Factor XII-dependent increases in thrombin activity induce carboxypeptidase-mediated attenuation of pharmacological fibrinolysis.

Activation of the contact system in patients treated with fibrinolytic agents may be an important source of thrombin that activates thrombin-activated fibrinolysis inhibitor (TAFI) and attenuates fibrinolysis. Factor (F)XIIa in plasma increased 2-fold over 60 min in patients given either tissue plasminogen activator (t-PA) or streptokinase (SK). To determine whether FXIIa-mediated generation of thrombin and activated TAFI (TAFIa) attenuates fibrinolysis in vitro, plasma clots were incubated with SK (250 U mL-1) or t-PA (2.5 g mL-1) and the rate of lysis was measured. Plasma FXIIa impaired lysis judging from marked acceleration when 2.5 micro m corn trypsin inhibitor were added (lysis increased by 172 +/- 144% for SK and 40 +/- 31% for t-PA vs. no inhibitor, n = 16, P < 0.01). Moreover, inhibition of thrombin with hirudin and TAFIa with carboxypeptidase inhibitor accelerated lysis. We conclude that activation of FXII increases thrombin generation, which promotes TAFIa-mediated attenuation of fibrinolysis.

Pharmacodynamics and pharmacokinetics of eptifibatide in patients with acute coronary syndromes: prospective analysis from PURSUIT.

BACKGROUND: Platelet deposition and aggregation are central to the pathogenesis of ischemic complications of acute coronary syndromes (ACS). Pharmacodynamic effects of the platelet glycoprotein IIb/IIIa antagonist eptifibatide have been delineated in healthy subjects but not in patients with ACS. We assessed effects of eptifibatide on ex vivo platelet aggregation in patients enrolled in the Platelet glycoprotein IIb/IIIa in Unstable angina: Receptor Suppression Using Integrilin (eptifibatide) Therapy (PURSUIT) trial of ACS. METHODS AND RESULTS: Patients were randomly assigned to an intravenous bolus (180 microgram/kg) and 72-hour infusion of eptifibatide (2.0 microgram/kg per minute, n=48) or placebo (n=50). We assessed correlations of plasma eptifibatide levels with receptor occupancy and inhibition of ex vivo platelet aggregation at 5 minutes and 1, 4, 24, 48, and 72 hours during treatment and 4 and 8 hours after termination of infusion. Blood was collected in buffered citrate and D-phenylalanyl-L-prolyl-L-arginine chloromethylketone anticoagulants. Although eptifibatide produced profound, prolonged inhibition of platelet aggregation during therapy, aggregation appeared to recover partially by 4 hours after the bolus. The aggregation response was greater with thrombin receptor agonist peptide versus ADP stimulation; inhibition of platelet aggregation was greater in blood samples anticoagulated with citrate versus D-phenylalanyl-L-prolyl-L-arginine chloromethylketone (PPACK). Plasma eptifibatide levels correlated significantly with receptor occupancy but not with inhibition of platelet aggregation. CONCLUSIONS: A bolus and infusion of eptifibatide inhibits platelet aggregation profoundly in patients with ACS and is followed by brief, partial recovery. These results enhance our understanding of the relation between pharmacodynamic and clinical effects of eptifibatide in such patients and may have important implications for its use in percutaneous interventions.

N-acetyl-cysteine reduces neointimal thickening and procoagulant activity after balloon-induced injury in abdominal aortae of New Zealand white rabbits.

BACKGROUND: Procoagulant activity and oxidative stress generated by balloon injury to normal vessels promote the migration of medial smooth muscle cells and their proliferation in the intima. We hypothesised that administering levo N-acetyl-cysteine (NAC) i.v. at the time of injury, and s.c. before and after injury would reduce neointimal formation 4 weeks later and would regulate procoagulant activity in vessels with neointima undergoing ballooning a second time. METHODS AND RESULTS: at the time of injury rabbits received: NAC, unfractionated heparin (HEP) or both (NAC + HEP). Neointimal thickening at 28 days, calculated as the ratio between the intimal and medial area, was attenuated after NAC, HEP and NAC+HEP by 39%, 30% and 47% respectively when compared to untreated injured animals (CONTROLS) (p <0.05). At 28 days, bound thrombin activity and platelet adhesion 1 h after a repeated balloon injury decreased in animals receiving NAC, HEP and NAC+HEP bv 54%, 63% and 64% for thrombin activity (p <0.05 vs CONTROLS), and by 56%, 66% and 75% respectively for 111Indium-platelet deposition (p <0.05 vs CONTROLS). CONCLUSIONS: NAC in-vivo was effective in reducing neointimal thickening and procoagulant response after balloon injury.

A novel synthetic inhibitor of factor Xa decreases early reocclusion and improves 24-h patency after coronary fibrinolysis in dogs.

Inhibition of factor Xa (FXa) attenuates thrombus progression. This study was designed to determine whether a novel, synthetic inhibitor of FXa (ZK-807834, molecular mass 527 Da, K(i) = 0.11 nM) administered during and briefly after pharmacologic coronary fibrinolysis increases 24-h patency. Either ZK-807834 (< or = 1.6 mg/kg, n = 10; 6.5 mg/kg, n = 8; or 13 mg/kg, n = 7); a peptide inhibitor of FXa, recombinant tick anticoagulant peptide (rTAP, 13.6 mg/kg, n = 7); heparin (150 U/kg bolus and 50 U/kg/h infusion) and aspirin (5 mg/kg) (n = 7); or saline as a control (n = 13) were administered i.v. over 135 min in conscious dogs after thrombotic occlusion induced by electrical injury to a coronary artery. Fibrinolysis was induced with recombinant human tissue-type plasminogen activator (1.0 mg/kg i.v. over 1 h), and patency was monitored continuously for 24 h with an implanted Doppler probe. Reocclusion occurred in all control and heparin/aspirin-treated dogs within 1 h after fibrinolysis. High dose ZK-807834 prevented reocclusion in five of six dogs and delayed reocclusion in the other dog (186 min after recanalization, p = 0.0005 versus heparin/aspirin). Reocclusion was delayed (406 +/- 329 min), but still occurred in three of six rTAP-treated dogs (p = 0.003 versus heparin/aspirin). Patency after 24 h was 100% in ZK-807834-treated and rTAP-treated dogs compared with 67% in control and 83% in heparin/aspirin-treated dogs. PT was increased 3.7-fold, activated partial thromboplastin time 4.9-fold, and bleeding time 2.5-fold by high dose ZK-807834 compared with 1.2-fold, 11.5-fold, and 2.3-fold, respectively, for heparin/aspirin. Inhibition of FXa with ZK-807834 decreases reocclusion and improves patency of recanalized arteries without increasing bleeding compared with heparin/aspirin.

Role of tissue factor-mediated coagulation in ischemia/ reperfusion-induced injury of Langendorf-perfused rabbit hearts.

BACKGROUND: Production of oxygen free radicals, and activation of neutrophils and plasma complement contribute to myocardial reperfusion injury, but the role of coagulation has not been assessed. OBJECTIVE: To characterize tissue-factor-mediated generation of thrombin and its association with tissue injury during reperfusion from normothermic ischemia of isolated, Langendorf-perfused rabbit hearts. METHODS: Activation of coagulation was assessed by addition of 12% rabbit plasma and human fibrinogen to Krebs-Henseleit-buffer perfusate with measurement of levels of human fibrinopeptide A (hFPA) in the heart effluent as an index of thrombin-mediated formation of fibrin. RESULTS: Concentrations of hFPA in the effluent were minimal during non-ischemic perfusion (5 +/- 5 ng/ml, n=6) and during 50 min of ischemia (13 +/- 3 ng/ml, n=6), but increased markedly during the first 20 min of reperfusion (to 41 +/- 29 ng/ml, P=0.03 versus before reperfusion). Addition to the perfusate of 10 microg/ml recombinant human tissue-factor-pathway inhibitor, the physiologic inhibitor of tissue-factor-mediated coagulation, abolished increases in the level of hFPA after reperfusion. However, indexes of myocardial injury manifested during reperfusion, including decrease in recovery of left ventricular pressure developed, increase in left ventricular end-diastolic pressure, and increase in activity of creatine kinase in the heart effluent, were not improved by anticoagulation with recombinant human tissue-factor-pathway inhibitor. CONCLUSION: Our results do not support the hypothesis that coagulation plays a major role in ischemia/reperfusion injury of Langendorf-perfused rabbit hearts.

Effects of ZK-807834, a novel inhibitor of factor Xa, on arterial and venous thrombosis in rabbits.

Inhibition of factor Xa (FXa) may interrupt thrombus progression. This study compared the antithrombotic activity of a novel FXa inhibitor, ZK-807834 [MW, 527 D; Ki (human FXa), 0.11 nM], with recombinant tick anticoagulant peptide [rTAP; MW, 6,685 D; Ki, (human FXa) = 0.28 nM], and DX-9065a [MW 445 D, Ki (human FXa), 40 nM] in rabbits with arterial thrombosis induced by electrical vascular injury. ZK-807834 also was compared with low molecular weight heparin (LMWH; MW, 5,500 D) during venous thrombosis induced by placing a copper wire and threads in the vena cava. Inhibitors were administered as an i.v. bolus and 2-h infusion. Total dosages of ZK-807834, > or =0.7 micromol/kg (n = 18); rTAP, > or =1 micromol/kg (n = 18); or DX-9065a, > or =11 micromol/kg (n = 18) decreased the incidence of arterial thrombotic occlusion compared with control animals (p < 0.05). However, five of six animals given the lowest effective dosage of rTAP and four of six animals given DX-9065a bled from a surgical incision >5 min, but only two of six animals given ZK-807834 bled >5 min. Venous clot weights were reduced compared with controls for dosages of ZK-807834 > or =0.007 micromol/kg (n = 36) or LMWH > or =0.2 micromol/kg (n = 18). Prothrombin time (PT) and activated partial thromboplastin time (aPTT) were unchanged from baseline at the minimally effective dose of ZK-807834, whereas aPTT was increased twofold at the effective dose of LMWH. Thus ZK-807834 may be useful to attenuate thrombosis at lower dosages and with less perturbation of systemic hemostasis compared with available agents.

Predictive value of a rapid semiquantitative D-dimer assay in critically ill patients with suspected venous thromboembolic disease.

OBJECTIVE: To evaluate the performance of a new, rapid semi-quantitative assay for the detection of circulating D-dimer in whole blood from critically ill patients with suspected venous thromboembolic disease. DESIGN: Prospective, blinded, single-center study. SETTING: Medical intensive care unit (ICU) of Barnes-Jewish Hospital, St. Louis, MO, a university-affiliated urban teaching hospital. PATIENTS: Two hundred thirty-nine adult patients with clinical suspicion of venous thromboembolic disease admitted to a medical ICU. INTERVENTIONS: Collection of blood samples within 24 hrs of clinical suspicion of venous thromboembolic disease. MEASUREMENTS AND MAIN RESULTS: The main outcome measures evaluated included the occurrence of venous thromboembolic disease (i.e., lower extremity venous thrombosis, pulmonary embolism, catheter-associated venous thrombosis) and hospital mortality. Fifty-seven patients (23.8%) were classified as having venous thromboembolic disease during their ICU stays (pulmonary embolism, 21 patients; lower extremity thrombosis, 44 patients; line-associated venous thrombosis, 3 patients). The semiquantitative whole-blood assay for circulating D-dimer had a 96.4% sensitivity and a negative predictive value of 92.1% for identifying patients with venous thromboembolic disease. The specificity of this assay was 19.7%, and its positive predictive value was 26.9%. There was a strong correlation between the semiquantitative assay for circulating D-dimer and the quantitative measurement of circulating D-dimer using an enzyme immunoassay (Spearman's correlation coefficient, 0.8643; p<.001). We also identified a strong correlation between both the quantitative and semiquantitative measurements of circulating D-dimer with the sepsis classification proposed by the American College of Chest Physicians/Society of Critical Care Medicine (i.e., systemic inflammatory response syndrome, sepsis, severe sepsis, septic shock) for patients without venous thromboembolic disease (n = 182; quantitative measure: Spearman's correlation coefficient, 0.207; p = .002; semiquantitative measure: Spearman's correlation coefficient, 0.3519; p<.001). CONCLUSIONS: These preliminary findings suggest that a rapid whole-blood assay for the semiquantitative detection of circulating D-dimer may be a useful diagnostic tool for the exclusion of venous thromboembolic disease among critically ill patients.

Adenosine as an adjunct to thrombolytic therapy for acute myocardial infarction: results of a multicenter, randomized, placebo-controlled trial: the Acute Myocardial Infarction STudy of ADenosine (AMISTAD) trial.

OBJECTIVES: The Acute Myocardial Infarction STudy of ADenosine (AMISTAD) trial was designed to test the hypothesis that adenosine as an adjunct to thrombolysis would reduce myocardial infarct size. BACKGROUND: Reperfusion therapy for acute myocardial infarction (MI) has been shown to reduce mortality, but reperfusion itself also may have deleterious effects. METHODS: The AMISTAD trial was a prospective, open-label trial of thrombolysis with randomization to adenosine or placebo in 236 patients within 6 h of infarction onset. The primary end point was infarct size as determined by Tc-99 m sestamibi single-photon emission computed tomography (SPECT) imaging 6+/-1 days after enrollment based on multivariable regression modeling to adjust for covariates. Secondary end points were myocardial salvage index and a composite of in-hospital clinical outcomes (death, reinfarction, shock, congestive heart failure or stroke). RESULTS: In all, 236 patients were enrolled. Final infarct size was assessed in 197 (83%) patients. There was a 33% relative reduction in infarct size (p = 0.03) with adenosine. There was a 67% relative reduction in infarct size in patients with anterior infarction (15% in the adenosine group vs. 45.5% in the placebo group) but no reduction in patients with infarcts located elsewhere (11.5% for both groups). Patients randomized to adenosine tended to reach the composite clinical end point more often than those assigned to placebo (22% vs. 16%; odds ratio, 1.43; 95% confidence interval, 0.71 to 2.89). CONCLUSIONS: Many agents thought to attenuate reperfusion injury have been unsuccessful in clinical investigation. In this study, adenosine resulted in a significant reduction in infarct size. These data support the need for a large clinical outcome trial.

The independence of maximal intimal thickening from severity of balloon injury in the rabbit aorta.

Endothelial injury induces intimal thickening, but whether more extensive injury increases the extent of neointimal proliferation in the rabbit aorta is not well defined. We induced graded injury in the abdominal aortas of rabbits and maximal intimal/medial (I/M) area and thickness ratios were calculated from aortic cross sections harvested 2 weeks after injury. The degree of injury was verified by blinded observers who graded the extent of disruption of the internal elastic laminae. Intimal thickening was not significantly different after severe injury (mean maximal I/M area ratio 0.32+/-0.02 [SE], n = 16) compared with moderate injury (0.23+/-0.02, n = 8, p = 0.24), but was greater than that induced by mild injury (0.08+/-0.01, n = 7, p <0.0001). The ratio of the maximal I/M thickness was similar in all groups (I/M thickness ratio 0.68+/-0.04, 0.73+/-0.04, and 0.56+/-0.04 for severe, moderate, and mild focal injury groups. respectively; p = 0.19). Thus, balloon injury of the rabbit aorta induces reproducible thickening of the intima by 2 weeks. The maximal I/M area ratio is dependent on the extent of injury, while the maximal intimal thickening is independent.

Platelet-dependent and procoagulant mechanisms in arterial thrombosis.

The results of experiment and clinical studies support the hypothesis that initiation and progression of arterial thrombosis are dependent on a dynamic interaction between platelet- and coagulation-dependent mechanisms. Although treatment of patients with unstable coronary syndromes acutely with glycoprotein IIb/IIIa inhibitors has already been shown to be remarkably effective in improving short- and long-term prognosis, strategies that inhibit factor thrombin and/or Xa activity may also have a role either alone or in combination with platelet inhibitors in treating patients with coronary thrombosis.

Early risk stratification of unstable angina/non-Q myocardial infarction: biochemical markers of coronary thrombosis.

Coronary thrombosis is an important determinant of prognosis in patients with acute coronary syndromes (ACS). However, the identification of patients at high-risk for progression of coronary thrombosis is difficult partly because we currently lack clinically meaningful laboratory methods for its detection. The most promising approaches involve the measurement in plasma of markers of fibrin formation and degradation. Thrombin activity, as reflected by plasma or urine concentrations of fibrinopeptide A, is increased in patients with ACS and is associated with adverse outcome. However, the use of fibrinopeptide A as a marker of fibrin formation is limited by the very short half-life of the compound, by artifact due to sample acquisition, and by extremely long turnaround times. To overcome these limitations, measurement of soluble fibrin has been proposed. We have recently explored the prognostic value of a new fibrin-specific ELISA assay for soluble fibrin in patients with ACS and found that patients with the highest levels had a 2-fold increased risk of early and late cardiac events. Increases in plasma concentrations of cross-linked fibrin degradation products (XL-FDPs), which reflect increased fibrin turn-over, are a marker of risk for complications of myocardial infarction. However, until recently, assays for XL-FDPs lacked specificity, because they did not distinguish between fibrin and fibrinogen degradation products. Recently, fibrin-specific ELISAs have been described and a rapid whole blood assay for D-dimer has been developed. We recently validated the prognostic value of this whole blood agglutination assay in patients with ACS. These results suggest that: (1) The detection of significant activation of the coagulation and/or fibrinolytic system may be important for rapid risk stratification of patients with ACS; (2) patients with biochemical evidence of ongoing coronary thrombosis may particularly benefit from aggressive antithrombotic strategies; (3) sequential measurement of these markers may be useful to guide antithrombotic treatment during the unstable phase of coronary artery disease.

Prior aspirin use predicts worse outcomes in patients with non-ST-elevation acute coronary syndromes. PURSUIT Investigators. Platelet IIb/IIIa in Unstable angina: Receptor Suppression Using Integrilin Therapy.

Aspirin is beneficial in the prevention and treatment of cardiovascular events, but patients who have events while taking aspirin may have worse outcomes than those not on aspirin. We investigated the association between prior aspirin use and clinical outcomes in 9,461 patients with non-ST-elevation acute coronary syndromes enrolled in the Platelet IIb/IIIa in Unstable angina: Receptor Suppression Using Integrilin Therapy (PURSUIT) trial, before and after adjustment for baseline factors. We also examined whether eptifibatide has a differential treatment effect in prior aspirin users. Prior aspirin users were less likely to have an enrollment myocardial infarction (MI) (vs unstable angina) (43.9% vs 48.8%, p = 0.001) but more likely to have death or MI at 30 days (16.1% vs 13.0%, p = 0.001) and at 6 months (19.9% vs 15.9%, p = 0.001). After adjustment, prior aspirin users remained less likely to have an enrollment MI (odds ratio 0.88, 95% confidence interval 0.79 to 0.97) and more likely to have death or MI at 30 days (odds ratio 1.16, 95% confidence interval 1.00 to 1.33) but not at 6 months (odds ratio 1.14, 95% confidence interval 0.98 to 1.33). In a multivariable model, eptifibatide did not have a different treatment effect in prior aspirin users compared with nonusers (p = 0.534). Prior aspirin users had fewer enrollment MIs but worse long-term outcomes than nonusers. We found no evidence for a different treatment effect of eptifibatide in prior aspirin users.

In vitro characterization of a novel, tissue-targeted ultrasonic contrast system with acoustic microscopy.

Targeted ultrasonic contrast systems are designed to enhance the reflectivity of selected tissues in vivo [Lanza et al., Circulation 94, 3334 (1996)]. In particular, these agents hold promise for the minimally invasive diagnosis and treatment of a wide array of pathologies, most notably tumors, thromboses, and inflamed tissues. In the present study, acoustic microscopy was used to assess the efficacy of a novel, perfluorocarbon based contrast agent to enhance the inherent acoustic reflectivity of biological and synthetic substrates. Data from these experiments were used to postulate a simple model describing the observed enhancements. Frequency averaged reflectivity (30-55 MHz) was shown to increase 7.0 +/- 1.1 dB for nitrocellulose membranes with targeted contrast. Enhancements of 36.0 +/- 2.3 dB and 8.5 +/- 0.9 dB for plasma and whole blood clots, respectively, were measured between 20 and 35 MHz. A proposed acoustic transmission line model predicted the targeted contrast system would increase the acoustic reflectivity of the nitrocellulose membrane, whole blood clot, and fibrin plasma clot by 2.6, 8.0, and 31.8 dB, respectively. These predictions were in reasonable agreement with the experimental results of this paper. In conclusion, acoustic microscopy provides a rapid and sensitive approach for in vitro chracterization, development, and testing of mathematical models of targeted contrast systems. Given the current demand for targeted contrast systems for medical diagnostic and therapeutic use, the use of acoustic microscopy may provide a useful tool in the development of these agents.

A rapid assay for the detection of circulating D-dimer is associated with clinical outcomes among critically ill patients.

OBJECTIVE: To determine whether the results of a rapid, semiquantitative assay for the detection of circulating D-dimer in whole blood (SRDD assay) are associated with the occurrence of clinical outcomes among critically ill patients. DESIGN: Prospective, blinded, single-center study. SETTING: Medical intensive care unit (ICU) of Barnes-Jewish Hospital, St. Louis, MO, a university-affiliated teaching hospital. PATIENTS: Three hundred twenty-three adult patients admitted to a medical ICU. INTERVENTIONS: Collection of blood samples within 24 hrs of ICU admission. MEASUREMENTS AND MAIN RESULTS: The main outcome measures evaluated included vascular thrombosis, hospital mortality, and the development of multiorgan dysfunction. Fifty (15.5%) patients were found to have increased concentrations of D-dimer as detected by the SRDD assay within 24 hrs of ICU admission. The concentrations of plasma D-dimer simultaneously measured by an enzyme immunoassay based on the same antibody were significantly greater among patients with a positive SRDD assay compared with patients with a negative SRDD assay (1214+/-483 vs. 405+/-407 ng/mL; p< .001). The hospital mortality rate was significantly greater among SRDD-positive patients compared with SRDD-negative patients (32.0% vs. 15.0%; p=.004). SRDD-positive patients also had significantly greater frequencies of acquired multiorgan dysfunction (48.0% vs. 17.6%; p < .001), severe sepsis or septic shock (56.0% vs. 20.9%; p< .001), and vascular thrombosis (14.0% vs. 4.0%; p=.005) compared with SRDD-negative patients. Multiple logistic regression analysis identified the presence of increased concentrations of D-dimer, detected by a positive SRDD assay, as being independently associated with vascular thrombosis (adjusted odds ratio 5.06; 95% confidence interval 2.96 to 8.65; p=.003) and the development of multiorgan dysfunction (adjusted odds ratio 1.51; 95% confidence interval 1.28 to 1.78; p=.012). CONCLUSIONS: Our preliminary investigation suggests that the results from a rapid whole blood assay for the semiquantitative detection of circulating D-dimer are associated with clinical outcomes among patients admitted to a medical ICU. In addition, the use of D-dimer to identify the presence of active intravascular thrombosis may identify patients likely to benefit from antithrombotic therapies in the ICU setting.

Prolonged activation of prothrombin on the vascular wall after arterial injury.

This study was designed to characterize the relative roles of bound Xa/Va and thrombin activity in vascular wall procoagulant activity after balloon-induced injury and the extent to which intravenous aspirin and heparin attenuate procoagulant activity associated with the vascular wall. Abdominal aortic injury was induced in rabbits by overinflation and multiple passages ofa 4F embolectomy catheter. Rabbits were killed 15 minutes or 4, 8, 24, 48, 72, 96, or 120 hours after injury. Aortic segments were incubated ex vivo to define bound procoagulant activity. Thrombin activity bound to the aorta was detected by 4 hours after injury and was most marked over the first 24 hours, as estimated by increases in concentration of fibrinopeptide A during incubation of segments with recalcified barium-adsorbed plasma or activity against the thrombin-synthetic substrate S-2238. Based on comparison with purified human thrombin incubated under the same conditions, a maximum of 0.04 to 0.1 nmol/L per square centimeter of thrombin activity was associated with the vascular wall during the first 24 hours and remained detectable for 72 hours. In contrast, bound Xa/Va complex activity to injured segments was detected within 15 minutes and induced activation of prothrombin added to recalcified barium-adsorbed plasma incubated with injured segments for 96 hours. Aspirin (15 mg/kg) administered 30 minutes before injury attenuated 111In-platelet deposition at 4 hours by 67%, with an associated decrease in bound Xa/Va and thrombin activity at 15 minutes and 4 hours. However, intravenous heparin did not attenuate bound Xa/Va activity at 15 minutes or thrombin activity at 15 minutes and 4 hours. Platelet-dependent bound Xa/Va activity occurs rapidly after arterial injury and may promote thrombin elaboration for up to 96 hours. Bound thrombin activity and de novo thrombin elaboration on the vascular wall may play an important role in the progression of thrombosis and vascular wall remodeling.

Rapid detection of D-dimer using a fiber optic biosensor.

We describe a rapid and sensitive method for detection and quantification of D-dimer and other crosslinked fibrin degradation products (XL-FDPs), which are present in elevated concentrations in patients with sepsis and thrombotic disorders. The method utilizes a sandwich fluoroimmunoassay immobilized in the sensing region of an evanescent wave biosensor. Physiological concentrations of D-dimer and high molecular weight XL-FDP could be determined in buffer and plasma samples on calibrated fibers in 11 min. Samples from septic patients were assayed using ELISA and the fiber optic method; concentrations determined by fiber optic assay were strongly correlated with those determined by ELISA (r = 0.918); intra- and inter-assay errors were comparable to those from ELISAs. Given its accuracy and rapid response time, this fiber optic biosensor shows great potential for development as a diagnostic tool.

Arterial thrombosis induces early upregulation of intercellular adhesion molecule in the media.

PURPOSE: Thrombosed peripheral vessels that are pharmacologically or mechanically recanalized have diminished long-term patency rates compared with vessels that are repaired before occlusion. We hypothesized that thrombosis induces proinflammatory changes in the arterial media that may contribute to postthrombotic vascular remodeling. METHODS: We studied expression of intercellular adhesion molecule (ICAM), a mediator of leukocyte recruitment, in the arterial wall after thrombosis. Thrombosis was induced in rabbit superficial femoral arteries by embolizing polystyrene beads (Thr-emb) or by ligation (Thr-lig). Control vessels were dissected but not ligated (C-dis) or were subjected to bead embolization and immediate removal (C-emb). Arterial wall ICAM expression was measured by indirect immunohistochemical analysis at 6 hours, 24 hours, and 1 week. Staining intensity was graded from 0 (none) to 4 (intense) by observers who were blinded to the experimental conditions. RESULTS: No increase in ICAM expression by thrombosed vessels was present at 6 hours. After 24 hours, ICAM expression in the media of thrombosed vessels was increased (Thr-emb, 2.3 +/- 0.5; Thr-lig, 2.0 +/- 0) compared with control vessels (C-dis, 0 +/- 0; C-emb, 0.8 +/- 0.5; p < 0.004). This difference became more marked at 1 week. ICAM staining localized to actin-staining regions of the media. CONCLUSIONS: Arterial thrombosis, but not surgical injury, induces pronounced early and sustained upregulation of ICAM expression in smooth muscle-containing regions of the arterial media. Upregulation of ICAM is likely to promote recruitment of inflammatory cells or mediate vascular remodeling after luminal thrombosis.