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 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.     

Inhibition of endothelial cell-mediated generation of activated protein C by direct and antithrombin-dependent thrombin inhibitors.

The present study investigated the effect of the thrombin inhibitors antithrombin (AT) (with and without unfractionated heparin or low molecular weight heparin), hirudin, inogatran and melagatran on thrombin-thrombomodulin-mediated generation of activated protein C (APC), in solution and on endothelial cells. Sequential incubation with thrombin, thrombin inhibitors and protein C was followed by measurement of APC by an amidolytic assay. The approximate concentrations resulting in 50% inhibition of endothelial cell-mediated APC generation for AT, AT-unfractionated heparin, AT-low molecular weight heparin, hirudin, melagatran and inogatran were 200, 4, 9, 1, 8 and 60 nmol/l, respectively. The normal plasma level of AT is 2800 nmol/l and relevant therapeutic concentrations from clinical trials are 200 nmol/l for hirudin, 500 nmol/l for melagatran and 1000 nmol/l for inogatran. The present study indicates that clinically relevant concentrations of the tested thrombin inhibitors interfere with endothelial-mediated APC generation, which may offer an explanation for the lack of a dose-response effect in clinical trials with thrombin inhibitors.     

Inhibition of thrombin generation by the zymogen factor VII: implications for the treatment of hemophilia A by factor VIIa

Factor VII circulates as a single chain inactive zymogen (10 nmol/L) and a trace ( approximately 10-100 pmol/L) circulates as the 2-chain form, factor VIIa. Factor VII and factor VIIa were studied in a coagulation model using plasma concentrations of purified coagulation factors with reactions initiated with relipidated tissue factor (TF). Factor VII (10 nmol/L) extended the lag phase of thrombin generation initiated by 100 pmol/L factor VIIa and low TF. With the coagulation inhibitors TFPI and AT-III present, factor VII both extended the lag phase of the reaction and depressed the rate of thrombin generation. The inhibition of factor Xa generation by factor VII is consistent with its competition with factor VIIa for TF. Thrombin generation with TF concentrations >100 pmol/L was not inhibited by factor VII. At low tissue factor concentrations (<25 pmol/L) thrombin generation becomes sensitive to the absence of factor VIII. In the absence of factor VIII, factor VII significantly inhibits TF-initiated thrombin generation by 100 pmol/L factor VIIa. In this hemophilia A model, approximately 2 nmol/L factor VIIa is needed to overcome the inhibition of physiologic (10 nmol/L) factor VII. At 10 nmol/L, factor VIIa provided a thrombin generation response in the hemophilia model (0% factor VIII, 10 nmol/L factor VII) equivalent to that observed with normal plasma, (100% factor VIII, 10 nmol/L factor VII, 100 pmol/L factor VIIa). These results suggest that the therapeutic efficacy of factor VIIa in the medical treatment of hemophiliacs with inhibitors is, in part, based on overcoming the factor VII inhibitory effect. (Blood. 2000;95:1330-1335)     

Inhibition of thrombin activity by prothrombin activation fragment 1.2

Prothrombin is the precursor of thrombin, the central enzyme in coagulation. Prothrombin is activated in vivo by the prothrombinase complex to form fragment 1.2 and thrombin. Fragment 1.2 has an amino-terminal gla domain and two kringle domains. The second kringle domain (kringle 2) binds to the exosite II on thrombin. Nascent thrombin generated on platelet surface remains non-covalently bound to fragment 1.2 by kringle 2-exosite II interaction. To determine whether this interaction can modulate coagulant activity of thrombin, we labeled thrombin at the active site with fluorescein-Phe-Pro-Arg chloromethylketone and monitored the fluorescence changes upon ligand binding. Anionic phospholipid-bound fragment 1.2 and fragment 2 bound to FPR-thrombin and induced changes in the active site with half maximal effects at 7.2 μM and 8.8 μM, respectively. We also tested the effect of anionic phospholipid-bound fragment 1.2 (0–10 μM) on thrombin clotting activity. Phospholipid-bound fragment 1.2 inhibited fibrinogen clotting in a concentration-dependent manner but had no significant effect on amidolytic activity towards S2238, suggesting a competitive inhibition of the fibrinogen binding site. Furthermore, fragment 1.2 inhibited FPR-thrombin binding to platelet. Consistent with these findings fragment 1.2 inhibited thrombin-induced aggregation of gel filtered platelets in a concentration-dependant manner. These results suggest that the membrane-bound prothrombin fragment 1.2 may play a role in hemostasis by down regulating the procoagulant activity of newly formed thrombin.     

Inhibition of thrombin generation by protein S at low procoagulant stimuli: implications for maintenance of the hemostatic balance

The activated protein C (APC)-independent anticoagulant activity of protein S on tissue factor-induced thrombin generation was quantified in plasma. In absence of APC, protein S significantly decreased the endogenous thrombin potential (ETP) in a concentration-dependent manner. The APC-independent anticoagulant activity of protein S in plasma was not affected by phospholipid concentrations but strongly depended on tissue factor concentrations: protein S inhibited the ETP from 6% at 140 pM tissue factor to 74% at 1.4 pM tissue factor. Plasma with both 60% protein S and 140% prothrombin showed an ETP of 240% compared to normal plasma, suggesting an APC-independent protective role of protein S in the development of thrombosis as a result of protein S deficiency and the prothrombin-G20210A mutation. At high tissue-factor concentrations, protein S hardly expressed APC-independent anticoagulant activity but exerted potent APC-cofactor activity when thrombomodulin or APC were added to plasma. Neutralization of protein S under these conditions resulted in a 20-fold reduction of the anticoagulant activity of APC. The present study shows that protein S effectively regulates coagulation at 2 levels: at low procoagulant stimuli, protein S maintains the hemostatic balance by directly inhibiting thrombin formation, and at high procoagulant stimuli, protein S restores the hemostatic balance via its APC-cofactor activity.     

Inhibition of some activities of thrombin by a collagen-derived octapeptide

The effect of a collagen-derived octapeptide on some properties of thrombin is presented. This peptide provoked a dose- and time-dependent prolongation of the thrombin-induced plasma and fibrinogen clotting time and inhibited the polymerization of fibrin generated from fibrinogen by thrombin. It did not affect the polymerization of fibrin monomers; it was also without effect on the coagulation of plasma or fibrinogen by reptilase. The prolongation of the fibrinogen clotting time depended on the duration of the incubation of thrombin and the octapeptide and not on the duration of the incubation of fibrinogen and the octapeptide. The inhibition was therefore ascribed to an interference with thrombin, rather than with fibrinogen. A preincubation of the octapeptide with thrombin resulted in an inhibition of the thrombin-induced platelet aggregation. The effect of the octapeptide on thrombin has been related to the presence of positively and negatively charged groups, because uncharged analogue sequences were without effect on these activities of thrombin.     

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 in plasma by heparin or arginine.

The inhibition of plasmatic thrombin is of clinical importance in a broad range of diseases. To obtain reliable data the assay system should be as similar to physiology as possible. Using a newly developed physiologic assay system for fibrinogen/thrombin interaction (the FIFTA), the inhibition of plasmatic thrombin by heparin or by arginine was studied. The standard fibrinogen functional turbidimetric assay (FIFTA) was performed, varying heparin or arginine concentrations and varying the time point the inhibitor was added to the FIFTA. Plasmatic heparin concentrations equal to or greater than 0.63 IU/mL completely inhibit thrombin in the assay system described. The IC(50) is 0.1 IU/mL heparin. Heparin can only inhibit fibrin generation within the first 2 minutes at room temperature (RT=23 degrees C). The 50% inhibitory time point, that is, the time point that a 10 IU/mL final concentration of heparin results in 50% inhibition of FIFTA, is 30 seconds at RT. A final arginine concentration of at least 125 mM in the first 100 seconds of the FIFTA reaction at RT completely inhibits turbidity increase. Half-maximal turbidity increase occurs at 63 mM arginine. Final arginine concentrations of at least 250 mM completely inhibit turbidity increase, when arginine acts in the first 4 minutes (RT) of the thrombin/ fibrinogen interaction. A final arginine concentration of 477 mM added at the 12-minute or 30-minute thrombin/ fibrinogen reaction time point decreases the resulting turbidity by 50% after an additional 30 minutes at RT. Pathologic disseminated intravascular coagulation occurs in a multitude of diseases; in common is always the generation of thrombin either by the contact phase or by the tissue factor phase of coagulation. Such pathologically elevated thrombin activity in blood or blood products must be prevented or inhibited. This study demonstrates the efficiency of two physiologic thrombin inhibitors: heparin and arginine.     

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.     

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.     

"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.     

Inhibition of annexin V binding to cardiolipin and thrombin generation in an unselected population with venous thrombosis

OBJECTIVE: To examine the effect on annexin V binding to cardiolipin (CL) and in vitro thrombin generation by plasma samples from an unselected population of patients with confirmed venous thrombosis and matched controls. The prevalence of autoimmune antiphospholipid antibodies (aPL) was also determined. METHODS: A total of 111 patients who presented to a single emergency room with symptoms suggestive of venous thromboembolic (VTE) disease were studied. In 34 patients the diagnosis of lower limb deep venous thrombosis (DVT) and/or pulmonary embolus (PE) was confirmed (VTE+ group). In the remaining 77 patients the diagnostic workup was negative (VTE- group). Plasma samples were collected prior to the initiation of anticoagulation and examined for IgG anticardiolipin (aCL), IgG anti-beta2-glycoprotein I (GPI), and IgG anti-prothrombin (aPT antibodies) by ELISA. In addition, the effect of individual patient and control plasma samples on annexin V binding to CL and on in vitro thrombin generation was determined by a competitive ELISA and a chromogenic assay, respectively. RESULTS: The prevalence and levels of IgG aCL, anti-beta2-GPI, and aPT antibodies were similar in the VTE+ and VTE- groups. However, plasma samples from the VTE+ group caused a significant inhibition of in vitro thrombin generation (mean +/- SD Z score: -0.66 +/- 0.97 vs 0.26 +/- 1.46; p < 0.001) and a concurrent but less impressive inhibition of annexin V binding to CL (mean +/- SD Z score: -2.53 +/- 1.44 vs -2.05 +/- 1.61; p = 0.123). Upon analyzing a panel of clinical and laboratory variables, only age and inhibition of thrombin generation were significantly associated with VTE disease. CONCLUSION: Our findings suggest that subtle abnormalities in annexin V physiology may contribute to the procoagulant state in patients with idiopathic venous thrombosis.