Distinct effects of recombinant desulfatohirudin (Revasc) and heparin on plasma levels of fibrinopeptide A and prothrombin fragment F1.2 in unstable angina. A multicenter trial

BACKGROUND: Thrombin plays an important role in the pathogenesis of acute coronary thrombosis. We studied the effects of a direct thrombin inhibitor, recombinant desulfatohirudin, and heparin on plasma levels (at 0, 4, 12, and 24 hours) of fibrinopeptide A (FPA), which reflects thrombin action, and prothrombin fragment F1.2, which reflects thrombin generation, in patients with unstable angina. METHODS AND RESULTS: Patients were randomized to one of two doses of heparin (n = 50) (target activated partial thromboplastin time, 65 to 90 seconds or 90 to 110 seconds) or one of four doses of r-hirudin (n = 113) (0.05, 0.10, 0.20, or 0.30 mg.kg-1.h-1 by infusion). r-Hirudin induced a dose-dependent decline in plasma FPA. At 24 hours, FPA levels with 0.1- to 0.3-mg.kg-1.h-1 r-hirudin regimens were significantly lower than with 0.05 mg.kg-1.h-1 r-hirudin; levels with 0.1- to 0.2-mg.kg-1.h-1 r-hirudin regimens were lower than with both heparin regimens. Plasma F1.2 did not decline significantly during therapy with heparin or hirudin except at 0.3 mg.kg-1.h-1 hirudin. At 24 hours, they were higher with the 0.05-mg.kg-1.h-1 r-hirudin regimen than with other regimens. For comparable levels of thrombin generation (F1.2 levels), FPA levels were higher in heparin patients than in hirudin patients. For the same FPA values, the corresponding F1.2 values were higher in the hirudin group. CONCLUSIONS: Our findings provide evidence for distinct in vivo effects of the two agents and suggest that r-hirudin is a relatively more potent inhibitor of thrombin action but a less effective inhibitor of thrombin generation than heparin. The lower FPA levels in hirudin patients may reflect its ability to inactivate clot-bound thrombin. The relative clinical efficacies of the two agents need to be defined by clinical trials in progress.     

Surface-bound heparin fails to reduce thrombin formation during clinical cardiopulmonary bypass

The hypothesis that heparin-coated perfusion circuits reduce thrombin formation and activity; fibrinolysis; and platelet, complement, and neutrophil activation was tested in 20 consecutive, randomized adults who had cardiopulmonary bypass. Twenty identical perfusion systems were used; in 10, all blood-contacting surfaces were coated with partially degraded heparin (Carmeda process; Medtronic Cardiopulmonary, Anaheim, Calif.). All patients received a 300 U/kg dose of heparin. Activated clotting times were maintained longer than 400 seconds. Cardiopulmonary bypass lasted 36 to 244 minutes. Blood samples for platelet count, platelet response to adenosine diphosphate, plasma beta-thromboglobulin, inactivated complement 3b, neutrophil elastase, fibrinopeptide A, prothrombin fragment F1.2, thrombin-antithrombin complex, tissue plasminogen activator, plasminogen activator inhibitor-1, plasmin alpha 2-antiplasmin complex, and D-dimer were obtained at these times: after heparin was given, 5 and 30 minutes after cardiopulmonary bypass was started, within 5 minutes after bypass was stopped, and 15 minutes after protamine was given. After cardiopulmonary bypass, tubing segments were analyzed for surface-adsorbed anti-thrombin, fibrinogen, factor XII, and von Willebrand factor by radioimmunoassay. Heparin-coated circuits significantly (p < 0.001) reduced platelet adhesion and maintained platelet sensitivity to adenosine diphosphate (p = 0.015), but did not reduce release of beta-thromboglobulin. There were no significant differences between groups at any time for fibrinopeptide A, prothrombin fragment F1.2, or thrombin-antithrombin complex or in the markers for fibrinolysis: D-dimer, tissue plasminogen activator, plasminogen activator inhibitor-1, and alpha 2-antiplasmin complex. In both groups, concentrations of prothrombin fragment F1.2 and thrombin-antithrombin complex increased progressively and significantly during cardiopulmonary bypass and after protamine was given. Concentrations of D-dimer, alpha 2-antiplasmin complex, and plasminogen activator inhibitor-1 also increased significantly during bypass in both groups. Fibrinopeptide A levels did not increase during bypass but in both groups increased significantly after protamine was given. No significant differences were observed between groups for levels of inactivated complement 3b or neutrophil elastase. Radioimmunoassay showed a significant increase in surface-adsorbed antithrombin on coated circuits but no significant differences between groups for other proteins. We conclude that heparin-coated circuits used with standard doses of systemic heparin reduce platelet adhesion and improve platelet function but do not produce a meaningful anticoagulant effect during clinical cardiopulmonary bypass. The data do not support the practice of reducing systemic heparin doses during cardiac operations with heparin-coated extracorporeal perfusion circuitry.     

Anticoagulant effects of hirulog, a novel thrombin inhibitor, in patients with coronary artery disease

Selective thrombin inhibitors are a new class of antithrombotic drugs that, unlike heparin, can effectively inhibit clot-bound thrombin and escape neutralization by activated platelets. Hirulog is a 20 amino acid hirudin-based synthetic peptide that has shown promise in experimental models of thrombosis. Little information is available about the effects of hirulog in patients with coronary artery disease. Forty-five patients undergoing cardiac catheterization, who were taking aspirin, were randomized to receive either (1) hirulog, 0.05 mg/kg intravenous bolus followed by 0.2 mg/kg/hour intravenous infusion until the end of the catheterization; (2) hirulog, 0.15 mg/kg intravenous bolus followed by 0.6 mg/kg/hour intravenous infusion; or (3) heparin; 5,000 U intravenous bolus. Serial activated partial thromboplastin time (APTT), prothrombin time, activated clotting time and fibrinopeptide A were measured. Hirulog produced a dose-dependent prolongation of all coagulation parameters; the 0.6 mg/kg/hour dose prolonged the APTT to 218 +/- 50% of baseline after 2 minutes and 248 +/- 50% of baseline after 15 minutes. The half-life of the effect on APTT was 40 minutes. The hirulog blood level correlated well with the APTT, prothrombin time and activated clotting time (r = 0.77, 0.73, and 0.82 respectively, all p < 0.001). Both doses of hirulog potently suppressed the generation of fibrinopeptide A (p < 0.05). There were no major hemorrhagic, thrombotic or allergic complications in patients treated with hirulog or heparin. Thus, hirulog, a direct thrombin inhibitor, provides a predictable level of anticoagulation and appears to have a potent yet well-tolerated anticoagulant profile in patients with coronary artery disease.     

Activation of the hemostatic mechanism after pharmacological cardioversion of acute nonvalvular atrial fibrillation

BACKGROUND: Given that the restoration of sinus rhythm after chronic atrial fibrillation is associated with embolic events, anticoagulation is prescribed before and after pharmacological and electrical cardioversion. However, the need for anticoagulation in patients with acute atrial fibrillation (lasting <48 hours) who undergo cardioversion is less clear. In addition, it is not known whether cardioversion to sinus rhythm determines a hypercoagulable state in these patients. METHODS AND RESULTS: In 21 patients with acute nonvalvular atrial fibrillation, plasma median concentrations of thrombin-antithrombin complex, a marker of thrombin generation, significantly increased from 2.8 ng/mL (interquartile range, 2.1 to 4.0 ng/mL) on hospital admission to 3.5 ng/mL (interquartile range, 2.9 to 6.0 ng/mL) after cardioversion to sinus rhythm obtained by means of infusion of antiarrhythmic drugs and decreased to 2.5 ng/mL (interquartile range, 2.0 to 3.5 ng/mL) at the 1-month follow-up visit (P=.04). Similarly, the levels of fibrinopeptide A, a marker of thrombin activity, increased from 1.1 nmol/L (interquartile range, 0.7 to 1.5 nmol/L) at baseline to 1.8 nmol/L (interquartile range, 1.1 to 3.0 nmol/L) after cardioversion and returned to 0.8 nmol/L (interquartile range, 0.6 to 1.1 nmol/L) at the 1-month follow-up visit (P=.02). CONCLUSIONS: A significant increase in plasma levels of the markers of thrombin generation and activity was observed in patients with acute atrial fibrillation early after pharmacological cardioversion to sinus rhythm. This is the first biochemical evidence that cardioversion of recent-onset atrial fibrillation determines a hypercoagulable state.     

The generation of fibrinopeptide A in clinical blood samples: evidence for thrombin activity

Plasma fibrinopeptide A (FPA) concentrations were measured in clinical blood samples incubated in the collecting syringe for different time periods before addition to heparin and Trasylol, and the rate of in vitro generation of FPA was calculated as the mean increment in FPA concentration per minute over the linear portion of the generation curve. 36 normal individuals had a mean plasma FPA level of 0.64 +/- 0.56 pmol/ml and an FPA generation rate of less than 0.5 pmol/ml per min. Clinical samples with elevated plasma FPA levels manifested slow (less than 1 pmol/ml per min) (28 patients) or rapid FPA generation (greater than 1 pmol/ml per min) (33 patients). Slow FPA generation was found in 10/10 patients with venous thrombosis, in 4/4 with aortic aneurysm, and in several patients with acquired hypofibrinogenemia. In one such patient, addition of fibrinogen resulted in rapid FPA generation whereas thrombin addition was without effect. Rapid FPA generation was generally linear, was usually associated with slower fibrinopeptide B generation and was inhibited by parenteral or in vitro heparin. It is thought to reflect increased thrombin activity and was seen in patients with pulmonary embolism, active systemic lupus erythematosus, renal transplant rejection, and after infusion of prothrombin concentrates. The initial rate of FPA cleavage by thrombin at fibrinogen concentrations from 0.05 to 4 mg/ml showed little change between 2 and 4 mg/ml with a Km of 2.99 muM. At a fibrinogen concentration of 2.5 mg/ml the FPA cleavage rate was 49.2 +/- 1.6 nmol/ml per min per U of thrombin. Exogenous thrombin added to normal blood generated 21.7 nmol/ml per U of thrombin FPA in the first minute with a nonlinear pattern reflecting inactivation of thrombin and the presence of alternative substrates. Hence, the thrombin concentration in the blood cannot be calculated from the FPA generation rate. The FPA generation rates in clinical samples with rapid generation (1-28 pmol/ml per min) could be produced by 2 X 10(-5) to 5.6 X 10(-4) thrombin U/ml acting on purified fibrinogen at physiological conditions of pH, ionic strength, and temperature.     

Purification and characterization of two cysteine proteinase inhibitors from the skin of Atlantic salmon (Salmo salar L.)

This study was designed to characterize hemostatic activation (using fibrinopeptide A (FPA), a marker of thrombin activity, and beta-thromboglobulin (BTG), a marker of platelet activation) sequentially in the coronary and peripheral circulation in patients during percutaneous coronary intervention (PCI) and several hours after PCI and discontinuation of heparin therapy. Heparin administered during PCI is known to nonuniformly suppress thrombin activity in the coronary. Persistent elevations of FPA in coronary sinus (CS) blood during PCI have been associated with subsequent ischemic events. As a related consideration, rebound thrombin activity has been demonstrated in peripheral blood samples several hours after cessation of heparin therapy in patients with acute coronary syndromes. Accordingly, we hypothesized that increased thrombin activity occurs in the coronary circulation after PCI and is induced by cessation of intravenous heparin to facilitate vascular sheath removal. Such a rebound prothrombotic effect, may contribute to suboptimal outcomes after PCI. In 21 patients undergoing PCI, heparin-bonded catheters were employed to obtain sequential CS and femoral vein (FV) blood samples for measurement in the coronary and peripheral circulation of plasma FPA, a marker of thrombin activity in vivo, and BTG released by platelets during degranulation. Following heparin administration samples were obtained immediately prior to (base) and during (start and end) PCI. Late samples were obtained several hours after PCI (284 +/- 46 min, mean +/- SD) following the cessation of heparin and prior to planned vascular sheath removal. Mean FPA concentration in CS blood was low at baseline (3.82 +/- 2.09 ng/ml) and did not increase during PCI. Mean FPA concentration in CS blood increased significantly several hours after cessation of heparin (3.42 +/- 2.36 vs. 7.82 +/- 9.98, end vs. late, P < 0.001). In contrast, mean FPA concentration in FV blood was highest at baseline following vascular sheath insertion, decreased during PCI (69%, P < 0.05, base vs. end), and trended upward after PCI and cessation of heparin. Mean FPA values were higher at all times in FV compared with CS blood samples and were not concordant after PCI. Elevation of coronary circulation FPA after PCI was maximal in patients with myocardial infarction within 7 days (13.7 +/- 12.4 vs. 5.6 +/- 7.9 ng/ml, P = 0.08), but was not influenced by heparin treatment prior to PCI, a history of unstable angina, or coronary stent placement during PCI (9 of 21 patients). BTG values showed less variation than did FPA values, and cessation of heparin after PCI was not associated with an increase in BTG in CS or FV blood samples. An increase in thrombin activity occurs in the coronary circulation after PCI following discontinuation of heparin. The extent of increased thrombin activity was greatest in patients with recent myocardial infarction and was not exacerbated by coronary stent placement during PCI. This phenomenon may contribute to the important minority of ischemic complications early after PCI.     

Current approach in antithrombotic treatment during coronary interventions: specific thrombocyte aggregation inhibitors and direct thrombin antagonists in high-risk patients

Patients undergoing angioplasty (PTCA) for unstable angina, postinfarction angina, or complex coronary lesions represent a high risk group for ischemic complications. High dose heparin and aspirin are used routinely to prevent thrombotic complications. However, new approaches designed to avoid platelet aggregation, including development of specific platelet GP IIb/IIIa receptor antagonists and specific thrombin inhibitors, demonstrate a significant reduction of thrombotic events following coronary interventions compared to heparin alone. Bleeding complications are not increased if conjunctive heparin administration is weight-adjusted. Pathophysiology of acute coronary closure, mechanisms of action of the new anti-thrombotic drugs, and current and future clinical applications are discussed.     

Thrombogenesis of different cell types seeded on vascular grafts and studied under blood-flow conditions

BACKGROUND: Small-diameter vascular grafts tend to have an early and high occlusion rate. Cell seeding on the luminal surfaces of small-diameter vascular prostheses may provide an antithrombotic lining and improve both the short-term and the long-term patency rates. We studied the net results of procoagulant and anticoagulant properties of seeded grafts under blood-flow conditions, and we compared the different available types of donor cells. METHODS: Monolayers of liposuction-derived cultured human microvascular endothelial cells (MVECs), human adult endothelial cells (HAECs), human umbilical vein endothelial cells (HUVECs), and human mesothelial cells (MCs) that had been seeded on expanded polytetrafluoroethylene (ePTFE) grafts were perfused with marginally anticoagulated blood (20 U/mL low molecular weight heparin; shear rate, 400/s, 10 minutes) or with non-anticoagulated blood (shear rate, 100/s, 5 minutes). The thrombin and fibrin generation in time was studied with the measurement of the plasma levels of prothrombin fragment 1 and 2 (F 1+2) and of fibrinopeptide A (FPA). The plain ePTFE graft was taken as a control. RESULTS: When the seeded MCs were perfused with recirculating anticoagulated blood, a linear generation of F 1+2 in time was seen, with high levels of F 1+2 and FPA after 10 minutes (4.38 nmol/L and 362 ng/mL, respectively). Allopurinol was added, and the MCs generated less F 1+2 than the HAECs (0.7 nmol/L vs 1.86 nmol/L; P <.05). No fibrin formation was seen. The MVECs generated low amounts of F 1+2 (0.7 nmol/L; 10 minutes), and the HUVECs and the plain ePTFE graft generated the lowest amounts of F 1+2 (0.26 and 0.25 nmol/L, respectively). When the MCs were perfused with non-anticoagulated blood, high amounts of thrombin and fibrin were generated immediately and constantly and could not be decreased with allopurinol. The perfusion of the plain ePTFE graft showed a dramatic increase in F 1+2 and FPA levels towards the end of the experiments. The seeded HAECs, HUVECs, and MVECs inhibited this increase. These results were confirmed by means of scanning electron microscopy. CONCLUSION: Vascular prostheses that are seeded with cultured MCs are highly procoagulant. Standard ePTFE graft prostheses also initiate coagulation, which supports the idea of cell seeding. The endothelial cells, of which the MVECs are the most readily available, seem to preserve their anticoagulant properties after being seeded on vascular grafts.     

Rapid activation of protein C by factor Xa and thrombin in the presence of polyanionic compounds

A recent study indicated that negatively charged substances such as heparin and dextran sulfate accelerate thrombin activation of coagulation factor XI by a template mechanism. Because the serine proteinase of the natural anticoagulant pathway, activated protein C, can bind heparin, it was reasonable to think that these compounds may also bind protein C (PC) and accelerate its activation by thrombin or other heparin binding plasma serine proteinases by a similar mechanism. To test this, PC activation by thrombin and factor Xa (fXa) was studied in the presence of these polysaccharides. With thrombin in the absence of thrombomodulin (TM), these polysaccharides markedly reduced the Km for PC and Gla-domainless PC (GDPC) activation in the presence of Ca2+. With TM containing chondroitin sulfate, heparin did not influence PC activation by thrombin, but with TM lacking chondroitin sulfate, the characteristic high-affinity PC interaction at low Ca2+ (approximately 50 to 100 micromol/L) was largely eliminated by heparin. In EDTA, heparin enhanced thrombin activation of GDPC by reducing the Km, but it inhibited PC activation by increasing the Km. PC activation in EDTA was insensitive to the presence of heparin if the exosite 2 mutant, R93,97,101A thrombin, was used for activation. These results suggest that, when the Gla-domain of PC is not fully stabilized by Ca2+, it interacts with the anion binding exosite 2 of thrombin and that heparin binding to this site prevents this interaction. Additional studies indicated that, in the presence of phospholipid vesicles, heparin and dextran sulfate dramatically accelerate PC activation by fXa by also reducing the Km. Interestingly, on phospholipids containing 40% phosphatidylethanolamine, the activation rate of near physiological PC concentrations ( approximately 80 nmol/L) by fXa in the presence of dextran sulfate was nearly comparable to that observed by the thrombin-TM complex. The biochemical and potential therapeutical ramifications of these findings are discussed.     

Substitution of tyrosine for phenylalanine in fibrinopeptide A results in preferential thrombin cleavage of fibrinopeptide B from fibrinogen

Phenylalanine at residue 8 in the Aalpha chain of fibrinogen is a highly conserved amino acid that is believed to be critical for binding and catalysis by the serine protease thrombin. We have examined the requirement for Phe at this position by constructing a variant recombinant fibrinogen with a conservative substitution of tyrosine for phenylalanine, Aalpha F8Y fibrinogen. We found that the variant fibrinopeptide A (F8Y 1-16) was cleaved by thrombin, in contrast to the lack of cleavage of an Aalpha 1-23 peptide and an Aalpha 1-50 fusion protein with the same substitution. This result indicates that fibrinogen residues other than Aalpha 1-50 participate in thrombin binding and fibrinogen proteolysis. We found, for the first time, that thrombin-catalyzed lysis of the fibrinogen Bbeta chain preceded lysis of the Aalpha chain, such that fibrinopeptide B (FpB) was released prior to F8Y 1-16. Kinetic analysis demonstrated that F8Y 1-16 was a very poor substrate for thrombin, with a specificity constant 280-fold lower than normal fibrinopeptide A. FpB was also a poor substrate, but the specificity constant for FpB was only 4-fold lower than normal. Consequently, FpB was preferentially released from Aalpha F8Y fibrinogen. This "role reversal" had a dramatic effect on polymerization, such that the rate of Aalpha F8Y fibrinogen polymerization was 13% of the rate of normal recombinant fibrinogen. These results confirm the importance of phenylalanine at Aalpha chain residue 8 for efficient thrombin-catalyzed proteolysis of fibrinogen, and further demonstrate that sequential fibrinopeptide release has an important role in normal polymerization.     

Dynamic imaging of the pancreas using real-time endoscopic ultrasonography with secretin stimulation

BACKGROUND: Tissue factor (TF) is a transmembrane glycoprotein that, after binding to factor VII/VIIa, initiates the extrinsic coagulation pathway, resulting in thrombin generation and its sequelae. Thrombin has been shown to induce TF mRNA in endothelium, monocytes, and smooth muscle cells, further perpetuating the thrombogenic cycle. This study was designed to determine the effect of specific inhibition of thrombin by recombinant hirudin (r-hirudin) on TF distribution after balloon angioplasty in the cholesterol-fed rabbit femoral artery and porcine coronary artery models. METHODS AND RESULTS: Thirty-five femoral arteries from 32 cholesterol-fed New Zealand White rabbits and 84 coronary arteries from 55 Yorkshire-Albino swine were studied by use of a recently developed in situ method of TF localization based on digoxigenin labeling of recombinant factor VIIa (Dig-VIIa), with correlative studies of TF immunoreactivity by use of anti-rabbit (AP-1) or anti-human (sTF) antibodies. At sites of balloon angioplasty in rabbit femoral or pig coronary arteries (double or single injury), TF-antibody and Dig-VIIa staining were noted in association with endothelial cells, smooth muscle cells, and foam cells and within the fibrous tissue matrix primarily of the adventitia and neointima. Staining was significantly greater after balloon angioplasty than in vessels that had not undergone angioplasty but was similar after single and double balloon injury. Animals treated with r-hirudin (rabbits, 1 mg/kg bolus plus 2-hour infusion; pigs, 1 mg/kg bolus plus 0.7 mg x kg(-1) x d(-1) infusion for 14 days with implantable pump) had diminished TF-antibody and Dig-VIIa staining 28 days after balloon angioplasty compared with controls (bolus heparin only). This effect was more prominent on the neointima and was more striking in the porcine than the rabbit model. CONCLUSIONS: TF expression, persistent 1 month after balloon angioplasty in rabbit femoral arteries and porcine coronary arteries, is attenuated by specific thrombin inhibition with hirudin. These results suggest that thrombin inhibition, in addition to its effect on acute thrombus formation and its effect on luminal narrowing by plaque in experimental animals, may result in a prolonged reduction in thrombogenicity of the restenotic plaque through this effect on TF expression.     

Experimental kallikrein gene therapy in hypertension, cardiovascular and renal diseases

Argatroban, a direct thrombin inhibitor, is used clinically because of its safe and effective antithrombotic action. This drug of low molecular weight shows reversible inhibition of thrombin irrespective of whether thrombin is fibrin-bound or soluble. Optimal anticoagulant effects can easily be attained by monitoring with the activated partial thromboplastin time or whole-blood activated clotting time when a therapeutic range of argatroban equivalent to that of heparin is used. The antithrombotic action is simply detected with a chromogenic substrate assay. The clinical use of the drug in Japan was approved for the treatment of chronic peripheral arterial obstructive disease and acute ischemic stroke. For coronary artery disease in patients with deficiency of antithrombin activities attributable to either antithrombin III or heparin cofactor II deficiency, argatroban is effective as an anticoagulant. Acute coronary occlusion during and after percutaneous transluminal coronary angioplasty can be treated by argatroban as an alternative to heparin. The presence of platelets activated by a trace amount of thrombin is evidenced by modified methods of platelet aggregometry in acute ischemic stroke. Therefore, argatroban can render the platelets insensitive against the platelet hyperaggregation enhanced by thrombin.     

Effect of nadroparin, a low-molecular-weight heparin, on clinical and angiographic restenosis after coronary balloon angioplasty: the FACT study. Fraxiparine Angioplastie Coronaire Transluminale

BACKGROUND: Experimental studies suggest that the antiproliferative effect of heparin after arterial injury is maximized by pretreatment. No previous studies of restenosis have used a pretreatment strategy. We designed this study to determine whether treatment with nadroparin, a low-molecular-weight heparin, started 3 days before the procedure and continued for 3 months, affected angiographic restenosis or clinical outcome after coronary angioplasty. METHODS AND RESULTS: In a prospective multicenter, double-blind, randomized trial, elective coronary angioplasty was performed on 354 patients who were treated with daily subcutaneous nadroparin (0.6 mL of 10,250 anti-Xa IU/mL) or placebo injections started 3 days before angioplasty and continued for 3 months. Angiography was performed just before and immediately after angioplasty and at follow-up. The primary study end point was angiographic restenosis, assessed by quantitative coronary angiography 3 months after balloon angioplasty. Clinical follow-up was continued up to 6 months. Clinical and procedural variables and the occurrence of periprocedural complications did not differ between groups. At angiographic follow-up, the mean minimal lumen diameter and the mean residual stenosis in the nadroparin group (1.37+/-0.66 mm, 51.9+/-21.0%) did not differ from the corresponding values in the control group (1.48+/-0.59 mm, 48.8+/-18.9%). Combined major cardiac-related clinical events (death, myocardial infarction, target lesion revascularization) did not differ between groups (30.3% versus 29.6%). CONCLUSIONS: Pretreatment with the low-molecular-weight heparin nadroparin continued for 3 months after balloon angioplasty had no beneficial effect on angiographic restenosis or on adverse clinical outcomes.     

Persistent thrombin generation in humans during specific thrombin inhibition with hirudin

BACKGROUND: The degree to which antithrombotic drugs suppress thrombin generation is unknown. Because hirudin, unlike antithrombin III, binds intravascular thrombin rapidly and selectively to yield a circulating inactive complex of 3- to 4-hour half-life, we used intravenous hirudin in humans to investigate the course of thrombin generation during and early after anticoagulation with this potent, direct antithrombin. METHODS AND RESULTS: Intravascular thrombin was measured with an ELISA for the thrombin-hirudin complex formed during and for 18 hours after stopping a 6-hour infusion of hirudin at 0.1, 0.2, and 0.3 mg.kg-1.h-1 in three groups of six patients each. With free hirudin in 20- to 10,000-fold molar excess of thrombin and peak activated partial thromboplastin times of 2.3 to 3.0 times baseline, mean plasma thrombin-hirudin complex increased from 794 +/- 85 pg/mL (mean +/- SEM) 15 minutes after the start of the infusion to 1617 +/- 151 pg/mL at 6 hours of infusion to 2667 +/- 654 pg/mL at 24 hours. During the 24-hour observation period, plasma concentration of fragment 1.2 (the peptide released during conversion of prothrombin to thrombin) never fell below baseline but rather increased transiently during the hirudin infusion. Plasma concentrations of thrombin-antithrombin III complex (in ng/mL) decreased from 4.34 +/- 0.40 at baseline to 1.64 +/- 0.13 at 6 hours (P < .001) and gradually increased after stopping the infusion to 5.7 +/- 0.87 at 24 hours (nonsignificant compared with baseline). CONCLUSIONS: Measurement of thrombin-hirudin complex may be used as a marker of thrombin generation in humans. Persistent accumulation of thrombin-hirudin complex and generation of fragment 1.2 during and after completion of potent anticoagulation with hirudin suggest thrombin generation is not blocked by high-affinity thrombin inhibition. The persistent formation of thrombin during declining plasma levels of hirudin may contribute to the pathogenesis of rethrombosis early after antithrombin therapy or during inadequate anticoagulation.     

Reduction in complications of angioplasty with abciximab occurs largely independently of baseline lesion morphology. EPIC and EPILOG Investigators. Evaluation of 7E3 for the Prevention of Ischemic Complications. Evaluation of PTCA To Improve Long-term Outcome with abciximab GPIIb/IIIa Receptor Blockade

OBJECTIVES: We investigated the hypothesis that abciximab might lead to a differential effect among patients with different lesion morphologies; hence, its cost/benefit ratio would be optimized if it were used selectively on the basis of baseline angiographic findings. BACKGROUND: Major complications of coronary angioplasty occur in 4% to 9% of patients. In the Evaluation of 7E3 for the Prevention of Ischemic Complications (EPIC) and Evaluation of PTCA To Improve Long-term Outcome with abciximab GPIIb/IIIa Receptor Blockade (EPILOG) trials, abciximab decreased the ischemic complications after intervention by 35% to 56%. However, the cost of this agent is appreciable, and there remain concerns about the safety of its readministration. METHODS: There were 1,362 patients in EPIC and 2,792 patients in EPILOG randomized to either bolus plus an infusion of abciximab or placebo, administered with aspirin and heparin at the time of the coronary intervention. Data from these studies were combined, and a differential effect of abciximab in relation to baseline lesion morphology on 30-day risk of death, myocardial infarction or urgent intervention was investigated using the Breslow Day test for statistical interaction. RESULTS: Abciximab consistently reduced the relative risk of complications across all lesion morphologies studied, with the possible exception of patients treated with degenerated saphenous vein grafts (risk with placebo 16.3% vs. risk with abciximab 18.6%, Breslow Day test for interaction, p=0.08). However, the absolute reduction of risk was somewhat greater in patients with more complex B2 or C lesions (7.6% and 5.8%, respectively) than in patients with morphologically simpler A or B1 lesions (3.7% and 3.2%, respectively). CONCLUSIONS: The reduction of early adverse ischemic events associated with angioplasty by abciximab occurs largely independent of pretreatment morphology.     

Structural examination of the influence of phosphorylation on the binding of fibrinopeptide A to bovine thrombin

Upon addition of thrombin, fibrinopeptides A and B are cleaved off from the N-termini of four chains of fibrinogen (Aalpha Bbeta gamma)2, and sites of polymerization are exposed, resulting in formation of a fibrin clot. For the fibrinogen Aalpha chain, cleavage occurs most prevalently at the Arg16-Gly17 peptide bond. About 25-30% of the human fibrinogen Aalpha chains are phosphorylated in nature at the position of Ser3, but the function for this modification is not understood. Previous NMR studies indicated that the N-terminal portion (1ADSGE5) of unphosphorylated fibrinopeptide A does not interact with the surface of bovine thrombin. Kinetic and NMR studies have now been carried out to assess whether phosphorylation at Ser3 allows the N-terminal segment (1ADSGEGDFLAEGGGVR16) to become anchored on the thrombin surface, leading to formation of a catalytically more efficient enzyme-substrate complex. Kinetic results indicate that phosphorylation leads to an approximately 65% increase in substrate specificity (kcat/Km) toward hydrolysis of fibrinogen Aalpha(1-20). 31P NMR studies reveal that the phosphorylated group does interact with thrombin, and 1H line broadening studies suggest that phosphorylation does promote binding of amino acids 1-5. Two-dimensional transferred nuclear Overhauser effect spectroscopy studies of bound fibrinopeptide A(1-16 Ser3P) indicate that phosphorylation allows new through-space interactions involving amino acid residues 1ADSGE5 to be observed. Computational docking of the peptide onto the X-ray structure of thrombin suggests that the phosphate may interact with basic residues at the rim of the heparin binding site of thrombin. As a result, the phosphate may serve as an anionic linker between the fibrinopeptide and the enzyme thrombin.     

Coronary artery and saphenous vein graft remodeling: a review of histologic findings after various interventional procedures--Part IV

Catheter balloon angioplasty is a well accepted form of nonsurgical treatment of acutely and chronically obstructed coronary artery vessels. It is also the centerpiece for various new intervention techniques. Their morphologic effects on the site of obstruction has been termed "remodeling." Part IV of this six-part series focuses on morphologic correlates of coronary angiographic patterns of remodeling after balloon angioplasty and discusses effects of angioplasty on adjacent, nondilated vessels.     

Differential inhibition of platelet aggregation induced by adenosine diphosphate or a thrombin receptor-activating peptide in patients treated with bolus chimeric 7E3 Fab: implications for inhibition of the internal pool of GPIIb/IIIa receptors

OBJECTIVES: This study sought to describe in detail the pharmacokinetics and pharmacodynamics of chimeric monoclonal 7E3 Fab (c7E3 Fab) and to compare platelet responses to adenosine diphosphate (ADP) and the 11-amino acid thrombin receptor-activating peptide (TRAP [SFLLRNPNDKY-NH2]) in patients undergoing elective coronary angioplasty. BACKGROUND: Inhibition of platelet aggregation with monoclonal antibody c7E3 Fab directed against glycoprotein (GP) IIb/IIIa has been shown to reduce ischemic complications after angioplasty and is being considered for treatment of other acute ischemic syndromes. METHODS: Patients undergoing elective coronary angioplasty received aspirin (325 mg orally), heparin (12,000 U intravenously) and a bolus of c7E3 Fab (0.25 mg/kg body weight). Surface GPIIb/IIIa receptor blockade and aggregation in response to 20 mumol/liter ADP, 5 micrograms/ml collagen and 7.5 and 15 mumol/liter TRAP were assessed. RESULTS: Surface GPIIb/IIIa receptor blockade by c7E3 Fab was 80% 2 h after injection and decreased to 50% at 24 h. Platelet aggregation in response to 20 mumol/liter ADP was inhibited by 73% at 2 h, and this inhibition decreased to 27% at 24 h. Platelet aggregation in response to 7.5 mumol/liter TRAP was inhibited by 53% at 2 h and 30% at 24 h. In contrast, aggregation in response to 15 mumol/liter TRAP was inhibited only 37% at 2 h and 10% at 24 h (p < 0.001 and p = 0.006, respectively vs. 20 mumol/liter ADP). Addition of exogenous c7E3 Fab to platelet-rich plasma led to more complete inhibition of 7.5 mumol/liter TRAP-induced aggregation. CONCLUSIONS: After c7E3 Fab treatment, inhibition of platelet aggregation depends on the agonist and can be overcome by increased thrombin activity but is restored if additional c7E3 Fab is added to block additional GPIIb/IIIa receptors. This phenomenon may be related to an internal pool of GPIIb/IIIa receptors joining the surface membrane and has implications concerning the duration of therapy with c7E3 Fab for patients with unstable angina or acute myocardial infarction.     

Clinical studies and thrombin generation in patients homozygous or heterozygous for the G20210A mutation in the prothrombin gene

A genetic variation in the prothrombin gene, the G-->A transition at nucleotide 20210, is a risk factor for venous thrombosis in heterozygotes and is associated with increased prothrombin activity. The homozygous phenotype and the extent of thrombin generation in heterozygous and homozygous subjects are unknown. We investigated a family that included 2 homozygous and 5 heterozygous carriers of the 20210 A allele. The homozygous propositus and his presumably heterozygous father suffered from deep-vein thrombosis. His presumably heterozygous mother and his homozygous sister had recurrent phlebitis at a young age. The remaining 5 affected family members are still asymptomatic. We studied thrombin generation in the family and in 22 unrelated carriers of the 20210 A allele by measuring (1) prothrombin fragment F1+2 (F1+2) as an index of ongoing thrombin generation and (2) the endogenous thrombin potential (ETP) as an index of the possible thrombin-forming capacity. Their F1+2 levels were not different from those of age-matched controls, and thus, ongoing hemostatic system activation was not detectable. A significantly increased ETP was found in the heterozygous carriers of the 20210A allele compared with the controls (527.8+/-114.9 versus 387+/-50.1 nmol/L x min, P<0.0001). In the 2 homozygotes, the ETP was almost twice (639 and 751 nmol/L x min, respectively) as high as in the controls. We conclude that homozygosity for the G20210A mutation in the prothrombin gene is associated with a severe, albeit more benign, thrombotic diathesis compared with homozygosity for deficiencies of antithrombin, protein C, or protein S. In carriers of the 20210 A allele, the pathomechanisms leading to thrombosis should be sought in the higher amounts of thrombin that may be formed once thrombin generation is triggered, rather than in ongoing thrombin generation in vivo.     

Therapeutic inhibition of thrombin activities, receptors, and production

Thrombin mediates acute vascular thrombosis following mechanical denuding injury or spontaneous rupture of atherosclerotic plaques. In the process of generating thrombin, factor VII/VIIa binds avidly with tissue factor exposed on cellular membranes, leading to sequential activation of coagulation serine proteases via macromolecular catalytic complexes on phospholipid surfaces. At sites of disrupted arteries thrombin activates platelets, blood leukocytes, endothelium, and vascular SMCs by cleaving G protein-coupled TRs, mediating SMC intimal proliferation in the formation of neointimal vascular lesions. Therapeutic strategies targeting thrombin include inactivation of bound thrombin, inhibition of TR activation by thrombin, and interruption of thrombin production. In patients having orthopedic surgery, inactivating bound thrombin with direct antithrombins markedly reduces venous thromboembolism as compared with heparin or its derivatives, without significant impairment of hemostasis. Antithrombotic effects in arterial thromboembolism, such as acute coronary syndrome, are not conclusively benefitted by systemic direct antithrombins when administered at safe levels, because interrupting TR-dependent platelet thrombosis demands systemic levels of direct antithrombins that compromise hemostatic function. Alternative safer strategies evolving from preclinical studies include (1) inhibiting thrombin activation of TRs, thereby abolishing platelet recruitment in arterial thrombogenesis, while sparing fibrin formation in hemostatic plugs; (2) enhancing the formation of endogenous activated protein C by protein C-selective thrombin mutants; and (3) preventing thrombin production by inhibiting precursor serine protease function and interrupting the formation of both acute thrombosis and vascular lesion formation. Tissue factor pathway antagonists are particularly promising because they exhibit both efficacy and safety in the prevention of thrombosis and vascular lesions.