 3.62 IU/ml (glass). (ii) Recalcification of preincubated whole blood generated up to about fivefold more thrombin. (iii) Thrombin generation is proportional to the plasmatic concentration of Hb-MP, 10 g/l Hb-MP generating about 4 IU/ml thrombin within 20 min CRT. (iv) The IC50 of aprotinin and corn typsin inhibitor on thrombin generation in RECA are about 2 KIU/ml and about 1 U/ml, respectively. The reaction wall, the preincubation temperature, and hemolysis influences thrombin generation. The RECA allows to diagnose the prothrombotic capacity of any material.     

Inhibition of thrombin-induced feedback activation of factor V: a potential pathway for inhibition of thrombin generation by melagatran.

The feedback mechanism by which melagatran, the active form of the oral direct thrombin inhibitor ximelagatran, inhibits thrombin generation was investigated in vitro, using an endogenous thrombin potential (ETP) assay. Melagatran decreased ETP in a concentration-dependent manner and increased the time to thrombin peak. FEIBA reversed the melagatran-induced reduction in ETP in a concentration-dependent manner and marginally reduced the prolongation of the time to thrombin peak. Similar results were observed for prothrombin as were seen with FEIBA. Both activated factor V and Russell's Viper Venom-factor V activator reversed the melagatran-induced prolongation in time to thrombin peak in a concentration-dependent manner and partially restored ETP. Prothrombin, in combination with Russell's Viper Venom-factor V or activated factor V, reversed both the melagatran-induced reduction in ETP and the prolongation in time to thrombin peak, in a concentration-dependent manner. These results indicate that inhibition of thrombin-mediated amplification reactions in blood coagulation is an effective way to delay or inhibit thrombin generation.     

Inhibition of thrombin generation in recalcified plasma.

Eight inhibitors of thrombin generation were compared in recalcified unfrozen plasma. Individual or pooled normal citrated plasma was supplemented on polystyrol flat-bottom wells (23 degrees C) with increasing concentrations of low-molecular-weight heparin, heparin, danaparoid, fondaparinux, hirudin, argatroban, corn trypsin inhibitor, or aprotinin. Thrombin was generated by addition of 5 microl fresh 250 mmol/l CaCl2 to 50 microl plasma in polystyrol flat-bottom wells and incubation for 20 min at 37 degrees C (recalcified coagulation activity assay). Arginine stopped hemostasis activation and then the generated thrombin activity was specifically quantified. The approximate 50% inhibitory concentrations of plasmatic anticoagulants for individual or pooled normal plasma are, respectively, 0.6 or 3.7 mIU/ml low-molecular-weight heparin, 0.3 or 1.6 mIU/ml heparin, 0.7 or 6.1 mU/ml danaparoid, 0.023 or 0.18 microg/ml fondaparinux, 75 or 230 pg/ml hirudin, 0.026 or 0.24 microg/ml argatroban, 1 or 2 U/ml corn trypsin inhibitor, and 2 or 4 KIU/ml aprotinin. The 50% inhibitory concentration values for corn trypsin inhibitor or aprotinin at plasmatic concentrations above 4-100 U/ml might increase pathologically the thrombin generation. The recalcified coagulation activity assay is a sensitive method to measure prothrombotic tendencies of blood or subtle concentrations of any plasmatic anticoagulant. It is suggested to analyze the individual patient's sensibility to certain plasmatic anticoagulants.     

Tissue factor pathway inhibitor is the main determinant of thrombin generation in haemophilic patients

The thrombin generation (TG) assay evaluates haemostatic balance, which is influenced by the levels of many coagulation factors and inhibitors. Our objective was to identify the determinant factors of TG in haemophilia A (HA) and haemophilia B (HB) patients and to compare them to those in healthy controls. Coagulation factor and inhibitor levels, and TG, were measured in platelet‐poor plasma from 40 patients with HA, 32 patients with HB and 40 healthy subjects. Data were analysed using multiple regression models. In HA patients, factor VIII was a positive determinant of endogenous thrombin potential (ETP) and peak, whereas tissue factor pathway inhibitor (TFPI) and factor V were negative determinants of ETP and peak. In HB patients, FIX was a positive determinant of ETP and peak, FVII being a positive determinant of peak. Antithrombin and protein S (PS) were negative determinants of ETP while FX was a negative determinant of peak. Above all, in HB patients, TFPI was a negative determinant of ETP and peak. In healthy subjects, FVIII was a positive determinant of ETP and peak, whereas FX and protein S were negative determinants of these parameters. TFPI was not a negative determinant of either peak or ETP. In haemophilic patients, the determinant factors of TG are all implicated in FXa generation and inhibition, the crucial determinant factor being TFPI whatever the type of haemophilia, A or B. These findings contribute to the rationale that recently place TFPI as a target for innovative therapies of haemophilia.     

Extracellular histones promote thrombin generation through platelet-dependent mechanisms: involvement of platelet TLR2 and TLR4

The release of histones from dying cells is associated with microvascular thrombosis and, because histones activate platelets, this could represent a possible pathogenic mechanism. In the present study, we assessed the influence of histones on the procoagulant potential of human platelets in platelet-rich plasma (PRP) and in purified systems. Histones dose-dependently enhanced thrombin generation in PRP in the absence of any trigger, as evaluated by calibrated automated thrombinography regardless of whether the contact phase was inhibited. Activation of coagulation required the presence of fully activatable platelets and was not ascribable to platelet tissue factor, whereas targeting polyphosphate with phosphatase reduced thrombin generation even when factor XII (FXII) was blocked or absent. In the presence of histones, purified polyphosphate was able to induce thrombin generation in plasma independently of FXII. In purified systems, histones induced platelet aggregation; P-selectin, phosphatidylserine, and FV/Va expression; and prothrombinase activity. Blocking platelet TLR2 and TLR4 with mAbs reduced the percentage of activated platelets and lowered the amount of thrombin generated in PRP. These data show that histone-activated platelets possess a procoagulant phenotype that drives plasma thrombin generation and suggest that TLR2 and TLR4 mediate the activation process.     

"Normal" thrombin generation.

We have investigated the influence of alterations in plasma coagulation factor levels between 50% and 150% of their mean values for prothrombin, factor X, factor XI, factor IX, factor VII, factor VIII, factor V, protein C, protein S, antithrombin III (AT-III), and tissue factor pathway inhibitor (TFPI) as well as combinations of extremes, eg, 50% anticoagulants and 150% procoagulants or 50% procoagulants and 150% anticoagulants in a synthetic "plasma" system. The reaction systems were constructed in vitro using purified, natural, and recombinant proteins and synthetic phospholipid vesicles or platelets with the reactions initiated by recombinant tissue factor (TF)-factor VIIa complex (5 pmol/L). To investigate the influence of the protein C system, soluble thrombomodulin (Tm) was also added to the reaction mixture. For the most extreme situations in which the essential plasma procoagulants (prothrombin, and factors X, IX, V, and VIII) and the stoichiometric anticoagulants (AT-III and TFPI) were collectively and inversely altered by 50%, a 28-fold difference in the total available thrombin generated was observed. Variations of most of these proteins 50% above and below the "normal" range, with the remainder at 100%, had only modest influences on the peak and total levels of thrombin generated. The dominant factors influencing thrombin generation were prothrombin and AT-III. When these 2 components were held at 100% and all other plasma procoagulants were reduced to 50%, there was a 60% reduction in the available thrombin generated. No increase in the thrombin generated was observed when the 150% level of all plasma procoagulants other than prothrombin was evaluated. When only prothrombin was raised to 150%, and all other factors were maintained at 100%, the thrombin generated increased by 71% to 121%. When AT-III was at 50% and all other constituents were at 100%, thrombin production was increased by 104% to 196%. The additions of protein C and protein S over the 50% to 150% ranges with Tm at 0.1 nmol/L concentration had limited influence on thrombin generation. Individual variations in factors VII, XI, and X concentrations had little effect on the duration of the initiation phase, the peak thrombin level achieved, or the available thrombin generated. Paradoxically, increases in factor IX concentration to 150% led to lowered thrombin generation, while decreases to 50% led to enhanced thrombin generation, most likely a consequence of factor IX as a competitive substrate with factor X for factor VIIa-TF. Reductions in factor V or factor VIII concentration led to prolongations of the initiation phase, while the reduction of TFPI to 50% led to shortening of this phase. However, none of these alterations led to significant changes in the available thrombin generated. Based on these data, one might surmise that increases in prothrombin and reductions in AT-III, within the normal range, would be potential risk factors for thrombosis and that algorithms that combine normal factor levels may be required to develop predictive tests for thrombosis.     

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.     

Inhibition of thrombin generation by simvastatin and lack of additive effects of aspirin in patients with marked hypercholesterolemia

OBJECTIVES: To assess the effects of aspirin compared with simvastatin on thrombin generation in hypercholesterolemic men, and to establish whether the reduction of elevated blood cholesterol by simvastatin would affect the action of aspirin on thrombin formation. BACKGROUND: Aspirin inhibits thrombin formation, but its performance is blunted in hypercholesterolemia. By virtue of altering lipid profile, statins could be expected to influence thrombin generation. METHODS: Thirty-three men, aged 34 to 61 years, with minimal or no clinical symptoms, serum total cholesterol >6.5 mmol/liter and serum triglycerides <4.6 mmol/liter, completed the study consisting of three treatment phases. First, they received 300 mg of aspirin daily for two weeks (phase I), which was then replaced by simvastatin at the average dose of 24 mg/d for three months (phase II). In phase III, aspirin, 300 mg/day, was added for two weeks to simvastatin, the dose of which remained unchanged. Thrombin generation was assessed: 1) in vivo, by measuring levels of fibrinopeptide A (FPA) and prothrombin fragment 1+2 (F1+2) in venous blood; and 2) ex vivo, by monitoring the rates of increase of FPA and F1+2 in blood emerging from standardized skin incisions of a forearm. A mathematical model was used to describe the kinetics of thrombin formation at the site of microvascular injury. RESULTS: Two-week treatment with aspirin had no effect on thrombin markers in vivo, while ex vivo it depressed the total amount of thrombin formed, though not the reaction rate. After simvastatin treatment, serum cholesterol decreased by 31% and LDL cholesterol by 42%, while thrombin generation became markedly depressed. In venous blood, FPA was significantly reduced. Concomitantly, the initial thrombin concentration and total amount of thrombin generated decreased significantly. Addition of aspirin to simvastatin (phase III) had no further effect on any of these parameters. CONCLUSIONS: In men with hypercholesterolemia, lowering serum cholesterol level by a three-month simvastatin treatment is accompanied by a marked reduction of thrombin generation both at basal conditions in venous blood and after activation of hemostasis by microvascular injury. Once blood cholesterol became reduced, adding aspirin to simvastatin did not enhance dampening of thrombin formation.     

Inhibition of tissue-factor-mediated thrombin generation by simvastatin

A previous study has shown that simvastatin reduces in vivo clotting activation and monocyte tissue factor (TF) expression. This effect, however, was only in part attributable to the reduction of serum cholesterol, suggesting that more than one mechanism may be involved. Furthermore, it was not investigated if the inhibition of clotting activation was dependent upon the reduced expression of monocyte TF. In order to assess if simvastatin directly affects clotting activation, we developed an in vitro method in which clotting system is activated by monocytes stimulated with LPS. Monocytes were prepared from blood taken from healthy volunteers or patients with hypercholesterolemia and incubated with heparinized plasma plus either simvastatin (0.01-10 microM) or medium as control. Samples were then stimulated with LPS (4 microg/ml) and after 6 h the rate of thrombin generation, assessed by prothrombin fragment (F) 1+2, was measured. In separate experiments, we measured the expression of TF by monocytes which were incubated with simvastatin and then stimulated with LPS. The study showed that compared to control, LPS-stimulated monocytes induced abundant formation of F1+2, which was inhibited by simvastatin in a dose-dependent manner. Simvastatin also inhibited dose dependently the monocyte expression of TF. This study suggests that simvastatin inhibits the rate of thrombin generation by directly interfering with the monocyte expression of TF.     

Influence of coagulation factors on extrinsic thrombin generation.

The extrinsic coagulation activity assay (EXCA) is a new thrombin generation test for the tissue factor pathway of coagulation. The EXCA was performed with 10 parts citrated plasma of different contents of fibrinogen. One part tissue factor, 250 mmol/l CaCl(2), generating about 1 IU/ml thrombin within 1 min (37 degrees C). After 0-30 min 2.5 mol/l arginine (pH 8.6) Generated thrombin was detected by addition of CHG-Ala-Arg-pNA and measurement of triangle upA/t. The EXCA is dependent on factors 10% of the factor VII norm in the sample achieves 70-80% of the thrombin generation norm. The EXCA is not dependent on factors VIII, IX, XI and XII. Even in antithrombin III-deficient plasma, a phase of thrombin inhibition appears after the thrombin peak. Supplemented purified fibrinogen resulted in decreased thrombin generation in the important. Fibrinogen seems to act as antithrombin I; thrombin might be entrapped in the nascent fibrin. The EXCA is suitable to diagnose the level of extrinsic factors in patient plasma.     

Platelet-dependent thrombin generation after in vitro fibrinolytic treatment.

BACKGROUND. Fibrinolytic therapy is associated with frequent rethrombosis. There is evidence of both increased coagulation and platelet activation. METHODS AND RESULTS. Platelet-rich plasma (PRP) or washed platelets were incubated with the fibrinolytic agents urokinase, recombinant tissue-type plasminogen activator (rt-PA), or plasmin at concentrations consistent with those in the plasma of patients treated for myocardial infarction. All of the fibrinolytic agents induced a more rapid generation of thrombin and decreased the clotting times of non-contact-activated PRP than in untreated PRP. This effect was not blocked by the inclusion of thrombin inhibitors during the fibrinolytic treatment. Washed platelets derived from rt-PA-treated PRP induced more rapid thrombin generation when resuspended in untreated plasma or treated plasma. Washed platelets were treated with plasmin, rt-PA, and urokinase and added to platelet-poor plasma. Platelets treated with either plasmin or rt-PA increased the ability of washed platelets to support thrombin generation, but urokinase was without significant effect. CONCLUSIONS. These results indicate not only that plasmin can cause increased platelet support of prothrombin activation but also that rt-PA in the absence of plasminogen can have a direct effect on the platelet, which increases thrombin generation.     

Evaluation of the hemostatic potential including thrombin generation of three different therapeutic pathogen-reduced plasmas

Background and Objectives Several pathogen inactivation methods currently applied to therapeutic plasma may result in products with different hemostatic properties. This study aims at evaluating and comparing the hemostatic potential of different therapeutic plasma preparations currently available in France. Materials and methods We studied three types of pathogen‐reduced plasma for transfusion (MB/light, Amotosalen/UVA, industrial S/D plasma). Quarantine, non‐pathogen‐reduced plasma, was used as a control. This study compared more specifically the content in FVIII, fibrinogen (clottable and antigen assays) and ADAMTS‐13 and evaluated the intrinsic hemostatic properties using a thrombin generation test [Calibrated Automated Thrombogram (CAT)] at high and low concentrations of tissue factor to assess the maximum quantity of thrombin generated or the contribution of FVIII and FIX in the amplification phase of thrombin generation, respectively. Results The median FVIII concentration was >70 IU/dl for each preparation. Endogenous thrombin potential values were significantly different among the methods of plasma preparation (P < 0·001) but were all in the range of the values measured in donors’ plasma. Control by the thrombomodulin‐activated protein C system was preserved in all preparations (>50% inhibition of endogenous thrombin potential). Fibrinogen concentrations were all within normal range but fibrinogen levels were lower in the plasmas treated with photochemical methods. ADAMTS‐13 levels were preserved. Conclusion The hemostatic potential appears well preserved in all therapeutic plasmas tested but there are some differences between preparations, the clinical relevance of which remains to be elucidated.     

Comparative studies on various assays for the laboratory evaluation of r-hirudin.

Several laboratory methods are available to measure r-hirudin, including clot-based, amidolytic, immunologic, and physicochemical techniques. The global tests, such as the PT, APTT, and Heptest, did not show an adequate response to r-hirudin in the range of 0.5 to 10.0 micrograms/ml, where full anticoagulation is achieved, as determined by animal models of thrombosis. The 10 U/ml thrombin time assay was very sensitive to r-hirudin, whereas the 10 U/ml calcium thrombin time gave a dose-dependent response from 0.15 to 10.0 micrograms/ml. Whole blood clotting assays (ACT, TEG) effectively measured r-hirudin levels up to 25 micrograms/ml. The amidolytic anti-Factor IIa assay, specific for evaluating direct thrombin inhibition, was very effective, particularly when modified to decrease the sample: thrombin ratio for higher r-hirudin concentrations. This assay may be useful in quality control, since it is biochemically defined and reagents are easily standardized. Thrombin generation assays based on synthetic substrates showed limited effect of r-hirudin; however, assays based on TAT complex and prothrombin fragment F1+2 generation showed a dose-dependent response. Immunologic methods (ELISA) are under development. Since these assays measure both complexed and noncomplexed hirudin, and since they are only sensitive to submicrogram levels, they may only be useful for the direct quantitation of absolute levels of r-hirudin but not for monitoring clinical anticoagulation. Thus, thrombin-based clotting, amidolytic, and immunologic assays can be used to evaluate and measure r-hirudin. However, optimization of each assay to respond to high and low concentrations of r-hirudin and their application to clinical monitoring, batch control, and standardization needs to be determined.